*Review* **A Review and Evaluation of the Data Supporting Internal Use of** *Helichrysum italicum*

**Katja Kramberger 1,2, Saša Kenig 1, Zala Jenko Pražnikar 1, Nina Koˇcevar Glavaˇc <sup>3</sup> and Darja Barliˇc-Maganja 1,\***


**Abstract:** *Helichrysum italicum* is a Mediterranean plant with various pharmacological activities. Despite extensive reports on the bioactivity of the plant, its clinically studied applications have not yet been reviewed. The aim of our study was to gather information on the internal use of *H. italicum* and its bioactive constituents to determine its efficacy and safety for human use. We reviewed research articles that have not been previously presented in this context and analyzed relevant clinical studies with *H. italicum*. Cochranelibrary.com revealed six eligible clinical trials with *H. italicum* that examined indications for pain management, cough, and mental exhaustion. Although the efficacy of *H. italicum* has been demonstrated both in in vitro tests and in humans, it is difficult to attribute results from clinical trials to *H. italicum* alone, as it has usually not been tested as the sole component. On the other hand, clinical trials provide positive information on the safety profile since no adverse effects have been reported. We conclude that *H. italicum* is safe to use internally, while new clinical studies with *H. italicum* as a single component are needed to prove its efficacy. Based on the recent trend in *H. italicum* research, further studies are to be expected.

**Keywords:** *Helichrysum italicum*; biological activity; internal use; clinical studies

## **1. Introduction**

Plants have a long history of use in medicine and have been used by all cultures or ethnic groups throughout history to improve human health [1]. They are considered to be the oldest form of medicine known to humankind but are, on the other hand, also an important source of modern medicines and govern synthetic drug development [1,2]. According to the World Health Organization (WHO), 70–95% of the world's population rely on traditional medicine for their primary health care [2]. This is especially true for the Mediterranean countries, where plants play a vital role in the diet habits, and sometimes there is no clear dividing line between food and medicinal plants, particularly in indigenous and local traditions [3].

The plants belonging to the genus *Helichrysum* (family Asteraceae) are known as everlasting flowers and are widely used in traditional medicine worldwide [4]. The plant species of this genus typically have inflorescences of a bright yellow color [5], which retain their form and color when dried, hence the name "everlasting" or "immortal" [4]. The stems are woody at the base and can reach 30–70 cm in height. The plant is well adapted to environments that lack water as it naturally grows on alkaline, dry, sandy and poor soil at the altitude from the sea level up to 2200 m [6]. The *Helichrysum* Miller genus includes more than a thousand of taxa, among them the most well-known and studied species are *Helichrysum italicum* (Roth) G. Don [7] (Figure 1), *Helichrysum stoechas* (L.) Moench [8], and *Helichrysum arenarium* (L.) Moench [9]. *H. italicum* and *H. stoechas* are distributed throughout the Mediterranean [10] but are especially characteristic to Adriatic region [11] and Iberian Peninsula [12], respectively, whereas *H. arenarium* is mostly found

**Citation:** Kramberger, K.; Kenig, S.; Jenko Pražnikar, Z.; Koˇcevar Glavaˇc, N.; Barliˇc-Maganja, D. A Review and Evaluation of the Data Supporting Internal Use of *Helichrysum italicum*. *Plants* **2021**, *10*, 1738. https:// doi.org/10.3390/plants10081738

Academic Editors: Sofia Rhizopoulou, Maria Karatassiou and Efi Levizou

Received: 23 July 2021 Accepted: 18 August 2021 Published: 23 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in the Central Europe [9,10]. Despite the long tradition in treatment of various disorders for all three before-mentioned species, their traditional use for treating digestive problems (e.g., fullness and bloating) has been approved by the WHO and the European Medicines Agency (EMA) for the species *H. arenarium* alone [8]. Nevertheless, *H. italicum* has been recently investigated quite extensively, especially in the Balkan countries. The focus of this review article will be from herein after on *H. italicum* only. Among the *H. italicum* species, there are also several subspecies (abbreviated ssp.), which are difficult to distinguish due to a strong polymorphism in morphology [10]. The explanation of *H. italicum* classification is beyond the scope of this review article, but the name of subspecies is included, if available in the referenced article.

**Figure 1.** Cultivated *Helichrysum italicum* plant in flowering stage (Photo: Katja Kramberger).

Characteristic yellow fade-resistant inflorescences (seen in Figure 1) as well as vegetative aerial organs of *H. italicum* are a treasury of bioactive secondary metabolites that result from the plants' adaptation to this challenging environment. Apart from the volatile terpenes present in essential oils, absolutes and supercritical CO2 extracts, *H. italicum* is also very rich in phenolic compounds, which are recognized as health promoting agents due to antioxidant properties they exert and their probable role in the prevention of various diseases associated with oxidative stress, such as cancer, cardiovascular and neurodegenerative diseases [13]. The health-beneficial potential of *H. italicum* has been reported in ethnopharmacological surveys and supported by numerous in vitro and in vivo experiments [7]. In the Greek-Roman system of medicine, *H. italicum* was used as an antiinflammatory and anti-infective plant, and both uses are still well rooted in traditional medicine today [14]. Antunes Viegas et al. [7] emphasized that, in contrast to animal studies, there is a severe lack of clinical studies investigating the effects of the *H. italicum* extracts, which undermines the possibility of validating the traditional uses of this plant. One of the reasons why clinical and other comprehensive studies with herbal products are scarce probably lies in the difficulty of interpreting the results of such studies [2].

The aim of our study was to gather all information reported in the literature on internal applications of *H. italicum* or its bioactive constituents in humans to support the efficacy and safety of *H. italicum* preparations for human use. We reviewed studies that have not been outlined in this context before and analyzed relevant clinical trials with *H. italicum* as the main or one of the studied components.

## **2. Methodology**

Research and review articles were searched via online databases including PubMed and Scopus until February 2021. Clinical trials on *H. italicum* and its constituents were searched through Cochrane Library (https://www.cochranelibrary.com/) and ClinicalTrials (https://clinicaltrials.gov/) (last accessed on 2 May 2021).

The reviewed literature on *H. italicum* use is presented in thematic sections; ethnopharmacological surveys and non-clinical research articles (Section 3), human clinical observations (Section 4), clinical trials (Section 5) and recently published articles (Section 6). The first section briefly reviews the use of *H. italicum* in traditional medicine, as well as the most prominent in vitro and in vivo scientific experiments. Section on human clinical observation summarizes the experiments in humans, which were not registered as clinical trials. Clinical trials section is divided in clinical trials with *H. italicum* (Section 5.1) and trials with isolated compounds commonly found in *H. italicum* (Section 5.2). Lastly, recently published articles on *H. italicum* are presented to provide insight into the current trend of *H. italicum* research.

## **3. Traditional Uses and Scientific Data**

Ethnopharmacological surveys on *H. italicum,* summarized in the study by Antunes Viegas et al. [7], show that the most frequently reported traditional uses are related to respiratory, digestive and skin inflammatory conditions. Depending on the application, *H. italicum* preparations are administered via inhalation, ingestion or topically. Other therapeutic applications include wound healing and antimicrobial uses, as well as gall and bladder disorders and analgesic use. Common types of preparations are mostly infusions and decoctions, for both oral and external use, followed by vapors, juices and powders [7]. Several in vitro as well as in vivo studies have confirmed biological activity of compounds isolated from *H. italicum* or its fractionated extracts, whereas for some indications such as digestive non-inflammatory disorders, pain in the gastrointestinal tract, alopecia, helminthic infections, and sleeplessness, scientific validation is still missing [6]. Moreover, studies investigating crude extracts, especially aqueous extracts, which are the most commonly used in traditional medicine, are rather scarce.

A large variety of *H. italicum* extracts can be prepared, and the resulting products differ in their chemical composition, yet the main compound classes are terpenes and phenolics. A systematic review of *H. italicum* bioactive compounds with regard to extraction procedure used for their isolation was conducted by Maksimovic et al. [15] and for the most characteristic compounds this information is summarized in Table 1. Research on *H. italicum* regained interest in the 1990s with the studies by Facino et al. [16,17], Pietta et al. [18,19] and by Zapesochnaya et al. [20,21] on isolation and identification of bioactive substances of Italian *H. italicum*. Numerous studies were performed in the following decades, in which additional bioactive constituents were isolated and in vitro and in vivo tests performed to support their bioactivity [14,22–42]. Nostro et al. [22–25] investigated anti-cariogenic potential of *H. italicum* diethyl ether and ethanolic extracts, which is probably attributed to flavonoids. Studies by Sala et al. [26–29] dealt with a class of acetophenone compounds and flavonoids pinocembrin, gnaphallin and tiliroside, isolated from *H. italicum*, and tested their anti-inflammatory action in mice. Noteworthy are also the studies by Appendino et al. [14], Rosa et al. [33,34] and Bauer et al. [35], who discovered and investigated a main anti-inflammatory compound present in *H. italicum*, arzanol. Its anti-inflammatory effects have also been proved in vivo [35]. Arzanol's activity and its mechanism of action are summarized in review by Kothavade et al. [43]. The following studies are worth mentioning due to validating traditional uses at in vivo level. Rigano et al. [39] proved that ethanol extract elicited antispasmodic action in the isolated mouse ileum and inhibited transit preferentially in the inflamed gut. The suitability of the traditional use of *H. italicum* ssp. *italicum* flowers for intestinal diseases was thereby confirmed. De la Garza et al. [38] showed that methanol-water extract decreased blood glucose levels and reduced postprandial glucose levels, as well as improved hyperinsulinemia in a dietary model of insulin resistance in rats. Furthermore, Pereira et al. [42] investigated anti-diabetic activity of water-based preparations (infusions and decoctions) of *H. italicum* ssp. *picardii* via inhibitory activity towards α-glucosidase and found moderate effects. Although diabetes is not one of the conditions mentioned in traditional medicine of *H. italicum*, this study opened another possible application—treatment of metabolic syndrome.

In recent years, increased interest for *H. italicum* was also observed in many Southern European countries, predominantly due to *H. italicum* essential oil and its use in the perfume and cosmetic industry. This topic is reviewed by Ninˇcevi´c et al. [10], who also focused on taxonomic classification and morphological characteristics arising from genetic diversity, in addition to bioactive compounds of *H. italicum* and their biological activity.

**Table 1.** The list of the most characteristic bioactive compounds found and investigated in *H. italicum*.



**Table 1.** *Cont.*

<sup>1</sup> Images of 2D structures of compounds were obtained from PubChem database. <sup>2</sup> Only the primary solvent used for extraction is mentioned. Solvents used in subsequent fractionation process are not listed but can be found in the referenced articles. <sup>3</sup> Extraction yields were calculated from the masses obtained after isolation procedures in response to the amount of the starting plant material. In addition, information on the plant part used in the extraction is included, if specified. <sup>4</sup> Preferentially only activity related to *H. italicum* investigation is reported, when available. NA—not available, NS—not specified.

## **4. Human Clinical Observations**

Despite several review articles on *H. italicum*, the following clinical observations are rarely mentioned. Systematic clinical studies on the anti-inflammatory properties of *H. italicum* were already carried out by Leonardo Santini, an Italian physician, in the 1940s. Despite the promising results, these investigations were largely overlooked at that time, but were later recognized as relevant for studies on anti-inflammatory activity of *H. italicum* [14]. The clinical experiments performed by Santini [49], Benigni [50], Vannini [51] and Campanini [52] are described in Italian literature and summarized in the

article by Appendino et al. [53]. These observations are presented in Table 2 along with additional studies by Facino et al. [16], Voinchet and Giraud-Robert [54], and a very recent one by Granger et al. [55]. These studies mainly cover treatments of respiratory and dermal conditions.

**Table 2.** Clinical observations in humans, listed in chronological order.


## **5. Registered Clinical Trials**

## *5.1. H. italicum Herb*

To date, no review of clinical trials including *H. italicum* has been conducted. Searching the Cochrane Library for the term "Helichrysum" in Title Abstract Keyword (Word variations have been searched) resulted in seven hits (https://www.cochranelibrary.com/, accessed on 4 January 2021). An additional record was identified through PubMed database. After inspection, one duplicate record was identified, and another one was excluded based on an inappropriate *Helichrysum* species investigated. Other six records addressed *H. italicum* alone or in combination with other herbs. The indications studied were pain (chronic prostatitis, post-surgical pain), cough and mental exhaustion. Consequently, diverse dosage forms were used: granules, syrup, inhalation preparation and suppositories. All relevant records were included in the qualitative synthesis, although few were missing full-text articles. The above-described process of literature search and article selection is shown in Figure 2. No meta-analysis could be performed, as there were too few trials

published, and the presented trials possessed too much heterogeneity both in studied conditions and in the formulations tested.

**Figure 2.** Flowchart of the trials' selection process. n—number of records/studies.

The reviewed clinical trials on *H. italicum* are summarized in Table A1 and described in more detail in the following paragraphs. The first trial, chronologically, was performed by Aboca S.p.A., an Italian company focused on innovative products based on natural substances. According to the EU clinical trials register record [56], two commercial products in the form of granules for oral suspension were administered as a pain treatment to adult patients with post-surgery pain. Freeze-dried extracts of *H. italicum* flowering tops and *Salvia officinalis* (sage) leaves were parallelly compared against placebo. Although trial status is "completed", the results are not available in the databases, and no articles have been published. We also tried contacting the company directly but were unsuccessful.

In the trial by Galeone et al. [57], *H. italicum* was incorporated in the medical device Proxelan® (Sala Bolognese, Italy) suppositories along with other plants: *Boswellia serrata*, *Centella asiatica* (Asiatic pennywort) and *Cucurbita pepo* (pumpkin) seeds. Altogether sixty subjects with bacterial and non-bacterial prostatitis were divided in two groups, one receiving antibiotic treatment and the other receiving antibiotics together with Proxelan® suppositories. Minor side effects were observed, but they did not cause trial interruption in any case. From a microbiological point of view, Proxelan® treatment was not better than antibiotics alone (*p* = 0.46). However, the combination of antibiotics and Proxelan® improved both symptoms associated to chronic prostatitis and urinary symptoms, which were two-fold decreased compared to control group after two months following the intervention (*p* = 0.028). The trial provides some relevant information on the safety since the rectal application can also have systemic effects. Conclusions regarding efficacy are not as straightforward, as *H. italicum* was not a single plant in that formulation.

Additional trial with the same product has been published more recently [58]. This time, authors aimed to investigate the effects of Proxelan® monotherapy on the pain symptoms of patients with a clinical diagnosis of chronic abacterial prostatitis or chronic pelvic pain syndrome. Proxelan® suppositories were prescribed to thirty male patients for a month with a daily dosage of one suppository at bedtime. Subjective pain relief was obtained in all the patients (*p* = 0.04). Urinary symptoms, investigated by questionnaire, decreased significantly (*p* = 0.04), and the quality of life improved (*p* = 0.04). Further seminal investigations were performed on a subset of patients. In a one-month follow-up, leukocytospermia decreased substantially or disappeared, IL-6 decreased by 11.55%, while IL-8 values did not show significant variation. The sperm motility increased by 17.3% and spermatozoa concentration remained unchanged. The medical device showed efficiency in pain reduction, as well as in improvement of semen quality by addressing the inflammatory component of this condition. This trial thus confirmed that Proxelan® monotherapy can be successfully used without antibiotics combination treatment to obtain comparable clinical outcomes in patients with chronic prostatitis or chronic pelvic pain syndrome symptoms. However, the results obtained should be investigated on a larger cohort of patients in randomized controlled trials.

Another trial was published by Varney and Buckle [59], who investigated the effect of *H. italicum* essential oil on mental exhaustion and moderate burnout. Patients were given a personal inhaler with mixture of essential oils (peppermint, basil, and everlasting) or placebo (rose water), which they administered themselves three times in each nostril every hour of the working day (approx. seven times per day) for the duration of five days. According to the authors, this mixture contained two stimulant essential oils to address the fatigue, and one balancing essential oil to address the anxiety. In aromatherapy, *Mentha* x *piperita* (peppermint) essential oil is used to increase alertness and mental clarity, and *Ocimum basilicum* ct *linalol* (basil) essential oil to reduce mental fatigue and achieve antidepressant properties. *H. italicum* essential oil is known for its calming and soothing properties. The participants self-assessed their feelings via questionnaire three times per day in the intervention week, as well as one week before and after. Both groups reported reduction in perception of mental exhaustion or moderate burnout, whereas for the aromatherapy group, reduction was two times greater. Although the results were encouraging, they may not be generalizable due to the small population tested and due to some reported inconsistency in the administration.

The trial led by Cohen et al. [60] was by far the most extensive and multicentered. It included 150 children over four pediatric clinics in Israel. The purpose was to determine if there is comparable efficacy between mucolytic substance carbocysteine and a protective cough syrup (Grintuss®, Sansepolcro, Italy) based on natural ingredients on children's cough due to upper respiratory tract infections, such as the common cold. Mucolytic agents have been shown to be helpful, but serious side effects have been reported, and the use has been prohibited for children under two years of age. Therefore, safer alternatives for cough management, which function via other mechanisms such as irritated pharynx mucosa protection, were explored. Grintuss® syrup contained a combination of specific substances such as resins, polysaccharides, saponins, flavonoids and sugars derived from *Grindelia robusta* (gumweed), *Plantago lanceolata* (ribwort plantain), *H. italicum*, and honey. The protective effect of the syrup on the mucosa of the upper respiratory tract was exerted by a local mechanical barrier (limiting cough stimuli with a non-pharmacological approach, but with an indirect anti-inflammatory action), as well as by radical scavenging action. A survey was conducted among parents on four consecutive days, where treatment was Grintuss® or Mucolit® (Kiryat Malachi, Israel), with single-blinded randomization, 3 times per day for 3 days. Both syrups were well tolerated, and the cough was alleviated. There was a significantly better result throughout for Grintuss® (*p* < 0.05) after one day for all the main outcome measures (cough frequency, cough severity, bothersome nature of cough, and sleep quality for both a child and a parent). The trend for improvement over the four days was steeper for Grintuss® (*p* < 0.05) for all cough parameters. Although both syrups were effective and safe treatments for children over two years of age, Grintuss® appeared to produce faster (first night) and more effective response (over four days of treatment) as to clinical cough symptoms. This trial reveals important information on the safety of *H. italicum* even for young children.

In the trial by Canciani et al. [61], Grintuss® syrup was compared to a placebo syrup in young children suffering from persisting cough. Both syrups were taken in four doses per day for eight days. None of the patients discontinued the trial for adverse events, or other safety reasons. The authors, however, state that Grintuss® should not be used in case of known hypersensitivity to the components of the medical device, but no other contraindications have been registered. It is worth mentioning that at the time of the research, Grintuss® syrup has been on the market for more than ten years, registered as a medical device (class IIa), during which the post-marketing surveillance system, in compliance with Directive 93/42/EC, has not registered incident or side effects related to the medical device, which further supports the safety of this device.

A similar trial by Calapai et al. [62] investigated the effect of KalobaTuss® (Egna–Neumarkt, Italy) syrup in children with persisting cough. This product contained *H. stoechas* as a component, therefore the record was not evaluated further. However, as the *H. stoechas* is closely related to *H. italicum* [7], these findings might also be relevant.

## *5.2. Individual Bioactive Substances*

Several factors, such as the growing conditions, drying, storage and extraction procedure, can greatly affect quality and the composition of an herbal preparation and hence its therapeutic outcome [63]. In modern phytotherapy and traditional medicine, mostly extracts with complex chemical composition are used, rather than isolated substances, favoring occurrence of synergistic effects and polyvalent activity. It needs to be stressed out, that also less abundant compounds can be potent. Consequently, identifying the active constituent in many herbal extracts has often proved to be difficult [64]. Several bioactive compounds were previously isolated from *H. italicum* and their activity investigated (already discussed in Section 3). Pereira et al. [42] investigated the composition of infusions and decoctions of *H. italicum* ssp. *picardii* and established that the main compounds were chlorogenic and quinic acids, dicaffeoylquinic acid isomers and flavonoid gnaphaliin A. According to Karaˇca et al. [65], the most abundant phenolic compounds present in the water extract prepared from commercially available *H. italicum* flowers, were caffeic acid, chlorogenic acid, and its derivatives. Kramberger et al. [66] found that caffeoylquinic acids and pyrones were the most prevalent compounds in *H. italicum* ssp. *italicum* water extracts. Composition of the essential oils, on the other hand, is completely different. In essential oil volatile terpenes predominate to the contrary of polar and semi-polar phenols, which are common in water-based preparations and organic solvent extracts [15]. Some terpenes can also be extracted with non-polar organic solvents or supercritical fluids, but this extraction procedure deviates from the traditional methods of preparations. In the following sub-section, clinical trials on the most relevant individual substances that are confirmed to be present in *H. italicum* water and hydroalcoholic extracts are briefly described.

## 5.2.1. Phenolic Acids

In contrast to clinical trials evaluating *H. italicum* extracts of a whole plant, trials on isolated substances are more numerous. One of the most studied isolated substances found in *H. italicum* is chlorogenic acid, an ester of caffeic and quinic acid. It is a widely distributed natural compound with many important activities. *In vitro* and in vivo studies have found that the main pharmacological effects of chlorogenic acid are antioxidant, antiinflammatory, antibacterial, antiviral, hypoglycemic, lipid lowering, anti-cardiovascular, antimutagenic, anticancer and immunomodulatory [45].

In the Cochrane Library, there are 160 Trials matching "chlorogenic acid" in Title Abstract Keyword (Word variations have been searched) (https://www.cochranelibrary. com/, accessed on 4 January 2021), and among them, 71 have been published in the recent four years [67]. The trials mostly investigated effects on cardiovascular system, weight loss, chronic inflammatory diseases, cognition, and lung cancer as well as bioavailability. Chlorogenic acid has been investigated alone or as the main component of some dietary supplements (i.e., green coffee extract). As this ubiquitous phenolic acid is present in largest

quantities in coffee [68], which is a widely consumed beverage, action in the *H. italicum* should not pose health concerns.

Similarly, caffeic acid—a very common phenolic acid with antioxidant, anti-inflammatory and anticarcinogenic activity [44], is also a well investigated compound (68 registered trials, accessed on 17.3.2021) [69]. Trials include effects on esophageal cancer, non-alcoholic fatty liver disease, photoprotection, immune thrombocytopenia, and bioavailability studies.

5.2.2. Flavonoids

Pinocembrin is a well investigated flavonoid of *H. italicum* with demonstrated antiinflammatory action in vivo [29]. To date, there are only two registered clinical trials on pinocembrin, and both have investigated its neuroprotective effect. Pinocembrin was injected into patients with ischemic stroke [70], and in another trial by Cao et al. [71], pharmacokinetics and safety of pinocembrin injection was investigated. When administered intravenously to healthy adults, pinocembrin was well tolerated up to 120 mg/d. Furthermore, no major safety concerns were identified that would preclude further clinical development of pinocembrin injection.

Quercetin is a versatile antioxidant known to possess protective abilities against tissue injury induced by various drug toxicities [46]. It is present in over twenty plants, in *H. italicum* mostly in the form of various glycosides. There are over 497 registered trials in Cochrane Library (https://www.cochranelibrary.com/, accessed on 17 March 2021) investigating versatile interventions [72]. These include effects on the vascular system (cerebral blood flow, vascular function, blood pressure, thalassemia), inflammation (sarcoidosis, asthma, chronic obstructive pulmonary disease), sex hormone disorders (prostate cancer, prostatitis, polycystic ovary syndrome and estrogen deficiency), metabolic disorders (dyslipidemia, glucose absorption, non-alcoholic fatty liver disease), performance (neuromuscular function, endurance, recovery) and other (immune response, stroke, myocardial infarction, hyperuricemia and oral mucositis).

Naringenin is a commonly found flavonoid in citrus fruits but is also found in its glycoside forms in *H. italicum*. Several biological activities have been ascribed to this phytochemical, above all cardioprotective action is the best investigated clinically [47]. Its effects on liver markers and blood pressure or on metabolic rate, insulin sensitivity and blood glucose have been studied in patients with non-alcoholic fatty liver disease or diabetes, respectively [73]. In the trial by Rebello et al. [74] on safety and pharmacokinetics of naringenin consumption, naringenin proved to be safe in healthy adults (up to 900 mg), and serum concentrations were proportional to the dose administered.

Clinical trials have been performed with luteolin as well. Effects on obesity and cardiometabolic risk factors in metabolic syndrome and on memory and behavior in children with autism were investigated [75]. In addition, its effect on exercise performance was also investigated.

## 5.2.2. Other Compounds

A triterpene ursolic acid, is one of the non-polar compounds of *H. italicum*, that has been isolated from acetone [37] and methanol extracts [26]. Although it possesses antiinflammatory, anticancer, antidiabetic, antioxidant and antibacterial effects, its bioavailability and solubility limit its clinical application [48]. Its activity has been investigated in patients with metabolic syndrome and on muscle function. Furthermore, ursolic acid was also injected to patients with solid tumors, where it was shown that ursolic acid liposome does not accumulate in the body. The administration was tolerable, had manageable toxicity, and could potentially improve patient remission rates [76].

Lastly, we want to mention also arzanol, which is probably the most characteristic compound for *H. italicum*. Although extensively investigated in vitro and in vivo, to date, no clinical trial has been registered.

## **6. Recent Advances in** *H. italicum* **Studies**

Concurrently with the clinical studies, other publications from the past three years were also evaluated. While the majority of the published articles is still focused on *H. italicum* essential oil, quite some of the recent research has been devoted to minimizing waste from the production process and herewith to follow emerging sustainability and upcycling approaches. Essential oil is produced preferentially from the flowerheads, while the rest of the plant, which contains significant amounts of secondary metabolites and could be used for extract production, is left in situ [53]. Dzamic et al. [77] investigated wastewater extracts of *H. italicum* produced after distillation. The highest phenolic concentration was measured in deodorized aqueous extract. The deodorized aqueous extract also possessed the highest antioxidant activity, followed by deodorized methanol extract, while essential oil had the lowest radical scavenging activity. Addis et al. [78] investigated wastewater extracts of aromatic plants, among them also *H. italicum*. They determined that water decoction not only retains antioxidant activity, but is also effective in wound healing, as it promotes tissue re-establishment after environmental stress exposure. Environmental topics continue to arise as Pili´c and Martinovi´c [79] investigated effect of *H. italicum* macerate on the corrosion of copper in simulated acid rain solution and Eksi et al. [80] assessed *H. italicum* as a green roof substrate.

Recently, detailed chemical composition and antioxidant activity of hydroalcoholic and water extracts has been evaluated and compared by Kramberger et al. [66]. In addition, further functional studies with water extracts (infusions) have been performed on cell models and gene expression of oxidative-stress related genes has been carried out [81]. In this comparative study, two morphologically distinct *H. italicum* subspecies were compared with a recognized medicinal plant of *H. arenarium*, on a genetic, chemical, and functional level. Both *H. italicum* subspecies exhibited superior antioxidant activity in vitro as well as cytoprotective activity. As it has been emphasized before [7], genetic or morphological description of the plant material used is often lacking in studies, probably due to the difficult characterization of *H. italicum*, arising from great diversity of the species and disunited classification keys available. In the recently published study by Baruca Arbeiter et al. [82], a set of new microsatellites as DNA markers was developed, which will serve for selection of most promising genotypes for propagation and their implementation in agricultural production.

## **7. Critical Perspective on Safety and Efficacy**

*H. italicum* toxicity has been investigated in some in vitro studies, but in general, this information is scarce. Pereira et al. [42] investigated cytotoxicity of *H. italicum* ssp. *picardii* tisanes towards different mammalian cell lines: hepatocarcinoma (HepG2), microglia (N9) and bone marrow stromal (S17) cell line. The extracts in the tested concentration of 100 μg/mL and after 72 h of exposure had low toxicity, with cell viability values similar or higher than those obtained for green (*Camellia sinensis*) and red bush (*Aspalathus linearis*) teas, which suggest that these aqueous extracts can be regarded as non-toxic beverages. Kramberger et al. [81] evaluated cell viability on lymphoma cell line (U937), adenocarcinoma cell line (Caco-2) and primary colon fibroblasts (CCD112CoN) after exposure to *H. italicum* infusions. Concerning U937 cells infusion was not toxic up to 5% *v/v* concentration, whereas for Caco-2 it was toxic at 1% *v/v*. Interestingly, higher concentration (2% *v/v*) was toxic for CCD112CoN cells, than for cancerous cell line Caco-2. Genotoxic activity of *H. italicum* has been evaluated by Nostro et al. [24], where diethyl ether extract showed no DNA-damaging activity at concentrations up to 2000 μg/disc. Some potential cytochrome P450 enzyme interactions have been discussed by Antunes Viegas et al. [7], more specifically for flavonoid tiliroside. It should be emphasized that the concentrations that are achievable via oral route in vivo may not be sufficient to cause medical important interactions due to low bioavailability. Such interactions are even less likely to occur when administering traditional preparations, where effect of one minor compound usually does not prevail. From the safety perspective, essential oils are potentially more problematic, as they are more concentrated mixtures. In this case, allergic reactions in hypersensitive individuals can occur [83].

From the current review of the clinical trials, no adverse events that could be attributed to the tested herbal products, were reported. Very importantly, the review also included trials on a large number of young children. Furthermore, *H. italicum* has a long traditional use and several products containing *H. italicum* are already on the market. However, the products are mostly not registered as therapeutics, but rather as dietary supplements or cosmetics. These products include oral supplements developed to favor venous circulation or cough treatment, while cosmetic products, claim the calming and antimicrobial properties of *H. italicum* essential oil incorporated in their formulas [7]. Appendino [84] strongly believes that the former focus on *H. italicum* essential oil and its application in cosmetics, should be turned to development of novel ingredients for medicinal and health-food products. The use of herbal products in general is very complex; apart from medicines, food supplements and cosmetics, botanicals could be marketed also as food (for example spices or herbal teas) or as medical devices, when the plant product can demonstrate a sole mechanical and non-pharmacological action, such as protection of mucous membranes or skin cooling/warming effect [85]. As these products are not proposed as treatments of diseases, demonstration of their clinical profile is not legally required [86]. Nevertheless, consumers usually choose the ones that better respond to their health needs, often ignoring the fact that diverse marketing categories imply profound differences in terms of manufacturing processes, chemical composition, quality controls, and studies of efficacy [85]. Based on these data all together, it can be concluded that *H. italicum* in the orally acceptable formulations is safe to use but would need further evaluation in the case of consideration as an herbal medicine with well-established use.

Efficacy of *H. italicum* has been established several times both in in vitro tests and in humans; first in human clinical observations and more recently also in clinical trials. Although, there are proper clinical trials that demonstrated its tested efficacy, findings are difficult to attribute to *H. italicum* alone, as it was usually not the only plant component tested. From the only one monotherapy trial performed by the company Aboca S.p.A., the findings were unfortunately not available. On the other hand, there are numerous studies and trials conducted with individual compounds present in *H. italicum*, which can contribute to identification of bioactive substances and elucidation of their mechanism of action. However, the findings can be misleading, as the effect of single isolated compound can be more potent due to higher concentrations achieved than in a plant, or even less manifested due to cumulative effects that occur in a compound mixture. For that reason, such findings cannot be directly extrapolated to whole plant extracts.

## **8. Conclusions**

With this review, *H. italicum* has been evaluated in terms of efficacy and safety for internal use. The clinical trials provide rather more insight into the safety profile than into the efficacy, due to lack of trials performed with *H. italicum* alone. The efficacy of *H. italicum*, however, is evident from reports on traditional use, human observational studies, or in vitro research. From the data gathered, particularly the trials in young children, we conclude that the ingestion of *H. italicum* does not pose a risk to human health. Although *H. italicum* is a plant with documented immense potential in several aspects of health, it still lacks regulatory recognition. *H. italicum* could be considered for evaluation by regulatory bodies such as the Committee on Herbal Medicinal Products under the EMA based solely on its traditional use. Although ethnopharmacological reports are available, for that purpose, traditional use of *H. italicum* in European territory would have to be evaluated more thoroughly. On the other hand, to meet the well-established medicinal use criteria, novel clinical studies with *H. italicum* as a single component are needed. Based on the recent trend in *H. italicum* research, it is evident that the current interest in *H. italicum*, especially at Balkan Peninsula, is considerable and expands far beyond cosmetic

applications. As various publications continue to emerge, further clinical trials can also be expected in the future.

**Author Contributions:** Conceptualization, K.K., S.K., D.B.-M. and N.K.G.; methodology, K.K.; validation, S.K.; investigation, K.K.; resources, K.K.; data curation, K.K.; writing—original draft preparation, K.K.; writing—review and editing, S.K., N.K.G., Z.J.P. and D.B.-M.; visualization, K.K.; supervision, S.K. and D.B.-M.; project administration, Z.J.P. and D.B.-M.; funding acquisition, Z.J.P. and D.B.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by the Slovenian Research Agency (research program P1-0386 and grant number 1000-18-1988 for junior researcher Katja Kramberger).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data is presented within the article.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## **Appendix A**

**Table A1.** Characteristics of included clinical trials, listed in chronological order.


NA—information not available, EO—essential oil, m—male, f—female, no.—number, comb.—combination, supp.—suppository, spec. specified, compos.—composition of a product when *H. italicum* is not the single component, y—years.

## **References**


## *Article* **Climatic Drivers of the Complex Phenology of the Mediterranean Semi-Deciduous Shrub** *Phlomis fruticosa* **Based on Satellite-Derived EVI**

**Aris Kyparissis \* and Efi Levizou**

Department of Agriculture Crop Production and Rural Environment, University of Thessaly, Fytokou Str., 384 46 Volos, Greece; elevizou@uth.gr

**\*** Correspondence: akypar@uth.gr

**Abstract:** A 21-year Enhanced Vegetation Index (EVI) time-series produced from MODIS satellite images was used to study the complex phenological cycle of the drought semi-deciduous shrub *Phlomis fruticosa* and additionally to identify and compare phenological events between two Mediterranean sites with different microclimates. In the more xeric Araxos site, spring leaf fall starts earlier, autumn revival occurs later, and the dry period is longer, compared with the more favorable Louros site. Accordingly, the control of climatic factors on phenological events was examined and found that the Araxos site is mostly influenced by rain related events while Louros site by both rain and temperature. Spring phenological events showed significant shifts at a rate of 1–4.9 days per year in Araxos, which were positively related to trends for decreasing spring precipitation and increasing summer temperature. Furthermore, the climatic control on the inter-annual EVI fluctuation was examined through multiple linear regression and machine learning approaches. For both sites, temperature during the previous 2–3 months and rain days of the previous 3 months were identified as the main drivers of the EVI profile. Our results emphasize the importance of focusing on a single species and small-spatial-scale information in connecting vegetation responses to the climate crisis.

**Keywords:** remote sensing; MODIS; enhanced vegetation index; temperature; precipitation; rain days; inter-annual variability; time-series; machine learning; climate change

## **1. Introduction**

Vegetation response to climate variability is becoming increasingly important, especially under the frame of the ongoing global climate change [1–3]. Our understanding of vegetation function, its interactions with the climate, the key controlling mechanisms, and its vulnerability to climate change are far from complete. Evidently, understanding climatic influences on processes and interactions enables the prediction of changes under different climatic scenarios [4,5].

The most consistent results of climate change experiments are the species-specific responses. Many experiments that have been conducted worldwide, including manipulations of the CO2 and UV-B environment, temperature rise, and N deposition, have manifested that plant species differ in their sensitivity to damage and their morphological, biochemical, and physiological responses to altered environmental factors [6,7]. The exact position held by a certain species in the sensitivity-tolerance continuum, as well as its specific responses, could cascade upwards to alter community and ecosystem composition and structure through changes in the competitive balance between species [8,9]. Additionally, the proposed conceptual frameworks for analysis of the species and ecosystem response to changing climate underline the importance of thresholds for interpreting experimental results and predicting effects [10]. Rapid, nonlinear changes in some plant processes or responses can be triggered by even small differences in environmental conditions if threshold values are exceeded. This evidence stresses the importance of studies focusing

**Citation:** Kyparissis, A.; Levizou, E. Climatic Drivers of the Complex Phenology of the Mediterranean Semi-Deciduous Shrub *Phlomis fruticosa* Based on Satellite-Derived EVI. *Plants* **2022**, *11*, 584. https:// doi.org/10.3390/plants11050584

Academic Editor: Stefan Zerbe

Received: 30 December 2021 Accepted: 20 February 2022 Published: 22 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

on direct and indirect effects of environmental change on plant species and not only on large formations and ecosystem-level spatial scales.

Plant phenology is considered an important factor that mediates vegetation and climate relationships, through affecting a diverse set of processes [2]. Phenology is not merely the succession of recurrent biological events in the plant's lifecycle, but it also greatly relates to plant activity, since different phenophases affect plant function and resource allocation patterns [11]. The phenology–climate feedbacks are bi-directional. At one end is the climate impact on the timing and duration of phenological events [3,12]. At the other end is the influence of phenological events and, moreover, transitions on vegetation feedbacks to the microclimate, i.e., humidity, temperature, wind speed, as well as soil moisture and topsoil temperature [2]. Scaling up at the community and ecosystem level, phenology influences processes and mechanisms such as water, CO2, and energy fluxes which feedback to large-scale vegetation–atmosphere interactions. The well-established sensitivity of phenology to year-to-year variability in climate could also serve as an indicator of the long-term biological impacts of climate change on terrestrial ecosystems [13,14].

Remotely sensed data proved to be a valuable tool in coupling climate and vegetation distribution/performance at large spatial and temporal scales. As a result, the objective of many studies was the assessment of the effects of certain environmental factors on remotesensing-derived vegetation parameters [15–17]. A common feature in most of the abovementioned research efforts is the large spatial scale used, i.e., regional, continental or global, exploiting satellite-derived simultaneous estimates of ecosystem function over wide areas. Indeed, remote sensing of vegetation offers a promising and urgently needed assessment of ecosystem function at a spatial scale that is comparable with the extent of human-caused environmental change. However, information on specific species performance, which is possibly incorporated in remote sensed data, is rather lost in the inevitably vague picture given by large spatial-scale studies [18]. Ecophysiological field surveys could address this issue, but because of laborious and time-consuming measurements, they have the disadvantage of temporal and spatial limitations. Alternatively, satellite imagery in the context of studying a particular species' behavior, i.e., at small spatial scale, may render an accurate picture of species responses to natural climate variability, as well as climate change [19,20].

The focus on species and use of satellite data to study species-level responses, from a phenological and especially an eco-physiological point of view, to climate forcing has an essential prerequisite: strong correspondence with ground-measured plant processes or features [21,22]. Indeed, established relationships between ground-determined characteristics and their satellite-derived surrogates in terms of vegetation indices allow for an explicit physiological meaning to the latter. This in turn permits understanding, monitoring, and explaining species behavior, as well as identifying broad patterns in space and time, including a species' relationship with environmental determinants. Collectively, established correlations enable exploiting the advantages of remote sensed data, i.e., large spatial and temporal scales, with direct reference to species phenological/physiological characteristics. The above-mentioned benefits justify the intensive research efforts of the last two decades devoted in establishing such correlations [22–25].

The Enhanced Vegetation Index (EVI) has been shown to be well correlated with LAI, biomass, canopy cover, and the fraction of absorbed photosynthetically active radiation [26,27], and is therefore useful for monitoring seasonal, inter-annual, and long-term variation of the vegetation structure and function [28]. EVI has been used instead of the Normalized Difference Vegetation Index (NDVI) because it reduces sensitivity to soil and atmospheric effects and remains sensitive to variation in canopy density where NDVI becomes saturated [29–31]. Given these characteristics, modelers have begun using EVI data to predict net primary production in ecosystem modelling applications [24,29].

Under the above-described framework, a 21-year EVI time-series is used in the present study to assess climatic effects on the growth cycle of the drought semi-deciduous Mediterranean shrub *Phlomis fruticosa*. *P. fruticosa* was chosen for the following reasons: (a) it is

a typical drought shrub of Mediterranean ecosystems and, moreover is considered a key species of the garrigue vegetation which dominates the most xeric parts of the Mediterranean basin [32]; (b) we consider this species an ecological indicator of overgrazed ecosystems in which it forms large, continuous, and undisturbed stands, exclusively covered by this particular species, because it is not eatable by major farm animals (sheep, goats); (c) it has a multi-phase intra-annual growth cycle, with distinct phases being possible targets to climatic effects, which makes *P. fruticosa* a good model plant for satellite-derived phenology studies; (d) its growth cycle has been extensively studied through eco-physiological field measurements [33,34]; thus, there are established relationships between growth/ecophysiological parameters and satellite indices [22,25]; (e) the plasticity and adaptability of *P. fruticosa*, although established by field eco-physiological measurements have not been validated in large space and time scales, as those provided by satellite imagery.

For the purposes of the present study, two distant *P. fruticosa* ecosystems, with differences in climatic characteristics were chosen. The aims were (a) to depict the complex phenological cycle of this species through satellite-derived EVI and extract metrics that analyze the phenological events and transitions, (b) to determine the climatic drivers that influence the phenophases and their possible differences between the two sites, and (c) to identify trends for change, which could further be used as diagnostic and prognostic tool for climate crisis effects.

## **2. Materials and Methods**

## *2.1. Study Sites*

Two ecosystems dominated by *P. fruticosa* with different climatic characteristics were chosen in order to study possible differences in climate control and plant responses (Figure 1): (a) Araxos area, the southern one (NW Peloponessos, Greece, 38.18◦ N, 21.37◦ E), characterized by a prolonged summer stress period where high temperature co-exists with a severe water shortage and (b) Louros area, the northern one (Epirus, Greece, 39.17◦ N, 20.85◦ E), with more favorable temperature and water availability conditions for plant growth.

**Figure 1.** Map of Greece with the two study sites indicated with red dots and the locations of the meteorological stations with blue dots.

## *2.2. Meteorological Data*

Meteorological data (average daily temperature and daily precipitation) of the 21-year study period (2000–2020) for Araxos site were recorded by a meteorological station situated in Andravida, about 29 km from the study area, while for Louros site, in Aktion airport, about 28 km from the study site. Data were downloaded from U.S. National Oceanic and Atmospheric Administration (NOAA) National Climatic Data Center (NCDC, www.ncdc. noaa.gov, accessed on 1 March 2021). In Figure 2, the annual profile of the average total monthly precipitation and the average monthly temperature for the 21-year study period is presented for both study sites. Increased rain amounts in Louros site throughout the year and a slightly lower temperature during the stressful summer period are evident (average temperature of June to August 25.93 ± 0.62 ◦C for Araxos and 25.25 ± 0.63 ◦C for Louros). For Araxos, the average annual temperature is 17.86 ± 0.35 ◦C and total annual precipitation 759.5 ± 151.8 mm, while for Louros, the temperature and precipitation were 17.64 ± 0.42 ◦C and 919.3 ± 217.2 mm, respectively.

**Figure 2.** Annual profile of the average total monthly precipitation (bars) and the average monthly temperature (points) for the 21-year study period (2000–2020) for Araxos and Louros study sites.

## *2.3. Species Description*

*Phlomis fruticosa* is a dimorphic, semi-deciduous shrub of the eastern Mediterranean areas. It bears two kinds of leaves—winter and summer ones—with different biochemical and structural characteristics [30]. Winter leaves and summer leaves of the previous growing season are massively shed during mid to late spring, resulting in a decrease in Leaf Area Index (LAI). Hereinafter, we refer to this event as spring drop. During the summer dry period, plants bear summer leaves developed during spring, which are smaller than winter leaves and have lower chlorophyll content. After the onset of the autumn rainy period, summer leaves absorb water rapidly (within days) and increase their area, while they alter their biochemical characteristics, including chlorophyll content increase. Additionally, new winter leaves with high chlorophyll content appear during November. Hereinafter, we refer to this event as autumn revival. The transformation of summer leaves and the appearance of winter leaves during autumn result in an increase in LAI, which remains almost stable during winter, until next spring. Even though this phenological/physiological cycle is repeated every year, the extent and/or the exact date for each particular phenological event seem to depend on the microenvironmental conditions [31]. The main physiological advantage of the semi-deciduous habit is the decrease in the transpiring leaf area during the summer dry months, resulting in more efficient water economy.

## *2.4. Satellite Data*

The Enhanced Vegetation Index (EVI) was used in the present study. For the calculation of EVI, data from the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Terra satellite (part of the NASA Earth Observing System) were used. MODIS scans the entire Earth surface every 1–2 days, acquiring data in 36 spectral bands. Out of the 36 spectral bands, 7 bands are designed for the study of vegetation and land surfaces: blue (459–479 nm), green (545–565 nm), red (620–670 nm), near infrared (NIR1: 841–875 nm, NIR2: 1230–1250 nm), and shortwave infrared (SWIR1: 1628–1652 nm, SWIR2: 2105–2155 nm). Several products with differences in spectral, spatial, and temporal resolution, as well as in correction levels are freely provided by the MODIS Land Science Team to users. In the present study, the geometrically and atmospherically corrected Surface Reflectance 8-Day L3 Global 500 m product (MOD09A1), available to the public from the US Geological Survey EROS Data Center (USGS EROS Center, http://eros.usgs.gov/, accessed on 1 March 2021), was used. EVI was calculated according to the equation:

$$EVI = 2.5 \frac{R\_{vir} - R\_{red}}{R\_{vir} + 6R\_{red} - 7.5R\_{blue} + 1} \tag{1}$$

where *Rnir* is reflectance between 841 and 875 nm, *Rred* between 620 and 670 nm and *Rblue* between 459 and 479 nm [35].

The MOD09A1 datasets (2000–2020), which have a 500 m spatial resolution and 8-day temporal resolution, were downloaded from the USGS EDC website using the geographical coordinates of each study site and 21 years EVI time-series were produced for 4 and 3 pixels for Araxos and Louros sites, respectively. These pixels were selected for each site after land observations and GPS recording, as being homogenous and dominated exclusively by *P. fruticosa*. The time-series of each pixel were corrected for erroneous values during cloudy dates or other reasons using the BISE (Best Index Slope Extraction) algorithm [32]. Accordingly, for each date the average of the corresponding pixels was calculated for each site and used for the construction of time-series over the 21-year study period. The timeseries of the two sites were further smoothed using an adjusted Fast Fourier Transform [36]. Additionally, the average annual EVI profile for each site was calculated by averaging EVI values for the same 8-day period between years (Figure 4).

## *2.5. Phenology Metrics*

The phenological cycle of *P. fruticosa*—as described above—may be depicted by the seasonal EVI fluctuation shown in Figure 2. High and rather stable values of EVI during winter (December to March), corresponding to high LAI and chlorophyll content values, are followed by the spring drop period (April to June). The steep reduction in EVI during that period corresponds to the massive loss of winter leaves and summer leaves of the previous year. During summer dry period (July to September), plants bear a small number (low LAI) of low chlorophyll content summer leaves (low and stable EVI values). The subsequent autumn revival period, coinciding with the onset of the autumn rainy period (October– November), is characterized by an abrupt rise of EVI, as a result of the "resurrection" of summer leaves (rapid increase in leaf area due to water absorption, accompanied by chlorophyll content increase) followed by the burst of new winter leaves at the end of autumn. This pattern is repeated every year in both study sites, but remarkable differences in parameters of spring drop, dry period and autumn revival may occur among sites and/or years.

In order to quantify the phenological events described above (spring drop, dry period, and autumn revival), the parameters presented in Table 1 were calculated for each site and year. For the calculation of spring drop and autumn revival related parameters, the 1st derivative of the EVI curve was used. Day of Year for spring drop onset (SDO) and autumn revival onset (ARO) are determined when the 1st derivative departs from near zero values (Figure 3) and spring drop end (SDE) and autumn revival end (ARE) when the 1st derivative returns to near-zero values, during the spring and autumn EVI steep

change period. The differences between SDE-SDO and ARE-ARO result in the spring drop duration (SDD) and autumn revival duration (ARD), respectively.


**Figure 3.** Typical profile of the annual fluctuation of EVI (thick line) and its 1st derivative (thin line). Red horizontal line corresponds to EVI = 0.3 and EVI 1st derivative = 0. Black vertical lines indicate the onset and end of the spring drop and the autumn revival period, when EVI 1st derivative departs or returns to zero values for onset and end correspondingly. Red vertical lines indicate the onset and end of the dry period, when EVI is lower than 0.3.

Concerning dry period parameterization, onset (DPO) and end (DPE) of the dry period were calculated according to the threshold method [37,38]. The EVI value of 0.3 was defined as the threshold for DPO and DPE and was chosen because it represents the midpoint between absolute maximum and absolute minimum (all 21 years) EVI values. Thus, DPO and DPE were quantified as the Day of Year at which EVI reaches or leaves 0.3, respectively, and the dry period duration as their difference (Figure 3).

Additionally, in an attempt to exploit all the information contained in the shape of EVI curve, annual maximum and minimum values and the date of their occurrence were also determined for each site and year (Table 1). Finally, mean monthly EVI was calculated and used as a surrogate of ecosystem dynamics in the assessment of climate control on growth features. All the above-mentioned extracted parameters were used as independent phenology metrics in the statistical analyses (see below) for the identification of the most influential climatic factor.

## *2.6. Statistical Analysis*

The relationships between the above described phenological events and climatic parameters were assessed using Pearson correlations and stepwise multiple linear regressions. The examined climatic parameters concern total monthly precipitation (P), monthly sum of rainy days (RD) and mean monthly temperature (T), of different time intervals-concurrent and lagged-in relation to the corresponding phenological event. More specifically, phenological events were examined against the following combinations:


The first step was to perform a Pearson correlation analysis for each phenological event and the above-mentioned climatic parameters. Accordingly, the independent variables that exhibited the maximum correlations in each case were employed in multiple linear regression analyses with stepwise selection. Collinearity of predictor variables was automatically detected by the statistical software and subsequently dealt by omitting variables and re-running the regression analysis.

The influence of climatic parameters on inter-annual EVI variation was examined following two different regression analysis methods, i.e., multiple linear regression and random forest machine learning. As in the case of phenology and climate control, all combinations of the climatic parameters (precipitation, rain days, temperature) of various time intervals and time lags of consecutive months up to six months before the event (EVI of a particular month) were considered. Initially, a monthly step EVI time-series was produced for each site, by averaging the analytical time-series data for each month. For the first approach, the most significant climatic parameters were determined through single linear regressions between monthly EVI and climatic parameters. Accordingly, combinations of the most important parameters were examined through stepwise multiple linear regression. Analyses showed that a high regression coefficient was achieved with two climatic parameters, i.e., adding additional parameters did not significantly enhance the efficiency of the regression (data not shown). On the second approach, all climatic parameters were used in a random forest machine learning procedure. During this procedure, data were randomly split into a training set (64% of data), a validation set (16% of data) for performance optimization and a test set (20% of data) for assessment of performance efficiency. However, for an overall comparison of machine learning with multiple linear regression all data were used in Figure 8. The efficiency of the two approaches was assessed through the coefficient of determination (R2) and Root Mean Square Error (RMSE) of the predicted EVI against actual EVI values.

All statistical analyses were performed with JASP v.0.16 software (JASP Team 2021 Computer Software), which includes a machine learning module.

## **3. Results**

## *3.1. Phenology*

In Figure 4, the annual EVI profile for the two study sites is presented as average ± SD from the 21-year study period data. In Araxos, spring drop as well as dry period (EVI < 0.3) starts earlier and dry period ends later in autumn, accompanied by a retarded autumn revival. Additionally, annual maximum and minimum EVI values appear higher for the Louros site compared with Araxos, possibly as a result of better physiological performance and/or higher shrub density under the more favorable conditions of Louros.

**Figure 4.** Annual EVI profile for the two study sites as average ± SD from the 21-year study period data. The red line marks EVI = 0.3, the value which was defined as the threshold for dry period onset and end.

These general differences among sites are usually followed every year throughout the 21-year study period (Figure 5). Additionally, it is clear from Figure 5 that the annual profiles for both sites show strong differences from year to year. These interannual variations may be rather large, especially during summer periods, as seen by comparing the very dry summer of 2001 with the wet summer of 2016 (Figure 5).

In an attempt to reveal the detailed differences between sites, the phenological events described in Table 1, were determined for all years and sites and their average values are presented in Table 2, whereas the most important among them, i.e., events concerning spring drop, dry period, and autumn revival, are depicted in Figure 6.

As shown in Table 2, onset and end of spring drop occur 15 and 27 days earlier in Araxos compared with Louros, respectively, with both events showing statistically significant differences. Additionally, spring drop in Araxos occurs more rapidly, as indicated by the smallest duration, compared with Louros. Dry period starts 40 days earlier and finishes 11 days later in Araxos compared with Louros, resulting in a significantly longer dry period duration by 51 days. Concerning autumn revival, onset and end appear 13 and 5 days later, respectively, in Araxos, even though only onset is significant. However, as in the case of spring drop, autumn revival occurs more rapidly in Araxos (shorter duration), compared with Louros. Maximum and minimum EVI values are both significantly higher in the Louros site, where more favorable climatic conditions prevail. Accordingly, the date of maximum EVI appears 22 days earlier in Araxos, whereas no statistical difference is evident for the date of minimum EVI.

**Figure 5.** Interannual EVI fluctuation for the 21-year study period for the two study sites (**a**); EVI fluctuation and the corresponding precipitation profile during a dry (2001, **b**) and a wet (2016, **c**) year in the two study sites. The blue line marks the threshold for dry period onset and end (EVI = 0.3).

**Table 2.** Average data (±SD) for the phenological events described in Table 1 for the two study sites, their difference (Araxos–Louros), and the significance of their difference (P, paired *t*-test). DOY, Day of Year; ND, Number of Days.


**Figure 6.** Graphical depiction of onset, duration, and end of the main phenological events (Spring Drop, Dry Period and Autumn Revival) for the two study sites. For all events, onset and end correspond to Day of the Year, whereas duration corresponds to number of days (data from Table 2).

## *3.2. Phenology and Climatic Control*

In order to account for climatic controls of phenology, average monthly temperature, total monthly precipitation and total monthly rain days of time windows relevant to each event and transition were examined. More specifically, phenological events were examined against climatic parameters of various time intervals and time lags up to six months before the event (see Section 2.6).

Spring drop onset for Araxos is influenced by precipitation of April (Figure 7), with more rain delaying SDO. Accordingly, Louros is similarly influenced by the rain days of April and March, whereas the temperature during January and February also plays a role; low temperature delays SDO, probably through sustaining higher soil water capacity.

In Araxos, where precipitation during summer months is minimal and with low interannual variability (July and August rainfall of 15 ± 33 mm), the main influential factor on SDE is temperature of July and August with high temperature delaying the SDE and resulting in lower minimum EVI values compared with Louros (Table 2). On the contrary, in the wetter and more variable Louros site (July and August rainfall of 29 ± 61 mm), SDE is mainly influenced by summer precipitation: the more it rains the earlier SDE appears and at higher minimum EVI value compared with Araxos.

Dry period onset for Araxos, is affected by the precipitation of March and April (higher precipitation delays onset) and the temperature of April and May (higher temperature advances onset). For Louros, DPO is mildly affected by the February temperature, with higher temperature delaying the event. DPE for Araxos is strongly affected by summerearly autumn rain, since both precipitation and rain days of July to October period influence the event, causing a delay at drier years. Similar effects of precipitation are evident for Louros, but for this site, the temperature in July also plays a minor role; a higher temperature results in earlier DPE and shorter duration.

**Figure 7.** Climatic control on phenological events and transitions of *P. fruticosa* in the two sites as derived by single or multiple linear regressions. The phenological metrics are: Spring drop onset (SDO); spring drop end (SDE); spring drop duration (SDD); Dry period onset (DPO); dry period end (DPE); dry period duration (DPD); Autumn revival onset (ARO); autumn revival end (ARE), autumn revival duration (ARD); MaxEVI the maximum value of EVI; MaxDate, the date it is achieved; MinEVI the minimum value of EVI; MinDate, the date it is achieved. For each phenological event, the partial regression coefficient(s) of the most significant climatic variable(s) (precipitation (Pr), number of rain days (RD) and temperature (T)) of single or multiple months (top line) is presented in the corresponding colored horizontal lines, according to the chromatic scale appearing in the right. The regression coefficient (R2) of the model which includes the factors influencing each event and the corresponding level of significance (p) is presented in the right column of each site.

Autumn revival onset is also strongly affected by rain (precipitation of July to October and rain days of August and September), with earlier onset during wetter years. A similar effect of rain is apparent for Louros, but only through precipitation (June to October). For both sites, ARE is affected by autumn rain days (July to October for Araxos and September to October for Louros). Nevertheless, a rather unreasonable effect of June rain days is evident in Araxos, where more rain days during June delay the ARE. However, this peculiar effect is also recorded for ARD for both sites (rain days of June for Araxos and July for Louros).

The value of maximum EVI seems to be positively affected by spring rain days for both sites (of March for Araxos and March–April for Louros). Winter precipitation affects the date in which the maximum EVI is achieved for both sites. More specifically, more rainfall during January to March for Araxos and January for Louros causes a delayed appearance of maximum EVI. Minimum EVI occurs during late August or early September in both sites. The rain days of August is the determinant of minimum EVI in Araxos, whereas precipitation over a longer period, February to June, positively affects the minimum EVI of Louros. Additionally, Louros seems to be affected by temperature of July, but in a rather unexpected way, since higher temperature results in higher EVI. Finally, for both sites the date that the minimum EVI appears is affected by the spring–summer precipitation (May–June for Araxos and March to August for Louros), with more rain transferring the date earlier.

Collectively, the 13 phenological events analyzed above are influenced mostly by rain related parameters for the Araxos site; more specifically 10 events by precipitation and/or rain days, 2 events by temperature and 1 event by both rain and temperature. On the contrary, both rain and temperature play crucial roles in Louros phenology, since 6 events are influenced by precipitation and/or rain days, 6 events by both rain and temperature, and 1 event by temperature.

## *3.3. Phenology and Climate Change*

All phenological events examined above could be potentially related to the ongoing climate change. Our dataset of 21 years is long enough to permit the analysis of the trends of phenological events' interannual fluctuation in the context of climate change. As shown in Figure 8, spring-drop-related events show significant trends for the Araxos site, but not for Louros, whereas no significant trends appear for the rest of the phenological events (data not shown). The trends appearing for Araxos seem to be explained by similar trends in the main climatic factors that these events are related to (Figure 8). SDO tends to commence earlier in the season by 1 day per year, whereas April precipitation—the main influential climatic parameter (Figure 7)—tends to decrease by 1.7 mm per year. Accordingly, SDE experiences a delay by 3.8 days per year and spring drop duration is elongated by 4.9 days per year. Both events are influenced by July–August temperature (Figure 7), which shows a similar trend, increasing by 0.06 ◦C per year during the study period (Figure 8).

**Figure 8.** Interannual fluctuation of the spring drop related phenological events (dots) and their trends (lines) for the two study sites during the study period: (**a**) Spring drop onset (SDO,), (**b**) spring drop end (SDE), (**c**) spring drop duration (SDD. Interannual fluctuation of the main climatic parameters influencing the phenological events of the Araxos site (dots) and their trends (lines) during the study period: (**d**) April precipitation, (**e**) July–August temperature.

## *3.4. EVI and Climatic Control*

Since all phenological parameters are extracted from the EVI time-series, the influence of climatic parameters on EVI per se was examined as a final integrating step. To that purpose, as in the case of climate control on phenological events, all combinations of the climatic parameters (precipitation, rain days, temperature) of various time intervals and time lags of consecutive months up to six months before the event (EVI of a particular month) were considered through two regression analysis methods, i.e., multiple linear regression and random forest machine learning.

As shown on Figure 9, EVI may be predicted by similar parameters for both sites through multiple linear regression analysis, i.e., temperature of the previous two months for Araxos and three months for Louros and rain days of the precious three months for both sites. However, the machine learning approach—in which all climatic parameters are included—results in much stronger models compared with the multiple linear regression approach, as judged by R2, RMSE and the regression line which is closer to the 1:1 line. It is worth to note, that the parameters determined by the multiple linear regression approach are among the most important ones determined by the machine learning approach, but the inclusion of additional parameters significantly enhances model efficiency.

**Figure 9.** Regressions between measured and modelled EVI through multiple linear regression (MLR, **a**,**c**) and random forest machine learning (RF, **b**,**d**) for Araxos (**a**,**b**) and Louros (**c**,**d**). In multiple linear regressions (**a**,**c**) the two climatic parameters participating in the models are shown in the right lower corner of each graph. In machine learning models (**b**,**d**) the ten most important parameters are shown. In both cases parameter importance decreases from top to bottom. Black lines correspond to the 1:1 lines and red ones to the regression lines. Climatic parameters are described by an acronym for parameter description (T for temperature, Pr for precipitation, and RD for rain days) followed by a number showing the number of the corresponding months and a number corresponding to the lag time, for example T\_2\_1 refer to temperature of two months before one month.

## **4. Discussion**

In this study, the phenological differences between two sites dominated by the semideciduous shrub *Phlomis fruticosa*, were examined using MODIS EVI time-series. *P. fruticosa* has been extensively studied from an ecophysiological point of view with both field measurements [33,34] and in combination with satellite data [22,25]. The most important characteristic of its growth pattern is the massive leaf shedding during spring, as an adaptation to the adverse conditions of the hot and dry Mediterranean summer, accompanied by autumn revival after the onset of the autumn rains.

## *4.1. EVI Tntra- and Inter-Annual Fluctuation and Phenology Metrics*

The first target of the present study was to monitor seasonal and inter-annual fluctuation of EVI and, subsequently, to identify key phenological events, in order to analyze the temporal dynamics of *P. fruticosa* community in two distinct sites.

The two sites examined in this study are located near to the shoreline of western Greece but have a latitude difference of about 1◦. Accordingly, during the 21-year study period, the southern site (Araxos) appeared more xeric compared with the northern site (Louros, Figure 2).

As shown in Figures 3–5, satellite data can capture and effectively describe the complex phenology of the semi-deciduous shrub. The inter-annual variability of EVI values is considerable and this is well depicted in two extreme years, the dry 2001 and wet 2016, which are presented in Figure 5 in relation to precipitation. Both sites exhibit an analogous profile concerning the prolonged drought phase (denoted by EVI < 0.3) at the dry year, which is considerably shortened during the wet year. In the relevant remote sensing literature, several studies have reported a detailed single-species phenology monitoring, emphasizing the importance of spatially explicit analyses and the study of phenological trends in small-scale level [15,39,40]. Especially in the fragmented and highly heterogeneous Mediterranean vegetation, this approach connects small- to large-scale information on community or ecosystem function, which would be otherwise lost if only regional level is considered [41].

The timing of certain phenological events that describe the annual cycle of *P. fruticosa* differs in various degrees between the two sites. In the southern Araxos, the earlier spring drop onset, the later autumn revival onset, the prolonged dry period and the earlier appearance of maximum EVI are important in shaping the annual picture and statistically significant. It is well documented that the above-described phenological transitions relate to greening-up or senescence processes which control the community function, state, and productivity [22,25]. Thus, the phenological transitions are influenced and in turn may influence the microclimate, generating gradients of humidity and temperature and affecting topsoil characteristics. Playing such a crucial role in microclimate modification the phenological patterns may have long-term feedback on larger-scale vegetation–climate interactions [2].

## *4.2. Climatic Control on Phenological Events*

In semi-arid Mediterranean ecosystems, the high inter-annual variability of EVI suggests that the "memory effects" of previous year's climate are minimized, as proposed by Catorci et al. [42]. Therefore, our analysis of the relationship between phenological events and climatic drivers was focused on up to 6 months preceding each certain transition. The spring and dry-period-related events were influenced by both precipitation and temperature of a short preceding period. The spring drop of winter leaves indicates the end of winter growth and the "preparation" of *P. fruticosa* to face summer adverse period. Spring precipitation was the major driver of SDO, whereas its duration was influenced by summer precipitation in Louros and summer temperature in the drier Araxos site. Spring phenology has been proved to be sensitive to climatic control in Mediterranean-type ecosystems, possibly due to the high variability of climatic parameters during spring [4,43]. Working with Mediterranean grasslands Catorci et al. [42] reported that rainfall in March and

drought stress in April and May were the main drivers of satellite-derived spring biomass production. Climatic parameters linked to moisture control are predominant in shaping vegetation response in Mediterranean, semi-arid and arid ecosystems [44]. Precipitation totals over the preceding three months have been found to correlate with the start of growth season in various Mediterranean regions [4]. Piedallu et al. [45] highlighted the positive correlation of spring temperature with vegetation greenness in an elevation gradient in South France, while stating that rainfall played a minor role in the overall region response, except for the most arid microsites.

The onset of the dry period for *P. fruticosa* was determined as the time-point at which the EVI reaches the 0.3 threshold, denoting a massive leaf loss thus a significant decrease in LAI. Increased spring precipitation retards DPO, but increased spring temperature advances it in Araxos, whereas the temperature of February is the only important parameter in Louros. The duration of dry period and the DPE is influenced mainly by precipitation in both study sites. The seasonal timing of rainfall events is important in determining their effects on phenology [43]. Especially in semi-arid systems and drylands, the precipitation during water-deficit periods is significantly more important in driving phenology than the rain during favorable moisture conditions. Broich et al. [31] suggested that the stronger correlation patterns with single-month compared with multi-month aggregated drivers indicate that rainfall at a specific time-point determines most phenological events. Corroborating this conclusion, our findings suggest that 7 out of 13 phenology metrics were influenced by single or double-month precipitation related parameters in the drier site of Araxos.

The autumn revival of *P. fruticosa* is characterized by the resurrection of summer leaves and the massive production of the winter leaves, thus it is related to an abrupt increase in EVI values. The ARO is primarily driven by the precipitation of the previous 5 months in Louros, whereas in Araxos an equal contribution of the previous 4 months precipitation and the rain days of previous 3 months is recorded. This result is in accordance with Cabello et al. [43], who reported that the earlier arrival of the first rains after the summer adverse period significantly account for the acceleration of growth period onset in Mediterranean drylands. To the same direction was the effect of precipitation on vegetation greening in semi-arid sites of Tunisia [17]. On the contrary, Horion et al. [46] stated that temperature of the last month was the major climatic constraint for growth from the start of vegetative growth to flowering in two studied sites in the Mediterranean basin.

The study of possible climate change effects on phenology was significantly advanced by the use of satellite remote sensing as an efficient tool for continuous vegetation monitoring in large temporal scales [47,48]. The present 21-year study covers an adequate period to evaluate trends in timing of phenological events. A significant trend for earlier spring drop onset in Araxos site was evident following the similar downward trend of April precipitation, which was found to be the most influential climatic factor at this particular event and site. On the contrary, the SDE showed a delay rate of 3.8 days year−1, in response to July-August temperature increase. Both SDO advancement and SDE delay resulted in a significant trend for prolonged SDD, at a rate of 4.9 days year−1. Spring events in Louros and the other phenophases of *P. fruticosa* showed weak or no trends for change. The advancement of spring phenology is one of the consistent observations across Northern Europe [18], North America [30], and China [5] during the two recent decades. In Mediterranean-type ecosystems, the spring phenology trends show scattered spatial pattern according to a comprehensive study of Ivits et al. [4]; over the southern Mediterranean region, an earlier start-of-season was observed, whereas over parts of the northern Mediterranean basin a growing season shift towards later dates was evident. Concerning the climate forcing of spring phenology trends, March rainfall was reported as the main driver of NDVI variability [42], whereas in accordance with our results Cabelo et al. [43] also reported a trend for reduced spring precipitation which accounted for spring phenology variations. Temperature rising has also well documented consequences in vegetation phenology, especially in semi-arid ecosystems [49–51]. An interesting outcome of the

analyses presented here is that the contribution of both temperature and precipitation is higher in shaping the Louros phenological profile, whereas the more xeric Araxos depend more on rainfall. This is in accordance with the relevant literature, where a positive link of precipitation and satellite-derived phenology has been generally observed, but a stronger relationship has been reported for xeric compared with colder and wetter areas [45,52].

Temperature and rain days proved to be the main climatic drivers of EVI profile for both Araxos and Louros sites, according to multiple linear regressions and machine learning approach (Figure 9). Specifically, the temperature of the previous two (Araxos) or three (Louros) months and the number of rain days for the preceding three months account for the overall variation of EVI in the 21-year period. The same climatic factors with variable time windows are present in the set of the 10 most influential parameters derived from the machine learning approach, which is increasingly adopted in studies involving time-series. It is interesting to examine these results in the context of climate change. Mediterranean ecosystems are vulnerable due to intense anthropogenic pressure, natural disturbances (i.e., drought and fires), and a highly fluctuating climate, with main characteristic the erratic precipitation patterns [4]. The scenarios of climate change impact on the existing precipitation and temperature regimes include a large reduction in annual precipitation and an increase in inter-annual variability. The latter is expected to result in more heavy rainfall concentrated in fewer rain days, thus prolonged and frequent drought events. Thus, it is crucial to include the parameter of rain days in the studies of climatic forcing on phenology and ecosystem productivity.

The findings of the present work revealed differences in both *P. fruticosa* phenology and its climatic drivers between two sites being only 1◦ apart with small differences in climate. We may expect even more significant differences between regions with completely different climatic profile, though the direction and magnitude of response cannot be predicted. The single-species sites examined in this work facilitated EVI signal analysis and drawing conclusions but simultaneously may be considered a study's limitation concerning the applicability of the methodology in more diverse and species-rich ecosystems. Future studies may examine distant Mediterranean sites where significantly different climatic conditions prevail. Moreover, incorporating other key Mediterranean species will be crucial for understanding the dynamics of Mediterranean species phenology in regard to climate. Since *P. fruticosa* is a key species in garrigue formations in Greece (also called phrygana), the findings of the present study may give valuable baseline information for future studies on more complex garrigue ecosystems phenology and the involved climatic drivers.

## **5. Conclusions**

The complex phenological cycle of the drought semi-deciduous *P. fruticosa* was clearly depicted in the satellite-derived EVI seasonal fluctuations in both studied sites, the southern Araxos and the northern Louros. The phenology metrics were differentiated between the two sites. The contribution of both temperature and precipitation is higher in shaping the Louros phenological profile, whereas the more xeric Araxos depends more on rainfall. In Araxos, a trend for SDO advancement and more prolonged SDD was recorded during the last 21 years, closely related to certain precipitation and temperature trends. The results of the present study revealed the importance of analyzing the seasonal timing of the phenological events in the lifecycle of a typical species of the Mediterranean ecosystem and of identifying the climatic drivers of their profiles changes. This approach, which focuses on a single species and explicit small-spatial-scale information, will be crucial in connecting small- and large-scale vegetation responses to climate crisis.

**Author Contributions:** Conceptualization, A.K. and E.L.; investigation, A.K. and E.L.; writing original draft preparation, A.K. and E.L.; writing—review and editing, A.K. and E.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is available from the authors upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **The Influence of the Partitioning of Sugars, Starch, and Free Proline in Various Organs of** *Cyclamen graecum* **on the Biology of the Species and Its Resistance to Abiotic Stressors**

**John Pouris 1, Efi Levizou 2, Maria Karatassiou 3, Maria-Sonia Meletiou-Christou <sup>1</sup> and Sophia Rhizopoulou 1,\***

	- 38446 Volos, Greece; elevizou@uth.gr <sup>3</sup> Laboratory of Rangeland Ecology (PO 286), School of Forestry and Natural Environnent, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; karatass@for.auth.gr

**Abstract:** The geophyte *Cyclamen graecum* is native to the eastern Mediterranean. Its beautiful flowers with upswept pink petals appear during early autumn, after the summer drought period and before leaf expansion in late autumn. The floral and leaf development alternates with their cessation in early winter and late spring, respectively. Ecophysiological parameters and processes underlining the life-cycle of *C. graecum* have not previously been published. Seasonal fluctuations of sugars, starch, and free proline have been investigated in tubers, leaves, pedicels, and petals, as well as petal and leaf water status. At the whole plant level, the seasonal co-existence of leaves and flowers is marked by an elevated soluble sugar content, which was gradually reduced as the above-ground plant parts shed. The sugar content of petals and pedicels was lower than that of leaves and tubers. Leaf starch content increased from late autumn to spring and was comparable to that of tubers. The starch content in petals and pedicels was substantially lower than that of tubers and leaves. In tubers, monthly proline accumulation was sustained at relatively constant values. Although the partitioning of proline in various organs did not show a considerable seasonal variation, resulting in an unchanged profile of the trends between tubers, leaves, and flowers, the seasonal differences in proline accumulation were remarkable at the whole plant level. The pronounced petal proline content during the flowering period seems to be associated with the maintenance of floral turgor. Leaf proline content increased with the advance of the growth season. The values of leaf relative water content were sustained fairly constant before the senescence stage, but lower than the typical values of turgid and transpiring leaves. Relationships of the studied parameters with rainfall indicate the responsiveness of *C. graecum* to water availability in its habitat in the Mediterranean ecosystem.

**Keywords:** *Cyclamen graecum*; geophyte; Mediterranean; phenology; seasonality

## **1. Introduction**

The geophytes exhibit a life-cycle associated with temporal separation of the vegetative phase from the flowering phase and possess perennial tubers, which support their annual growth [1–3]. *Cyclamen* L. (Primulaceae) is commercially important and a very popular horticultural genus native to the area around the Mediterranean Basin [4–6]. Wild cyclamens are perennial geophytes of woods and rocky areas. *Cyclamen* was mentioned as kyklaminos (κυκλ*α*´μινo*ς*) in Theophrastus' writings (4th century BC) [2,7]. The Cyclamen Society [8] recognizes 20 *Cyclamen* species. The cultivated cyclamens are usually hybrids of the spring flowering species *Cyclamen persicum.*

**Citation:** Pouris, J.; Levizou, E.; Karatassiou, M.; Meletiou-Christou, M.-S.; Rhizopoulou, S. The Influence of the Partitioning of Sugars, Starch, and Free Proline in Various Organs of *Cyclamen graecum* on the Biology of the Species and Its Resistance to Abiotic Stressors. *Plants* **2022**, *11*, 1254. https://doi.org/10.3390/ plants11091254

Academic Editors: Jess K. Zimmerman and Matthew Paul

Received: 29 March 2022 Accepted: 3 May 2022 Published: 5 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Cyclamen graecum* Link grows in the wild; it is a perennial, tuberous geophyte, naturally distributed in bushy, stony, and sunlit ground in southern parts of the mainland of Greece and Aegean islands, as well as in coastal areas of Turkey and Cyprus, sometimes being confined into crevices in rocks. Environmental conditions, especially temperature, control annual development and florogenesis in geophytes [9]. *C. graecum* is an autumn flowering geophyte. Floral pedicels and leaf petioles arise from the upper part and/or the crown of the over summering, large, perennial tubers that have nodes marked by small buds [10,11]. It has been published that *Cyclamen* species with more than 30 chromosomes, e.g., *C. persicum* (2n = 48) and *C. graecum* (2n = 84), can develop very large tubers [12,13]. Actually, cyclamens do not produce sister tubers, but their tubers enlarge with age [14,15]. In *C. graecum*, thick anchor roots are developed between fibrous roots, from the center of the base of the tuber [8], but their role is not yet fully clarified; it is expected that they penetrate deep into the texture of stony substrate [4,5], which is a habitat of distinct seasonal aridity and moistness. The dark green, heart-shaped leaves of *C. graecum* appear in November. The leaves are slightly angular with variegated green spots on the adaxial surface; parts of the adaxial leaf surface are light green and other parts are dark green, while the abaxial leaf surface is violet-mauve.

Partitioning of total sugars between plant parts is directly linked to developmental flux of carbon molecules, osmolytes, and energy [16]. Moreover, soluble sugars and proline play pivotal roles in plants' stress responses [16–18]. In geophytes, the mobilization of total sugars and starch from below-ground organs generates crucial metabolites to support vegetative and reproductive growth, and synthesize essential compounds. Starch as a storage compound is easily hydrolyzed to soluble sugars that can be transported to growing plant organs. Subsequently, after leaf shedding, total sugars and starch accumulate in below-ground plant parts to sustain metabolism and serve as transient energy storage during the oncoming above-ground vegetative stage [16]. It may be worth noting that sowbread (*panis porcinus*), a common name for the genus *Cyclamen*, is a reference to tubers supposedly being a favorable food for pigs [19,20].

Proline has proven to be a multi-functional tool in plant metabolism during the last two decades. Beyond its well-established roles as an osmoticum and generally a protectant against abiotic stresses [21], its involvement in various developmental processes has also been recognized [18,22]. Its contribution to stress adaptation extends from the protection of photosynthetic apparatus and the involved enzymes to the stabilization of redox balance in the chloroplast, along with reactive oxygen species detoxification. Proline's involvement in re-adjustment of growth once the stress is relieved relates to its catabolism to support new growth with energy [22]. Under normal non-stress conditions, proline acts as a metabolic signal impacting plant growth and development via regulating metabolite pools and the expression of several genes [18]. Moreover, the widespread phenomenon of proline accumulation in reproductive organs has been attributed to its role as a developmental regulator and flowering signal [18,23]. Interestingly, proline-rich floral nectar has been found in ornamental tobacco flowers and connected with the attraction and reward of visiting pollinators with an energy source to sustain insect flight [24].

The objective of this study was to identify and compare the partitioning of soluble sugars, starch, and proline among above- and below-ground plant parts of Cyclamen graecum, which are exposed to fluctuating environmental conditions, as well as the water status of petals and leaves, which according to the best of our knowledge has not hitherto been published, in order to evaluate ecophysiological traits of tissues exposed to ambient conditions.

## **2. Results**

## *2.1. Phenological Stages*

Within the context of seasonality, the life-cycle of *C. graecum* is characterized by two phenological stages in the course of a year: the active phase (from flower emergence in September to leaf senescence in April) and the dormant phase spanning the prolonged drought period (from May to August) in the eastern Mediterranean, when the aboveground plant parts are not visible. Concerning the active phenological stage, the flowers splay open in September (Figures 1 and 2) before leaf emergence in November (Figure 2). The seasonal floral and leaf development of *C. graecum* in early and late autumn (Figure 2) respectively alternates with cessation of flowers and leaves in winter and before summer, respectively. There is a marked variation in the length of the period of flowering and leafing that affects the capacity of this species for resource acquisition.

**Figure 1.** The flowers of *C. graecum* that attract the eye consist of five upswept petals.


**Figure 2.** Iconographic presentation of the annual cycle and the phenological stages of *C. graecum* (using illustrations of flowers ( ), leaves ( ) and tubers ( )), i.e., the active phase indicated by the ivory area that includes flower initiation and longevity (September–December), and leaf emergence, development, longevity, and senescence (November–April), and the dormant phase indicated by the gray area that includes only the subterranean tubers, because above-ground growth is not visible.

## *2.2. Sugars*

Tubers accumulated large quantities of soluble sugars throughout the year (Figure 3). A gradual reduction of stored sugars in the tubers was detected in October, during the flowering stage, and continued during November and December, when flowers and leaves co-existed. Sugar content of petals and pedicels was lower than that of leaves and tubers, reaching maxima in October. Strong monthly fluctuation of soluble sugars in leaves was detected from November to April, with the maximum values being achieved by mature leaves of December. The considerable sugar content, which was estimated in tubers, declined during the coexistence of floral and leaf stage in November and December. At the whole plant level, the co-existence of leaves and flowers marked the highest sugar content, which was gradually reduced as the above-ground plant parts shed, eventually entering the dormant phase. Monthly values of petal sugar content were positively related with those of tubers (y = 0.523x − 3.882, R<sup>2</sup> = 0.889, *<sup>p</sup>* < 0.05) and pedicels (y = 1.227x − 30.461, R2 = 0.609, *p* < 0.05), while leaf sugar content was negatively related with that of tubers (y = −0.494x + 337.030, R2 = 0.591). In addition, the seasonal rainfall was negatively correlated with the sugar content of petals (y = −0.213x + 87.849, R2 = 0.619, *<sup>p</sup>* < 0.05) and positively correlated with the sugar content of leaves (y = 0.098x + 17.322, R2 = 0.709, *p* < 0.05).

**Figure 3.** Sugar content in tubers (brown bars), leaves (green bars), pedicels (gray bars), and petals (purple bars) of *Cyclamen graecum*, from September (S) to August (A). Each column denotes means of five replicates ± standard error. Standard Errors smaller than the line thickness of the columns are not shown. Significant differences (*p* < 0.05) of mean values are marked using lowercase letters.

## *2.3. Starch*

The highest starch content in tubers of *C. graecum* was observed in April (256 mg g<sup>−</sup>1) when the climatic conditions were favorable for photosynthesis in the Mediterranean ecosystems, and before the cessation of foliar growth of *C. graecum*. Thereafter, from May to October, starch content declined (Figure 4). The lowest values of starch content in tubers (80 mg g−1) were measured from June to October; actually, this period coincides the three-month summer drought (June to August), the cessation of foliar growth, and the early stage of flower formation in September, before the development of new expanding leaves in November (Figure 2). Intermediate values of starch content were detected in tubers from November to January during the development of new leaves. The values of starch content in leaves increased from November to April and were comparable to those of the tubers (Figure 4); in fact, seasonal variation of leaf starch content was positively related with that of tubers (y = 1.027x + 13.422, R2 = 0.784, *p* < 0.05). The starch content in petals and pedicels was substantially lower than that of tubers and leaves during the flowering stage (from September to December) (Figure 2). The relatively elevated values of starch content in petals in September and October decreased in November and December (Figure 4), coinciding with elevated starch content in leaves (approximately from 100 to 140 mg g−<sup>1</sup> d.w.). Negative relationships were detected between petal starch content with the corresponding tuber starch content (y = −0.632x + 125.930, R<sup>2</sup> = 0.892, *<sup>p</sup>* < 0.05) and the precipitation in the study site (y = −1.146x + 117.010, R<sup>2</sup> = 0.839, *<sup>p</sup>* < 0.05).

**Figure 4.** Starch content in tubers (brown bars), leaves (green bars), pedicels (gray bars), and petals (purple bars) of *Cyclamen graecum,* from September (S) to August (A). Each column denotes means of five replicates ± standard error; Standard Errors smaller than the line thickness of the columns are not shown. Significant differences (*p* < 0.05) of mean values are marked using lowercase letters.

## *2.4. Proline*

The partitioning of free proline in tubers, leaves, pedicels, and petals are presented in Figure 5. The tubers sustained an almost stable free proline content throughout the year, with a slight but statistically significant increase during the cold months (November to March). The pronounced free proline accumulation in petals was evident during the flowering stage showing a substantial enhancement during October and November, the period of full blossom. The pedicels contained a quantity of proline at a comparative level with that of the tubers. The proline content found in leaves during the first two months of their appearance is comparable with that of tubers, but it significantly increased during the next four months of the leaf-stage for this species. Accordingly, the partitioning of proline in the various plant parts did not show a considerable seasonal variation resulting in an unchanged profile of the trends between tubers, leaves, and flowers. Nevertheless, seasonal differences in proline accumulation were obvious at the whole plant level, with the contribution of the proline-rich petals being remarkable.

**Figure 5.** Proline content in tubers (brown bars), leaves (green bars), pedicels (gray bars), and petals (purple bars) of *Cyclamen graecum*, from September (S) to August (A). Each column denotes means of

five replicates ± standard error; Standard Errors smaller than the line thickness of the columns are not shown. Significant differences (*p* < 0.05) of mean values are marked using lowercase letters.

## *2.5. Water Status of above-Ground Plant Parts*

Low values of petal water potential (Ψ) were detected in September (Table 1), after the summer drought period. In October Ψ, osmotic potential (Ψs) and turgor (Ψp) values were increased (less negative) (Table 1), and the same holds true in November. In December, the elevated values of Ψ and Ψ<sup>s</sup> coincided with the lowest values of Ψ<sup>p</sup> (Table 1), most probably due to cease of flowering by late December. Regarding Ψ, Ψs, and Ψ<sup>p</sup> values of petals, significant differences (*p* < 0.05) were found among sampling dates (Table 1).

**Table 1.** Seasonal water potential (Ψ), osmotic potential (Ψs), and turgor (Ψp) of petals. The values are means of five replicates ± SE.


Significant differences (*p* < 0.05) of mean values are marked using lowercase superscript letters that are given separately on each column variable.

In petals, significant positive correlations were detected between Ψ and rainfall (y = 0.0123x − 1.2499, R2 = 0.954, *<sup>p</sup>* < 0.05) and <sup>Ψ</sup><sup>s</sup> (y = 0.0122x − 1.3865, R2 = 0.811, *<sup>p</sup>* < 0.05); also, negative relations were detected between soluble sugars and Ψ (y = −0.0029x − 0.1193, R2 = 0.730, *<sup>p</sup>* < 0.05), and <sup>Ψ</sup><sup>s</sup> (y = −0.0034x − 0.6634, R2 = 0.843, *<sup>p</sup>* < 0.05), as well as between starch and <sup>Ψ</sup> (y= −0.0149x + 0.2407, R<sup>2</sup> = 0.908, *<sup>p</sup>* < 0.05) and <sup>Ψ</sup><sup>s</sup> (y = −0.0164x + 0.1905, R2 = 0.936, *p* < 0.05), while a positive relationship between Ψ<sup>p</sup> and proline content was found (y = 0.022x − 0.1168, R<sup>2</sup> = 0.822, *<sup>p</sup>* < 0.05), as well as <sup>Ψ</sup><sup>p</sup> and soluble sugars (y = 0.0007x + 0.00146, R2 = 0.785, *p* < 0.05).

The relative water content (RWC) of leaves was sustained fairly constant until March and declined in April (Table 2). Additionally, a positive relationship was detected between RWC and rainfall (y = 0.2768x + 63.088, R2 = 0.835, *p* < 0.05).


**Table 2.** Relative water content (RWC) of leaves. The values are means of five replicates ± SE.

Significant differences (*p* < 0.05) of mean values are marked using lowercase superscript letters.

## **3. Discussion**

The seasonal accumulation of starch and soluble sugars in the tubers of *C. graecum* confirms to their role in storing photoassimilates and providing a supply of energy to drive new growth [17,25]. The distribution between soluble carbohydrates and starch differed between leaves and tubers. The partitioning of starch to tubers was reasonably similar to that of leaves and a positive linear relationship was detected between tubers and leaves (y = 1.027x + 13.422, R2 = 0.784, *p* < 0.05); starch partitioning may be linked to source-sink relationships; the photosynthetically active leaves (the source) provide assimilated carbon (available for transport) to a storage organ (sink), which will utilize it to support metabolic requirements. Furthermore, the elevated values of starch from February to April in both tubers and leaves coincide with the leaf photosynthetic efficiency, and the mild winter temperatures in the Mediterranean area that favor photosynthetic rates. It seems likely that in *C. graecum*, starch may represent a commitment of resources that are acquired by the above-ground tissues simultaneously with the growth of those drawn from stored reserves. This finding is in contrast to data published elsewhere for the summer flowering geophyte *Pancratium maritimum*, where starch was mainly stored in underground bulbs [26].

At the phenological stage of flowering, when the ambient temperatures begin to fall, the concentration of soluble sugars in the tubers rapidly decreased to support the metabolically demanding reproductive growth with carbon source and energy [27,28]; this is also indicated by the positive linear relationship between sugar content between tubers and petals (y = 0.523x − 3.882, R<sup>2</sup> = 0.881, *<sup>p</sup>* < 0.05). Floral growth during the early autumn takes precedence over allocation. A further decrease of soluble sugars in tubers at the period of leaf emergence indicates a translocation of stored sugars to sustain leaf development and floral exhibition, when winter lies ahead, via a transition from sink to source. The leaf sugar content during winter and spring denotes an active photosynthetic machinery for this species grown under ambient, environmental conditions, coinciding with the values of leaf RWC before wilting; leaf RWC may also be considered as a measure of the relative cellular volume, affecting interactions among macromolecules. Usually, levels of RWC below 70% imply a water potential at the order of −1.5 MPa or less, and this would cause changes in the metabolism with ceasing of photosynthesis [29], concomitantly with leaf senescence in April. Leaf sugar content may also be associated with the argument that leaves of cyclamen species are a static export pool of sucrose, and the sugar transport is probably linked to a time lag in the export of newly fixed carbon from leaves and low velocity of phloem transport [30]. Concerning the sugar partitioning, some geophytes seem to follow a pattern of relatively higher sugar concentration in the subterranean organs compared to leaves and in some cases, with flowers [31]. In petals, the reduced osmotic potential was significantly related with increased soluble sugar content (y = −0.0034x − 0.1663, R2 = 0.834, *<sup>p</sup>* < 0.05), presumably contributing to turgor maintenance, expansion and water status of these tissues [32–34]. Actually, anthesis appears to be due to a pulsed increase in the concentration of soluble sugars [35–38]. A relationship linked to transfer of sugars between leaves and petals, which might be interesting, was not evaluated, because in *C. graecum*, flower and leaf development are concomitantly exhibited only during a two-month period, i.e., November and December.

Proline content in tubers of *C. graecum* showed a small but statistically significant increase from November to March, compared to the rest of the year. This accumulation may be driven by the low temperatures of the corresponding months and may be considered a stress-related response. An analogous profile was followed by its leaves, resulting in unchanged partitioning of proline between leaves and tubers, when leaves are coming through during the life-cycle of C. graecum; nevertheless, the greatest variation was found between petal and leaf concentrations. It has been published that *C. graecum* is a coldtolerant species [37,39]. The increased proline biosynthesis and accumulation may partly account for a cold hardiness feature in both tubers and leaves, due to its protectiveness regarding stress and radical scavenging role [40]. Concerning the latter, proline has been related to scavenging of hydroxyl radicals (OH) and possibly other ROS [41], while indirectly modifies the plant's antioxidant response through increasing the capacity of the involved enzymes, especially ascorbate peroxidase [22]. Additionally, proline accumulation patterns may have implications on nitrogen storage and partitioning, especially under stress conditions [22,27,42]. Proline pool has been reported to expand during transition phase, i.e., from vegetative to reproductive growth [43–46].

Ecophysiological traits of plant organs that are seasonally either renewed or shed may be a suitable criterion of plant's adaptation to environmental conditions. *C. graecum* survives the hot summer in a state of dormancy. *C. graecum* blooms in autumn, before leaf emergence. Thereafter, leaves grow and accumulate metabolic reserves throughout the wet and cool season, until the dormancy period, which begins in late spring. In geophytes, long days can initiate the transition to bud dormancy [47,48]. The summer dormancy protects plants from negative effects of water shortage and elevated temperatures on vegetative and reproductive organs, and forces their active development in a more favorable season. *C. graecum* is released from dormancy when the ambient temperatures decrease; hence, shifts from the vegetative to reproductive stage, and floral initiation and differentiation occur in the mature tubers [8,49].

Free proline accumulation in petals was remarkable and significantly increased compared to the other plant organs. Flowers exhibited a 2.5- to 5-fold enhanced proline content in comparison to tubers and 2- to 3-fold compared to leaves. The transportation of proline into the reproductive organs, even under non-stress conditions, has been repeatedly reported [49]. Corroborating our results, a 60-fold higher proline concentration was detected in tomato flowers than in all the vegetative tissues [50]. Enhanced proline content was also documented in petals of *P. maritimum* and was attributed to a requirement for osmotic adjustment, because this geophyte is exposed to dry and saline ambient environmental conditions [26,46]. Multiple explanations of proline accumulation in flowers have been published. For example, the increased proline content has been connected to the high yield of ATP resulting from its oxidation, thus considering proline a molecule well-suited to sustain high energy-demanding processes in reproductive tissues [44]. Low values of petal osmotic potential coincided with enhanced proline accumulation. Additionally, the protective role of proline, which was positively correlated with the turgor of petals of *C. graecum*, has been highlighted during floral developmental processes that include dehydration, as spontaneously occurring during pollen formation or embryogenesis [51,52]. Furthermore, proline may provide a convenient source of energy and nitrogen during immediate post-stress metabolism [46,49,53].

## **4. Material and Methods**

## *4.1. Research Site and Plant Phenology*

The study was conducted in naturally occurring patches of *Cyclamen graecum* Link distributed in the campus of the National and Kapodistrian University of Athens in Greece (latitude: 37.9664, longitude: 23.756971, altitude 260 m), at the foothills of Hymettus Mountain, where travertine limestone appears along discontinuities of strongly fractured gray dolomite limestone; also, father soil characteristics, texture, and composition have been previously published [54,55]. Concerning perennial geophytes, most bulbs and tubers must reach a critical size before floral induction can occur [56]. In addition, large bulbs and tubers generally produce vigorous above-ground organs and/or many flowers. Therefore, the active and the dormant phenological stage (Figure 2) were monitored, via detailed field observations in distinct niches of *C. graecum* [57], on a monthly basis for two consecutive years (2017 and 2018). The selected at random plants were growing under natural conditions; tubers, leaves, and flowers of *C. graecum* were sampled from a single stand of *C. graecum* surrounded by uniform Mediterranean phryganic vegetation [54], at monthly intervals during the course of a year, i.e., from September of 2018 to August of the next year (2019). The first flowers of *C. graecum* [58] appear in September, while the heart-shaped leaves are coming through in November, arising from the crown of the tubers [59]. Values of mean monthly precipitation and temperature, obtained from a meteorological enclosure, provided by the National Observatory of weather conditions in Greece, are presented in Figure 6a (annual data during the study period) and Figure 6b (multiannual data).

**Figure 6.** (**a**) Ombrothermic diagram (Precipitation scale = 2 × Temperature scale) for the study site; the order to months is from September (S) of 2018 to August (A) of 2019. Mean monthly precipitation is indicated by blue bars and mean monthly temperature by closed circles and the black line; (**b**) A multiannual ombrothermic diagram (Precipitation scale = 2 × Temperature scale) for the study site from September of 1955 to August of 2010; the order to months is from September (S) to August (A). Mean monthly precipitation is indicated by shaded bars, and mean monthly temperature by closed circles and the black line.

## *4.2. Determination of Total Soluble Sugar and Starch*

Soluble sugars were extracted from dry, finely powdered samples (leaves, tubers, pedicels, petals) that were placed in 10 mL 80% ethanol (*v*/*v*), in a shaker, and the extracts were filtered using Whatman # 2 filter paper. Soluble sugar concentration was investigated colorimetrically, according to a modified phenol-sulphuric acid method [60,61], at 490 nm, using a spectrophotometer (Novaspec III+ Spectrophotometer, Biochrom, Cambridge, UK). The determination of starch was made in the residue after the extraction of sugars, using the anthrone method [26,62]. D-glucose (Serva, Heidelberg, Germany) aqueous solutions were used for the standard curve. The values are expressed as mg g−<sup>1</sup> d.w.

## *4.3. Determination of Proline*

Free proline content was determined colorimetrically on 4 mL samples of the condensed fluid extracted from the plant material [63,64]. The extraction procedure from plant samples (finely powdered dried tubers, leaves, pedicels, and petals) and colorimetric determination were carried out as we have analytically published [26]. Dried, powdered samples were homogenized with aqueous sulphosalicylic acid (20 mL, 3% *v*/*v*), and the

homogenate filtered through Whatman # 2 filter paper; 2 mL of the filtrate reacted with acid-ninhydrin solution (2 mL) and glacial acetic acid (2 mL) in test tubes, which were placed in a water bath at 100 ◦C for 1 h, and the reaction terminated in an ice bath. After cooling, the reaction mixture was extracted with 4 mL toluene and homogenized in a vortex. The chromophore containing the toluene was aspirated from the aqueous phase and the absorbance was read at 520 nm; immediately after, the terminated reaction in glass tubes placed in an ice bath, using toluene as a blank sample and the spectrophotometer mentioned in paragraph 5.2. The proline concentration was estimated using a standard curve of relevant L-proline solutions (Serva, Heidelberg, Germany) and calculated on a dry weight basis.

## *4.4. Determination of Water Status*

Petal water potential (Ψ) was measured psychometrically on 6 mm diameter fresh discs (five replicates) from fully expanded petals, which were placed in five C-52 psychrometric chambers (Wescor Inc., Logan, UT, USA) attached to a dew point psychrometer (HR-33T, Wescor Inc.) using the psychrometer switchbox (PS-10, Wescor); the time required for equilibration between the water vapor pressure of leaf sample and that of the psychrometer chamber was 2 h. The osmotic potential (Ψs) was measured using the same leaf discs after freezing and thawing [65,66]. Turgor pressure (Ψp) was calculated as the algebraic difference between Ψ and Ψs. The relative water content (RWC) of fully expanded leaves was determined according to the disc method [29], using the equation RWC (%) = [(FW − DW)/(TW − DW)] × 100, where: FW is the sample fresh weight, TW is the sample turgid weight, and DW is the sample dry weight.

## *4.5. Statistical Analysis*

The results are presented as mean ± Standard Error (SE). In order to determine differences in the studied parameters of plant parts of *C. graecum*, a two-way analysis of variance (ANOVA) was performed on the studied parameters at *p* < 0.05 and the Duncan's multiple range test was applied for comparing the means. All statistical tests were performed using the SPSS statistical v. 23.0 (SPSS Inc., Chicago, IL, USA). Regression analysis was used to determine relationships among results obtained by plant tissues of *C. graecum* and precipitation.

## **5. Conclusions**

The life form of *C. graecum* is characterized by two phenological stages in the course of a year, the active phase (from flower emergence in September to leaf senescence in April), and the dormant phase spanning the prolonged drought period, when above-ground plant parts are not exposed to the severity of summer in the eastern Mediterranean. Partitioning patterns of soluble sugars, starch, and free proline in above- and below-ground parts of *C. graecum* contribute to the maintenance of its annual rhythm and phenophases in fluctuating environmental conditions. The remarkable concentration of proline in petals, in comparison to other plant parts during autumn, seems to be associated with the maintenance of their turgor; without turgor, the exposed petals to ambient environmental conditions of the sharply reflexed corolla of *C. graecum* could not be standing so firm and erect. The leaf relative water content was found lower than the typical values of turgid and transpiring leaves; this may indicate that leaves of *C. graecum* subjected to ambient conditions are not susceptible to low temperatures. However, further work will be required to fully test this hypothesis.

**Author Contributions:** Conceptualization: J.P. and S.R.; methodology: J.P., M.-S.M.-C. and S.R.; investigation: J.P.; resources: S.R.; writing—original draft preparation. J.P., E.L., M.K., M.-S.M.-C. and S.R.; writing—review and editing: E.L., M.K., M.-S.M.-C. and S.R.; visualization: J.P., M.-S.M.-C. and S.R.; supervision: S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received funding from the National and Kapodistrian University of Athens (Greece).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are available from the authors upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Brief Report* **Effect of Temperature on the Germination of Five Coastal Provenances of** *Nothofagus glauca* **(Phil.) Krasser, the Most Representative Species of the Mediterranean Forests of South America**

**Rómulo E. Santelices-Moya 1, Marta González Ortega 2, Manuel Acevedo Tapia 2, Eduardo Cartes Rodríguez <sup>2</sup> and Antonio M. Cabrera-Ariza 3,\***


**Abstract:** Temperature is one of the most important abiotic factors affecting seed germination, and it is strongly influenced by local site conditions. Seeds of *Nothofagus glauca*, an endemic and vulnerable species of the Mediterranean region of Chile and the most representative of the Mediterranean forests of South America, were collected. In this study, we evaluated the effect of temperature on different germinative attributes of five *N. glauca* provenances representative of their natural distribution. The seeds were treated at a constant temperature (i.e., 18 ◦C, 22 ◦C, 26 ◦C, or 30 ◦C) in the absence of light for 40 days. The results show that in all the provenances, the germination ratio and energy increase linearly with temperature until reaching an optimum temperature (i.e., 22 ◦C), above which they decrease severely. At 22 ◦C, the response of average germination speed and germination vigor was significantly higher than with the other temperatures (performance of germination start day was not clear). The base temperature was around 18 ◦C and the maximum, above 30 ◦C, which may be close to thermo-inhibition. Given the threat of climate change, it is necessary to increase research in terms of the possible adaptation of this species to increased temperatures and prolonged periods of drought

**Keywords:** hualo; Mediterranean plants; seeds

## **1. Introduction**

*Nothofagus glauca* (Phil.) Krasser (common name, hualo or roble maulino) is an endemic species of Central Chile that belongs to the Nothofagaceae family and is the most representative of the Mediterranean forests of its genus in South America. It is a deciduous, monoecious tree that can reach up to 30 m in height and 2 m in diameter [1], although at present it is difficult to find individuals that are more than 40 cm in diameter. The species is listed as vulnerable, and its populations are currently severely fragmented and trending toward decreasing [2].

The *N. glauca* forests have a discontinuous distribution in a latitudinal range of about 400 km, from 33◦58 S, 71◦05 W to 37◦27 S, 71◦58 W, although they are mostly concentrated in the Maule Region [3]. They are a transitional system between xerophytic formations and the southernmost temperate forests. This type of deciduous forest has adapted to the prolonged dry periods of summer and plays very important roles in the conservation of water and organic soil and in the biogeochemical carbon cycle as well as offering a great variety of ecological niches and habitats to the flora, fauna, and associated microbiota [4]. The

**Citation:** Santelices-Moya, R.E.; González Ortega, M.; Acevedo Tapia, M.; Cartes Rodríguez, E.; Cabrera-Ariza, A.M. Effect of Temperature on the Germination of Five Coastal Provenances of *Nothofagus glauca* (Phil.) Krasser, the Most Representative Species of the Mediterranean Forests of South America. *Plants* **2022**, *11*, 297. https://doi.org/10.3390/ plants11030297

Academic Editors: Sofia Rhizopoulou, Maria Karatassiou and Efi Levizou

Received: 12 January 2022 Accepted: 21 January 2022 Published: 24 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

natural range of this species has been considered a hotspot of biodiversity for conservation and is characterized by a great diversity of endemic species, although this has decreased to critical levels in terms of dominance and variability mainly because of anthropogenic factors [5]. The highest population density in Chile is concentrated in *N. glauca* distribution area, with the consequent pressure on natural resources, including the *N. glauca* forests.

The anthropogenic pressure has strongly shaped the landscape in the natural distribution area of *N. glauca* in recent years, affecting its spatial distribution, among other variables. Forty-five years ago, Urzúa [6] reported that there were 900,000 ha of these forests, whereas today, there are only 157,000 ha [3]. Another threat looming over these and other forests in the region is global climate change. Indeed, an increase in both temperatures and prolonged periods of drought has been recorded, factors that predispose the vegetation to being more prone to damage caused by biotic and abiotic agents. As an example, it can be noted that in the summer of 2017, 184,000 ha of the forest were consumed in a single fire, which affected an important part of this forest system [7]. In addition, considering that its regeneration by natural seeding is currently almost nonexistent, it is necessary to study its propagation by seeds to provide the necessary background to produce plants intended for afforestation and/or restoration of its populations. It is also important to consider the temperatures that limit the germination of seeds, especially if climate change is a factor that is affecting ecosystems.

Temperature is a crucial factor in the germination process of seeds [8]. It plays an important role in determining the periodicity of seed germination and the distribution of species (among other factors, it affects enzymatic activity) [9]. The germination rate generally increases linearly with temperatures up to an optimum temperature; subsequently, germination decreases severely with higher temperatures. Moreover, in the seed germination process, there are three levels of temperature: minimum, optimal, and maximum. The minimum, or base, temperature is the lowest temperature at which the seeds can germinate. The optimum temperature is the temperature at which the seeds reach their highest germination rate, and the maximum, or ceiling, temperature is the temperature above which the seeds cannot germinate [10]. The germination rate will increase between the minimum and optimum temperatures, whereas temperatures ranging from optimum to maximum will lead to a decrease in this attribute [11]. If the temperature is changing because of global climate change, it is evident that some species will have to adapt to these new environmental conditions, among other aspects, in the germination process.

*N. glauca* is a species whose seed production cycles are becoming longer, with years without seed production (unpublished data) and has seeds that present endogenous dormancy. There are mechanisms to break this condition, for example, through variable stratification periods at 4 ◦C (i.e., between 4 and 6 weeks) or by soaking them in gibberellic acid in concentrations from 0.1 to 0.8 g L−<sup>1</sup> [12], reaching a germination ratio even above 95% using only viable seeds. However, the germination percentage may be different depending on the origin of the seeds [13]. Until today, no report has been made on the effect of temperature on the germination of any population of *N. glauca*. Therefore, this study aimed to analyze the effect of temperature in the germination process of seeds from five coastal provenances of *N. glauca*.

## **2. Results**

The results show that temperature has a significant effect on the germination of *N. glauca* seeds from different provenances (Table 1, Figures 1 and 2). In all the provenances, the highest germination percentage was obtained at 22 ◦C, although very different values were recorded in the germination ratio, from 34.0 ± 0.6% in the Los Ruiles provenance to 78 ± 0.3% in the Las Cañas provenance, observing the same tendency in the germinative energy. In general, in the northernmost provenances, a higher germination ratio was observed. These results show that *N. glauca* has a significant germination potential at 22 ◦C. In addition, the highest slope in the germination curves was observed at this temperature (Figure 2), which is an indicator that with endogenous application of gibberellic

acid at 22 ◦C, latency is better overcome; it could be considered, then, that the optimum germination temperature for most provenances of *N. glauca* is around 22 ◦C (in the Las Cañas provenance, there were no significant differences between 22 ◦C and 26 ◦C).


**Table 1.** Effect of temperature on different germination parameters of *Nothofagus glauca* (mean ± SE). Different letters indicate significant differences by Tukey's multiple comparison test (*p* < 0.05).

**Figure 1.** Cumulative germination percentage during 40 days for five *Nothofagus glauca* provenances treated at different temperatures in the absence of light.

**Figure 2.** Final germination percentage of five provenances of *Nothofagus glauca* seeds treated at different temperatures in the absence of light.

In most of the provenances, once the seeds had begun to germinate, higher temperatures stimulated germination to an optimum temperature (22 ◦C), after which at 26 ◦C and 30 ◦C, the germination rate decreased. A clear pattern was also observed on the germination start day because of temperature, although seeds treated at 18 ◦C generally took longer to germinate. In general, average germination speed and vigor were higher at 22 ◦C, to later decrease with increasing temperatures; at 18 ◦C the lowest values were recorded in these two variables, although in some provenances, no significant differences were observed regarding those seeds treated at the highest temperature (30 ◦C).

## **3. Discussion**

Seed germination is a process in which endogenous and exogenous factors intervene. On the one hand, two growth regulators that play a fundamental role in this process are gibberellic acid, a promoter of germination, and abscisic acid (ABA), which is an inhibitor. On the other hand, in addition to humidity and oxygen availability, temperature is the environmental factor with the greatest effect on the germination process of seeds, directly affecting their metabolism and germination speed [8]. With super-optimal temperatures, seed germination is delayed because a high level of ABA is maintained in the embryo and endosperm and the synthesis of gibberellic acid is suppressed [14,15]. The results of our research show a significant drop in germination at 30 ◦C, and it is likely that above this temperature, the seeds of *N. glauca* approach the thermo-inhibition process and prevent gibberellin biosynthesis.

The cardinal temperatures for germination are related to the environmental range of adaptation of a particular species and serve to match the germination time with the favorable conditions for the growth and subsequent development of the seedlings [10]. In our research in all the provenances studied, it was observed that from 18 ◦C, as the temperature increased, so did the germination until reaching a maximum at 22 ◦C, and then decreased. Thus, the optimum germination temperature for *N. glauca* would be at 22 ◦C, whereas the minimum, or basal, temperature would be around 18 ◦C, and the maximum, or ceiling, temperature would be above 30 ◦C. While the optimum temperature germination rate may vary between seed lots of the same species as a result of different genetic and environmental conditions [9], in the five provenances of *N. glauca*, this was not the case; the optimum temperature was clearly 22 ◦C, although for the Las Cañas provenance, it would be between 22 ◦C and 26 ◦C. It is striking that in the Los Ruiles provenance (area

protected by the state), germination at 18 ◦C was practically null and the basal temperature for that provenance would likely be above that level. In the case of the southernmost provenance (Quirihue), at 30 ◦C, germination fell to levels below those reached at 18 ◦C; in this case, it would likely be closer to thermo-inhibition at that temperature. According to these results, it is probable that there is a genetic effect that conditions the germination process of the seeds.

Temperature not only plays a fundamental role in capacity and energy germination, but it also affects the germination start day, average germination speed, and germination vigor (Table 2). Once the seed has been rehydrated after imbibition, under suitable temperature conditions, physiological processes are triggered that allow the germination process to develop [8]. Although germination occurred at 18 ◦C, with variable rates according to the geographic origin of the seeds, the process is significantly slower at that temperature, taking at least 24 days to begin germination; instead, with the optimum temperature (i.e., 22 ◦C), it begins in some cases at 12 days. This same trend is also observed in both the average germination speed and vigor, meaning that the germination process of *N. glauca* is strongly conditioned by temperature. The germination ratio at the optimal temperature (i.e., 22 ◦C), is in the range found by other authors for the same species [12,13].

**Table 2.** Geographic and climatic data for provenances sampled.


<sup>1</sup> M.A.T.: mean annual temperature; <sup>2</sup> M.A.R.: mean annual rainfall.

In the northernmost provenances, a higher percentage of germination was recorded, although no relationship was observed with the weight and morphometric characteristics of the seeds. In other species of the genus *Nothofagus*, it has been observed that the larger and heavier seeds have a greater germination ratio than those that are smaller and lighter, observing a clinal variation, although in a broader latitudinal distribution than that of this study [16]. This pattern was not observed in our study.

All species have a temperature range in which the germination process occurs. It has been described that for *N. glauca,* this range is likely between 10 ◦C and 30 ◦C [17]. On the one hand, in our research, we observed that the minimum temperature would be around 18 ◦C, especially for the Los Ruiles provenance, which is why it would be advisable to investigate the behavior of different provenances of this species under 18 ◦C. On the other hand, at 30 ◦C, germination strongly decreases, and above that level would be the maximum temperature and occurrence of thermo-inhibition. Consequently, temperatures under 18 ◦C and above 30 ◦C in the germination process of *N. glauca* should be evaluated.

Temperature is one of the main environmental factors that regulate seed physiology across plant taxa [10]. Because of climate change (i.e., increased temperatures and prolonged periods of drought), plants in general are being subjected to greater water stress, and therefore, their physiological processes are being affected. In the Mediterranean region of Chile, where *N. glauca* is distributed, there has been a significant increase in extremely hot events affecting the average temperature, and a deficit in rainfall [18]. *N. glauca* is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year, and it has been observed that the cycles in seed production are becoming longer, with years without seed production (unpublished data). Given the threat of climate change on the reproductive cycle of the species and considering the effect that temperature has on the germination of *N. glauca* seeds, it is urgent and necessary to study in greater depth the adaptation capacity that this species would have to these new environmental conditions. Santelices, Espinoza, Magni, Cabrera, Donoso, and Peña [13] reported that the intra-provenance variability of *N. glauca* is systematically greater than that of inter-provenance, indicating a high potential capacity of the species to adapt to climate change. However, these authors affirm that there are differences in germination between Andean and coastal origins (in our research, we evaluated only costal provenances); this reaffirms the need to deepen research on the potential adaptation of *N. glauca* to climate change and the capacity of the species to regenerate and self-perpetuate.

## **4. Material and Methods**

## *4.1. Seed Collection and Preparation*

In March 2017, mature seeds were collected from different provenances of *N. glauca* in the Maule Region of Chile (Table 3, Figure 3), except those from Licantén, which were collected in March 2015. Seeds were transported to the laboratory, where they were manually separated from the rest of the plant material and the damaged seeds were discarded; then, they were weighed, dried, and stored in the dark in glass containers in an environment at 4 ◦C until they were used (i.e., January 2020). The standards of the International Seed Testing Association (ISTA) were followed to characterize the seeds [19]. One hundred seeds were weighed separately for eight repetitions to determine the weight of the seeds, which was expressed as the average weight of 1000 seeds. Then, the average weight of 1000 seeds and its equivalence in number of seeds per kilogram were calculated. In addition, the dimerous seeds were measured for length and width, and the trimerous seeds for length, width, and thickness (Table 2). To break the dormancy, the seeds were soaked in a 200 mg L−<sup>1</sup> gibberellic acid solution (Giberplus® Tabletas, Anasac Chile S.A., Santiago, Chile) for 24 h before starting the germination tests [12].

The mean annual precipitation and temperature were obtained from WorldClim (version 2) at a spatial resolution of 30 s (~1 km2) by interpolation of the records of meteorological stations from 1970 to 2000 [20].

**Figure 3.** Seed provenances of *Nothofagus glauca*.


**Table 3.** Weight and morphometric characterization of the seeds from five provenances of *Nothofagus glauca* (mean ± Standard Error).

## *4.2. Germination Experiments*

The study was carried out in a laboratory at the Universidad Católica del Maule, Talca, Chile (35◦26 10 S, 71◦37 13 W, 131 m a.s.l.), during January and February 2020. The seeds were soaked in a Gibberellic Acid (GA3) solution at 200 mg L−<sup>1</sup> for 24 h using distilled water, and those that floated were excluded as they were considered unviable. To determine the effect of temperature on the germination of *N. glauca* seeds from different provenances, four different temperatures were tested: 18 ◦C, 22 ◦C, 26 ◦C, and 30 ◦C. The cultivation was carried out in germinating chambers in the absence of light, maintaining fixed temperatures according to each treatment and using filter paper as the substrate. To not interfere with the treatments, the ambient temperature of the laboratory was constantly maintained below 16 ◦C. Irrigation was manual, and care was taken that the seeds were always wet.

The germination process was monitored daily until germination ceased over a period of 40 days. Seeds were considered to have germinated when the emerging radicles were over 2 mm long. The following germination parameters, adapted from [21,22], were calculated:

$$\text{GR} = \left[\frac{\text{Sg}}{\text{Ss}}\right] \times 100 \tag{1}$$

Germination ratio (GR) (1): represents the final percentage of seeds that germinate (Sg) in relation to the total number of seeds sown (Ss):

Germination energy (GE): accumulated percentage of germination on the day when the maximum value occurs (maximum value is maximum ratio from cultivated germination percentage on day X divided by X).

Germination start day (GSD): the time elapsed from the sowing of the seeds to the germination of 5% of the sown seeds.

Average germination speed (AGS) [2]: corresponds to the average number of germinated seeds per day, calculated by the expression:

$$\text{AGS} = \sum\_{1}^{k} \frac{m}{t\dot{i}} \tag{2}$$

where *ni* corresponds to the number of seeds germinated in the *i*th data collection, *ti* is the time (in days) of the *i*th data collection, and *k* is the time (in days) of the germination test duration.

Germination vigor (GV): reflects in a single value the changes in the germination peak, the total germination, and the germination speed, calculated as the product between the maximum value and the average germination speed.

## *4.3. Trial Design and Statistical Analysis*

There were two factors tested in the trial: temperature (four levels) and provenance (five levels). The factorial combination of 20 treatments (4 × 5) was replicated three times in a split-plot design. Fixed effects were randomly assigned within subplots (considering the homogeneity of the temperature, whole plots were not randomized). There were 50 viable seeds per factorial combination, giving a total of 150 seeds per treatment. Temperature was applied to the whole plots and provenance, to the subplots. The treatments imposed follow:


Analyses of variance (ANOVAs) and comparisons of the means were conducted using the general linear model (GLM) procedure from the statistical software SPSS for Windows (SPSS, Chicago, IL, USA). Mean values with significant differences were compared using the Tukey test at the 5% significance level.

## **5. Conclusions**

Based on the results of this research, it can be concluded that temperature is an abiotic factor that significantly affects the germination of *N. glauca*. In the absence of light conditions, the optimum germination temperature for all the provenances studied was 22 ◦C. By maintaining the temperature at 18 ◦C, germination was induced, although at a low percentage. At 30 ◦C, the germination percentage was also low, and above this level is the maximum germination temperature and risk of thermo-inhibition.

**Author Contributions:** Conceptualization, R.E.S.-M.; methodology, R.E.S.-M. and A.M.C.-A.; software, R.E.S.-M. and A.M.C.-A.; validation, R.E.S.-M. and A.M.C.-A.; formal analysis, E.C.R.; investigation, R.E.S.-M. and A.M.C.-A.; resources, R.E.S.-M., M.G.O., M.A.T. and E.C.R.; data curation, R.E.S.-M. and A.M.C.-A.; writing—original draft preparation, R.E.S.-M.; writing—review and editing, A.M.C.-A., M.G.O., M.A.T. and E.C.R.; visualization, R.E.S.-M. and A.M.C.-A.; supervision, R.E.S.-M.; project administration, R.E.S.-M.; funding acquisition, R.E.S.-M., M.G.O., M.A.T. and E.C.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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