Epidermal Patterning Research Articles

StEPF2 and StEPFL9 Play Opposing Roles in Regulating Stomatal Development and Drought Tolerance in Potato (Solanum tuberosum L.)

Stomata are essential for photosynthesis and water-use efficiency in plants. When expressed in transgenic Arabidopsis thaliana plants, the potato (Solanum tuberosum) proteins EPIDERMAL PATTERNING FACTOR 2 (StEPF2) and StEPF-LIKE9 (StEPFL9) play antagonistic roles in regulating stomatal density. Little is known, however, about how these proteins regulate stomatal development, growth, and response to water deficit in potato. Transgenic potato plants overexpressing StEPF2 (E2 plants) or StEPFL9 (ST plants) were generated, and RT-PCR and Western blot analyses were used to select two lines overexpressing each gene. E2 plants showed reduced stomatal density, whereas ST plants produced excessive stomata. Under well-watered conditions, ST plants displayed vigorous growth with improved leaf gas exchange and also showed increased biomass/yields compared with non-transgenic and E2 plants. E2 plants maintained lower H2O2 content and higher levels of stomatal conductance and photosynthetic capacity than non-transgenic and ST plants, which resulted in higher water-use efficiency and biomass/yields during water restriction. These results suggest that StEPF2 and StEPFL9 functioned in pathways regulating stomatal development. These genes are thus promising candidates for use in future breeding programs aimed at increasing potato water-use efficiency and yield under climate change scenarios.

Negative feedback regulation of GLABRA1 contributes to epidermal cell patterning in the Arabidopsis root

GLABRA1 (GL1), which encodes an R2R3 MYB transcription factor, is a key regulator of trichome patterning in the aerial organs of Arabidopsis thaliana. Although it has been generally assumed that GL1 functions exclusively in shoots and is not expressed in roots, reverse transcription polymerase chain reaction (RT-PCR) analysis has revealed that GL1 is indeed expressed in roots. To investigate whether GL1 plays a role in root epidermal patterning, we analyzed the effects of gl1 mutations in sensitized genetic backgrounds. Our findings show that gl1 mutants enhance the root epidermal phenotype of a weak allele of the werewolf (wer) mutant and suppress the phenotype of the caprice (cpc) mutant. We also demonstrate that the GL1 promoter is active in N-position epidermal cells, and that the GFP-GL1 fusion protein is predominantly localized in the nucleus of N-position cells. Furthermore, we provide evidence that GL1 expression is positively regulated by WER, GLABRA3, ENHANCER OF GLABRA3, and TRANSPARENT TESTA GLABRA1, while negatively regulated by CPC, TRIPTYCHON, and GLABRA2 (GL2). Notably, GL2, which is positively regulated by GL1, moderately represses GL1 expression, and both GL1 and GL2 are positively regulated by WER in N-position cells. These findings suggest that a negative feedback regulation of GL1 expression via GL2 contributes to the fine-tuning of non-hair cell fate determination in Arabidopsis root epidermis.

Comparative Genomics Analysis of the Populus Epidermal Pattern Factor (EPF) Family Revealed Their Regulatory Effects in Populus euphratica Stomatal Development.

Drought stress seriously threatens plant growth. The improvement of plant water use efficiency (WUE) and drought tolerance through stomatal regulation is an effective strategy for coping with water shortages. Epidermal patterning factor (EPF)/EPF-like (EPFL) family proteins regulate stomatal formation and development in plants and thus contribute to plant stress adaptation. Here, our analysis revealed the presence of 14 PeEPF members in the Populus euphratica genome, which exhibited a relatively conserved gene structure with 1-3 introns. Subcellular localisation prediction revealed that 9 PeEPF members were distributed in the chloroplasts of P. euphratica, and 5 were located extracellularly. Phylogenetic analysis indicated that PeEPFs can be divided into three clades, with genes within the same clade revealing a relatively conserved structure. Furthermore, we observed the evolutionary conservation of PeEPFs and AtEPF/EPFLs in certain domains, which suggests their conserved function. The analysis of cis-acting elements suggested the possible involvement of PeEPFs in plant response to multiple hormones. Transcriptomic analysis revealed considerable changes in the expression level of PeEPFs during treatment with polyethylene glycol and abscisic acid. The overexpression of PeEPF2 resulted in low stomatal density in transgenetic lines. These findings provide a basis for gaining insights into the function of PeEPFs in response to abiotic stress.

Dual roles of the MPK3 and MPK6 mitogen-activated protein kinases in regulating Arabidopsis stomatal development.

An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)-MKK4/MKK5-MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4DD or MKK5DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development.

Decoding stomatal characteristics regulating water use efficiency at leaf and plant scales in rice genotypes.

Stomatal traits in rice genotypes affect water use efficiency. Low-frequency small-size stomata correlate with whole plant efficiency, while low-frequency large-size stomata show intrinsic efficiency and responsiveness to vapour pressure deficit. Leaf surface and the patterning of the epidermal layer play a vital role in determining plant growth. While the surface helps in determining radiation interception, epidermal pattern of stomatal factors strongly regulate gas exchange and water use efficiency (WUE). This study focuses on identifying distinct stomatal traits among rice genotypes to comprehend their influence on WUE. Stomatal frequency ranged from 353 to 687 per mm2 and the size varied between 128.31 and 339.01 μm2 among 150 rice germplasm with significant variability in abaxial and adaxial surfaces. The cumulative water transpired and WUE determined at the outdoor phenomics platform, over the entire crop growth period as well as during specific hours of a 24h-day did not correlate with stomatal frequency nor size. However, genotypes with low-frequency and large-size stomata recorded higher intrinsic water use efficiency (67.04μmol CO2 mol-1 H2O) and showed a quicker response to varying vapour pressure deficit that diurnally ranged between 0.03 and 2.17kPa. The study demonstrated the role of stomatal factors in determining physiological subcomponents of WUE both at single leaf and whole plant levels. Differential expression patterns of stomatal regulatory genes among the contrasting groups explained variations in the epidermal patterning. Increased expression of ERECTA, TMM and YODA genes appear to contribute to decreased stomatal frequency in low stomatal frequency genotypes. These findings underscore the significance of stomatal traits in breeding programs and strongly support the importance of these genes that govern variability in stomatal architecture in future crop improvement programs.

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw). However, the quantitative relationship between reduced SD, gsw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r2=0.60-0.98), but this response is damped relative to the change in anatomy.

Reducing stomatal density by expression of a synthetic Epidermal Patterning Factor increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop.

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (gs) has improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to limit AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, led to reduced stomatal densities, reduced gs, reduced plant water use and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to AN. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes.

Genome-wide identification and phylogenetic and expression pattern analyses of EPF/EPFL family genes in the Rye (Secale cereale L.)

Rye (Secale cereale L.) is one of the major cereal crop species in the Triticeae family and is known to be most tolerant to diverse abiotic stresses, such as cold, heat, osmotic, and salt stress. The EPIDERMAL PATTERNING FACTOR (EPF) and EPF-LIKE (EPFL) families of small secreted peptides act to regulate many aspects of plant growth and development; however, their functions are not widely characterized in rye. In this study, we identified 12 ScEPF/EPFL genes, which can be divided into six groups and are evenly distributed on six rye chromosomes. Further examination of the gene structure and protein conservation motifs of EPF/EPFL family members demonstrated the high conservation of the ScEPF/EPFL sequence. Interactions between ScEPF/EPFL proteins and promoters containing hormone- and stress-responsive cis-acting elements suggest that the regulation of ScEPF/EPFL expression is complex. Expression profiling analyses revealed that ScEPF/EPFL genes exhibited tissue-specific expression patterns. Notably, ScEPFL1,ScEPFL7, ScEPFL9, and ScEPFL10 displayed significantly higher expression levels in spikelets compared to other tissues. Moreover, fluorescence quantification experiments demonstrated that these genes exhibited distinct expression patterns in response to various stress conditions, suggesting that each gene plays a unique role in stress signaling pathways. Our research findings provide a solid basis for further investigation into the functions of ScEPF/EPFLs. Furthermore, these genes can serve as potential candidates for breeding stress-resistant rye varieties and improving production yields.

Genome-wide identification and expression analysis of EPF/EPFL gene family in Populus trichocarpa.

The Epidermal Patterning Factor/EPF-like (EPF/EPFL) family encodes a specific type of secreted protein in plants and plays an important role in plant growth and development, especially in the process of morphogenesis. To investigate the characteristics of EPF/EPFL gene family members and their regulatory functions in stomatal development of Populus trichocarpa, a total of 15 EPF/EPFL family genes were identified. Then the gene structure, chromosome location, phylogenetic relationship, protein conserved domain and gene expression profile were analyzed. According to phylogenetic analysis, PtEPF/EPFL can be classified into four groups. The gene structure and protein conservation motifs within the EPF family indicate the high conservation of the PtEPF/EPFL sequence. The promoter region of PtEPF/EPFL was found to contain cis-elements in response to stress and plant hormones. In addition, RT-qPCR results indicated that the PtEPF/EPFL have a differentially expressed in different tissues. Under drought stress treatment, a substantial upregulation was observed in the majority of PtEPF/EPFL members, suggesting their potential involvement in drought response. These results provide a theoretical basis for future exploration of the characteristics and functions of more PtEPF/EPFL genes.

A 1-bp deletion in the MC04g1399 is highly associated with failure to produce fruit wart in bitter gourd (Momordica charantia L.)

Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd. The molecular genetic mechanisms underlying fruit wart formation in bitter gourd are largely unknown. In this study, genetic analysis based on four generations showed that fruit wart formation in bitter gourd was controlled by a single dominant locus named as Fwa. The Fwa locus was initially mapped into a 4.82 Mb region on pseudochromosome 4 by BSA-seq analysis and subsequently narrowed down to a 286.30 kb region by linkage analysis. A large F2 population consisting of 2 360 individuals was used to screen recombinants, and the Fwa locus was finally fine mapped into a 22.70 kb region harboring four protein-coding genes through recombination analysis. MC04g1399, encoding an epidermal patterning factor 2-like protein, was proposed as the best candidate gene for Fwa via sequence variation and expression analysis. In addition, a 1-bp insertion and deletion (InDel) variation within MC04g1399 was converted to a cleaved amplified polymorphic sequence (CAPS) marker that could precisely distinguish between the warty and non-warty types with an accuracy rate of 100 % among a wide panel of 126 bitter gourd germplasm resources. Our results not only provide a scientific basis for deciphering the molecular mechanisms underlying fruit wart formation but also provide a powerful tool for efficient genetic improvement of fruit wart via marker-assisted selection.

Genome-Wide Identification and Analysis of the EPF Gene Family in Sorghum bicolor (L.) Moench.

The EPIDERMAL PATTERNING FACTOR (EPF) plays a crucial role in plant response to abiotic stress. While the EPF has been extensively studied in model plants such as Arabidopsis thaliana, there is a lack of research on identifying EPF genes in the whole sorghum genome and its response to drought stress. In this study, we employed bioinformatics tools to identify 12 EPF members in sorghum. Phylogenetic tree analysis revealed that SbEPFs can be categorized into four branches. Further examination of the gene structure and protein conservation motifs of EPF family members demonstrated the high conservation of the SbEPF sequence. The promoter region of SbEPFs was found to encompass cis-elements responsive to stress and plant hormones. Moreover, real-time fluorescence quantitative results indicated that the SbEPFs have a tissue-specific expression. Under drought stress treatment, most SbEPF members were significantly up-regulated, indicating their potential role in drought response. Our research findings establish a foundation for investigating the function of SbEPFs and offer candidate genes for stress-resistant breeding and enhanced production in sorghum.

Differential gene expression during floral transition in pineapple.

Pineapple (Ananas comosus var. comosus) and ornamental bromeliads are commercially induced to flower by treatment with ethylene or its analogs. The apex is transformed from a vegetative to a floral meristem and shows morphological changes in 8 to 10days, with flowers developing 8 to 10weeks later. During eight sampling stages ranging from 6h to 8days after treatment, 7961 genes were found to exhibit differential expression (DE) after the application of ethylene. In the first 3days after treatment, there was little change in ethylene synthesis or in the early stages of the ethylene response. Subsequently, three ethylene response transcription factors (ERTF) were up-regulated and the potential gene targets were predicted to be the positive flowering regulator CONSTANS-like 3 (CO), a WUSCHEL gene, two APETALA1/FRUITFULL (AP1/FUL) genes, an epidermal patterning gene, and a jasmonic acid synthesis gene. We confirm that pineapple has lost the flowering repressor FLOWERING LOCUS C. At the initial stages, the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was not significantly involved in this transition. Another WUSCHEL gene and a PHD homeobox transcription factor, though not apparent direct targets of ERTF, were up-regulated within a day of treatment, their predicted targets being the up-regulated CO, auxin response factors, SQUAMOSA, and histone H3 genes with suppression of abscisic acid response genes. The FLOWERING LOCUS T (FT), TERMINAL FLOWER (TFL), AGAMOUS-like APETELAR (AP2), and SEPETALA (SEP) increased rapidly within 2 to 3days after ethylene treatment. Two FT genes were up-regulated at the apex and not at the leaf bases after treatment, suggesting that transport did not occur. These results indicated that the ethylene response in pineapple and possibly most bromeliads act directly to promote the vegetative to flower transition via APETALA1/FRUITFULL (AP1/FUL) and its interaction with SPL, FT, TFL, SEP, and AP2. A model based on AP2/ERTF DE and predicted DE target genes was developed to give focus to future research. The identified candidate genes are potential targets for genetic manipulation to determine their molecular role in flower transition.

Allelic Diversity of OsEPF2 Governing Stomatal Regulation in Rice

Rice production has been significantly hindered by frequent drought events in recent times, directly impacting food security and agricultural sustainability. In the current study, a set of 100 diverse rice accessions from 3K RG panel were evaluated for Epidermal patterning factor 2 (OsEPF2) loci which governs the stomatal density on the matured leaves by preventing excessive stomatal development. This plays an important role in drought tolerance in plants. Hence, this gene was selected for analyzing haplotype variation which revealed 27 SNPs and 59 INDELs. Seven significant non-synonymous SNPs, grouped the one hundred accessions into three haplotype groups with the allelic combination CGATGCG for H1, TTGGAAA for H2 and CGGGGCG for H3. This shows the allelic diversity of the population that can be potentially employed in breeding of rice varieties with improved drought tolerance and water use efficiency by assembling superior haplotype.

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Bna.EPF2 Enhances Drought Tolerance by Regulating Stomatal Development and Stomatal Size in Brassica napus

Drought stress severely affects global plant growth and production. The enhancement of plant water-use efficiency (WUE) and drought tolerance by the manipulation of the stomata is an effective strategy to deal with water shortage. However, increasing the WUE and drought tolerance by manipulation on the stomata has rarely been tested in Brassica napus. Here, we isolated Bna.EPF2, an epidermal patterning factor (EPF) in Brassica napus (ecotype Westar), and identified its role in drought performance. Bna.EPF2 overexpression lines had a reduction average of 19.02% in abaxial stomatal density and smaller stomatal pore size, leading to approximately 25% lower transpiration, which finally resulted in greater instantaneous WUE and enhanced drought tolerance. Interestingly, the reduction in stomatal density did not affect the CO2 assimilation or yield-related agronomic traits in Bna.EPF2 overexpression plants. Together with the complementation of Bna.EPF2 significantly decreasing the stomatal density of Arabidopsis epf2, and Bna.EPF2 being expressed in mature guard cells, these results suggest that Bna.EPF2 not only functions in stomatal density development, but also in stomatal dimension in Brassicas. Taken together, our results suggest that Bna.EPF2 improves WUE and drought tolerance by the regulation of stomatal density and stomatal size in Brassica without growth and yield penalty, and provide insight into the manipulation of this gene in the breeding of drought tolerant plants with increased production under water deficit conditions.

A conserved gene regulatory network controls root epidermal cell patterning in superrosid species.

In superrosid species, root epidermal cells differentiate into root hair cells and nonhair cells. In some superrosids, the root hair cells and nonhair cells are distributed randomly (Type I pattern), and in others, they are arranged in a position-dependent manner (Type III pattern). The model plant Arabidopsis (Arabidopsis thaliana) adopts the Type III pattern, and the gene regulatory network (GRN) that controls this pattern has been defined. However, it is unclear whether the Type III pattern in other species is controlled by a similar GRN as in Arabidopsis, and it is not known how the different patterns evolved. In this study, we analyzed superrosid species Rhodiola rosea, Boehmeria nivea, and Cucumis sativus for their root epidermal cell patterns. Combining phylogenetics, transcriptomics, and cross-species complementation, we analyzed homologs of the Arabidopsis patterning genes from these species. We identified R. rosea and B. nivea as Type III species and C. sativus as Type I species. We discovered substantial similarities in structure, expression, and function of Arabidopsis patterning gene homologs in R. rosea and B. nivea, and major changes in C. sativus. We propose that in superrosids, diverse Type III species inherited the patterning GRN from a common ancestor, whereas Type I species arose by mutations in multiple lineages.

Paralog editing tunes rice stomatal density to maintain photosynthesis and improve drought tolerance.

Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. We explored stomatal developmental genetics as a mechanism for improving drought resilience in rice while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of the positive regulator of stomatal development STOMAGEN and its paralog EPIDERMAL PATTERNING FACTOR-LIKE10 (EPFL10) yielded lines with ∼25% and 80% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal density were able to conserve water to similar extents as stomagen lines but did not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal density achieved by editing EPFL10 present a climate-adaptive approach for safeguarding yield in rice. Editing the paralog of STOMAGEN in other species may provide a means for tuning stomatal density in agriculturally important crops beyond rice.

Optimization of rice panicle architecture by specifically suppressing ligand–receptor pairs

Rice panicle architecture determines the grain number per panicle and therefore impacts grain yield. The OsER1–OsMKKK10–OsMKK4–OsMPK6 pathway shapes panicle architecture by regulating cytokinin metabolism. However, the specific upstream ligands perceived by the OsER1 receptor are unknown. Here, we report that the EPIDERMAL PATTERNING FACTOR (EPF)/EPF-LIKE (EPFL) small secreted peptide family members OsEPFL6, OsEPFL7, OsEPFL8, and OsEPFL9 synergistically contribute to rice panicle morphogenesis by recognizing the OsER1 receptor and activating the mitogen-activated protein kinase cascade. Notably, OsEPFL6, OsEPFL7, OsEPFL8, and OsEPFL9 negatively regulate spikelet number per panicle, but OsEPFL8 also controls rice spikelet fertility. A osepfl6 osepfl7 osepfl9 triple mutant had significantly enhanced grain yield without affecting spikelet fertility, suggesting that specifically suppressing the OsEPFL6–OsER1, OsEPFL7–OsER1, and OsEPFL9–OsER1 ligand–receptor pairs can optimize rice panicle architecture. These findings provide a framework for fundamental understanding of the role of ligand–receptor signaling in rice panicle development and demonstrate a potential method to overcome the trade-off between spikelet number and fertility.

An EPFL peptide signaling pathway promotes stamen elongation via enhancing filament cell proliferation to ensure successful self-pollination in Arabidopsis thaliana.

Proper stamen filament elongation is essential for plant self-pollination and reproduction. Several phytohormones such as jasmonate and gibberellin play important roles in controlling filament elongation, but other endogenous signals involved in this developmental process remain unknown. We report here that three EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family peptides, EPFL4, EPFL5 and EPFL6, act redundantly to promote stamen filament elongation via enhancing filament cell proliferation in Arabidopsis thaliana. Knockout of EPFL4-6 genes led to shortened filaments due to defective filament cell proliferation, resulting in pollination failure and male sterility. Further genetic and biochemical analyses indicated that the ERECTA family and the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family RLKs form receptor complexes to perceive EPFL4-6 peptides and promote filament cell proliferation. Moreover, based on both loss- and gain-of-function genetic analyses, the mitogen-activated protein kinase cascade MKK4/MKK5-MPK6 was shown to function downstream of EPFL4-6 to positively regulate cell proliferation in stamen filaments. Together, this study reveals that an EPFL peptide signaling pathway composed of the EPFL4-6 peptide ligands, the ERECTA-SERK receptor complexes and the downstream MKK4/MKK5-MPK6 cascade promotes stamen filament elongation via enhancing filament cell proliferation to ensure successful self-pollination and normal fertility in Arabidopsis.