Diurnal stomatal apertures and density ratios affect whole-canopy stomatal conductance, water-use efficiency and yield

Key physiological traits of plants, such as transpiration and stomatal conductance, are usually studied under steady-state conditions or modeled using only a few measured data points. Those measurements do not reflect the dynamic behavior of the plant in response to field conditions. To overcome this bottleneck, we used a gravimetric functional phenotyping platform and a reverse-phenotyping method to examine the dynamic whole-plant water regulation responses of tomato introgression lines and compared those responses with several years of yield performance in commercial fields. Ideotype lines had highly plastic stomatal conductance and high abaxial to adaxial stomatal density ratios and the size of their stomatal apertures peaked early in the day under water-deficit conditions. These traits resulted in dynamic daily water-use efficiency, which allowed for the rapid recovery of transpiration when irrigation was resumed after a period of imposed drought. We found that stomatal density, the abaxial to adaxial stomatal density ratio and the time of maximum stomatal apertures are crucial for plant adaptation and productivity under drought stress conditions. Abaxial stomatal density was also found to be strongly correlated with the expression of the stomatal-development genes SPCH and ZEP. This study demonstrates how a reverse functional phenotyping approach based on field yield data, continuous and simultaneous whole plant waterbalance measurements and anatomical examination of individual leaves can help us to understand and identify dynamic and complex yield-related physiological traits.

CRISPR/Cas9 knockout of EPFL10 reduces stomatal density while maintaining photosynthesis and enhancing water conservation in rice

Rice production is of paramount importance for global nutrition and potential yields will be detrimentally affected by climate change. Rice stomatal developmental genetics were explored as a mechanism to improve water use efficiency while maintaining yield under climate stress. Gene-editing of STOMAGEN and its paralog, EPFL10, using CRISPR/Cas9 in rice cv. Nipponbare yielded lines with altered stomatal densities that were functionally characterized. CRISPR/Cas9 mediated knockouts of EPFL10 and STOMAGEN yielded lines with c. 80% and 25% of wild-type stomata, respectively. epfl10 lines with small reductions in stomatal densities are able to conserve water to similar extents as stomagen lines with large stomatal density reductions but do not suffer from any concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation. The duplicate of STOMAGEN, EPFL10, is a weak positive regulator of stomatal development in rice. epfl10 lines maintained wild-type physiological characteristics while conserving more water. Modest reductions in stomatal densities may be a climate-adaptive approach in rice that can safeguard yield.

The SPEECHLESS-induced stomatal increase is required for the salt tolerance of oil palm

Oil palm is the most productive oil producing plant. Salt stress leads to growth damage and decrease in yield of oil palm. However, the physiological responses of oil palm to salt stress and their underlying mechanisms are not clear. RNA-Seq for leaf samples from young palms challenged under three levels of salts (100, 250 and 500 mM NaCl) and control for 14 days was conducted. Diverse signalling pathways were involved in responses to different levels of salt stress. All the three levels of salt stress activated EgSPCH expression and induced stomatal density of oil palm, which was contrasting to that in Arabidopsis. Under strong salt stress group, oil palm removed excessive salt via stomata. Overexpression of EgSPCH in Arabidopsis increased the stomatal production but lowered the salt tolerance. These data suggest that in oil palm, salt activates EgSPCH to generate more stomata in response to salt stress. Our results shed a light on the cellular response to salt stress of oil palm and provide new insights into the mechanisms of different salt-induced stomatal development between halophytes and glycophytes.

Activation of 1-Aminocyclopropane-1-Carboxylic Acid Synthases Sets Stomatal Density and Clustered Ratio on Leaf Epidermis of Arabidopsis in Response to Drought

The adjustment of stomatal density and clustered ratio on the epidermis is the important strategy for plants to respond to drought, because the stoma-based water loss is directly related to plant growth and survival under drought conditions. But the relevant adjustment mechanism still needs to be explored. 1-Aminocyclopropane-1-carboxylate (ACC) is disclosed to promote stomatal development, while in vivo ACC levels depend on activation of ACC synthase (ACS) family members. Based on the findings of ACS expression involving in drought response and several ACS activity inhibitors reducing stomatal density and cluster in drought response, here we examined how ACS activation is involved in the establishment of stomatal density and cluster on the epidermis under drought conditions. Preliminary data indicated that activation of ACS2 and/or ACS6 (ACS2/6) increased stomatal density and clustered ratio on the Arabidopsis leaf epidermis by accumulating ACC under moderate drought, and raised the survival risk of seedlings under escalated drought. Further exploration indicated that, in Arabidopsis seedlings stressed by drought, the transcription factor SPEECHLESS (SPCH), the initiator of stomatal development, activates ACS2/6 expression and ACC production; and that ACC accumulation induces Ca2+ deficiency in stomatal lineage; this deficiency inactivates a subtilisin-like protease STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1) by stabilizing the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression, resulting in an increases of stomatal density and cluster ratio on the leaf epidermis. This work provides a novel evidence that ACS2/6 activation plays a key role in the establishment of stomatal density and cluster on the leaf epidermis of Arabidopsis in response to drought.

Arabidopsis Spliceosome Factor SmD3 Modulates Immunity to Pseudomonas syringae Infection

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of major biotic stress response factors were also altered upon treatment with Pseudomonas effectors. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst, verified by northern and RT-qPCR, showed that lack of SmD3-b protein deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Importantly, we show that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. We propose that it is the malfunction of the stomata that is the primary cause of an altered mutant response to the pathogen. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

Role of Basal ABA in Plant Growth and Development

Abscisic acid (ABA) regulates various aspects of plant physiology, including promoting seed dormancy and adaptive responses to abiotic and biotic stresses. In addition, ABA plays an im-portant role in growth and development under non-stressed conditions. This review summarizes phenotypes of ABA biosynthesis and signaling mutants to clarify the roles of basal ABA in growth and development. The promotive and inhibitive actions of ABA in growth are characterized by stunted and enhanced growth of ABA-deficient and insensitive mutants, respectively. Growth regulation by ABA is both promotive and inhibitive, depending on the context, such as concentrations, tissues, and environmental conditions. Basal ABA regulates local growth including hyponastic growth, skotomorphogenesis and lateral root growth. At the cellular level, basal ABA is essential for proper chloroplast biogenesis, central metabolism, and expression of cell-cycle genes. Basal ABA also regulates epidermis development in the shoot, by inhibiting stomatal development, and deposition of hydrophobic polymers like a cuticular wax layer covering the leaf surface. In the root, basal ABA is involved in xylem differentiation and suberization of the endodermis. Hormone crosstalk plays key roles in growth and developmental processes regulated by ABA. Phenotypes of ABA-deficient and insensitive mutants indicate prominent functions of basal ABA in plant growth and development.

SCARECROW is deployed in distinct developmental contexts during rice and maize leaf development

The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell-types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is down-regulated in Osscr1;Osscr2 mutants indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.

Stomatal Lineage Control by Developmental Program and Environmental Cues

Stomata are micropores that allow plants to breathe and play a critical role in photosynthesis and nutrient uptake by regulating gas exchange and transpiration. Stomatal development, therefore, is optimized for survival and growth of the plant despite variable environmental conditions. Signaling cascades and transcriptional networks that determine the birth, proliferation, and differentiation of a stomate have been identified. These networks ensure proper stomatal patterning, density, and polarity. Environmental cues also influence stomatal development. In this review, we highlight recent findings regarding the developmental program governing cell fate and dynamics of stomatal lineage cells at the cell state- or single-cell level. We also overview the control of stomatal development by environmental cues as well as developmental plasticity associated with stomatal function and physiology. Recent advances in our understanding of stomatal development will provide a route to improving photosynthesis and water-stress resilience of crop plants in the climate change we currently face.