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.

Plasticity in stomatal behavior across a gradient of water supply is consistent among field-grown maize inbred lines with varying stomatal patterning

Stomata regulate leaf CO2 assimilation (A) and water loss. The Ball-Berry and Medlyn models predict stomatal conductance (gs) with a slope parameter (m or g1) that reflects sensitivity of gs to A, atmospheric CO2 and humidity, and is inversely related to water use efficiency (WUE). This study addressed knowledge gaps about what the values of m and g1 are in C4 crops under field conditions, as well as how they vary among genotypes and with drought stress. m and g1 were unexpectedly consistent in four inbred maize genotypes across a gradient of water supply. This was despite genotypic variation in stomatal patterning, A and gs. m and g1 were strongly correlated with soil water content, moderately correlated with pre-dawn leaf water potential (Ψpd), and weakly correlated with midday leaf water potential (Ψmd). This implied that m and g1 respond to long-term water supply more than short-term drought stress. The conserved nature of m and g1 across anatomically diverse genotypes and water supplies suggests there is flexibility in structure-function relationships underpinning WUE. This evidence can guide simulation of maize gs across a range of water supply in the primary maize growing region and inform efforts to improve WUE.

Misregulation of MYB16 causes stomatal cluster formation by disrupting polarity in asymmetric cell division

Stomata and leaf cuticle regulate water evaporation from the plant body and balance the trade-off between photosynthesis and water loss. We identified MYB16, a key transcription factor controlling cutin biosynthesis, from previous stomatal lineage ground cell (SLGC)-enriched transcriptome study. The preferential localization of MYB16 in SLGCs but not meristemoids suggests a link between cutin synthesis and stomatal development. Here, we showed that downregulation of MYB16 in meristemoids was directly mediated by the stomatal master transcription factor, SPEECHLESS (SPCH). The suppression of MYB16 before asymmetric division was crucial for stomatal patterning because overexpression or ectopic expression of MYB16 in meristemoids increased impermeability and elevated stomatal density and clusters. The aberrant pattern of stomata was due to reduced and disrupted establishment of polarity during asymmetric cell division. Manipulating polarity by growing seedlings on hard agar rescued stomatal clusters and polarity defects in MYB16 ectopic lines. By expressing a cutinase in MYB16 ectopic lines, stomatal clustering was reduced, which suggests that the ectopic accumulation of cuticle affects the polarity in asymmetrically dividing cells and causes clustered stomata. Taken together, inhibiting MYB16 expression by SPCH in early stomatal lineage is required to correctly place the polarity complex for proper stomatal patterning during leaf morphogenesis.

Correlation and co-localization of QTL for stomatal density, canopy temperature and productivity with and without drought stress in Setaria

Mechanistic modeling indicates that stomatal conductance could be reduced to improve water use efficiency (WUE) in C4 crops. Genetic variation in stomatal density and canopy temperature was evaluated in the model C4 genus, Setaria. Recombinant inbred lines (RIL) derived from a Setaria italica x Setaria viridis cross were grown with ample or limiting water supply under field conditions in Illinois. An optical profilometer was used to rapidly assess stomatal patterning and canopy temperature was measured using infrared imaging. Stomatal density and canopy temperature were positively correlated but both were negatively correlated with total above-ground biomass. These trait relationships suggest a likely interaction between stomatal density and the other drivers of water use such as stomatal size and aperture. Multiple QTLs were identified for stomatal density and canopy temperature, including co-located QTLs on chromosomes 5 and 9. The direction of the additive effect of these QTLs on chromosome 5 and 9 were in accordance with the positive phenotypic relationship between these two traits. This along with prior experiments suggests a common genetic architecture between stomatal patterning and WUE in controlled environments with canopy transpiration and productivity in the field, while highlighting the potential of Setaria as a model to understand the physiology and genetics of WUE in C4 species.

Correlation and co-localization of QTL for stomatal density and canopy temperature under drought stress in Setaria

Mechanistic modeling indicates that stomatal conductance could be reduced to improve water use efficiency (WUE) in C4 crops. Genetic variation in stomatal density and canopy temperature was evaluated in the model C4 genus, Setaria. Recombinant inbred lines (RIL) derived from a Setaria italica x Setaria viridis cross were grown with ample or limiting water supply under field conditions in Illinois. An optical profilometer was used to rapidly assess stomatal patterning and canopy temperature was measured using infrared imaging. Stomatal density and canopy temperature were positively correlated but both were negatively correlated with total above-ground biomass. These trait relationships suggest a likely interaction between stomatal density and the other drivers of water use such as stomatal size and aperture. Multiple QTLs were identified for stomatal density and canopy temperature, including co-located QTLs on chromosomes 5 and 9. The direction of the additive effect of these QTLs on chromosome 5 and 9 were in accordance with the positive phenotypic relationship between these two traits. This suggests a common genetic architecture between stomatal patterning in the greenhouse and canopy transpiration in the field, while highlighting the potential of setaria as a model to understand the physiology and genetics of WUE in C4 species.HighlightThis article reports a phenotypic and genetic relationship between two water use related traits operating at leaf level and canopy level in a C4 model crop species.

Optical topometry and machine learning to rapidly phenotype stomatal patterning traits for QTL mapping in maize

Stomata are adjustable pores on leaf surfaces that regulate the trade-off of CO2 uptake with water vapor loss, thus having critical roles in controlling photosynthetic carbon gain and plant water use. The lack of easy, rapid methods for phenotyping epidermal cell traits have limited the use of quantitative, forward and reverse genetics to discover the genetic basis of stomatal patterning. A new high-throughput epidermal cell phenotyping pipeline is presented here and used for quantitative trait loci (QTL) mapping in field-grown maize. The locations and sizes of stomatal complexes and pavement cells on images acquired by an optical topometer from mature leaves were automatically determined. Computer estimated stomatal complex density (SCD; R2 = 0.97) and stomatal complex area (SCA; R2 = 0.71) were strongly correlated with human measurements. Leaf gas exchange traits correlated with the dimensions and proportion of stomatal complexes but, unexpectedly, did not correlate with SCD. Genetic variation in epidermal traits were consistent across two field seasons. Out of 143 QTLs in total, 36 QTLs were consistently identified for a given trait in both years. 24 hotspots of overlapping QTLs for multiple traits were identified. Orthologs of genes known to regulate stomatal patterning in Arabidopsis were located within some, but not all, of these regions. This study demonstrates how discovery of the genetic basis for stomatal patterning can be accelerated in maize, a model for C4 species where these processes are poorly understood.One sentence summaryOptical topometry and machine learning tools were developed to assess epidermal cell patterning, and applied to analyze its genetic architecture alongside leaf photosynthetic gas exchange in maize.

Effective range of non-cell autonomous activator and inhibitor peptides specifying plant stomatal patterning

ABSTRACTStomata are epidermal valves that facilitate gas exchange between plants and their environment. Stomatal patterning is regulated by the EPIDERMAL PATTERING FACTOR (EPF) family of secreted peptides: EPF1 enforces stomatal spacing, whereas EPIDERMAL PATTERNING FACTOR-LIKE9 (EPFL9), also known as Stomagen, promotes stomatal development. It remains unknown, however, how far these signaling peptides act. Utilizing Cre-lox recombination-based mosaic sectors that overexpress either EPF1 or Stomagen in Arabidopsis cotyledons, we reveal a range within the epidermis and across the cell layers in which these peptides influence patterns. To determine their effective ranges quantitatively, we developed a computational pipeline, SPACE (stomata patterning autocorrelation on epidermis), that describes probabilistic two-dimensional stomatal distributions based upon spatial autocorrelation statistics used in astrophysics. The SPACE analysis shows that, whereas both peptides act locally, the inhibitor EPF1 exerts longer range effects than the activator Stomagen. Furthermore, local perturbation of stomatal development has little influence on global two-dimensional stomatal patterning. Our findings conclusively demonstrate the nature and extent of EPF peptides as non-cell autonomous local signals and provide a means for quantitative characterization of complex spatial patterns in development.This article has an associated ‘The people behind the papers’ interview.

Basil interspecific hybridization and transcriptome study indicates altered developmental and metabolic gene expression

Background In order to understand the developmental modulation of transcriptome and associated gene expression in inter-genomic combinations, a systematic study was planned using two diverse yet closely related species of Ocimum, targeting their hybrid F1 and derived amphidiploid (colchiploid of F1 hybrid). The existing developmental alterations between F1 and amphidiploid through phenotypical and anatomical assessments were analyzed. Results Study of several genes and transcription factors putatively involved in the growth and developmental processes of plants clearly amalgamates the transcriptome data linking the phenotypic differences in F1 and amphidiploid. Additionally, differentially expressed genes of stomatal patterning and development revealed their involvement leading to higher density of stomata in F1 while larger size of stomata in the amphidiploid. Absence of 8,330 transcripts of interspecific hybrid F1 in its amphidiploid and exclusive presence of two detected transcripts in amphidiploid provides a set of genes to analyze the suppressed or activated functions between F1 and amphidiploid. Estimation of chlorophyll, lignin, flavonoid and phenylpropenes (eugenol and methyleugenol) content were correlated with the average FPKM and digital gene expression values in F1 and amphidiploid. Conclusion This is the first investigation which describes the genes and transcription factors influenced by interspecific hybridization leading to developmental changes and alleviation of intergenomic instability in amphidiploid.