Drought Stress Effects and Ways for Improving Drought Tolerance in Impatiens walleriana Hook.f.—A Review

Abstract

Drought is one of the main abiotic stresses affecting plant growth and development. Reduced plant yield and quality are primarily caused by the reductions in photosynthesis, mineral uptake, metabolic disorders, damages from the increased production of reactive oxygen species, and many other disruptions. Plants utilize drought resistance mechanisms as a defense strategy, and the systems’ activation is dependent upon several factors, including plant genotype, onthogenesis phase, drought intensity and duration, and the season in which the drought occurs. Impatiens walleriana is a worldwide popular flowering plant recognized for its vibrant flower colors, and is an indispensable plant in pots, gardens and other public areas. It prefers well-draining, moisturized soil, and does not perform well in overly dry or waterlogged conditions. Consequently, inadequate water supply is a common problem for this plant during production, transportation, and market placement, which has a substantial impact on plant performance overall. This review article outlines certain features of morphological, physiological, and molecular alterations induced by drought in ornamental, drought-sensitive plant species I. walleriana, as well as research carried out to date with the aim to improve the drought tolerance. Stress proteins aquaporins and dehydrins, whose molecular structure was described for the first time in this plant species, are highlighted specifically for their role in drought stress. Furthermore, the effective improvement of drought tolerance in I. walleriana by exogenous application of Plant Growth Regulators and Plant Growth-Promoting Bacteria is discussed in detail. Finally, this review can provide valuable insights for improving plant resilience and productivity in the face of water scarcity, which is critical for sustainable agriculture and horticulture.

1. General Introduction

The term drought usually refers to the physical lack of water in the substrate caused by an insufficient amount of atmospheric precipitation or an intense process of transpiration in plants [1]. However, there are other different types and definitions of drought [2]. One of them is meteorological drought, which characterizes drought as deficiency of water in soil, plants and atmosphere. A hydrological drought is characterized by a shortage of water in streams and rivers, typically measured by the number of days when the water level or discharge falls below a predetermined threshold. Agricultural drought is characterized as deficiency of water that negatively affects crop production. On the other hand, physiological drought is the state of soil and water in plants that limits plant growth and production. The relationship between physiological and other types of drought is not clear-cut; a meteorological drought does not always imply a physiological or hydrological drought. As a result, the type of plant and, in particular, its ontogenesis stage determines the physiological drought stage. Physiological drought could occur as an effect of low temperatures, which inhibit the activity of the root system. In addition, an increased concentration of salt in the substrate or flooding can negatively affect water uptake. At the global level, drought is a major problem and a limiting factor in the production of numerous crops and commercially important plant species. Drought induces tissue dehydration, inhibition of cell division, elongation and differentiation, oxidative stress manifested by increment in reactive oxygen species (ROS), and the slowing down of basic physiological and metabolic processes such as photosynthesis, transpiration, and mineral uptake, thus leading to decrease in overall plant growth and yield (Figure 1) [3,4]. The sensitivity of a plant species to drought depends on the genotype, ontogenetic stage of development, additional stress factors, frequency, intensity and drought duration [1,3,5]. Plant species respond to drought stress with different resistance mechanisms. Drought resistance mechanisms include drought escape or “ephemeroid strategy”, drought avoidance, and drought tolerance (Figure 1) [6,7]. In the genus Impatiens, there are proposed mechanisms of selection for drought stress tolerance and avoidance in Impatiens capensis, depending on the season when drought occurs [8]. Plants react to the negative effects of drought using different strategies within the resistance mechanisms, at the physiological, biochemical and molecular level. Depending on the genotype, we can talk about drought-tolerant and drought-intolerant plant species [9,10]. However, prolonged and intense drought can lead to death of even the most tolerant plant species, and considering this, various measures are taken in the production of commercially important plant species whose productivity can be limited by drought [11,12,13,14,15].

Drought escape or “ephemeroid strategy” is observed in plants at the end of the season when there is not enough rainfall in the habitat, so the plants are forced to finish their life cycle faster [16]. Drought avoidance implies the maintenance of high water potential in tissues through rapid accumulation of abscisic acid (ABA) which affects stomatal closure and transpiration reduction [6]. However, drought avoidance is very often related to the activation of drought tolerance mechanisms, especially in cases where drought periods are intense and prolonged [17]. Drought tolerance implies the plant’s ability to maintain physiological processes and functions even at a lower water potential in tissues. The accumulation of osmoprotectants, components of antioxidant protection, proteins with a protective role, transporters and various products of secondary metabolism, genes expression modulation and changes in phytohormonal signalization, underly the tolerance of plants to drought [6,7,18,19,20,21]. The accumulation of amino acid proline is often considered as drought stress indicator, since it has roles as osmoprotectant and antioxidant therewithal [22]. In addition to proline, the components with strong antioxidant activity are different phenolic compounds [23], as well as photosynthetic pigments such as carotenoids [24]. The antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate-gluthatione cycle enzymes (dehydroascorbate reductase—DHAR, monodehydroascorbate reductase—MDHAR, and glutathione reductase—GR) represent the main classes of enzymatic antioxidants and some of the most investigated components of the antioxidant defense system in general [7]. The cause of all changes at the physiological and biochemical levels lies in the expression of certain genes during drought. The perception of a drought stimulus by the receptor initiates a signaling cascade that leads to the activation/deactivation of genes associated with the plants’ responses to drought stress. Gene expression can be associated with the synthesis of ion transporters, water channels—aquaporins (AQPs), stress proteins, and enzymes involved in the biosynthesis of osmoprotectants and antioxidant components, as well as many transcription factors that regulate the expression of other genes [20]. In a complex network of interactions, the altered phytohormones concentrations under drought play a major role in the regulation of gene expression and coordination of the plant physiological response [21].

2. Impatiens walleriana—Species Characteristics

Impatiens walleriana belongs to the genus Impatiens (Balsaminaceae), which includes more than 1000 species and is one of the largest genera among flowering plants [25]. Species of the genus Impatiens are mainly herbaceous, annual or perennial, with laying or upright shoots, while woody representatives of this genus are rare [26]. The Impatiens species are the most abundant in the tropical parts of Africa, Madagascar, the Himalayan region, India and Sri Lanka. They represent the typical members of tropical and subtropical forests at altitudes of 500 to 800 m, and sometimes up to 5000 m [25]. In addition, the species can be found in the temperate regions of Asia, Europe and North America [27]. Although there are some exceptions, most of Impatiens species do not tolerate prolonged drought conditions or prolonged exposure to direct sunlight. Therefore, these species are mostly restricted in their distribution to wet habitats, such as tropical rain forests, places next to rivers, streams or wetlands [25]. Due to their beautiful appearance and long flowering period, many species of the genus Impatiens are cultivated as decorative plants worldwide.

I. walleriana (Figure 2) is an annual herb, and represents one of the most popular species of the Impatiens genus. The height of the plant ranges between 30 and 70 cm. The shape of the leaves is simple, with serrated ends and a spiral arrangement, but, often at the tips of the shoots there are leaves facing each other, i.e., oppositely arranged. The flowers are zygomorphic, with three sepals and five sepals [27]. The corolla consists of one upright petal, and the remaining four are grouped into two lateral pairs. Both corolla and sepals exhibit variability in size, shape and color, as an adaptation to different pollinators [26]. Fruits are berries or explosive capsules. The color of the flower can be red, white, orange, purple and pink. The plants are very branched, with a high water content, which makes them tender and fragile. Impatiens species require a great presence of water in the substrate. The lack of water leads to a rapid decrease in the cells’ turgor and dehydration of the tissues. Inadequate water supply in the process of plant production and during transport to sales facilities can slow down growth and affect the decorative properties of I. walleriana. Because of the relationship between their morphological characteristics and their native environments, proper cultivation of plants in the context of water availability requires careful consideration. I. walleriana is one of the three plant species (Impatiens hawkeri and Impatiens balsamina) of the genus Impatiens which have been commercially produced in Serbia for many years, and, due to its decorative properties and long flowering period, it is considered one of the most popular horticultural species around the world.

The earliest investigations concerning I. walleriana started more than 15 years ago in our laboratory. In one of the first reports, a successful regeneration protocol for I. walleriana in vitro was conducted using urea-type cytokinins [28]. Subsequent studies were focused on the production of Tomato spotted wilt virus—free plants using the meristem-tip culture technique in vitro [29,30], as well genetic transformation in order to obtain the virus-resistant plants [31,32]. As will be covered in more details in the following paragraphs, the majority of the following studies mainly focused on I. walleriana responses to drought and ways to improve the drought-tolerance. The aim of this review is to explore and summarize the current advancements in understanding and improving drought tolerance in I. walleriana through various biotechnological approaches. Given the plant’s popularity and sensitivity to water stress, this review will synthesize findings related to the plant’s responses to drought, with an emphasis on identifying effective strategies for enhancing its resilience in conditions of inadequate water supply. Ultimately, this review aims to provide valuable insights for improving plant growth and development under water scarcity, which could be of importance also for other plant species in light of the improvement in their tolerance to abiotic stress.

3. Morphological Changes in Drought-Stressed I. walleriana

The first reaction of plants to drought is a biophysical response based on changes at the cellular level [33]. Plant growth is impaired primarily by inhibition of cell division, elongation and differentiation, due to loss of cell turgor and reduced water potential [1,3,33]. Cell growth is considered as one of the most sensitive physiological processes under stressful conditions. Decreasing the water potential in cells during dehydration reduces the value of turgor, which under usual physiological conditions exerts pressure on the cell wall and increases its plasticity, enabling cell growth. By inhibiting the process of growth and development of individual cells, the growth and development of organs and therefore the organism as a whole is reduced [1]. The number and size of leaves per plant are reduced, which directly affects the reduced intensity of photosynthesis and the assimilation of nutrients [4]. Therefore, growth is reduced, as well as the fresh and dry weight of plants. Depending on the intensity and duration of the dry period, which is in nature usually related with high temperatures and intensity, many plants twist the leaves (leaf rolling) or change their orientation [34,35]. In this way, the water released into the atmosphere through transpiration is reduced. On the other hand, drought can contribute to increasing the water use efficiency (WUE) by the root system. Namely, when plants suffer a lack of water in the substrate, the growth relationship between the aerial part and the root system in rice is in favor of the roots, in order to improve the absorption of water and nutrients [36]. Faster growth of the root system increases the possibility for the plant to reach water in the deeper layers of the soil where available water exists, thus mitigating the negative effect of drought on growth and development [37]. Certainly, the growth of the root system during drought depends on the plant genotype, but also on the intensity and duration of the drought [38]. Plant species that have a shallow root system, such as potatoes, are more sensitive to the effects of drought compared to plant species with a deeper root system, such as corn or clover [38].

Several studies conducted on I. walleriana in the last 20 years, have showed its sensitivity to drought stress regarding the changes in growth and development. One of the earliest studies pointed that I. wallerana Hook ‘Rosebud Pink’ variety was one of the most dehydration-intolerant herbaceous plants among 30 tested taxa (ornamental tree, shrub, and herbaceous annual and perennial species), exposed to water withholding until they reached the lethal point (fewer than eight live leaves remained) [39]. Two years later, a study was conducted in which drought stress in I. walleriana was simulated using different concentrations of polyethylene glycol (PEG₈₀₀₀). The results indicated an altered morphology and nutrient uptake in hydroponically grown I. walleriana ‘Dazzler pink’ [40]. Namely, plants were shorter, narrower, and with reduced root length when grown in solutions containing increasing PEG₈₀₀₀ concentrations. A following study included the evaluation of drought stress responses of 17 ornamental herbaceous plants, among which was I. walleriana ‘Tempo White’ variety [41]. Five irrigation regimes were tested (0, 25, 50, 75 and 100% of the reference evapotranspiration—ET0, required for optimum Kentucky bluegrass growth), and the authors concluded that I. walleriana ‘Tempo White’ performed poorest among all the tested genera. That means that I. walleriana plants grow well only under 100% ET0, with respect to the measurement of plant biomass, changes in percent cover, and visual evaluations. A year later, the authors in [42] offered a more thorough analysis of I. walleriana’s ‘Deep Rose’ reactions to drought, including changes in morphological features. The experimental design included three irrigation points—80 (control), 60 (mild drought) and 30% (severe drought) of soil water content (SWC), with three 10-day drought cycles separated by a 10-day period of standard watering. Drought stress slightly reduced plant height, increased root length under 30% SWC, and significantly decreased number of flowers at both drought intensities. Another research study [43] described the effects of competition and reduced water content within the container on I. walleriana ‘Cajun Violet’ growth and flowering. The results indicated that reduced water content significantly reduced height, shoot number, dry weight, and flower number of I. walleriana. In another interesting report, it has been concluded that water stress did not significantly affect the growth of the I. walleriana ‘DeZire’ variety, despite an obvious tendency to decrease plant height, number of flower buds, and fresh and dry weight [44]. The water stress effect on I. walleriana ‘DeZire’ growth did not show statistical significance, which the authors attributed to the experimental design that used degrees of freedom that are more conservative in the test of the complete plot component. Polyethylene glycol-imposed drought (0, 1, 2, and 3% PEG₈₀₀₀) in vitro reduced plant height, fresh weight, and the number of leaves and shoots per plant of I. walleriana ‘Busy Lizzie’, with the strongest effect of 3% PEG₈₀₀₀ [45]. Similar results were described where 3% PEG₈₀₀₀ decreased fresh weight and proliferation rate of in vitro-grown I. walleriana ‘Xtreme Scarlet’ [46]. Drought imposed to potted I. walleriana ‘Accent Premium Red’ plants grown in the plant growth chamber induced wilting and floral abortion [47], while drought-stressed I. walleriana ‘Xtreme Scarlet’ plants showed a significant drop in water potential in leaves, which was reflected in reduced fresh and dry weight, as well as total leaf area [48,49]. In two I. walleriana cultivars, the drought imposed in the greenhouse significantly reduced plant height, the number of leaves per plant, leaf area, leaf fresh and dry weight, stem diameter, stem fresh and dry weight, the number of axillary shoots, root volume, root fresh and dry weight, and also total plant fresh and dry weight [50]. Despite that, by monitoring flowering, the authors noticed that drought reduced the number, diameter and longevity of flowers. Within the genus Impatiens, the negative consequences of drought stress in combination with high temperature were noted also for Impatiens glandulifera morphological traits [51]. Namely, drought in combination with high temperatures induced leaf senescence and decreased leaf number, as well as dropping flower production and shortening their life span. In one similar research study using different Impatiens clones, the authors defined drought-tolerance among their characteristics [52]. More drought-tolerant clones were less affected by the stress, had less reduced fresh and dry weight, and produced more flowers than the others. One research study also indicated leaf and stem anatomical anomalies in Impatiens balsamina under drought stress [53].

Figure 3 shows morphological differences between well-watered and drought-stressed I. walleriana shoots and roots. It can be observed that drought induced leaves wilting and rolling, and significantly reduced plant growth and flowering. On the other hand, drought caused obvious differences in root growth, where drought-stressed I. walleriana had better root development as a mechanism for plant adaptation to drought. The similar results for better root growth of drought-stressed I. walleriana were described earlier [42].

4. Physiological Changes in Drought-Stressed I. walleriana

4.1. Photosyntesis, Photosyntetic Pigments and Osmotic Adjustment

Inhibited growth and division of cells due to loss of turgidity leads to disruption of numerous physiological and biochemical processes such as photosynthesis, respiration, uptake of nutrients and other metabolic processes [1,3,54,55]. The greatest impact of drought can be seen through the reduction in the photosynthesis process. The plant’s ability to adapt to changed environmental conditions is directly or indirectly related to changes at the level of the photosynthetic apparatus. Lower activity of the photosynthetic apparatus is a common consequence of water deficiency in plant tissues, due to both limited stomatal conductivity and numerous damages attributed to “non-stomatal mechanisms” [1,54]. During dehydration, plants close their stomata by the action of the ABA, in order to limit the release of water through transpiration, and at the same time limit the influx of CO₂. Non-stomatal mechanisms include disruption of the biosynthesis of the main photosynthetic pigment—chlorophyll, functional and structural changes of chloroplasts, photosynthetic enzymes and disruption of the process of accumulation, transport and distribution of assimilates [1]. Both mechanisms are equally important and the dominance of one over the other depends on the plant species, developmental stage, and intensity and duration of stress, as well as the influence of other external factors. Stomatal closure ensures that plants reduce leaf water loss (LWL), and maintain a certain relative water content (RWC), which usually drops because of water deficiency. Despite that, since photosynthesis is altered, it is also common to expect changes in plant nutritional composition, since the production of assimilates is reduced.

Lack of water in the substrate reduces the concentration of photosynthetic pigments and the activity of photosynthetic enzymes, which results in disruption of the entire electron transport chain. It is known that the concentration of chlorophyll is positively correlated with the intensity of photosynthesis [1]. The decrease in chlorophyll concentration during drought is a direct consequence of photo-oxidation and degradation of pigments [3,33]. Most research on the effects of drought on the concentration of photosynthetic pigments indicates a reduction in the concentration of chlorophyll in plant leaves [56,57]. Depending on the genotype and sensitivity to drought, in different plant species the concentration of chlorophyll was reduced or even increased during drought [58,59,60]. On the other hand, carotenoids, as auxiliary photosynthetic pigments, play a role in protecting the photosynthetic apparatus from oxidative stress [61]. In addition, carotenoids are precursors in the biosynthesis of the phytohormone ABA, which plays a very important role in the mechanisms of plant resistance to drought [61]. In many plant species, increased concentration of carotenoids during drought is detected [62,63], or even decreased carotenoid concentration [64,65,66].

With decreasing water potential in cells and tissue, plants start to accumulate substances with an osmotic regulation role [67]. The accumulation of such compounds lowers the water potential inside the cell, and thus creates the difference in water potential between the cell and its surroundings. Accordingly, water is allowed to be taken in by the cells, since the external water potential is higher and provides water flow through the concentration gradient. One of the most investigated substances with an osmoprotection role is amino acid proline, which is often considered as an indicator of drought stress tolerance in plants [68,69]. Proline is an essential amino acid whose accumulation in plants increases under the various abiotic stressogenic factors, including drought. The effect of proline during tissue dehydration caused by drought can be twofold. As an osmoprotectant, proline affects the change in the osmotic and water potential in the cells and thus promotes the transport of water from a place of higher to a place of lower water potential. As an antioxidant, proline can “catch” free radicals or prevent the production of ROS [70,71].

Research conducted on I. walleriana points at drought-sensitivity regarding some physiological processes, such as nutrient composition changes. It has been described that PEG₈₀₀₀-treated I. walleriana ‘Dazzler pink’ had less zink, copper, nitrogen and calcium, and more phosphorus and nickel in the leaves, despite showing no visible nutritional deficiency symptoms [40]. In accordance with this, it has been concluded that changes in I. walleriana morphology were influenced by PEG₈₀₀₀ decreasing the water potential of the substrate and water absorption in that way. Mild and severe drought stress (60 and 30% SWC) caused a decrease in RWC and pigment content (chlorophyll a + b) in I. walleriana ‘Deep Rose’, especially during plant exposure to 30% SWC [42]. In contrast, drought-stressed I. walleriana increased free amino acid and ammonium content in leaves under the increasing water stress and after every drought period. However, since these parameters were also increased in control plants, it is concluded that the increment range was more time than drought dependent. The similar results were described for in vitro-grown I. walleriana ‘Busy Lizzie’ subjected to increasing concentration of PEG₈₀₀₀, which caused significant reduction in RWC, chlorophyll and total pigment content, as well as an increment in LWL [45]. On the other hand, the plants exposed to PEG₈₀₀₀ had considerably higher proline content in the leaves, indicating the role of proline in osmotic adjustment and osmoprotection. Slightly different results were described for I. walleriana ‘Xtreme Scarlet’ subjected to drought in the growth chamber, with a noticeable reduction in proline content, but, on the other hand, increased total chlorophyll and carotenoids, indicating that different physical conditions, stress duration and variety could affect plant response to drought [48]. Similar results regarding the increment in pigment composition in I. walleriana leaves were described three years later [72]. The same authors reported similar results for in vitro-grown I. walleriana ‘Xtreme Scarlet’ exposed to PEG₈₀₀₀ induced drought, and indicated significantly increased total chlorophyll and carotenoid content and proline, as well as total amino acid content [46]. Additionally, a significant increment in proline accumulation in two I. walleriana cultivars ‘Tempo’ and ‘Salmon’ subjected to drought was described, while plants showed lower pigment content, including chlorophyll a and b, total chlorophyll and carotenoids [50]. Among the genus Impatiens, it has been described that drought-tolerance of some Impatiens clones was related to the stomatal closure mechanisms which prevent excessive water release, but, at the same time, it did not negatively affect plant growth [52]. Similarly, drought-tolerance of invasive Impatiens parviflora was correlated with high WUE and adjusted water potential, which provides adequate water status in the cells [73].

4.2. Changes in Endogenous Abscisic Acid

The generally accepted role of ABA during the exposure of plants to various stressogenic factors leading to tissue dehydration characterized this hormone as a “stress hormone”. In mechanisms to avoid drought, ABA plays a key role because it affects stomatal closure and prevents water loss from the leaves through transpiration.

The endogenous concentration of ABA in plant tissue is regulated by biosynthesis, degradation, translocation and conjugation with other compounds. During tissue dehydration, ABA is synthesized de novo, while during tissue rehydration the concentration of ABA decreases. The key gene in ABA biosynthesis is NCED (9-cis-epoxycarotenoid dioxygenase), which encodes an enzyme with a role in converting cis-neoxanthin and cis-violaxanthin to xanthoxin [74]. In addition to NCED, the AAO (abscisic aldehyde oxidase) gene, which encodes the enzyme responsible for the last step in the synthesis of the biologically active form of ABA in cytoplasm, plays a very important role, that is, converting ABA aldehyde to ABA. Among genes involved in biosynthesis, the genes responsible for ABA catabolism are also important, since the phytohormone concentration is dependent on both biosynthesis and catabolism. ABA degradation is catalyzed by a cytochrome P450 monooxygenase (P450) encoded by the CYP707As gene family, in which ABA is converted to phaseic acid [74]. During tissue dehydration, it was shown that NCED gene expression increased in Cistus creticus, followed by increased endogenous ABA concentration [75]. Similar results were described for barley [76], and for three different carrot cultivars, in which the expression of NCED1 and NCED2 genes was significantly increased during tissue dehydration [77]. Also, it was shown that increased overexpression of NCED3 gene in soybean improved drought tolerance and increased the expression of AAO3, as well as the genes involved in ABA signal transduction [78]. The reduced expression of catabolic gene CYP707A2 under drought was described in Petunia × hybrida, but without changes in the expression of biosynthetic genes NCED1, NCED2, AAO31 and AAO32 [79]. Similar findings were reported in [80], demonstrating that when A. thaliana was subjected to dehydration, decreased expression of the ABA catabolic genes CYPT707A1/3 was detected.

Similarly to the available literature data, the changes in ABA metabolic genes in drought-stressed I. walleriana were investigated. Namely, the changes in I. walleriana ‘Xtreme Scarlet’ ABA metabolic genes (two genes involved in biosynthesis—IwNCED4 and IwAAO2, and one gene involved in catabolism—ABA8ox3, which belongs to the CYP707As gene family) upon exposure to different drought stress regimes in a growth chamber, were described [48]. The obtained results indicated that drought altered the expression level of these genes mainly by increasing it, which was followed by increased ABA content in I. walleriana leaves. The level of ABA in I. walleriana leaves increased by 2.92 and 4.3 times compared to control plants grown in moderate and intense drought, indicating an ABA role in mediating drought signal, depending on the stress intensity.

4.3. Changes in Stress-Related Proteins

Specifically, our group carried out two important studies on the molecular characterization of stress-related proteins—AQPs and dehydrins (DHNs), as well as the variations in their gene expression linked to I. walleriana drought-tolerance responses.

4.3.1. Aquaporins

Aquaporins (AQPs) are transmembrane proteins, which belongs to the Major Intrinsic Protein (MIP) family, and are responsible for water and small-molecule transport across membranes [81,82,83]. They are crucial for plant adaptation to environmental alterations which imply changes in water status in plant tissues, and many researches have pointed to their significant role [84,85,86,87]. With the exception of intracellular bacteria and thermophilic archaea, MIPs are found in every kingdom of living organisms [82,83,88]. All MIPs are composed of six transmembrane domains, with the protein’s N and C terminal ends found in the cytoplasm. Transmembrane regions are connected with five loops, of which two highly conserved ones (loops B and E) carry a conserved Asn-Pro-Ala (NPA) motif, found deep in the pore. Indeed, the most noticeable aquaporin trademark sequence is the NPA motif, which forms a pore for water and other solutes transport. Studies on biochemistry and functionality have also indicated that aquaporins are active in tetrameric formation, including four monomers in a holoprotein structure [83]. Plants comprise five AQP subfamilies, separated by intracellular localization and the functionality of their isoforms [82,83]. The subfamily with the highest number of members is represented by the Plasma membrane Intrinsic Proteins (PIPs), and, as implied by the subfamily name, PIPs are mainly found in the plasma membrane [83,88]. Two evolutionary groups, PIP1 and PIP2, comprise the PIP subfamily, and their water permeability properties, as well N- and C-terminal lengths, vary. Usually, PIP2 members produce a higher water permeability compared to PIP1 members. Another very important subfamily for regulation of water transport is the Tonoplast Intrinsic Proteins (TIPs), whose members are localized on the vacuole membrane—the tonoplast. Given the role of the vacuole in controlling turgor and osmotic adjustment in cells, the presence and activity of TIP isoforms in water transport across the vacuole is crucial [83,88].

Molecular characterization and expression profiling of four AQP isoforms in drought-stressed I. walleriana ‘Xtreme Scarlet’ have been performed [89]. Since members of the PIP and TIP subfamilies are recognized for their capacity for water transport, I. walleriana isoforms, which belong to these families, were selected for inclusion in the investigation. The sequenced leaf transcriptome of I. walleriana (RNASeq) was searched for sequences, and several bioinformatics methods were used to perform that. Gel electrophoresis was used to confirm that four of the identified isoforms (IwPIP1;4, IwPIP2;2, IwPIP2;7, and IwTIP4;1) exhibited the stable expression, and those isoforms were further tested in expression analysis. Before the expression analysis, detailed insight into molecular structure (sequence length, number of amino acids, molecular weight, isoelectric point, instability index, subcellular localization, transmembrane helices, stereochemical properties and 3D models of structures) of I. walleriana AQPs was performed using different available bioinformatical tools, and in Figure 4, in the first row, 3D models of IwAQPs holoproteins are presented. The authors explained in detail the IwAQPs’ structure, pointing to the crucial characteristics of MIP family members. All of the analyzed IwAQPs included the tetrameric structure of holoprotein, conserved NPA motifs, and transmembrane helices (TMh’s). Additionally, the isoelectric point corresponded to their predicted subcellular localization, while the instability index indicated the stable structures. A 3D model of the pore’s characteristics was obtained, and this model showed that the composition of amino acid residues in the pore of I. walleriana aquaporins vary, which could affect hydropathy and suggests that different solutes may be transported. Stereochemical analysis showed that most of the amino acids of the analyzed I. walleriana AQPs fall into regions which are energetically allowed, while the holoprotein structure of all IwAQPs indicated the presence of four monomers in each.

Drought stress and recovery affected the AQP gene expression in I. walleriana leaves differently [89]. The expression analysis of genes coding I. walleriana AQPs have revealed that the IwPIP2;7 gene was highly responsive to drought and recovery from drought, demonstrating its role in drought resistance mechanisms. On the other hand, it was concluded that other three AQP isoforms (IwPIP1;4, IwPIP2;2, and IwTIP4;1) could also be important in recovery after stress, maintaining the optimal water flow through the cells.

4.3.2. Dehydrins

Dehydrins are thermostable and highly hydrophilic proteins which are involved in plant responses to drought stress [90,91,92]. They could protect cell membranes and proteins from dehydration, thus protecting membrane integrity, regulating ROS homeostasis, antioxidant activity, photosynthetic pigment content and photosynthesis rates and osmotic adjustment, as well as morphological traits [93,94,95]. Dehydrins belong to group 2 of LEA (Late Embryogenesis Proteins) proteins, and are widely distributed in plants [91,96]. They consist of various conservative motifs such as K, Y and S and, according to their presence, could be divided in five structural subgroups—Kn, SKn, KnS, YnSKn, and YnKn [96]. The model of the DHN protection role of proteins under water deficit was proposed, indicating two possible mechanism [97]. In the first one, DHNs protect proteins via a molecular shield, in which the presence of DHN molecules maintains the quantity and arrangement of water molecules necessary to preserve the integrity of the enzymes without necessarily interacting with them. In the second one, DHNs must interact with proteins, as monomers, dimers or in any other oligomeric form.

The following study provided the insight into molecular structure of the dehydrin isoform characterized in the I. walleriana transcriptome [47]. In the I. walleriana transcriptome, three dehydrin isoforms—IwDhn1, IwDhn2.1 and IwDhn2.2—were detected (Figure 4, the second row), and their architecture, length, number of amino acids and protein isoelectric point were characterized [46]. In terms of IwDHNs architecture, there are two types: IwDhn2.1 (Y3SK1) and IwDhn2.2 (Y3SK2), which have a YnSKn composition, and IwDhn1, which is an SKn type. Upon exposure to drought in the plant growth chamber, I. walleriana ‘Accent Premium Red’ had slightly increased IwDhn1 gene expression, while the expression of other two isoforms—IwDhn2.1 and IwDhn2.2—was strongly induced [47]. Authors have concluded that the IwDhn1 protein is important, because of its almost constitutive expression under normal and stress conditions, but its expression profile implies that it is not crucial for drought stress protection in I. walleriana. Based on its structure (SK2 type), this isoform is not dehydration-inducible, but more likely to be responsive to other types of abiotic stresses, according to the literature data [47]. On the other hand, based on the strong induction of isoforms IwDhn2.1 and IwDhn2.2 during drought, it could be assumed that they have a role in plants for coping with stress. This could be explained by the specificities in their architecture and structure, and presence of amino acids which provide membrane binding and protection.

5. Biochemical Changes in Drought-Stressed I. walleriana

Reactive Oxygen Species and Antioxidant Defense

Regarding the drought effects on a plant’s growth and development, one of the most studied is secondary oxidative stress, i.e., the excessive production of ROS, which overwhelms the capacity of the antioxidant system to remove those chracteristics [98]. Depending on their nature and quantity, ROS can be toxic to the plants, but can also play a role as secondary messengers in the initiation of signaling pathways that activate defense mechanisms in plants [99]. The most frequently mentioned ROS are singlet oxygen (O₂¹), hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^•−), hydroxyl radical (OH•), perhydroxyl radical (HO₂•), peroxyl radical (RO₂•) and nitrogen monoxide (NO). Among the mentioned ROS, O₂¹, H₂O₂, O₂•⁻ and OH• are the most represented, with mutual differences in stability, reactivity and the possibility of transport through the cell membranes [100]. The consequences of oxidative stress are damage to the protein and nucleic acid structures through oxidative modifications, as well as membrane lipid peroxidation, which leads to the accumulation of oxidative stress indicators such as malondialdehyde (MDA) [98]. Considering proteins, the oxidative modifications of methionine, thiol groups of cysteine, site-specific amino-acid modification, fragmentation of peptide chains, and the aggregation of cross-linked reaction products, as well as carbonylation, are the most frequently mentioned effects of ROS [101,102,103]. The oxidative modifications of DNA usually involve modifications of nitrogenous bases and breaking the sugars’ bonds [104]. Lipid peroxidation of cell membranes is one of the most investigated consequences of ROS action on the structure and function of membrane lipids [19,63,98]. Precursors for the products of lipid peroxidation are polyunsaturated fatty acids, the main components of membrane lipids. Through the phases of initiation, propagation and termination, polyunsaturated fatty acids react with ROS, forming the different intermediers and final products such as MDA [105]. Malondialdehyde is a tricarbon dialdehyde whose concentration is often analyzed, and is considered as an indicator of cell membrane damage during oxidative stress [105,106,107,108,109]. Reactive oxygen species production in plants was confirmed in chloroplasts, peroxisomes, mitochondria, cell membranes and the apoplast [110].

Antioxidant enzymes are responsible for removing ROS and detoxifying plant tissues under stressful conditions. Among them, SOD, POX, CAT, DHAR, MDHAR, and GR represent the main classes of enzymatic antioxidants [111]. Superoxide dismutase catalyzes the reaction of converting O₂•⁻ to H₂O₂ in chloroplasts, peroxisomes, cytoplasm and mitochondria [110]. Based on the presence of metals as cofactors and sites of activity, four SOD isoforms have been detected in living organisms: Fe-SOD, Mn-SOD, Cu/Zn-SOD and Ni-SOD, while the Ni-SOD isoform was not found in higher plants [110,111]. Catalase degrades H₂O₂ to water and oxygen primarily in peroxisomes, but also in cytoplasm and mitochondria [110,111], and, until now, three genes encoding CAT isoforms (CAT1-3) have been identified in the A. thaliana genome. Peroxidase, such as ascorbate peroxidase (APX), DHAR, MDHAR, and GR maintain H₂O₂ balance in the cell via the ascorbate-glutathione cycle [110,111,112]. Firstly, APX converts H₂O₂ to H₂O by oxidizing ascorbate to monodehydroascorbate (MDHA), and ascorbate can be regenerated by the enzyme MDHAR, which uses NAD(P)H, as a cofactor. Monodehydroascorbate can spontaneously convert to dehydroascorbate (DHA), and the enzyme DHAR, which oxidizes glutathione (GSH) to glutathione disulfide (GSSG), regenerates ascorbate from dehydroascorbate. In the final step, the enzyme GR regenerates GSH from GSSG using NAD(P)H as a cofactor. Among components with strong antioxidant potential there are also some non-enzymatic compounds, such as phenolics [113,114]. Phenolics represent a group of secondary plant metabolites that contain aromatic rings with one or more hydroxyl groups in their structure [113]. The antioxidant activity of phenolic compounds is reflected by their ability to “capture” free radicals, to donate electrons or protons, or to bind metals that participate in the production of ROS. By binding metals, they can prevent, for example, the Fenton reaction, which produces the most reactive forms of oxygen [113,115].

Several studies with I. walleriana have shown that drought can cause oxidative stress and alterations in the antioxidant defense system. The changes in oxidative status were usually manifested as an increment in oxidative stress indicators—H₂O₂, MDA—while on the other hand, the activities of antioxidant enzymes such as SOD, POX and CAT were also altered. In one such report, the authors documented a significant increment of H₂O₂ and MDA in I. walleriana ‘Busy Lizzie’ leaves exposed to increasing PEG₈₀₀₀ concentration in vitro [45]. However, the highest increment in oxidative stress indicators was detected when plants were grown on media with the highest PEG₈₀₀₀ concentration (3%). Due to disturbed oxidative status in cells, increased activities of antioxidant enzymes—SOD, POX, and CAT—were determined, with the highest values observed in plants exposed to strongest drought stress, namely 3% PEG₈₀₀₀. In addition, 3% PEG₈₀₀₀ caused the induction of all eight POX isoforms, but particularly isoforms B and I. As a response to drought induced by PEG₈₀₀₀ (3%) in vitro, in I. walleriana ‘Xtreme Scarlet’ leaves the increased content of total polyphenol content, H₂O₂, and MDA was also detected [46]. Recently the authors [47] conducted an experiment with potted I. walleriana ‘Accent Premium Red’ exposed to drought in the plant growth chamber, and recorded the increased MDA content, as well as the induction of chloroplastic Cu/ZnSOD and two peroxidase isoforms. Also, upon exposure of I. walleriana ‘Xtreme Scarlet’ to drought stress in a growth chamber, an increment in total polyphenol and flavonoid content was detected in leaves, as well as the increment in total antioxidant capacity [48]. Such results indicated the significant role of phenolic compounds in plant responses to drought stress. Additionally, drought-stressed plants exhibited changes in oxidative stress indicators and antioxidant enzymes. Namely, depending on the drought intensity, an increment in H₂O₂ and MDA was observed, as well as the increased activity of SOD, POX, and CAT. Drought-stressed plants expressed one Mn-SOD and two Cu/Zn-SOD (A and B) isoforms, similar to those in control plants, while a total of four POX isoforms were detected and, among them, the D isoforms was specific to non-stressed I. walleriana plants. Three years later, the same authors described similar results for drought-stressed I. walleriana ‘Xtreme Scarlet’ grown in a growth chamber, and observed increased H₂O₂ and MDA content, as well as increased activity of antioxidant enzymes and phenolic content [72]. In similar research, as responses to drought, I. walleriana increased the activity of enzymatic antioxidants (guaiacol peroxidase—GPX, pyrogallol peroxidase—PPX, ascorbate peroxidase—APX, and CAT) and total phenolic and flavonoid contents, as well as the total antioxidant capacity of leaves [116]. Drought stress also increased the MDA, POX and APX in two I. walleriana cultivars grown under controlled conditions in a greenhouse [50].

6. Mitigation Strategies for Drought Stress

Elicitation is the process of applying certain compounds with the aim of inducing the signaling pathways associated with the stimulating of any type of plant defense [117]. Based on the elicitor’s nature, the common classification includes two groups—abiotic and biotic elicitors [118,119]. However, inside these two major groups of elicitors there are some differences in classification of individual groups of certain compounds. For example, according to one classification [118], abiotic elicitors include physical, chemical and hormonal factors, while biotic elicitors include substances of biological origin, such as plant cell-wall polysaccharides and microorganisms. In contrast to this classification, another one [119] classifies hormonal factors in a biotic group of elicitors as substances generated in living organisms, namely plants.

Plant hormones are naturally occurring, low-molecular-weight substances produced in plants. Phytohormones are responsible for coordinating all aspects of plant growth and development, including the responses to abiotic and biotic stress factors. As synthetic compounds, plant hormones are commonly used in the elicitation process to improve plant defense against different stressors. Along with some other compounds, synthetic plant hormones are usually referred to as Plant Growth Regulators (PGRs) [120,121]. The most used and investigated PGRs in plant drought tolerance are abscisic acid (ABA), salycilic acid (SA), and jasmonates—jasmonic acid (JA), methyl jasmonate (MeJA), and jasmonil–isoleucine (JA-Ile). Undoubtedly, Plant Growth Promoting Bacteria (PGPB) are the biotic elicitors that require the greatest consideration in future research. Interestingly, recently isolated Pseudomonas, Pantoea, Acinetobacter and Chryseobacterium bacteria from the rhizosphere of various Mediterranean medicinal plants (Thymus, Sarcocornia, Mentha), are hypothesized to increase the drought and salinity tolerance of these plant species through indole3-acetic-acid (IAA) production, phosphorus solubilization, and ACC (1-aminocyclopropane-1-carboxylate) deaminase activity [122]. It is estimated that as much as 80% of rhizosphere-associated bacteria are the source of IAA [122,123]. Since mechanisms of drought and salinity tolerance often overlap, the total number of PGPB that alleviate drought is probably much higher. In our laboratory, we identified four rhizosphere-associated Pseudomonas strains with the dual ability to produce and degrade IAA, and monitored their PGPB effects on duckweed (Lemna minor) [124]. The most promising is P. gessardii C31-106/3, which increases biomass production, modulation of duckweeds’ antioxidant enzymatic activity, and reduction in H₂O₂ content [125]. In fact, there is an abundance of studies with different PGPB strains that may also have the potential to improve drought stress tolerance in ornamental plants, including I. walleriana (see Section The PGRs and PGPB Application for Drought-Tolerance Improvement in I. walleriana).

Table 1. The effects of PGRs and PGPBs on drought-tolerance improvement in different plant species. ABA—abscisic acid, ASA—ascorbate, Chl—chlorophyll, DW—dry weight, FW—fresh weight, GSH—glutathione, H₂O₂—hydrogen peroxide, JA—jasmonic acid, MDA—malondialdehyde, MeJA—methyl jasmonate, O₂^•−—superoxide anion radical, PGPB—Plant Growth-Promoting Bacteria, PGRs—Plant Growth Regulators, RWC—Relative Water Content, WUE—Water Use Efficiency.

Elicitor	Plant Species	Concentration/ Bacterial Strains	Way of Application	Effects	References
ABA	Triticum aestivum	10 µM	Soil drenching	Increased ABA concentration, reduced stomatal conductance, decreased oxidative stress damages, increased antioxidant enzyme activities	[126]
	Ulmus minor	50 and 100 μM	Foliar application	Increased DW, reduced water loss, enhancement of antioxidant capacity	[127]
	Camellia sinensis	50 mg L−1	Foliar application	Leaf proteome changes	[128]
	Vicia faba	0.1 mM	Foliar application	Improved morphological and anatomical characters, Chl concentrations, and yield	[129]
	Triticum aestivum	10 μM	Supplemented in Hoagland solution	Increased shoot length, and shoot and root dry weights; decreased H2O2 and MDA, increased content of GSH and ASA	[130]
	Agrostis stolonifera	5 μM	Foliar application	Lower electrolyte leakage, greater RWC, accumulation of organic acids	[131]
	Lolium perenne	0.054 kg ai ha−1	Foliar application	Enhanced RWC, decreased electrolyte leakage and H2O2, increased soluble sugar content and antioxidant enzyme activity	[132]
	Axonopus compressus	100 μM	Foliar application	Reduced oxidative stress, increased pigment content, osmolyte accumulation	[133]
	Camellia sinensis	50 mg L−1	Foliar application	Metabolic changes, increased phenolic content	[134]
	Pennisetum glaucum	100 μM	Supplemented in Hoagland solution	Improved Chl and RWC, increased activities of antioxidative enzymes	[135]
SA	Brassica juncea	50 μM	Foliar application	Increased RWC, Chl content, antioxidant enzyme activity, decreased H2O2 and lipid peroxidation level	[136]
	Vicia faba	1 mM	Foliar application	Improved morphological and anatomical characters, Chl concentrations, and yield	[129]
	Artemisia aucheri	0.01 and 0.1 mM	In vitro	Improved DW and FW, Chl and carotenoid contents, increased soluble carbohydrates, increased biosynthesis of phenolic compounds	[137]
	Agrostis stolonifera	10 μM	Foliar application	Lower electrolyte leakage, greater RWC, accumulation of amino acids and carbohydrates	[131]
	Carthamus tinctorius	250 μM	Foliar application	Activation of non-enzymatic antioxidant defense system, increased proline content, decreased oxidative stress	[138]
	Brassica napus	1 mM	Foliar application	Increased antioxidant enzyme activities, water content, membrane integrity and Chl, improvement in grain and oil yields	[139]
	Hordeum vulgare	0.5 mM	Foliar application	Increased stem length, DW, Chl, RWC, activity of antioxidant enzymes, and grain yield; decreased lipid peroxidation, electrolyte leakage, O2·− and H2O2	[140]
	Abelmoschus esculentus	80, 160 and 240 mgL−l	Foliar application	SA at 240 mgL−1 improved the best all of the growth and yield attributes; had minimum days to flowering and picking, and maximum single-pod weight, average pod length, plant height, number of leaves per plant, number of pods per plant, stem diameter and yield	[141]
	Oryza sativa	250, 500, 750 and 1000 µM m−2	Foliar application	Improved grain yield and harvest index when 750 µM m−2 SA applied	[142]
	Triticum aestivum	140 mg L− 1	Foliar application	Increased grain yield, decreased MDA, H2O2 and O2•−, increased proline, soluble sugars and antioxidant enzyme activity	[143]
Jasmonates	Triticum aestivum	0.5 mM MeJA	Foliar application	Improved dry biomass, number of grains per spike, and grain weight and yield	[144]
	Glycine max	20 µM MeJA	Foliar application	Increased growth parameters, RWC, photosynthetic pigments, cell wall components, unsaturated fatty acids, and phenolic compounds	[145]
	Beta vulgaris	0.01, 0.1, 1 or 10 μM MeJA	Foliar application	1 and 10 μM MeJA reduced moderate- and severe-drought effects on RWC, photosynthesis rate, substomatal CO2 concentration and WUE, and altered drought-induced changes in proline accumulation	[146]
	Verbascum sinuatum	200 µM MeJA	In vitro	MeJA negatively affected growth parameters and increased the content of MDA, H2O2, total saponin and activity of peroxidase and polyphenol oxidase	[147]
	Fragaria × ananassa	0.01 and 0.05 mM JA	In vitro	Improved growth, RWC, and pigment content	[148]
	Dracocephalum kotschyi	0.5 mM MeJA	Foliar application	Higher FW and DW, lower electrolyte leakage, MDA, H2O2, total phenol content, total antioxidant activity and antioxidant power assay	[149]
	Triticum sativum	0.1 mM JA	Foliar application	Improved growth, restoration of shoot/root ratio, accumulation of osmolytes, regulated activity of antioxidant enzymes	[150]
	Pennisetum glaucum	100 μM	Supplemented in Hoagland solution	Improved Chl and RWC, increased activities of antioxidative enzymes	[135]
	Brassica rapa	10 µM JA	Foliar application	Improved photosynthetic rate, photosynthetic pigments, stomatal conductance, transpiration rate and antioxidant defence, increased osmolyte accumulation, and decreased membrane damage	[151]
	Oryza sativa	100 μmol L−1 MeJA	Foliar application	Increased grain yield and quality	[152]
PGPBs	Zea mays	Proteus penneri, Pseudomonas aeruginosa, and Alcaligenes faecalis	Seed priming	Improved plant biomass, root and shoot length, leaf area, RWC, protein and sugar content	[153]
	Oryza sativa	Pseudomonas sp., Bacillus cereus, Arthrobacter nitroguajacolicus	Soil inoculation	Enhanced growth, higher proline content and antioxidant enzyme activities, lower MDA and H2O2	[154]
	Triticum aestivum	Klebsiella sp., Enterobacter ludwigii, Flavobacterium sp.	Seed priming, soil inoculation	Transcriptomic changes, improved root length and number, shoot DW, root FW and DW, and physiological and biochemical parameters	[155]
	Setaria italica	Pseudomonas fluorescens, P. migulae, Enterobacter hormaechei	Seed priming, soil inoculation	Stimulated seed germination and seedling growth	[156]
	Trema micrantha, Cariniana estrellensis	Azospirillum brasilense, Bacillus sp., Azomonas sp., Azorhizophillus sp.	Seed priming, soil inoculation	Increased drought tolerance, growth parameters, and physiological and biochemical attributes	[157]
	Ziziphus jujuba	Pseudomonas, Bacillus, Serratia	Soil inoculation	Pseudomonas lini and Serratia plymuthica increased plant height, shoot and root dry matter, RWC, and antioxidant enzyme activities; decreased MDA and ABA	[158]
	Thymus, Sarcocornia, Mentha	Pseudomonas, Pantoea, Acinetobacter	Soil inoculation	in vitro PGP-associated traits, including phosphate solubilization, indole-3-acetic acid production, and 1-aminocyclopropane-1-carboxylate deaminase activity; increased tolerance to salinity and drought	[122]
	Petunia, Pelargonium	Pseudomonas, Arthrobacter, Herbaspirillium,	Soil inoculation	Increased biomass; increased number of flowers; mitigation of the reduction in photosynthetic parameters of water-stressed P. hybrida and Pelargonium × hortorum	[123]
	Haloxylon ammodendron	Bacillus sp., Pseudomonas sp.	Seed priming, soil inoculation	Improved root system and growth, RWC, photosynthetic capacity, antioxidant enzyme activities and regulated ABA content	[159]
	Triticum aestivum	Bacillus megaterium, B. licheniformis	Seed priming, soil inoculation	Increased germination index, promptness index, seedling vigor index, FW and DW, RWC, photosynthetic pigments, osmolytes, and activities of defense-related antioxidant enzymes	[160]
	Hordeum vulgare	four bacterial isolates (MFC1, MFE3, MFF2, and MFF5)	Soil inoculation	Increased shoot dry weight, RWC, Chl content, photosynthesis efficiency, and proline content; decreased MDA and H2O2	[161]
	Festuca ovina	Azotobacter vinelandii, Pantoea agglomeran, Pseudomonas putida	Seed priming	Improved seed germination, plant growth, and nutrient uptake	[162]

presents the selected publications from the last decade describing the effects of ABA, SA, jasmonates, and PGPBs on plant drought-tolerance improvement, conducted in controlled ex vitro or in vitro conditions.

The PGRs and PGPB Application for Drought-Tolerance Improvement in I. walleriana

One of the earliest reports that described I. walleriana drought-tolerance improvement was conducted fourteen years ago [163]. In this experiment, drench and spray ABA (0 or 500 mg_L⁻¹) applications were implemented for several drought-stressed bedding plants, including I. walleriana ‘Xtreme Lavender’. The application of ABA significantly delayed visible wilting symptoms and increased shelf life of I. walleriana, with more effectiveness with drench application than spraying. The next study evaluated the effect of a commercial extract of Giant Knotweed (Fallopia sachalinensis F. Schmidt) on drought tolerance of I. walleriana ‘Super Elfin XP [164]. The commercial extract, named Regalia^®, was foliar-applied in different concentrations and under different substrate volumetric water contents in two independent experiments. Foliar application of Regalia^® at a certain concentration improved some morphological (root dry weight) and physiological attributes (soluble protein content, leaf greenness, net photosynthesis etc.) in I. walleriana. Despite this being observed, the authors concluded that these results could not be related to elevated drought tolerance because overall growth of plants was not enhanced in either of the two applied experiments. Two years later, the efficacy of several commercially available antitranspirants in enhancing drought stress tolerance in bedding plants, including I. walleriana ‘Double Fiesta Ole Purple Stripe’, was evaluated [165]. While three physiological antitranspirants—two sugar alcohol-based compounds (SACs) and a biologically active ABA (s-ABA)—were added into the substrate, two physical antitranspirants—β-pinene polymer (βP) and vinyl–acrylic polymer (VP) were sprayed on the plants grown in greenhouse conditions when they reached a marketable stage of at least one open flower per plant. After that, half of the plants in each group were water stressed until all treated plants attained a visual wilt status rating of three or lower, defined previously as unmarketable [166]. The results of the study indicated that βP and s-ABA were effective in enhancing shelf life and delaying wilting symptoms of I. walleriana, with more effectiveness noticed for s-ABA. Based on these data, it appears that foliar application of βP or drenching s-ABA could allow I. walleriana to withstand a water shortage during transport and/or retailing. The potential role of SA as a stress-ameliorating agent in I. walleriana ‘Buzzy Lizy’, grown in vitro under PEG-induced drought, was also proposed [45]. Salycilic acid in graded concentrations (0, 1, 2, and 3 mM) was added in media supplemented with PEG₈₀₀₀ (0, 1, 2, and 3%), and consequently enhanced growth and improved some physiological and biochemical parameters in drought-stressed I. walleriana. For this purpose, the authors recommended the use of 2–3 mM SA for improving I. walleriana drought tolerance, with no growth-retarding, but only beneficial, effects. In the followed experiment, the authors had chosen 2 mM of SA as foliar treatment for drought-stressed I. walleriana ‘Accent Premium Red’ grown in the plant growth chamber [47]. Salycilic acid was applied on the second day of the experimental setup to well-watered plants, and plants were further subjected to water deprivation until the fourteenth day of the experiment. In the same way as in the first two experimental groups, two more plant groups received distilled-water spraying instead of SA, and were also well-watered and drought stressed. Samples were taken from five different points: the start of the experiment, 24 h after SA/distilled-water application, on the 11th and 14th day of drought duration, and 24 h after rehydration. The study’s findings showed that SA had no effects on well-watered plants, but had ameliorating effects on drought-exposed plants. That was manifested by preventing wilting, preserving RWC, increasing proline accumulation, modulating antioxidative activities, and significantly lowering lipid peroxidation, but had no effect on flower preservation. Additionally, the application of SA altered the expression of three DHNs isoforms in plants under drought stress, with one isoform being slightly enhanced after the treatment. However, under drought, the alterations in dehydrin expression were the same as in plant foliar sprayed with distilled water, so the SA effects were not considered noteworthy. The authors considered this plant response as an SA drought-ameliorating effect, so DHNs are not needed as much as in drought-stressed plants without SA treatment.

Recently, it was described how PGPB can stimulate I. walleriana ‘Super Elfin Ruby’ growth under drought stress by enhancing plant drought tolerance [167]. Namely, two Pseudomonas sp. strains—P. poae 29G9 and P. fluorescens 90F12-2—stimulated the increment of shoot biomass and flower numbers of I. walleriana grown under low-nutrient conditions and after recovery from drought stress in greenhouse conditions.

Further investigations also refer to the potential role of SA in ameliorating drought effects. Recent research has also indicated the power of exogenous SA application on morpho-physiological and molecular characteristics of two I. walleriana cultivars (‘Tempo and Salmon’) grown under water deficit stress [50]. Three SA concentration (0, 1, and 2 mM) were applied weekly as foliar treatment to I. walleriana grown in a greenhouse under three levels of soil moisture content (95, 85, and 75% of field capacity). The results indicated that SA, especially in 2 mM concentration, improved the drought-tolerance of I. walleriana through increased plant height, leaf area, number of auxiliary shoots, root volume, stem diameter, number of leaves, chlorophyll content, and antioxidant enzymes activities, and on the other hand, reduced electrolyte leakage and oxidative damage. In addition, the authors noted that the Salmon cultivar is more drought-tolerant than the Tempo cultivar, and, accordingly, Salmon is recommended for cultivation in areas with low water supply. Additionally, the potential of foliar and drench application of a secondary messenger molecule, calcium, in increasing shelf life and delaying wilting symptoms, was reported in drought-stressed I. walleriana [168].

The latest investigations of our group have revealed the role of exogenously applied MeJA as a potential regulator of drought tolerance of in vitro- and ex vitro-grown I. walleriana ‘Xtreme Scarlet’ [46,49,72]. I. walleriana plants grown on media supplemented with 5 μM MeJA for seven days had better morpho-physiological performance during a further twenty days on the media supplemented with 3% PEG [46]. In water-stressed plants, pretreatment with 5 μM MeJA for seven days increased growth parameters (fresh weight, height, number of leaves per plant and proliferation rate) compared to control plants without MeJA pretreatment. In these plants, decreased photosynthetic pigment content was detected, as well as proline and total amino acid, total polyphenol and DPPH (1,1′-diphenyl-2-picrylhydrazyl) activity, and H₂O₂ and MDA content. Accordingly, the activities of antioxidant enzymes (SOD, POX, and CAT) were also affected. It is interesting to note that the application of 5 μM MeJA also provided the best growth (fresh weight and height) of non-stressed plants in comparison to the plant group grown on hormone-free media during the entire experimental period. Namely, when MeJA was applied in pretreatment for seven days, and, subsequently, in treatment for twenty days, plant growth was improved and visual characteristics of tested plants were the best. In this research, the authors also investigated the effects of pretreatment with higher MeJA concentration (50 and 100 μM) on drought ameliorating effect on I. walleriana, but it seems that these concentrations had mainly inhibitory effects. Based on the mentioned in vitro research, MeJA (5 and 50 μM) was applied as a foliar treatment to drought-stressed I. walleriana grown ex vitro in a growth chamber under controlled conditions [72]. The highest concentration (100 μM MeJA) was omitted in this experiment, since it was assumed that it would suppress I. walleriana growth. Foliar treatment with 20 mL of each MeJA concentration was applied seven days before and on the day when drought stress (15 and 5% SWC) was imposed. The control plants and one drought-stressed plant group were treated with distilled water. The obtained results indicated that foliar application of 50 μM MeJA had the strongest effect on physio-biochemical responses of drought-stressed plants, increasing pigment content, and decreasing oxidative stress in plant cells. Additionally, in drought-stressed plants sprayed with 50 µM MeJA increased expression of ABA metabolic genes was detected, while of the four analyzed aquaporin gene isoforms, the expression of IwPIP1;4 and IwPIP2;7 was strongly induced. Foliar application of 50 µM MeJA also increased the chlorophyll index and Nitrogene Balance Index (NBI) of I. walleriana at 5% SWC, indicating the effect of the elicitor on plant drought tolerance [49]. The latest research aimed to evaluate the potential role of sodium nitroprusside (SNP) on alleviating water deficit caused by PEG in vitro in I. walleriana ‘Xtreme Scarlet’, monitoring the changes at a physiological and biochemical level [169]. The findings of this investigation suggested that SNP might be advantageous for biochemical parameters when applied only in combination with PEG, but not solely, to I. walleriana shoots cultivated in vitro. The summarized effects of exogenously applied elicitors on I. walleriana drought-tolerance improvement are presented in the Figure 5.

7. Conclusions and Future Perspectives

Drought stress affects plants, and ways to cope with this abiotic stress factor have been reported in numerous studies in recent years. Whether they are conducted in the laboratory, greenhouse or in the field, undoubtedly all of them demonstrated the harmful effects of this stressor on different aspects of plant growth and development. On the other hand, plants have evolved drought resistance mechanisms, and, depending on the genotype, duration and stress intensity, respond differently at a morphological, physiological, biochemical, and molecular level. One of the most important ornamental plants in horticultural production, I. walleriana, known for its sensitivity to drought, has been the object of investigation in recent years. In the last 20 years, many studies have investigated the responses of I. walleriana plants to drought, and at the same time, the diverse ways to improve drought tolerance. Drought sensitivity of I. walleriana has been reported at different levels of organization, indicating also drought-tolerance dependence on the variety of plant. It has been reported that many exogenously applied chemicals could improve I. walleriana tolerance, such as hormones ABA, SA, and MeJA, as well as antitranspirants, calcium and PGPB. Additionally, improvements in understanding of I. walleriana drought-tolerance mechanisms has been achieved by molecular characterization and understanding the role of some crucial proteins in drought stress response—AQPs and DHNs.

Further investigations will focus on the phytohormonal network involved in drought-stress responses of I. walleriana, as well as finding additional ways for drought-tolerance improvement. A promising strategy is to test various PGPBs in improving the drought tolerance of I. walleriana, especially those strains that were tested on other plants. Moreover, the effects of other types of abiotic stress factors (e.g., osmotic stress, temperature stress) on I. walleriana growth and development should be examined in future studies.