EPIDERMAL PATTERNING FACTOR Research Articles

Duplicated antagonistic EPF peptides optimize grass stomatal initiation.

Peptide signaling has emerged as a key component of plant growth and development, including stomatal patterning, which is crucial for plant productivity and survival. Although exciting progress has been made in understanding EPIDERMAL PATTERNING FACTOR (EPF) signaling in Arabidopsis, the mechanisms by which EPF peptides control different stomatal patterns and morphologies in grasses are poorly understood. Here, by examining expression patterns, overexpression transgenics and cross-species complementation, the antagonistic stomatal ligands orthologous to Arabidopsis AtEPF2 and AtSTOMAGEN/AtEPFL9 peptides were identified in Triticum aestivum (wheat) and the grass model organism Brachypodium distachyon. Application of bioactive BdEPF2 peptides inhibited stomatal initiation, but not the progression or differentiation of stomatal precursors in Brachypodium. Additionally, the inhibitory roles of these EPF peptides during grass stomatal development were suppressed by the contrasting positive action of the BdSTOMAGEN peptide in a dose-dependent manner. These results not only demonstrate how conserved EPF peptides that control different stomatal patterns exist in nature, but also suggest new strategies to improve crop yield through the use of plant-derived antagonistic peptides that optimize stomatal density on the plant epidermis.

Martin McAinsh.

Martin McAinsh.

Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling.

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

A single-cell analysis of the Arabidopsis vegetative shoot apex.

The shoot apical meristem allows for reiterative formation of new aerial structures throughout the life cycle of a plant. We use single-cell RNA sequencing to define the cellular taxonomy of the Arabidopsis vegetative shoot apex at the transcriptome level. We find that the shoot apex is composed of highly heterogeneous cells, which can be partitioned into 7 broad populations with 23 transcriptionally distinct cell clusters. We delineate cell-cycle continuums and developmental trajectories of epidermal cells, vascular tissue, and leaf mesophyll cells and infer transcription factors and gene expression signatures associated with cell fate decisions. Integrative analysis of shoot and root apical cell populations further reveals common and distinct features of epidermal and vascular tissues. Our results, thus, offer a valuable resource for investigating the basic principles underlying cell division and differentiation in plants at single-cell resolution.

ERECTA family signaling constrains CLAVATA3 and WUSCHEL to the center of the shoot apical meristem.

The shoot apical meristem (SAM) is a reservoir of stem cells that gives rise to all post-embryonic above-ground plant organs. The size of the SAM remains stable over time owing to a precise balance of stem cell replenishment versus cell incorporation into organ primordia. The WUSCHEL (WUS)/CLAVATA (CLV) negative feedback loop is central to SAM size regulation. Its correct function depends on accurate spatial expression of WUS and CLV3 A signaling pathway, consisting of ERECTA family (ERf) receptors and EPIDERMAL PATTERNING FACTOR LIKE (EPFL) ligands, restricts SAM width and promotes leaf initiation. Although ERf receptors are expressed throughout the SAM, EPFL ligands are expressed in its periphery. Our genetic analysis of Arabidopsis demonstrated that ERfs and CLV3 synergistically regulate the size of the SAM, and wus is epistatic to ERf genes. Furthermore, activation of ERf signaling with exogenous EPFLs resulted in a rapid decrease of CLV3 and WUS expression. ERf-EPFL signaling inhibits expression of WUS and CLV3 in the periphery of the SAM, confining them to the center. These findings establish the molecular mechanism for stem cell positioning along the radial axis.

SPEECHLESS and MUTE Mediate Feedback Regulation of Signal Transduction during Stomatal Development.

Stomatal density, spacing, and patterning greatly influence the efficiency of gas exchange, photosynthesis, and water economy. They are regulated by a complex of extracellular and intracellular factors through the signaling pathways. After binding the extracellular epidermal patterning factor 1 (EPF1) and 2 (EPF2) as ligands, the receptor-ligand complexes activate by phosphorylation through the MAP-kinase cascades, regulating basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (SPCH), MUTE, and FAMA. In this review, we summarize the molecular mechanisms and signal transduction pathways running within the transition of the protodermal cell into a pair of guard cells with a space (aperture) between them, called a stoma, comprising asymmetric and symmetric cell divisions and draw several functional models. The feedback mechanisms involving the bHLH factors SPCH and MUTE are not fully recognized yet. We show the feedback mechanisms driven by SPCH and MUTE in the regulation of EPF2 and the ERECTA family. Intersections of the molecular mechanisms for fate determination of stomatal lineage cells with the role of core cell cycle-related genes and stabilization of SPCH and MUTE are also reported.

Higher Stomatal Density Improves Photosynthetic Induction and Biomass Production in Arabidopsis Under Fluctuating Light.

Stomatal density (SD) is closely associated with photosynthetic and growth characteristics in plants. In the field, light intensity can fluctuate drastically within a day. The objective of the present study is to examine how higher SD affects stomatal conductance (gs) and CO2 assimilation rate (A) dynamics, biomass production and water use under fluctuating light. Here, we compared the photosynthetic and growth characteristics under constant and fluctuating light among three lines of Arabidopsis thaliana (L.): the wild type (WT), STOMAGEN/EPFL9-overexpressing line (ST-OX), and EPIDERMAL PATTERNING FACTOR 1 knockout line (epf1). ST-OX and epf1 showed 268.1 and 46.5% higher SD than WT (p < 0.05). Guard cell length of ST-OX was 10.0% lower than that of WT (p < 0.01). There were no significant variations in gas exchange parameters at steady state between WT and ST-OX or epf1, although these parameters tended to be higher in ST-OX and epf1 than WT. On the other hand, ST-OX and epf1 showed faster A induction than WT after step increase in light owing to the higher gs under initial dark condition. In addition, ST-OX and epf1 showed initially faster gs induction and, at the later phase, slower gs induction. Cumulative CO2 assimilation in ST-OX and epf1 was 57.6 and 78.8% higher than WT attributable to faster A induction with reduction of water use efficiency (WUE). epf1 yielded 25.6% higher biomass than WT under fluctuating light (p < 0.01). In the present study, higher SD resulted in faster photosynthetic induction owing to the higher initial gs. epf1, with a moderate increase in SD, achieved greater biomass production than WT under fluctuating light. These results suggest that higher SD can be beneficial to improve biomass production in plants under fluctuating light conditions.

The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate.

Receptor endocytosis is important for signal activation, transduction, and deactivation. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting plant stomatal differentiation, undergoes dynamic subcellular behaviors in response to different EPIDERMAL PATTERNING FACTOR (EPF) peptides. Activation of ERL1 by EPF1 induces rapid ERL1 internalization via multivesicular bodies/late endosomes to vacuolar degradation, whereas ERL1 constitutively internalizes in the absence of EPF1. The co-receptor, TOO MANY MOUTHS is essential for ERL1 internalization induced by EPF1 but not by EPFL6. The peptide antagonist, Stomagen, triggers retention of ERL1 in the endoplasmic reticulum, likely coupled with reduced endocytosis. In contrast, the dominant-negative ERL1 remained dysfunctional in ligand-induced subcellular trafficking. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

Influence of red light on the expression of genes on stomatal formation in maize seedlings

Red light significantly affects the expression of plant photoreceptor genes and influences stomatal development through crosstalk of the constitutive photomorphogenic 1–cryptochrome–phytochrome signaling pathway. When blue light was replaced with red light, the expression levels of ZmCry1, ZmPhyB1, ZmEPF2, and ZmEPFL9 were enhanced, whereas that of ZmCOP1 was restricted. Moreover, the expression levels of ZmSPCH and ZmMUTE were also enhanced, but they were generally lower than those under white light. Consequently, stomatal formation, which was determined by net photosynthesis, stomatal conductance, intercellular CO2 concentration, and transpiration rate, was inhibited through decreased stomatal index and stomatal density. We conclude that red light positively regulates EPFL9 in the intercellular signaling but reduces the positive regulation of blue light on COP1 and epidermal patterning factor 2 in the intracellular and intercellular signaling; therefore, though red light promotes the gene’s function on stomatal development of seedling maize, blue light maybe dominant to red light in seedling stage.

Overexpression of the potato StEPF2 gene confers enhanced drought tolerance in Arabidopsis

Epidermal patterning factor 2 (EPF2) is a negative regulator of stomatal development, and is essential for plant growth, development, and environmental stress responses. However, the role of EPF2 in potato (Solanum tuberosum) has not been investigated to date. Here, we cloned and characterized the potato EPF2-like gene (StEPF2). StEPF2 is predominantly intercellular space localized and its transcripts were rhythmically expressed, and showed the highest expression in apical unexpanded leaves. Expression of StEPF2 was markedly down-regulated in response to abscisic acid and sodium chloride treatments; however, upon the application of polyethylene glycol, the expression of StEPF2 peaked at 4 h and then decreased gradually. Overexpression of StEPF2 in Arabidopsis (OE) substantially reduced stomatal density and photosynthetic rate, but had little effects on plant growth. Under drought stress, OE lines maintained higher photosynthetic rates, photosystem II efficiency, and instantaneous water use efficiency than wild-type (WT) plants. Moreover, OE lines showed less water loss and hydrogen peroxide accumulation in detached leaves compared with WT plants. Thus, our results suggest that StEPF2 acts as a negative regulator of stomatal development in potato, indicating that the role of EPF2 is conserved across plant species. Overall, StEPF2 represents an important target for the development of drought-tolerant potato cultivars via genetic engineering.

The barley miR393 has multiple roles in regulation of seedling growth, stomatal density, and drought stress tolerance

microRNA393 (miR393) and its target module have been implicated as comprising a conserved mechanism to regulate developmental processes and plant growth in response to environmental signals through the auxin signaling pathway. Our previous work identified miR393 and its two targets in barley. In this study, we further investigated the expression pattern of miR393 and its biological functions in seedling growth and drought tolerance. We showed that the miR393 overexpressing line (OE) exhibited increased stomatal density with decreased guard cell length, while the miR393 knockdown line (MIM) displayed the opposite phenotype, which might be due to the effects of miR393 on AUXIN RESPONSE FACTOR5 (ARF5) and three stomatal development-related genes, such as EPIDERMAL PATTERNING FACTOR1 (EPF1), SPEECHLESS (SPCH), and MUTE. In addition, the MIM line conferred enhanced drought tolerance, with alleviated leaf chlorosis and lipid peroxidation after 22 days drought treatment. In contrast, the OE line was more sensitive to drought stress and accumulated more malondialdehyde and hydrogen peroxide than the wild type. Furthermore, polyethylene glycol (PEG) treatment-induced abscisic acid (ABA) accumulation in leaves was suppressed in the OE line, indicating that miR393 might regulate drought stress response and tolerance through its interaction with ABA biosynthesis. Overall, these data suggest that miR393 might be a potential target for manipulation of stomatal density and improvement of drought tolerance in barley.

Ectopic expression of secretory peptide PdEPF3 in Arabidopsis confers drought tolerance with reduced stomatal density

Drought is one of the most prevalent environmental stresses that affect plant growth and development. Improvement in drought tolerance is associated with reduced stomatal density and higher water use efficiency (WUE). In this study, an epidermal patterning factor (EPF), PdEPF3, from a fast-growing poplar clone, NE-19 [&lt;em&gt;Populus nigra&lt;/em&gt; × (&lt;em&gt;Populus deltoides&lt;/em&gt; × &lt;em&gt;Populus nigra&lt;/em&gt;)], was characterized. Quantitative reverse transcription polymerase chain reaction showed that the transcription of &lt;em&gt;PdEPF3&lt;/em&gt; was induced by drought. We further found that the transgenic &lt;em&gt;Arabidopsis&lt;/em&gt; overexpressing &lt;em&gt;PdEPF3&lt;/em&gt; had an earlier seedling germination and longer primary roots under osmotic stress treatments, compared with the WT and mutant &lt;em&gt;epf1-1&lt;/em&gt;. In addition, ectopic overexpression of &lt;em&gt;PdEPF3&lt;/em&gt; in &lt;em&gt;Arabidopsis&lt;/em&gt; plants was able to enhance drought tolerance. This observation was associated with the reduced stomatal density of transgenic plants, which would limit transpiration and reduce water loss, consequently improving the WUE of plants. Interestingly, the reduction of stomatal density in transgenic plants overexpressing &lt;em&gt;PdEPF3&lt;/em&gt; did not affect their photosynthetic capacity. These results indicate that &lt;em&gt;PdEPF3&lt;/em&gt; could be used in transgenic breeding to enhance plant drought tolerance.

The effect of light quality on the expression of stomatal development genes of maize

Light-induced stomatal development is mediated through a crosstalk between cryptochrome (CRY), phytochrome (phy), and constitutive photomorphogenic 1 (COP1)-signaling systems. In maize, crosstalk is a complicated phenomenon that is accomplished by COP1-signaling pathway. Epidermal patterning factor 2 (EPF2) and epidermal patterning factor-like 9 (EPFL9)-regulated path signals rely on CRY and phy under blue and red light, respectively. Our experimental results showed that, in maize, ZmCOP1 expressed downstream CRY under monochromatic blue light. Blue light, which induced ZmEPF2 expression, was restricted, thereby increasing stomatal formation through the increase in stomatal index and density. Moreover, blue light reduced the expression of ZmSPCH and ZmMUTE. By contrast, red light promoted the expression of ZmEPF2 and ZmEPFL9, and mainly acted as a negative signal for regulation. As a consequence, the activator-inhibitor system of light was induced by blue-and-red light cross-effect on the EPF/EPFL peptides. The mediating action of monochromatic light was reduced under polychromatic light.

Rice plants overexpressing OsEPF1 show reduced stomatal density and increased root cortical aerenchyma formation

Stomata are adjustable pores in the aerial epidermis of plants. The role of stomata is usually described in terms of the trade-off between CO2 uptake and water loss. Little consideration has been given to their interaction with below-ground development or diffusion of other gases. We overexpressed the rice EPIDERMAL PATTERNING FACTOR1 (OsEPF1) to produce rice plants with reduced stomatal densities, resulting in lowered leaf stomatal conductance and enhanced water use efficiency. Surprisingly, we found that root cortical aerenchyma (RCA) is formed constitutively in OsEPF1OE lines regardless of tissue age and position. Aerenchyma is tissue containing air-spaces that can develop in the plant root during stressful conditions, e.g. oxygen deficiency when it functions to increase O2 diffusion from shoot to root. The relationship with stomata is unknown. We conclude that RCA development and stomatal development are linked by two possible mechanisms: first that reduced stomatal conductance inhibits the diffusion of oxygen to the root, creating an oxygen deficit and stimulating the formation of RCA, second that an unknown EPF signalling pathway may be involved. Our observations have fundamental implications for the understanding of whole plant gas diffusion and root-to-shoot signalling events.

Overexpression of MdEPF2 improves water use efficiency and reduces oxidative stress in tomato

Increasing drought frequency and diminishing groundwater resources are critical environmental constraints that seriously reduce global plant production. However, these limitations might be partially addressed by improving crop drought tolerance and water-use efficiency (WUE). We isolated an epidermal patterning factor (EPF), MdEPF2, from apple (Malus domestica). Transcript levels of that gene were higher after plants were treated with abscisic acid and drought. To investigate its function in drought tolerance, we ectopically expressed MdEPF2 in Solanum lycopersicum cultivar ‘Micro-Tom’. When compared with the wild type (WT), stomatal density was significantly lower in the leaves from the transgenics, which enabled those plants to avoid dehydration. Under drought stress, transgenic plants had higher values for relative leaf water content, chlorophyll, photosynthetic rates, and WUE than did WT. The former also had lower accumulations of reactive oxygen species and malonyldialdehyde but greater activities of antioxidant enzymes, thereby leading to less oxidative damage. Our results suggested that MdEPF2 is a functional ortholog of EPF2 in Arabidopsis and can be potentially used in apple breeding to improve WUE and drought tolerance in this economically important fruit crop.

Homologous genes of epidermal patterning factor regulate stomatal development in rice

Stomata are microscopic pores on the surface of leaves through which water as vapor passes to the atmosphere and CO2 uptake for the photosynthesis. The signaling peptides of the epidermal patterning factor (EPF) family regulate stomatal development and density in Arabidopsis. Several putative homologs of EPF/EPFL exist in rice genome. To understand their possible involvement in stomatal formation, in this study we generated a series of transgenic lines including reporter promoter fusions, down-regulation and overexpression and demonstrated drastic differences in stomatal densities between different genotypes, as elevated expression of OsEPF1 or OsEPF2 greatly reduced stomatal density in rice, whereas ectopic overexpression of either OsEPF1 or OsEPF2 significantly decreased the high stomatal frequency of both mutant lines of epf2 and epf1epf2 Arabidopsis. Conversely, knocking down OsEPFL9 transcription conferred transgenic plants with fewer stomata than WT in rice, whereas overexpressing rice OsEPFL9 gene could cause excessive production of stomata in Arabidopsis. In conclusion, homologs of EPF/EPFL regulate stomatal development in a generally highly conserved way yet there exist function distinctions between dicot and monocot plants.

Rice with reduced stomatal density conserves water and has improved drought tolerance under future climate conditions.

Summary Much of humanity relies on rice (Oryza sativa) as a food source, but cultivation is water intensive and the crop is vulnerable to drought and high temperatures. Under climate change, periods of reduced water availability and high temperature are expected to become more frequent, leading to detrimental effects on rice yields.We engineered the high‐yielding rice cultivar ‘IR64’ to produce fewer stomata by manipulating the level of a developmental signal. We overexpressed the rice epidermal patterning factor OsEPF1, creating plants with substantially reduced stomatal density and correspondingly low stomatal conductance.Low stomatal density rice lines were more able to conserve water, using c. 60% of the normal amount between weeks 4 and 5 post germination. When grown at elevated atmospheric CO 2, rice plants with low stomatal density were able to maintain their stomatal conductance and survive drought and high temperature (40°C) for longer than control plants. Low stomatal density rice gave equivalent or even improved yields, despite a reduced rate of photosynthesis in some conditions.Rice plants with fewer stomata are drought tolerant and more conservative in their water use, and they should perform better in the future when climate change is expected to threaten food security.

Molecular control of stomatal development.

Plants have evolved developmental plasticity which allows the up- or down-regulation of photosynthetic and water loss capacities as new leaves emerge. This developmental plasticity enables plants to maximise fitness and to survive under differing environments. Stomata play a pivotal role in this adaptive process. These microscopic pores in the epidermis of leaves control gas exchange between the plant and its surrounding environment. Stomatal development involves regulated cell fate decisions that ensure optimal stomatal density and spacing, enabling efficient gas exchange. The cellular patterning process is regulated by a complex signalling pathway involving extracellular ligand–receptor interactions, which, in turn, modulate the activity of three master transcription factors essential for the formation of stomata. Here, we review the current understanding of the biochemical interactions between the epidermal patterning factor ligands and the ERECTA family of leucine-rich repeat receptor kinases. We discuss how this leads to activation of a kinase cascade, regulation of the bHLH transcription factor SPEECHLESS and its relatives, and ultimately alters stomatal production.

Disruption of stomatal lineage signaling or transcriptional regulators has differential effects on mesophyll development, but maintains coordination of gas exchange.

Summary Stomata are simultaneously tasked with permitting the uptake of carbon dioxide for photosynthesis while limiting water loss from the plant. This process is mainly regulated by guard cell control of the stomatal aperture, but recent advancements have highlighted the importance of several genes that control stomatal development.Using targeted genetic manipulations of the stomatal lineage and a combination of gas exchange and microscopy techniques, we show that changes in stomatal development of the epidermal layer lead to coupled changes in the underlying mesophyll tissues. This coordinated response tends to match leaf photosynthetic potential (V cmax) with gas‐exchange capacity (g smax), and hence the uptake of carbon dioxide for water lost.We found that different genetic regulators systematically altered tissue coordination in separate ways: the transcription factor SPEECHLESS (SPCH) primarily affected leaf size and thickness, whereas peptides in the EPIDERMAL PATTERNING FACTOR (EPF) family altered cell density in the mesophyll. It was also determined that interlayer coordination required the cell‐surface receptor TOO MANY MOUTHS (TMM).These results demonstrate that stomata‐specific regulators can alter mesophyll properties, which provides insight into how molecular pathways can organize leaf tissues to coordinate gas exchange and suggests new strategies for improving plant water‐use efficiency.

A receptor-like protein acts as a specificity switch for the regulation of stomatal development.

Stomata are microscopic openings that allow for the exchange of gases between plants and the environment. In Arabidopsis, stomatal patterning is specified by the ERECTA family (ERf) receptor kinases (RKs), the receptor-like protein (RLP) TOO MANY MOUTHS (TMM), and EPIDERMAL PATTERNING FACTOR (EPF) peptides. Here we show that TMM and ER or ER-LIKE1 (ERL1) form constitutive complexes, which recognize EPF1 and EPF2, but the single ERfs do not. TMM interaction with ERL1 creates a binding pocket for recognition of EPF1 and EPF2, indicating that the constitutive TMM-ERf complexes function as the receptors of EPF1 and EPF2. EPFL9 competes with EPF1 and EPF2 for binding to the ERf-TMM complex. EPFL4 and EPFL6, however, are recognized by the single ERfs without the requirement of TMM. In contrast to EPF1,2, the interaction of EPFL4,6 with an ERf is greatly reduced in the presence of TMM. Taken together, our data demonstrate that TMM dictates the specificity of ERfs for the perception of different EPFs, thus functioning as a specificity switch for the regulation of the activities of ERfs.