Effects of Cold Acclimation on Morpho-Anatomical Traits of Heteroblastic Foliage in Pinus massoniana (Lamb.) Seedlings

Abstract

Cold acclimation before winter has been shown to enhance the cold tolerance of evergreen conifers, including Pinus massoniana Lamb., a characteristic heteroblastic foliage tree in the conifer. In the initial growing season of P. massoniana, both primary needle seedlings (PNSs) and secondary needle seedlings (SNSs) are generated. While previous research has highlighted differences in the morphological structure and photosynthetic physiological functions of primary and secondary needles, their response to cold acclimation remains poorly understood. This study aimed to investigate the changes in morpho-anatomical structure, starch grain accumulation, and lignin deposition in the roots, stems, and leaves of PNSs and SNSs during cold acclimation using solid potassium iodide and hydrochloric acid phloroglucinol double-staining techniques. The results revealed that, during cold acclimation, the leaves and stems of PNSs exhibited sensitivity to low-temperature stress, resulting in noticeable shrinkage and fracture of mesophyll and cortical parenchyma cells. Furthermore, the early stages of cold acclimation promoted the accumulation of starch grains and lignin in the seedling tissues. In contrast to PNSs, the leaves and stems of SNSs exhibited a shorter cold acclimation period, attributed to the hydrolysis of starch grains in the epidermal cell walls and the transformation of xylem lignin, which supports cell structure stability and enhances cold resistance. In conclusion, these findings suggest that SNSs displayed a superior cold resistance potential compared to PNSs following cold acclimation, providing a significant theoretical basis for the further screening of cold-tolerant germplasm resources of P. massoniana and the analysis of cold resistance traits in heteroblastic foliage.

1. Introduction

Coniferous trees, classified as evergreen species, adapt to cold winter climatic conditions by initiating a process known as cold acclimation in early autumn, triggered by shorter day lengths and temperatures slightly above freezing. This adaptive mechanism involves the regulation of physiological and developmental processes to enhance their resilience against low-temperature stress and freezing damage [1]. Research has shown that cold acclimation significantly improves the cold tolerance of plants, enabling them to better withstand the increasing frequency of global climate fluctuations [2]. In recent years, the rise in global average temperatures and the subsequent shift in climate zones have led to the emergence of extreme weather events in new regions, resulting in changes to the seasonal activities and growth patterns of many plant species [3]. Specifically, the rise in autumn temperatures will delay the down-regulation of photosynthesis and affect the development of autumnal overwintering defense mechanisms [4]. Consequently, needles become more susceptible to photo-oxidative damage during autumn frost periods and winter freeze events [5]. In the context of climate warming, evergreen conifers in subtropical and temperate regions are increasingly prone to cold or freeze damage [6]. Therefore, there is an urgent need to identify the cold-tolerance traits of conifer species through cold acclimation.

Cold acclimation is a multifaceted process through which plants adapt to low temperatures, involving structural, physiological, metabolic, and molecular adjustments, along with noticeable changes in morphological structure [7]. When exposed to cold temperatures, structural changes within cellular compartments occur as a response to the cold, activating a series of protective mechanisms [8]. These mechanisms include alterations in stomatal frequency [9], increases in mesophyll cell size, reductions in epidermal cell density, and enhanced lignification [10]. In Arabidopsis thaliana L., parenchyma cells exhibit significant ultrastructural alterations after 6 to 24 h of low-temperature adaptation, including membrane invagination and an abundance of microvesicles, plastidospherules, and ER fragments near the cell membrane [11]. The regulation of cell wall mechanical properties plays a crucial role in influencing tissue growth and frost resistance during cold acclimation. As plants adapt to cold environments, they often experience delayed leaf expansion, which subsequently contributes to the development of frost resistance [12]. Additionally, cold acclimation stimulates polyphenol metabolism in Populus tremula L. seedlings, enhancing the protective capacity of the cell wall by increasing lignin content, which is a vital mechanism for helping plants withstand environmental stress [13]. Furthermore, in colder environments characterized by reduced metabolic demands, Juniperus virginia L. utilizes soluble sugars as both cryoprotectants and osmotic regulators to perform non-metabolic functions. This adaptive process enhances cold resistance by preserving a high sugar-to-starch ratio [14]. The interaction between β-amylolytic enzymes and starch particles in plant parenchyma cells is significantly influenced by temperature. Cold-induced starch hydrolysis can result in elevated sucrose levels during cold acclimation, which ultimately bolsters the plant’s cold tolerance [15]. During cold acclimation, specialized transcriptional programs activated by cold-induced nucleoproteins are associated with lignin biosynthesis. This process facilitates cell wall remodeling by reducing lignin content, ultimately enhancing freeze tolerance [16].

Pinus massoniana Lamb. is widely distributed in the southern subtropical regions of China and serves as the primary tree species for developing the national timber strategic reserve production base, playing a crucial role in maintaining the integrity of the southern mountain forest ecosystem [17]. Cold resistance is the main driver of genetic differentiation in P. massoniana [18]. Despite its significance, the physiological mechanisms underlying cold acclimation in seedlings remain poorly understood. This lack of comprehensive understanding hampers the precise and timely evaluation of traits associated with low-temperature resistance in P. massoniana. Moreover, P. massoniana is a notable heteroblastic foliage species, exhibiting both primary needle seedlings (PNS) and secondary needle seedlings (SNS) by the end of the first growing season [19,20]. Heteroblasty refers to the developmental changes that occur within a plant as its growth and is not necessarily linked to environmental changes [21]. This phenomenon is driven by maturation or phase transitions within the apical meristem, leading to significant morphological differences in leaf primordia as the tree ages [22]. These ongoing ontogenetic changes in form and function are intrinsic to the meristematic tissues and reflect internal transformations rather than external environmental influences [23]. Previous studies have highlighted significant distinctions in the non-photochemical quenching mechanism, photosynthetic product accumulation, and drought resistance between primary and secondary needles of P. massoniana [24]. These differences are evident in leaf structure, photosynthesis, and the accumulation of chlorophyll and carbohydrates. However, the processes underlying low-temperature resistance development in P. massoniana seedlings during cold acclimation remain unexplained. In this study, we analyzed the morphological structure, starch grain distribution, and lignin characteristics of PNSs and SNSs to investigate the differences in phenotypic morphology and cold adaptation of heteroblastic foliage seedlings during cold acclimation under varying durations of 4 °C low-temperature stress. This research holds significant importance in establishing a scientific and theoretical foundation for the screening of cold-resistant germplasm resources, as well as for the development of superior germplasm and the breeding of robust seedlings of P. massoniana.

2. Materials and Methods

2.1. Seedling Culture and Temperature Treatment

The seeds were sourced from the seed orchard at the national base of improved tree varieties (26°9′ N, 107°18′ E, 920 m a.s.l.) in Duyun, Guizhou Province, China. This region is characterized by a subtropical humid climate, with an average annual temperature of 15.8 °C and an average annual rainfall of approximately 1400 mm. Container-based seedling cultivation began in late March 2022, employing methods for seedling cultivation and management as described by Wang et al. [24].

Our initial observations indicated that PNSs and SNSs could be clearly distinguished starting in September. From September to October, the seedlings undergo a period of rapid growth, followed by a wintering phase that begins in December. In this study, we selected PNSs and SNSs in December as our experimental materials. Two hundred seedlings from each category, PNSs and SNSs, were chosen and relocated to an indoor artificial climate chamber for cold acclimation. The treatment parameters were defined as follows: a 14 h light period followed by 10 h of darkness, with a consistent temperature of 4 °C maintained throughout the day and night cycles. Root, stem, and leaf samples from both PNSs and SNSs were collected at 0 h, 24 h, 72 h, 168 h, and 336 h after cold acclimation (Figure 1). To ensure reliable experimental outcomes, four biological replicates were performed for each time point and treatment.

2.2. Preparation and Staining of Paraffin Sections

Following the methods outlined by Chen [25], fresh seedling tissues comprising roots, stems, and leaves were placed in Carnoy’s solution (95% ethanol/glacial acetic acid, v/v, 3:1). After a 12 h vacuum treatment, the materials were transferred to a 70% ethanol solution. For transparency, a baso dewaxing agent was selected. Crushed wax was added directly to the container in a 1:1 ratio with the dewaxing agent, and the mixture was left in an oven at 42 °C overnight. After appropriate trimming, the samples were embedded in conventional paraffin sections and sectioned to a thickness of 8 µm using a microtome (RM2235; Leica, Wetzlar, Germany).

Starch staining observation: solid green potassium iodide staining was conducted by immersing the sections in potassium iodide solution for 10 min. Following this, the samples were washed twice with distilled water and then treated with 95% ethanol for 2 min. They were subsequently stained in a 1% solid green dye solution for 1 min. A third-stage decolorization using anhydrous ethanol was then performed. The samples were observed and photographed under a microscope (AmScope, Irvine, CA, USA) in conjunction with a Dual Pixel camera (SM-G955, Samsung, Suwon-si, Republic of Korea), revealing that the starch appeared green [26].

Lignin staining observation: lignin staining was performed using phloroglucinol. After rehydration, a drop of concentrated hydrochloric acid was applied to the sections and allowed to sit for 5 min to facilitate cell acidification. Subsequently, phloroglucinol solution was introduced, and the samples were kept for 4 min before being examined under a microscope. The lignin was observed to display a red color [27].

3. Results

3.1. Response of Structure and Starch Grain Accumulation in Heteroblastic Foliage Seedlings to Cold Acclimation

3.1.1. Leaf Morphological Structure and Starch Grain Accumulation

In Figure 2, we demonstrate that after 24 h of cold acclimation, the cell structure of leaves from both PNSs and SNSs exhibited notable alterations. As the duration of cold acclimation progressed, the epidermal tissue structure of PNSs displayed damage, with the mesophyll cell tissue becoming fragmented and deformed. In contrast, the mesophyll cells of SNSs underwent a gradual spreading process, transitioning from a tightly packed state to a more dispersed formation while maintaining a relatively intact organizational structure. Moreover, we observed that both the mesophyll and vascular tissues of PNSs and SNSs accumulated a significant amount of starch grains within 24 h of cold acclimation. Our hypothesis suggests that seedlings undergoing early-stage cold acclimation develop resistance to low-temperature stress by accumulating substantial quantities of starch grains. After 72 h of cold acclimation, the starch grains in PNS leaves showed a continuous decrease until starch accumulation resumed at 336 h. Conversely, the starch grains in SNS leaves decreased after 72 h of cold acclimation but recovered by 168 h. These findings indicate that cold tolerance in seedlings improves through the conversion of starch grains into sucrose during cold acclimation. Furthermore, it was observed that the cold acclimation period for PNS leaves was longer than that of SNS leaves, with SNSs being able to restore their physiological regulatory functions in a shorter timeframe.

3.1.2. Stem Morphological Structure and Starch Grain Accumulation

After 24 h of cold acclimation, the gap between the epidermal and cortical tissues in the stems of PNSs and SNSs began to widen (Figure 3). By the 72 h mark, the stem cells of PNSs showed signs of disorganization, particularly in the epidermal layer, compared to those of SNSs. In PNSs, the epidermal cells experienced water loss and atrophy, leading to an increase in intercellular space and partial detachment of the epidermis from the cortex. In contrast, the gap between the epidermal and cortical cells in SNSs gradually narrowed, resulting in a more compact cell arrangement as the cold acclimation period progressed. Furthermore, we observed a significant accumulation of starch grains in the parenchyma of the stem cortex at the onset of cold acclimation. Notably, PNSs demonstrated a sustained accumulation of starch grains even after 336 h of cold acclimation. Conversely, SNSs exhibited a decrease in starch grain accumulation after 336 h, followed by a gradual recovery.

3.1.3. Root Morphological Structure and Starch Grain Accumulation

By anatomically examining the roots of normally growing P. massoniana seedlings, we observed a distinct meristem cell structure at the root tip, characterized by closely spaced stele cells. The cortical parenchyma cells exhibited a well-organized and compact arrangement, featuring dense cytoplasm and robust division capabilities (Figure 4a,f). After 24 h of cold acclimation, the apical structures of both PNSs and SNSs showed signs of atrophy, while the cortical and meristem cells exhibited damage and began to deform. The epidermal cells appeared visibly wrinkled and disordered, with some cells broken, while the central column cells were loosely arranged, resulting in expanded spaces. These observations suggest structural damage to the cell wall due to low-temperature stress, alongside cavity formation resulting from inadequate cell support during cold acclimation. However, after 168 h of cold acclimation, the epidermal and cortical parenchyma cells of PNS roots recovered to a tightly organized arrangement with minimal spacing between the central column cells. In contrast, the root structure of SNSs remained chaotic. Before cold acclimation, only a small amount of starch grain was observed in the meristem and cortical parenchyma cells within the elongation region of both PNSs and SNSs. After 72 h of cold acclimation, starch grain accumulation in SNS roots progressively increased with the duration of exposure. Conversely, following 336 h of cold acclimation, there was a significant decline in starch grain accumulation in the PNS roots, which subsequently returned to initial levels.

3.2. Response of Structure and Lignin in Heteroblastic Foliage Seedlings to Cold Acclimation

3.2.1. Leaf Morphological Structure and Lignin Accumulation

The degree of lignin accumulation was assessed by analyzing the depth of xylem staining (Figure 5). Throughout the cold acclimation process, xylem staining and lignin accumulation in PNS leaves exhibited a progressive increase. In contrast, lignin accumulation in SNS leaves showed a gradual decrease after 168 h of cold acclimation. These results highlight that cold acclimation promotes the buildup of lignin in the epidermal cell walls and xylem of leaves, enabling SNSs to efficiently utilize lignin to enhance cold resistance.

3.2.2. Stem Morphological Structure and Lignin Accumulation

In Figure 6, the stem xylem of both PNSs and SNSs exhibited prominent red coloration during cold acclimation, indicative of increased lignin accumulation induced by cold exposure. Interestingly, there was a notable disparity in the timing of color change between the stems of PNSs and SNSs. Lignin deposition in the stems of PNSs began after 72 h of cold acclimation, followed by a decrease in xylem lignin accumulation at 336 h, while lignin continued to accumulate in the epidermal cell walls. In comparison, SNS displayed an earlier onset of xylem lignin accumulation at 24 h post-cold acclimation, but with prolonged exposure to cold, lignin levels decreased in the xylem and shifted toward the stem epidermal cell walls. These findings suggest that the physiological response of lignin in SNS stems is more sensitive during cold acclimation compared to PNSs.

3.2.3. Root Morphological Structure and Lignin Accumulation

Through root structure and staining analysis of the seedlings (Figure 7), it was observed that the red areas were predominantly present in the cortical tissues during cold acclimation at both 0 h and 24 h, indicating that lignin primarily accumulated in the cortical parenchyma cells during the initial stages of cold acclimation. As cold acclimation progressed, lignin accumulation in PNS roots gradually decreased and normalized after 336 h. Conversely, cold acclimation facilitated the gradual diffusion of lignin from the cortical tissue to the xylem in SNS roots, resulting in sustained high lignin content even after 336 h of cold acclimation.

4. Discussion

4.1. Morpho-Anatomical Changes of Heteroblastic Foliage Seedlings during Cold Acclimation

The morphological and anatomical characteristics of plants are the result of long-term adaptation to environmental conditions and represent stable hereditary traits. Analyzing these traits offers valuable insights into how plants respond to specific environmental challenges [28]. Microscopic observations at the cytological level provide a direct view of how stress influences cellular structure, making this technique essential for understanding plant stress tolerance [29]. Research on leaf tissues from diverse plant species has revealed that factors such as the prominence of leaf veins, as well as the density and porosity of leaf tissues, are associated with a plant’s cold tolerance [30]. In this study, we observed variations in the duration of cold acclimation among different organ tissues in both PNSs and SNSs. Leaves, as the primary photosynthetic organs, exhibit high vulnerability to environmental changes [31]. In studying the cold tolerance of Brassica napus L., researchers have found that leaves exhibit greater sensitivity to temperature drops compared to stems and roots. Long-term cold stress significantly alters the ultrastructure of leaf mesophyll, resulting in damage to the membrane system, the destruction of chloroplasts, and the swelling of mitochondria [32]. In our study, we observed a similar phenomenon. At the onset of cold acclimation, the leaves of PNSs exhibited increased sensitivity to temperature, resulting in initial damage and deformation of the epidermal tissue. Despite an extended cold acclimation period, the damage in PNS leaves persisted even after 336 h. Conversely, SNS leaves quickly adapted to the cold through mesophyll diffusion, maintaining a largely intact leaf structure. Some studies indicate that mesophyll diffusion under cold stress is closely linked to photosynthesis, with plants exhibiting higher cold tolerance maintaining elevated photosynthetic enzyme activity and transcription levels, as well as increased concentrations of reducing sugars and sucrose [33]. In our previous research, we also observed that SNS leaves exhibit a greater accumulation of non-structural carbohydrates [19]. Additionally, both the stems and roots of PNSs and SNSs exhibited disorganization and atrophy after 24 h of cold acclimation. After 72 h of acclimation, the epidermal tissue and xylem in SNS stems recovered, whereas the cell gaps in PNS stems continued to widen with prolonged acclimation. This phenomenon may be attributed to the low water content and elevated levels of solutes (sugars and antifreeze proteins) in the stem tissues of SNSs. The stem water content of plants with high cold tolerance is stable and low, and the low tissue water content combined with high solutes slows the rate at which water inside the cell freezes and migrates to water outside the cell [34]. Intriguingly, the cold acclimation response in roots differed from that of stems in both seedlings. The root epidermis and cortical parenchyma cells in PNSs gradually reorganized into a denser arrangement after 168 h, while the root structure in SNSs remained disordered even after 336 h of acclimation. We hypothesize that this phenomenon may be attributed to the robust division capabilities of the apical meristem in roots [35]. Upon successful cold acclimation, there was an increase in the rate of cell division, facilitating the rapid replacement of dead cells with newly generated ones [36]. Notably, this specific structural change at the cellular level was exclusively observed in the root tip of PNSs, whereas the cell walls in the leaves and stem cortex regions of SNSs exhibited a dynamic interplay between relaxation and rigidity. This observation implies that the leaves and stems of PNSs are more vulnerable to low temperatures during sustained cold acclimation. The cell wall structure of Ranunculus glacialis L. and Broussonetia papyrifera L. exhibited comparable changes in response to cold conditions [37,38]. Additionally, some researchers have noted in their evaluation of the freezing resistance of primary and secondary needles in various ecotypes of Mediterranean pine that primary needles are significantly more sensitive to freezing than secondary needles in Pinus halepensis Mill., P. brutia Ten., P. pinaster Ait., and P. nigra Arn.. Secondary needles demonstrate a greater adaptive capacity, and this complex response can be understood from the perspectives of a seedling’s individual development and species’ ecological niches [39]. Our study of cold acclimation in PNSs and SNSs yielded similar conclusions regarding the morpho-anatomical changes observed in both primary and secondary needles following cold acclimation.

4.2. Changes of Starch Grain in Heteroblastic Foliage Seedlings during Cold Acclimation

When plants are exposed to low-temperature stress, they accumulate specific nutrients and metabolites to enhance their cold tolerance [40]. Among these, the storage of sugars in tissues plays a critical role. Typically, these sugars are stored in the form of starch grains [41]. Starch is recognized for its role in supplying energy and carbon under potentially limiting conditions for plant photosynthesis, particularly during stress. The increase in starch content within leaves during cold acclimation may reflect an important adaptation to cold environments [42]. Furthermore, as low-temperature stress intensifies, these starch granules can be metabolized into sugars (sucrose, glucose, and fructose), which then participate in the osmoregulatory pathway [43]. In this study, the leaves, stems, and roots of PNSs and SNSs exhibited significant accumulation of starch grains during 24 h of cold acclimation. It is speculated that in the initial stages of cold acclimation, seedlings store starch through photosynthesis in response to low-temperature stress. However, as cold acclimation progresses, the photosynthetic enzyme activity in the leaves is inhibited, leading to the hydrolysis of starch into triose phosphate. This triose phosphate is then transported to synthesize sucrose, with ATP being supplied by the TCA cycle to facilitate cold acclimation [44]. In addition, the energy derived from starch decomposition during cold acclimation is utilized to enhance plant resistance to low-temperature stress or to compensate for reduced enzyme activity caused by cold conditions [45]. In our study, after 336 h cold acclimation, the leaves and stems of PNSs continued to show starch accumulation. Contrarily, after 168 h of cold acclimation, the starch accumulation in the leaves and stems of SNSs decreased, signifying the gradual completion of cold acclimation. Some researchers propose that during the process of cold acclimation, excessive accumulation of starch can lead to a reduction in the total amounts of sucrose and glucose. This limitation hampers carbon allocation within leaf tissues during cold exposure, which can negatively impact photosynthesis and metabolic processes at low temperatures [46]. Notably, the roots of PNSs exhibited a significant decrease in starch grains accumulation, while SNS roots maintained high levels of starch grains. Therefore, we hypothesized that the leaves and stems of SNSs enhance cold resistance by increasing starch accumulation during the early stages of cold acclimation. As cold acclimation progresses, some of this starch is hydrolyzed into soluble sugars, facilitating metabolic transport within the plant, such as the movement of sucrose to the roots for the resynthesis of starch. However, the excessive accumulation of starch in the leaves and stems during cold acclimation severely limits the metabolism of PNSs in the roots. These observations align with the morpho-anatomical changes observed during cold acclimation in the leaves, stems, and roots of PNSs and SNSs.

4.3. Changes of Lignin in Heteroblastic Foliage Seedling during Cold Acclimation

Lignin is a vital secondary metabolite synthesized by plant cells through the phenylalanine metabolic pathway [47]. It is predominantly found in the cell walls of transport and lignified tissues, providing essential structural support [48]. Lignin not only fortifies cell walls but also enhances plant resistance to various stresses [49]. Studies at the cellular level have revealed that freeze resistance is closely linked to cell permeability and cell wall properties, particularly lignin content, which allows water to exit the cell and form ice in the extracellular space without damaging the cell structure [50]. In Eriobotrya japonica Thunb., low temperatures trigger the synthesis of lignin, its precursors, and related enzymes to facilitate fruit lignification [51]. Our research shows that cold acclimation facilitated lignin accumulation in the epidermal cell walls and xylem of leaves. It is noteworthy that the lignin content in SNS leaves gradually increased during the initial stages of cold acclimation; however, this accumulation began to decline after 168 h of acclimation. This trend suggests that SNS leaves effectively utilize lignin synthesis to maintain cell wall integrity during cold exposure, thereby enhancing cold resistance. Importantly, the lignin content does not continue to accumulate indefinitely. Studies have shown that elevated phenylalanine aminase activity during cold acclimation decreases lignin accumulation, underscoring the direct correlation between lignin content, cell wall permeability, and freeze resistance [52,53]. In stems, the cold acclimation response of SNSs preceded that of PNSs, resulting in a reduction in xylem lignin levels over time, with a transference to the stem’s epidermal cell wall. Conversely, lignin levels in PNS roots progressively decreased after 24 h of cold acclimation, while SNS roots maintained high lignin levels throughout the cold acclimation process. To sum up, these findings suggest that lignin serves as crucial support for SNS leaves and stems during cold acclimation, preserving cellular integrity and enhancing cold resistance.

5. Conclusions

The process of cold acclimation resulted in significant morpho-anatomical changes in the leaves, stems, and roots of P. massoniana seedlings, as well as in the accumulation of starch grains and lignin in their heteroblastic foliage. This study revealed that the leaves and stems of PNSs exhibited more sensitivity to low-temperature stress during cold acclimation, leading to damage and deformation in mesophyll and cortical parenchyma cells. Additionally, cold acclimation triggered early-stage accumulation of starch grains and lignin in various tissues of the seedlings. In comparison to PNSs, the leaves and stems of SNSs demonstrated a shorter cold acclimation period, which was attributed to the hydrolysis of starch grains in the epidermal cell walls and the transformation of lignin in the xylem. These processes contributed to the stability of cell structure and enhanced cold resistance during cold acclimation in SNSs. In summary, this study provides a theoretical foundation for analyzing the cold acclimation resistance traits of heteroblastic foliage in conifers.