Morphology, anatomical structure and leaf development regulation

The morphology

Leaf morphology of a P. euphratica tree sequentially changes according with the development of linear, lanceolate, ovate, and serrated broad-ovate.^18,19 The shape of P. euphratica leaves transitions from linear to serrated broad-ovate, so the leaf area was increased, even one branch has a similar change. As shown in Figure 1, the types of transitions between linear and serrated fan-shaped leaf can be characterized into three major groups through hierarchical cluster analysis: young, developing and mature leaves. This indicates that the change of leaf types is gradual, and the related developmental regulatory factors are differentially expressed in various transition types of leaves.¹⁸ A distinct shape polymorphism occurs during the leaf development of the species, which is named as leaf heterophylly.¹⁸ Therefore, it can be used as an ideal model plant for studying the development of leaf shape.²⁰ The relationships between leaf biomass, area, volume and petiole biomass of the three leaf shape were investigated by a field research. The results showed that the allometric relationship was not consistent in different stages of leaf development. The study suggested that allometric relationships were not significant at young leaf stage. During leaf developing stage, there were significant allometric relationships only in ovate leaves. At the maturity stage, the allometric relationships between leaf mass, area, volume and petiole biomass were all significant in three types of leaf.²¹ It has been reported that with the increase in BDH (diameter at breast height) and crown level from the base to the top, the leaf width, area and thickness of leaves in P. euphratica gradually increased, while the leaf length and leaf index (leaf length/leaf width) steadily decreased, indicating that the morphological change of P. euphratica leaves is closely related to the ontogenetic stage.²²

Progress in studying heteromorphic leaves in Populus euphratica: leaf morphology, anatomical structure, development regulation and their ecological adaptation to arid environments

All authors

Zhihan Song, Xinbo Ni, Jian Yao & Fang Wang

https://doi.org/10.1080/15592324.2020.1870842

Published online:

11 January 2021

Figure 1. Heteromorphic leaves of Populus euphratica tree from the bottom to top of the crown (adapted from Liu et al., 2015)

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Figure 1. Heteromorphic leaves of Populus euphratica tree from the bottom to top of the crown (adapted from Liu et al., 2015)

The anatomical structure

The structure of the leaves is closely associated with its various functions. Therefore, it is crucial to study the anatomical features of heteromorphic leaves in P. euphratica.²³ P. euphratica is a typical xerophyte detected from the leaf cross-section anatomical characteristics.¹⁸ For example, the morphology of heteromorphic leaves of P. euphratica is closely connected with the proportion of sponge and palisade tissues, as well as the number of cell layers. As the leaf blade changed from lanceolate to ovate, the thickness of palisade tissue increased, and the sponge tissue decreased. In addition, the increase in the epidermal layer also increased the thickness of ovate leaves.¹⁸ In another field research, the cell number and length of epidermal cells, the thickness, length and width of palisade tissue increased with the increase of DBH and crown level (from base to top), but the thickness of spongy tissue decreased with the increase of DBH.²² The lamina thickness (LT), ratio of epidermal cell thickness to lamina thickness (REL) and ratio of palisade tissue to lamina thickness (TCR) all increased from Pe1 (period 1) to Pe8 (Figure 1). Moreover, the leaves from Pe9 to Pe12 still maintained a higher level of thickness.¹⁸ It is considered that the high palisade packing of the thick leaves help them avoid rapid wilting during water stress under drought conditions.²⁴ Furthermore, epidermal cells may function as a buffer layer and may be useful for the reflectance and absorption properties.¹⁸ Based on the correlation analysis, it was revealed that there were significant positive correlations between BDH and leaf width, area and thickness, and significant positive correlations were also found between BDH and thickness of palisade tissue, as well as cell length, number and thickness of epidermal cells. However, a significant negative correlation between the thickness of spongy tissue and BDH was discovered.²² The traits of xerophytic anatomy in heteromorphic leaves of P. euphratica enable them to adapt to the drought in the arid area.

Crystal idioblasts have a strong capacity for water absorption. This means they can store water in the beneficial environment, and provide a relatively humid environment to surrounding cells under drought conditions.¹⁸ Mucilage plays a critical role in the water supply of apical leaves, and it was considered more important than water uptake by the roots.¹⁸ Cuticular wax as a hydrophobic layer act as a protective barrier against water stress, which is considered to play a critical role in minimizing transpirational water loss through the epidermis and trichomes.²⁴ The cuticular wax coverage on the leaf surface achieved more than 90%, and the cuticular wax thickness increased from Pe1 to Pe12; crystal idioblasts appeared in palisade tissue, epidermal cells, and phloem of vascular bundles, and the number of crystal idioblasts increased significantly from Pe1 to Pe12; meanwhile, large amount of mucilage cells appeared in the palisade tissue and the quantities of mucilage cells increased from Pe3 to Pe12.¹⁸ This means by combining the regulation of water uptake by mucilage and crystal idioblasts, and assistant defense of transpiration reduction through leaf epidermal appendages such as wax,¹⁸ mature ovate leaves have a high ability to resist the stress of drought for P. euphratica growing in a drought environment.

The chloroplasts were elongated and become fusiform shaped from Pe3 to Pe12; the number and size of chloroplast and starch grains increased from Pe3 to Pe8 where the chloroplasts were nearly full of starch grains; the width of chloroplast increased, which was due to the large size of starch grains during the leaf-developing process.¹⁸ This indicates the developing leaves of P. euphratica have maximal photosynthetic activity. Based on the chloroplast ultrastructural characters and subordinate function values, the authors deduced that the older the age of P. euphratica trees, the higher proportion of mature leaves that evolved in response to adverse environmental conditions.¹⁸

Leaf development regulation

At present, the information of the molecular mechanism for the heteromorphic leaf development of P. euphratica is limited, and most of the experiments are still at the level of high-throughput sequencing analysis and bioinformatics without elaborating the specific metabolic pathways and the relationship between various regulatory elements. Currently, research on the molecular mechanism of heteromorphic leaf development primarily focuses on the regulation of non-coding RNA (ncRNAs). NcRNAs are composed primarily of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), and the latter two can be regarded as attractants that snare miRNAs and play an important role in the development of animals and plants.^19,25–27 A comparison of the levels of expression of miRNAs between the lanceolate and broad-ovate leaves identified nine miRNAs and their 113 target genes that participate in the development of apical-basal and medial-lateral axes.²⁰ LncRNA plays a complex and precise regulatory role inanimal and plant development and gene expression.¹¹ A total of 2,448 lncRNAs were detected that may participate in the regulation of heteromorphic leaf development, and 86 lncRNAs could be involved in antagonizing the functions of miRNA to regulate heteromorphic leaf morphogenesis in P. euphratica.²⁸ The 586 differentially expressed genes and 54 lncRNAs were identified in the high-throughput RNA sequencing analysis of the lanceolate and broad-ovate leaves.²⁹ Although the analysis results of lncRNAs were different in the two studies, they both revealed that various lncRNAs could eventually function in the development of profile-shaped leaves by regulating the activity of mRNA. Besides, circRNAs may also play a regulatory role in the circRNAs-miRNAs-mRNAs to participate in the regulation of miRNAs.¹³ Eighteen differentially expressed circRNAs (DECs) among the linear, lanceolate, ovate and broad-ovate leaves of P. euphratica antagonized 23 miRNAs and ultimately affected the levels of expression of 84 mRNAs. These DECs were hypothesized to participate in leaf developmental processes and respond tohormones. Abscisic acid (ABA) might play a crucial role in the formation of lanceolate and broad-ovate leaves.³⁰ In this study, the targets of two circRNAs (circRNA_0168 and circRNA_0351) were regulated by ABA, which means that circRNAs may play a vital role in the development of profile-shaped leaves of P. euphratica by affecting the expression of hormone abscisic acid (ABA).¹⁴ However, more detailed regulatory mechanisms require additional clarification.

In addition to certain ncRNAs, some related genes may be involved in the regulation of heteromorphic leaf morphogenesis, such as the rot3, rot4, an and spk1 genes. The expression of rot3 and spk1 in lanceolate leaves was remarkably higher during the early development of leaves compared with those in broad-ovate leaves, while the expression of rot4 and an genes in broad-ovate leaves was significantly higher than that in the lanceolate leaves.¹¹ This suggests that rot3 and spk1 can promote the development of leaf shapes to lanceolate during the heteromorphic leaf development, particularly during the early development stage, while rot4 and an may further promote the development of leaf shape to wide ovate. The previous studies about the function of the rot3, rot4, an and spk1 genes in Arabidopsis have mentioned that the rot3 and rot4 genes can regulate cell division and proliferation in the apical-basal and medial-lateral axes. Moreover, an and spk1 can affect leaf shape development by adjusting the arrangement of the cytoskeleton. Accordingly, the rot3, rot4, an and spk1 genes also have a notable effect on regulating leaf type development of the heteromorphic leaf in P. euphratica. The rot3 and spk1 genes are highly expressed in lanceolate leaves. The rot3 gene may promote the generation of lanceolate leaves by regulating cell amplification along the apical-basal axis, while spk1 inhibits the development of leaves toward the shape of broad-ovate by regulating the cytoskeleton rearrangement of the medial-lateral axes. Besides, the expression of rot4 and an genes in broad-ovate leaves was significantly higher than that in the lanceolate leaves. The rot4 gene may limit cell extension along the apical-basal axis and an could accelerate the process for the leaves toward the shape of broad-ovate by regulating the arrangement of the cytoskeleton.¹¹ The profiles of expression of P. euphratica leaves at indifferent developmental stages were also sequenced. The results showed that, among all the differentially expressed genes, cytochrome P450, LG1 (LIGULELESS1), FLC (FLOWERING LOCUS C) and 11 other genes related to leaf morphogenesis may be involved in leaf formation in P. euphratica.³¹ The response to stimulus were the genes that were most distinctively expressed that participated in biological processes after metabolic processes and cellular processes. An analysis of metabolic pathways indicated that some genes participated in the enlargement of cells and plant growth, such as the hormone transduction. The functions of these genes in leaf morphogenesis of P. euphratica require further validation.

In conclusion, during the development of heteromorphic leaves, many factors are involved in the regulation process. However, the developmental difference of the medial-lateral axes might be an important factor for the development of heteromorphic leaves. spk1, an genes and various miRNAs have a comprehensive effect on the development of the medial-lateral axes.

Ecological adaptation to arid environments in heteromorphic leaves

Chlorophyll fluorescence traits of heteromorphic leaves under high light and temperature

The absorption of an excessive amount of light can exceed the amount of photochemical reactions in the high light environments of the desert. If the light energy is not dissipated, it will cause photoinhibition. Severe photoinhibition can lead to the irreversible destruction of the photosynthetic reaction center.⁴³ Chlorophyll fluorescence kinetic techniques can be used to evaluate the ability of plants to withstand stress environments.^44,45 It has been shown that three representative heteromorphic leaves of P. euphratica have evolved in different manners to adapt to the strong solar radiation in desert areas. For example, the oval leaves improved the PS ll effective photochemical quantum efficiency (F_v’/F_m’) and the PS ll effective actual photochemical quantum efficiency (Φ_PSll) to maintain a higher capacity of photosynthesis to dissipate excess light energy and reduce the effects of excess excitation energy on the photosynthetic apparatus.⁴³ The serrated broad-ovate leaves maintained a high electron transport rate (ETR) to relieve the reduced pressure of photosynthetic membrane and dissipate thermally to protect the photosynthetic apparatus, avoid light damage and maintain a higher net photosynthetic rate (P_n). However, compared with oval and serrated broad-ovate leaves, lanceolate leaves had a weak resistance to strong light.⁴³ In conclusion, the serrated broad-ovate leaves can efficiently resist extreme environmental habitats owing to their effective allocation of energy.¹⁸

The tolerance of plants in desert areas to high temperatures is a key factor that determines their distribution and even survival.⁴⁵ High temperature can decrease the activity of PS ll reaction center and the rate of photochemical electron transfer and suppress the heat dissipation and distribution balance of excitation energy between PS □and PS ll, which results in structural damage to the photosynthetic apparatus and a functional decrease in photosynthetic ability.^45,46 The chlorophyll fluorescence parameters of the heteromorphic leaves of P. euphratica varied remarkably from each other during high temperature processing. Serrated broad-ovate leaves can reduce their photochemical efficiency to maintain the machinery that functions in the photochemical reaction of PS □ and initiate the heat loss dispersion mechanism to protect its photosynthetic machinery with strong thermal stability and tolerance to high temperatures (>45°C). However, the lanceolate leaves were more sensitive to high temperatures, and the ability to dissipate heat and the function of photochemical reaction were weaker at high temperature compared with those of the serrated broad-ovate leaves.⁴⁵ This is consistent with the distribution of heteromorphic leaves in P. euphratica plants, i.e., lanceolate leaves are primarily in the lower part of the crown, and the serrated broad-ovate leaves are primarily in the upper part of the crown, which are subjected to high temperature and strong light and are more resistant to high temperature.^45,47

Responses of heteromorphic leaves to water stress

In arid areas, water use efficiency (WUE) is an important factor that affects the survival of plants. The WUE in broad ovate leaves is higher than that of the lanceolate leaves in P. euphratica.^48,49 With the increase in light intensity and CO₂ concentration, the WUE in broad-ovate leaves is clearly enhanced, and it can adapt better to the variation in environment. Therefore, when the near-surface air becomes dry and ground water level decreases, or with climate warming owing to increasing concentrations of atmospheric CO₂, the number of lanceolate leaves may be likely to decrease or even disappear.⁵⁰

The significant differences in WUE were closely related to the anatomical structure of the heteromorphic leaves in P. euphratica. The results of scanning electron microscopy (SEM) showed that the lanceolate leaf surfaces produced a deposition of wax plate-like structures, with a lower density of crystals, while broad-ovate leaf surfaces were covered by a thickened amorphous wax layer.^18,51 The protective effect of waxy crystals enables P. euphratica to avoid damage from bright light under high temperatures and high radiation conditions in the desert and utilize the high light intensity to perform normal photosynthesis.^16,51,52 The expression of genes associated with cuticle lipids will increase to reduce the loss of water under drought conditions.¹⁶ Moreover, in comparison with the lanceolate leaves, the broad-ovate leaves have a larger blade width, leaf area, leaf thickness, thicker cuticle, weaker sponge tissue and more mucous cells in the mesophyll. These anatomical differences provide the broad-ovate leaves with a greater tolerance to water scarcity and bright light in arid zones.^18,53

Stable hydraulic conductivity in forest trees maintains the survival of trees that contribute to productivity in forest ecosystems. Determining the hydraulic parameters in P. euphratica in a P. euphratica time-series of drought demonstrated that could enhance hydraulic transport in severe drought stress under athreshold of soil water content.¹⁷ The average water conductivity of the branches with serrated broad-ovate leaves was higher than that in the lanceolate leaves, and the water conductivity of the former branches was significantly lower than that of the latter under water loss stress. In addition, the serrated broad ovate leaves strongly restricted the content of water; the free water content was small; the water potential was low, and the leaves had a strong capacity to absorb water. Moreover, the serrated broad-ovate leaf is more tolerant to drought owing to its thick cell walls, high mechanical strength and more developed vascular bundles.^47,54,55

Increasing the ability to adjust osmotically is an important strategy to ensure water uptake when the plants are subjected to water stress and salinity. Water stress can induce protein hydrolysis and amino acid accumulation, stimulate the glutamate synthesis of proline and its compounds, and inhibit its oxidation and protein synthesis. The content of free amino acids and proline in the broad-ovate leaves was higher than those in the lanceolate leaves of P. euphratica.^15,55 Moreover, the broad-ovate leaves contained more Na⁺ and Cl⁻ than the lanceolate leaves, and the broad-ovate leaves have a higher level of H⁺-ATPase activity and are better able to compartmentalize the Na⁺ in vacuoles.¹⁵ This indicates that the broad-ovate leaves are better able to make osmotic adjustments than the lanceolate leaves of P. euphratica under combined water and salinity stress in an arid region. These anatomical and physiological characteristics in the heteromorphic leaves of P. euphratica clearly indicate that the species can adapt well to arid environments by developing heteromorphic leaves with different capacities for photosynthesis and different ecological adaptability to high light and temperature, as well as water deficiency. This is the reason why the species can survive in the desert ecosystem by exhibiting a tolerance to extreme drought stress (Figure 2).

Progress in studying heteromorphic leaves in Populus euphratica: leaf morphology, anatomical structure, development regulation and their ecological adaptation to arid environments

All authors

Zhihan Song, Xinbo Ni, Jian Yao & Fang Wang

https://doi.org/10.1080/15592324.2020.1870842

Published online:

11 January 2021

Figure 2. The outline of leaf morphogenesis and ecological adaptation to arid environments in heteromorphic leaves of Populus euphratica. Lanceolate and ovate leaves are provided as examples

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Conclusion and perspectives

P. euphratica can produce heteromorphic leaves, which have different anatomical and physiological characteristics. Compared with lanceolate leaves, broad-ovate leaves can obtain WUE to resist the drought stress owing to their histological structures. Certain miRNAs, lncRNAs and circRNAs, as well as genes, such as cytochrome P450, LG1 and FLC, were related to leaf morphogenesis in P. euphratica. A higher photosynthetic efficiency and activity of broad oval leaves of P. euphratica at the mature stage can maintain a higher rate of photosynthesis under the desert environment of high temperature and strong light.

To date, the molecular mechanism of heteromorphic leaf development of P. euphratica is not clear. Interestingly, circRNAs are involved in morphogenesis and stress responses in the heteromorphic leaves of P. euphratica, which provides a basis for further research on the molecular mechanism of leaf shape development and stress response of plant circRNAs. Therefore, some regulatory factors and genes related to morphogenesis and stress responses in heteromorphic leaves of P. euphratica merit additional study.