Habitat differentiation and environmental adaptability contribute to leaf size variations globally in C3 and C4 grasses.

The grass family (Poaceae) dominates ~43 % of Earth's land area and contributes 33 % of terrestrial primary productivity that is critical to naturally regulating atmosphere CO2 concentration and global climate change. Currently grasses comprise ~11,780 species and ~50 % of them (~6000 species) utilize C4 photosynthetic pathway. Generally, grass species have smaller leaves under colder and drier environments, but it is unclear whether the primary drivers of leaf size differ between C3 and C4 grasses on a global scale. Here, we analyzed 34 environmental variables, such as latitude, elevation, mean annual temperature, mean annual precipitation, and solar radiation etc., through a comparatively comprehensive database of ~3.0 million occurrence records from 1380 C3 and 978 C4 grass species (2358 species in total). Results from this study confirm that C4 grasses have occupied habitats with lower latitudes and elevations, characterized by warmer, sunnier, drier and less fertile environmental conditions. Grass leaf size correlates positively with mean annual temperature and precipitation as expected. Our results also demonstrate that the mean temperature of the wettest quarter of the year is the primary control for C3 leaf size, whereas C4 leaf size is negatively correlated with the difference between summer and winter temperatures. For C4 grasses, phylogeny exerts a significant effect on leaf size but is less important than environmental factors. Our findings highlight the importance of evolutionarily contrasting variations in leaf size between C3 and C4 grasses for shaping their geographical distribution and habitat suitability at the global scale.

Transcriptome and Metabolome Reveal Key Genes from the Plant Hormone Signal Transduction Pathway Regulating Plant Height and Leaf Size in Capsicum baccatum.

Plant structure-related agronomic traits like plant height and leaf size are critical for growth, development, and crop yield. Defining the types of genes involved in regulating plant structure size is essential for the molecular-assisted breeding of peppers. This research conducted comparative transcriptome analyses using Capsicum baccatum germplasm HNUCB0112 and HNUCB0222 and their F2 generation as materials. A total of 6574 differentially expressed genes (DEGs) were detected, which contain 379 differentially expressed transcription factors, mainly including transcription factor families such as TCP, WRKY, AUX/IAA, and MYB. Seven classes of DEGs were annotated in the plant hormone signal transduction pathway, including indole acetic acid (IAA), gibberellin (GA), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), ethylene (ET), and salicylic acid (SA). The 26 modules were obtained by WGCNA analysis, and the MEpink module was positively correlated with plant height and leaf size, and hub genes associated with plant height and leaf size were anticipated. Differential genes were verified by qRT-PCR, which was consistent with the RNA-Seq results, demonstrating the accuracy of the sequencing results. These results enhance our understanding of the developmental regulatory networks governing pepper key traits like plant height and leaf size and offer new information for future research on the pepper plant architecture system.

CsRAXs negatively regulate leaf size and fruiting ability through auxin glycosylation in cucumber.

Leaves are the main photosynthesis organ that directly determines crop yield and biomass. Dissecting the regulatory mechanism of leaf development is crucial for food security and ecosystem turn-over. Here, we identified the novel function of R2R3-MYB transcription factors CsRAXs in regulating cucumber leaf size and fruiting ability. Csrax5 single mutant exhibited enlarged leaf size and stem diameter, and Csrax1/2/5 triple mutant displayed further enlargement phenotype. Overexpression of CsRAX1 or CsRAX5 gave rise to smaller leaf and thinner stem. The fruiting ability of Csrax1/2/5 plants was significantly enhanced, while that of CsRAX5 overexpression lines was greatly weakened. Similarly, cell number and free auxin level were elevated in mutant plants while decreased in overexpression lines. Biochemical data indicated that CsRAX1/5 directly promoted the expression of auxin glucosyltransferase gene CsUGT74E2. Therefore, our data suggested that CsRAXs function as repressors for leaf size development by promoting auxin glycosylation to decrease free auxin level and cell division in cucumber. Our findings provide new gene targets for cucumber breeding with increased leaf size and crop yield.

Heterotic growth of hybrids of Arabidopsis thaliana is enhanced by elevated atmospheric CO2.

PREMISE: With the global atmospheric CO2 concentration on the rise, developing crops that can thrive in elevated CO2 has become paramount. We investigated the potential of hybridization as a strategy for creating crops with improved growth in predicted elevated atmospheric CO2. METHODS: We grew parent accessions and their F1 hybrids of Arabidopsis thaliana in ambient and elevated atmospheric CO2 and analyzed numerous growth traits to assess their productivity and underlying mechanisms. RESULTS: The heterotic increase in total dry mass, relative growth rate and leaf net assimilation rate was significantly greater in elevated CO2 than in ambient CO2. The CO2 response of net assimilation rate was positively correlated with the CO2 response of leaf nitrogen productivity and with that of leaf traits such as leaf size and thickness, suggesting that hybridization-induced changes in leaf traits greatly affected the improved performance in elevated CO2. CONCLUSIONS: Vegetative growth of hybrids seems to be enhanced in elevated CO2 due to improved photosynthetic nitrogen-use efficiency compared with parents. The results suggest that hybrid crops should be well-suited for future conditions, but hybrid weeds may also be more competitive.

Overexpression of SLIM1 transcription factor accelerates vegetative development in Arabidopsis thaliana.

The transcription factor Sulfur Limitation 1 (SLIM1) belongs to the plant-specific Ethylene Insenstive3-Like transcription factor family and is known to coordinate gene expression in response to sulfur deficiency. However, the roles of SLIM1 in nutrient-sufficient conditions have not been characterized. Employing constitutive SLIM1 overexpression (35S::SLIM1) and CRISPR/Cas9 mutant plants (slim1-cr), we identified several distinct phenotypes in nutrient-sufficient conditions in Arabidopsis thaliana. Overexpression of SLIM1 results in plants with approximately twofold greater rosette area throughout vegetative development. 35S::SLIM1 plants also bolt earlier and exhibit earlier downregulation of photosynthesis-associated genes and earlier upregulation of senescence-associated genes than Col-0 and slim1-cr plants. This suggests that overexpression of SLIM1 accelerates development in A. thaliana. Genome-wide differential gene expression analysis relative to Col-0 at three time points with slim1-cr and two 35S::SLIM1 lines allowed us to identify 1,731 genes regulated directly or indirectly by SLIM1 in vivo.

Introduction of a terminal electron sink in chloroplasts decreases leaf cell expansion associated with higher proteasome activity and lower endoreduplication.

Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell numbers per leaf. Proteomic analysis showed that components of the light-harvesting systems accumulated before electron-transport proteins, suggesting a mechanism for the early oxidative event. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide build-up through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.

CsHLS1-CsSCL28 module regulates compact plant architecture in cucumber.

Increased planting densities boost crop yields. A compact plant architecture facilitates dense planting. However, the mechanisms regulating compact plant architecture in cucurbits remain unclear. In this study, we identified a cucumber (Cucumis sativus) compact plant architecture (cpa1) mutant from an ethyl methane sulfonate (EMS)-mutagenized library that exhibited distinctive phenotypic traits, including reduced leaf petiole angle and leaf size. The candidate mutation causes a premature stop codon in CsaV3_1G036420, which shares similarity to Arabidopsis HOOKLESS 1 (HLS1) encoding putative histone N-acetyltransferase (HAT) protein and was named CsHLS1. Consistent with the mutant phenotype, CsHLS1 was predominantly expressed in leaf petiole bases and leaves. Constitutive overexpressing CsHLS1 in cpa1 restored the wild-type plant architecture. Knockout of CsHLS1 resulted in reduces leaf petiole angle and leaf size and as well as decreased acetylation levels. Furthermore, CsHLS1 directly interacted with CsSCL28 and negatively regulated compact plant architecture in cucumber. Importantly, CsHLS1 knockout increased the photosynthesis rate and leaf nitrogen in cucumbers, thereby maintaining cucumber yield at normal density. Overall, our research provides valuable genetic breeding resource and gene target for creating a compact plant architecture for dense cucumber planting.

Genome-wide identification of GROWTH-REGULATING FACTORs in Liriodendron chinense and functional characterization of LcGRF2 in leaf size regulation.

GROWTH-REGULATING FACTORs (GRFs) play a pivotal role in the regulation of leaf size in plants and have been widely reported in plants. However, their specific functions in leaf size regulation in Liriodendron chinense remains unclear. Therefore, in this study, we identified GRF genes on a genome-wide scale in L. chinense to characterize the roles of LcGRFs in regulating leaf size. A total of nine LcGRF genes were identified, and these genes exhibited weak expression in mature leaves but strong expression in shoot apex. Notably, LcGRF2 exhibited the highest expression level in the shoot apex of L. chinense. Further RT-qPCR assay revealed that the expression level of LcGRF2 gradually decreased along with the leaf development process, and also displayed a gradient along the leaf proximo-distal and medio-lateral axes. Furthermore, overexpression of LcGRF2 in Arabidopsis thaliana resulted in increased leaf size, and significantly up-regulated the expression of genes involved in cell division like AtCYCD3;1, AtKNOLLE, and AtCYCB1;1, indicating that LcGRF2 may influence leaf size by promoting cell proliferation. This work contributes to a better understanding of the roles and molecular mechanisms of LcGRFs in the regulation of leaf size in L. chinense.

Overview of molecular mechanisms of plant leaf development: a systematic review.

Leaf growth initiates in the peripheral region of the meristem at the apex of the stem, eventually forming flat structures. Leaves are pivotal organs in plants, serving as the primary sites for photosynthesis, respiration, and transpiration. Their development is intricately governed by complex regulatory networks. Leaf development encompasses five processes: the leaf primordium initiation, the leaf polarity establishment, leaf size expansion, shaping of leaf, and leaf senescence. The leaf primordia starts from the side of the growth cone at the apex of the stem. Under the precise regulation of a series of genes, the leaf primordia establishes adaxial-abaxial axes, proximal-distal axes and medio-lateral axes polarity, guides the primordia cells to divide and differentiate in a specific direction, and finally develops into leaves of a certain shape and size. Leaf senescence is a kind of programmed cell death that occurs in plants, and as it is the last stage of leaf development. Each of these processes is meticulously coordinated through the intricate interplay among transcriptional regulatory factors, microRNAs, and plant hormones. This review is dedicated to examining the regulatory influences of major regulatory factors and plant hormones on these five developmental aspects of leaves.

Natural variation in the SVP contributes to the pleiotropic adaption of Arabidopsis thaliana across contrasted habitats.

Coordinated plant adaptation involves the interplay of multiple traits driven by habitat-specific selection pressures. Pleiotropic effects, wherein genetic variants of a single gene control multiple traits, can expedite such adaptations. Until present, only a limited number of genes have been reported to exhibit pleiotropy. Here, we create a recombinant inbred line (RIL) population derived from two Arabidopsis thaliana (A. thaliana) ecotypes originating from divergent habitats. Using this RIL population, we identify an allelic variation in a MADS-box transcription factor, SHORT VEGETATIVE PHASE (SVP), which exerts a pleiotropic effect on leaf size and drought-versus-humidity tolerance. Further investigation reveals that a natural null variant of the SVP protein disrupts its normal regulatory interactions with target genes, including GRF3, CYP707A1/3, and AtBG1, leading to increased leaf size, enhanced tolerance to humid conditions, and changes in flowering time of humid conditions in A. thaliana. Remarkably, polymorphic variations in this gene have been traced back to early A. thaliana populations, providing a genetic foundation and plasticity for subsequent colonization of diverse habitats by influencing multiple traits. These findings advance our understanding of how plants rapidly adapt to changing environments by virtue of the pleiotropic effects of individual genes on multiple trait alterations.

Model-based estimation of the leaf area of ozone-indicator tobacco (Nicotiana tabacum L.) plants under ambient ozone conditions.

Bel-W3 is an ozone-sensitive tobacco (Nicotiana tabacum L.) cultivar widely used worldwide for ozone biomonitoring. Despite its extensive use, there is no comprehensive predictive model to non-destructively estimate the leaf area using only a common ruler, yet leaf area is a major evaluative trait in plants under ozone stress and of economic value in tobacco plants. In this method, we aimed at developing a predictive model to estimate leaf area using the product between leaf length and leaf width. To this end, we conducted a field experiment with ground-grown Bel-W3 plants treated with different solutions under ambient ozone conditions. The solutions were water, the antiozonant ethylenediurea (EDU; 500 ppm), and the antitranspirant pinolene (Vapor Gard; 1%, 5%, 10%). The chemical treatments were introduced to enhance leaves pool and capture different conditions that can occur in ozone biomonitoring projects.•A simple linear predictive model was developed and validated using data from a previous chamber experiment with small seedlings.•Overestimation of the model led to the integration of data from both experiments and development of another simple linear predictive model.•This integrated model provides improved estimation of leaf area and can be used for representative estimation of the area of Bel-W3 leaves of any sizes.

Construction of the first high-density genetic linkage map and QTL mapping of flavonoid and leaf-size related traits in Epimedium.

BACKGROUND: Leaves are the main medicinal organ in Epimedium herbs, and leaf flavonoid content is an important criterion of Epimedium herbs. However, the underlying genes that regulate leaf size and flavonoid content are unclear, which limits the use of breeding for Epimedium development. This study focuses on QTL mapping of flavonoid and leaf-size related traits in Epimedium. RESULTS: We constructed the first high-density genetic map (HDGM) using 109 F1 hybrids of Epimedium leptorrhizum and Epimedium sagittatum over three years (2019-2021). Using 5,271 single nucleotide polymorphism (SNP) markers, an HDGM with an overall distance of 2,366.07 cM and a mean gap of 0.612 cM was generated by utilizing genotyping by sequencing (GBS) technology. Every year for three years, 46 stable quantitative trait loci (QTLs) for leaf size and flavonoid contents were discovered, including 31 stable loci for Epimedin C (EC), one stable locus for total flavone content (TFC), 12 stable loci for leaf length (LL), and two stable loci for leaf area (LA). For flavonoid content and leaf size, the phenotypic variance explained for these loci varied between 4.00 and 16.80% and 14.95 and 17.34%, respectively. CONCLUSIONS: Forty-six stable QTLs for leaf size and flavonoid content traits were repeatedly detected over three years. The HDGM and stable QTLs are laying the basis for breeding and gene investigation in Epimedium and will contribute to accelerating the identification of desirable genotypes for Epimedium breeding.

Integrative analysis of physiology, biochemistry and transcriptome reveals the mechanism of leaf size formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Introduction: The leaf, the main product organ, is an essential factor in determining the Chinese cabbage growth, yield and quality. Methods: To explore the regulatory mechanism of leaf size development of Chinese cabbage, we investigated the leaf size difference between two high-generation inbred lines of Chinese cabbage, Y2 (large leaf) and Y7 (small leaf). Furtherly, the transcriptome and cis-acting elements analyses were conducted. Results and Discussion: According to our results, Y2 exhibited a higher growth rate than Y7 during the whole growth stage. In addition, the significant higher leaf number was observed in Y2 than in Y7. There was no significant difference in the number of epidermal cells and guard cells per square millimeter between Y2 and Y7 leaves. It indicated that cell numbers caused the difference in leaf size. The measurement of phytohormone content confirmed that GA1 and GA3 mainly play essential roles in the early stage of leaf growth, and IPA and ABA were in the whole leaf growth period in regulating the cell proliferation difference between Y2 and Y7. Transcriptome analysis revealed that cyclins BraA09g010980.3C (CYCB) and BraA10g027420.3C (CYCD) were mainly responsible for the leaf size difference between Y2 and Y7 Chinese cabbage. Further, we revealed that the transcription factors BraA09gMYB47 and BraA06gMYB88 played critical roles in the difference of leaf size between Y2 and Y7 through the regulation of cell proliferation. Conclusion: This observation not only offers essential insights into understanding the regulation mechanism of leaf development, also provides a promising breeding strategy to improve Chinese cabbage yield.

Canopy height, rather than neighborhood effects, shapes leaf herbivory in a tropical rainforest.

Factors shaping the interspecific variations in herbivory have puzzled ecologists for decades and several hypotheses have been proposed to explain interspecific variation in leaf herbivory. In a tropical rainforest in Yunnan Province, China, we collected 6732 leaves from 129 species with canopy heights ranging from 1.6 to 65.0 m above the ground. We tested the role of canopy height, the diversity, composition and structural heterogeneity of neighbors and leaf traits in shaping the interspecific variations in herbivory. Results show that leaf herbivory decreased with canopy height and specific leaf area (SLA) and increased with leaf size. However, neighboring species' diversity, composition, and structural heterogeneity showed no association with herbivory. Therefore, neither the visual apparency effect nor the associational resistance effect was detected in this hyperdiverse tropical rainforest. These findings highlight the importance of vertical structure in shaping herbivory patterns in natural communities.

GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum).

KEY MESSAGE: We proposed a working model of BR to promote leaf size through cell expansion. In the BR signaling pathway, GhBES1 affects cotton leaf size by binding to and activating the expression of the E-box element in the GhEXO2 promoter region. Brassinosteroid (BR) is an essential phytohormone that controls plant growth. However, the mechanisms of BR regulation of leaf size remain to be determined. Here, we found that the BR deficient cotton mutant pagoda1 (pag1) had a smaller leaf size than wild-type CRI24. The expression of EXORDIUM (GhEXO2) gene, was significantly downregulated in pag1. Silencing of BRI1-EMS-SUPPRESSOR 1 (GhBES1), inhibited leaf cell expansion and reduced leaf size. Overexpression of GhBES1.4 promoted leaf cell expansion and enlarged leaf size. Expression analysis showed GhEXO2 expression positively correlated with GhBES1 expression. In plants, altered expression of GhEXO2 promoted leaf cell expansion affecting leaf size. Furthermore, GhBES1.4 specifically binds to the E-box elements in the GhEXO2 promoter, inducing its expression. RNA-seq data revealed many down-regulated genes related to cell expansion in GhEXO2 silenced plants. In summary, we discovered a novel mechanism of BR regulation of leaf size through GhBES1 directly activating the expression of GhEXO2.

Ectophagous folivores do not profit from rich resources on phylogenetically isolated trees.

Resource use by consumers across patches is often proportional to the quantity or quality of the resource within these patches. In folivores, such proportional use of resources is likely to be more efficient when plants are spatially proximate, such as trees forming a forest canopy. However, resources provided by forest-trees are often not used proportionally. We hypothesised that proportional use of resources is reduced when host trees are isolated among phylogenetically distant neighbours that mask olfactory and visual search cues, and reduce folivore movement between trees. Such phylogenetically distant neighbourhoods might sort out species that are specialists, poor dispersers, or have poor access to information about leaf quality. We studied individual oaks, their leaf size and quality, their folivory and abundance of folivores (mostly Lepidopteran ectophages, gallers and miners), and parasitism of folivores. We found that leaf consumption by ectophages hardly increased with increasing leaf size when host trees were phylogenetically isolated. We found a similar effect on host use by parasitoids in 1 year. In contrast, we found no consistent effects in other folivore guilds. Relative abundances of specialists and species with wingless females declined with phylogenetic isolation. However, resource use within each of these groups was inconsistently affected by phylogenetic isolation. We suggest that phylogenetic isolation prevents ectophages from effectively choosing trees with abundant resources, and also sorts out species likely to recruit in situ on their host tree. Trees in phylogenetically distant neighbourhoods may be selected for larger leaves and greater reliance on induced defences.

Differences in Leaf Morphology and Related Gene Expression between Diploid and Tetraploid Birch (Betula pendula).

Plant polyploidization changes its leaf morphology and leaf development patterns. Understanding changes in leaf morphology and development patterns is a prerequisite and key to studying leaf development in polyploid plants. In this study, we quantified and analyzed the differences in leaf morphology, leaf growth polarity, and leaf size between diploid and tetraploid birches (Betula pendula subsp. pendula), and preliminarily investigated genes involved in leaf growth and development in birch. The results showed significant changes in leaf morphology in tetraploid birches, especially the basal part of the leaf. In addition, the proximal growth rate of tetraploid leaves was altered. The changed proximal growth rate did not affect the growth polarity pattern of tetraploid leaves. The leaf area of tetraploid was significantly larger than that of diploid birch. The difference in leaf size was mainly due to differences in their growth rates in the middle and late stages of leaf development. Increased cell expansion capacity was the major reason for the enormous leaves of tetraploid birch; however, cell proliferation did not contribute to the larger tetraploid leaf. The gene expression of ATHB12 was associated with cell size and leaf area, and may be a critical gene affecting the leaf size in diploid and tetraploid birches. The results will provide valuable insights into plant polyploid leaf development and a theoretical basis for later investigations into the molecular mechanisms underlying the gigantism of tetraploid birch leaves.

Multivariate analysis of eshnan (Seidlitzia rosmarinus Boiss.) based on morphological characterizations.

Seidlitzia rosmarinus Boiss. has been identified as one of the potential species that could be used for rehabilitating degraded desert rangelands and salt-affected soils due to its high salinity resistance and soil-stabilizing ability. Morphological variation of 144 accessions of this species from 14 regions of the Isfahan province, Iran was investigated. The accessions studied were significantly different in terms of the traits recorded. Three forms of plant growth habit were observed, including spreading bush, erect bush, and shrub. The range of leaf dimensions was as follows: terminal leaf length: 1.57-7.22 mm, terminal leaf width: 0.91-3.34 mm, basal leaf length: 11.84-45.27 mm, and basal leaf width: 1.32-4.18 mm. Fruit diameter (with wings) ranged from 0.19 to 12.91 mm, and 100-fruits dry weight varied between 0.11 and 0.76 g. A dendrogram created using Euclidean distances and the Ward's method revealed two main clusters. The obtained data revealed the morphological diversity within the studied populations. The reason for such a high diversity can be explained by a low probability of gene flow among the studied accessions. This is the first report on the application of morphological characteristics in the evaluation of the phenotypic variation of S. rosmarinus. This study presented a high phenotypic diversity of S. rosmarinus germplasm that could provide useful information for conservation and selection of cross-parents in breeding.

Genome-Wide Identification, Characterization, and Transcriptomic Analysis of the Cyclin Gene Family in Brassica rapa.

Cyclins are involved in cell division and proliferation by activating enzymes required for the cell cycle progression. Our genome-wide analysis identified 76 cyclin genes in Brassica rapa, which were divided into nine different types (A-, B-, C-, D-, H-, L-, P-, T-, and SDS-type). Cyclin genes were unevenly scattered on all chromosomes, with a maximum of 10 on A08 and a minimum of 2 on A04. The gene structure and conserved motif analysis showed that the cyclins which belonged to the same type or subgroup have a comparable intron/exon pattern or motif. A total of 14 collinear gene pairs suggested that the B. rapa cyclin genes experienced a mass of segmental duplication. The Ka/Ks analysis revealed that the Brcyclin gene family has undergone an extensive purifying pressure. By analyzing the cis-elements in the promoters, we identified 11 cis-elements and five of them are related to the hormone response. We observed 48 potential miRNAs targeting 44 Brcyclin genes, which highlighted the involvement of miRNAs in the regulation of cyclin genes. An association analysis between the leaf size and SNPs in mutants and a transcriptome analysis of two Chinese cabbage-cabbage translocation lines also showed that the Brcyclin gene family was involved in the development of the leaves. The functional characterization of the B. rapa cyclin gene family will provide the foundation for future physiological and genetic studies in the regulation of leaf growth.