Regulation of stomatal development by epidermal, subepidermal and long-distance signals.

Plant leaves consist of three layers, including epidermis, mesophyll and vascular tissues. Their development is meticulously orchestrated. Stomata are the specified structures on the epidermis for uptake of carbon dioxide (CO2) while release of water vapour and oxygen (O2), and thus play essential roles in regulation of plant photosynthesis and water use efficiency. To function efficiently, stomatal formation must coordinate with the development of other epidermal cell types, such as pavement cell and trichome, and tissues of other layers, such as mesophyll and leaf vein. This review summarizes the regulation of stomatal development in three dimensions (3D). In the epidermis, specific stomatal transcription factors determine cell fate transitions and also activate a ligand-receptor- MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) signaling for ensuring proper stomatal density and patterning. This forms the core regulation network of stomatal development, which integrates various environmental cues and phytohormone signals to modulate stomatal production. Under the epidermis, mesophyll, endodermis of hypocotyl and inflorescence stem, and veins in grasses secrete mobile signals to influence stomatal formation in the epidermis. In addition, long-distance signals which may include phytohormones, RNAs, peptides and proteins originated from other plant organs modulate stomatal development, enabling plants to systematically adapt to the ever changing environment.

How and why do species break a developmental trade-off? Elucidating the association of trichomes and stomata across species.

PREMISE: Previous studies have suggested a trade-off between trichome density (Dt) and stomatal density (Ds) due to shared cell precursors. We clarified how, when, and why this developmental trade-off may be overcome across species. METHODS: We derived equations to determine the developmental basis for Dt and Ds in trichome and stomatal indices (it and is) and the sizes of epidermal pavement cells (e), trichome bases (t), and stomata (s) and quantified the importance of these determinants of Dt and Ds for 78 California species. We compiled 17 previous studies of Dt-Ds relationships to determine the commonness of Dt-Ds associations. We modeled the consequences of different Dt-Ds associations for plant carbon balance. RESULTS: Our analyses showed that higher Dt was determined by higher it and lower e, and higher Ds by higher is and lower e. Across California species, positive Dt-Ds coordination arose due to it-is coordination and impacts of the variation in e. A Dt-Ds trade-off was found in only 30% of studies. Heuristic modeling showed that species sets would have the highest carbon balance with a positive or negative relationship or decoupling of Dt and Ds, depending on environmental conditions. CONCLUSIONS: Shared precursor cells of trichomes and stomata do not limit higher numbers of both cell types or drive a general Dt-Ds trade-off across species. This developmental flexibility across diverse species enables different Dt-Ds associations according to environmental pressures. Developmental trait analysis can clarify how contrasting trait associations would arise within and across species.

Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana.

Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.

Measuring stomatal and guard cell metrics for plant physiology and growth using StoManager1.

Automated guard cell detection and measurement are vital for understanding plant physiological performance and ecological functioning in global water and carbon cycles. Most current methods for measuring guard cells and stomata are laborious, time-consuming, prone to bias, and limited in scale. We developed StoManager1, a high-throughput tool utilizing geometrical, mathematical algorithms, and convolutional neural networks to automatically detect, count, and measure over 30 guard cell and stomatal metrics, including guard cell and stomatal area, length, width, stomatal aperture area/guard cell area, orientation, stomatal evenness, divergence, and aggregation index. Combined with leaf functional traits, some of these StoManager1-measured guard cell and stomatal metrics explained 90% and 82% of tree biomass and intrinsic water use efficiency (iWUE) variances in hardwoods, making them substantial factors in leaf physiology and tree growth. StoManager1 demonstrated exceptional precision and recall (mAP@0.5 over 0.96), effectively capturing diverse stomatal properties across over 100 species. StoManager1 facilitates the automation of measuring leaf stomatal and guard cells, enabling broader exploration of stomatal control in plant growth and adaptation to environmental stress and climate change. This has implications for global gross primary productivity (GPP) modeling and estimation, as integrating stomatal metrics can enhance predictions of plant growth and resource usage worldwide. Easily accessible open-source code and standalone Windows executable applications are available on a GitHub repository (https://github.com/JiaxinWang123/StoManager1) and Zenodo (https://doi.org/10.5281/zenodo.7686022).

Brassinosteroid regulates stomatal development in etiolated Arabidopsis cotyledons via transcription factors BZR1 and BES1.

Brassinosteroids (BRs) are phytohormones that regulate stomatal development. In this study, we report that BR represses stomatal development in etiolated Arabidopsis (Arabidopsis thaliana) cotyledons via transcription factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS SUPPRESSOR1 (BES1), which directly target MITOGEN-ACTIVATED PROTEIN KINASE KINASE 9 (MKK9) and FAMA, 2 important genes for stomatal development. BZR1/BES1 bind MKK9 and FAMA promoters in vitro and in vivo, and mutation of the BZR1/BES1 binding motif in MKK9/FAMA promoters abolishes their transcription regulation by BZR1/BES1 in plants. Expression of a constitutively active MKK9 (MKK9DD) suppressed overproduction of stomata induced by BR deficiency, while expression of a constitutively inactive MKK9 (MKK9KR) induced high-density stomata in bzr1-1D. In addition, bzr-h, a sextuple mutant of the BZR1 family of proteins, produced overabundant stomata, and the dominant bzr1-1D and bes1-D mutants effectively suppressed the stomata-overproducing phenotype of brassinosteroid insensitive 1-116 (bri1-116) and brassinosteroid insensitive 2-1 (bin2-1). In conclusion, our results revealed important roles of BZR1/BES1 in stomatal development, and their transcriptional regulation of MKK9 and FAMA expression may contribute to BR-regulated stomatal development in etiolated Arabidopsis cotyledons.

Efeito de sistemas de consórcio e inseticida na formação dos estômatos em plântulas de erva-doce (Foeniculum vulgare Mill. ) / Effects of intercropping systems and insecticide in formation of stomata in fennel seedlings

Foeniculum vulgare Mill., pertencente à família Apiacea, é conhecida como erva-doce e apresenta grande importância medicinal e comercial, tanto no Brasil como em vários outros países. Objetivou-se com esta pesquisa, estudar o desenvolvimento dos estômatos em plântulas de F. vulgare oriundas de sementes produzidas em sistemas de consórcio erva-doce X algodão e com aplicação do inseticida monocrotofós. A erva-doce foi cultivada em consórcio com algodão colorido cultivar BRS Safira, sendo utilizados os seguintes tratamentos: 1A2E, uma fileira de algodão e duas de erva-doce; 2A1E, duas fileiras de algodão e uma de erva-doce; ES, erva-doce solteira; onde foram distribuídos com e sem aplicação de inseticida, totalizando seis tratamentos. As sementes colhidas foram semeadas em areia e mantidas em casa de vegetação por 25 dias. Partes das plântulas (zona de transição, caule, cotilédones e folhas) foram seccionadas à mão livre, coradas e montadas em lâminas com glicerina para observação em microscópio. Foram avaliadas as seguintes características: número de estômatos, diâmetro polar e equatorial dos estômatos e número de cloroplastos nas células-guarda. Os dados foram analisados em delineamento inteiramente casualizado e distribuídos em arranjo fatorial 3X2; sendo realizado teste de Tukey a 5% de probabilidade. Na zona de transição e no caule observou-se aumento do número e do diâmetro polar dos estômatos quando foram utilizados sistemas de consórcio. Nos cotilédones, a erva-doce solteira proporcionou maior número de estômatos, porém com menor diâmetro e com menor quantidade de cloroplastos. Já na folha, os consórcios influenciaram positivamente o número de estômatos e de cloroplastos. De forma geral, os sistemas de consórcio e o inseticida influenciaram positivamente o desenvolvimento dos estômatos das plântulas de erva-doce.

Foeniculum vulgare Mill., belonging to the family Apiaceae, is known as fennel and has great medicinal and commercial importance, both in Brazil and in several other countries. The objective of this research was to study the development of stomata of F. vulgare seedlings grown from seeds produced in intercropping systems fennel and cotton, with application of insecticide monocrotophos. The fennel was grown in association with colored cotton BRS Safira, with the following treatments: 1A2E, one rows of cotton and two fennel; 2A1E, two rows of cotton and one fennel; ES, fennel single; were distributed with and without application of insecticide, total six treatments. The seeds were sown in sand and kept in a greenhouse for 25 days. Parts of seedlings (transition zone, stem, cotyledons and leaves) were cut freehand, stained and mounted on slides with glycerol for observation under microscope. Were evaluated the following characteristics: stomata number, polar and equatorial diameter of the stomata and chloroplasts number in guard cells. The data were analyzed in completely randomized and distributed in factorial 3x2, being conducted Tukey test at 5% probability. The transition zone and stem showed an increase of the stomata number and polar diameter the when consortium systems were used. In cotyledons, fennel single provided the highest stomata number, but with smaller diameter and fewer chloroplasts. In leaf, the consortia have positively influenced the stomata and chloroplasts number. In general, the intercropping systems and insecticide positively influenced the development of stomata in fennel plants.