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HDG2 is highly enriched in meristemoid population of stomatal cell lineages. (A) HDG2 absolute and relative expression levels among wild-type and stomatal mutants enriched in specific epidermal cell populations. Absolute expressions (dark gray) are from ATH1 microarray data; relative expressions (light gray) are from qRT-PCR analysis. Data are mean values of triplicates; error bars indicate s.e.m. Col, wt; spch, pavement-cell only; mute scrm-D, overwhelmingly enriched in meristemoids; scrm-D, stomata-only epidermis. Below each graph are confocal images of cotyledons from corresponding genotypes. (B) Stomatal-lineage accumulation of HDG2 transcriptional reporter (HDG2pro::nls-3xGFP) in seedling epidermis. (C) Stomatal-lineage accumulation of HDG2 translational reporter (HDG2pro::HDG2-GFP) in 10-day-old abaxial cotyledon epidermis. Scale bars: 20 μm. (D) Expression levels of AtML1 and PDF2 compared with HDG2 among wild-type and stomatal mutants.
Arabidopsis homeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis
The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of ‘master regulatory’ transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.
A novel-type phosphatidylinositol phosphate-interactive, Ca-binding protein PCaP1 in Arabidopsisthaliana: stable association with plasma membrane and partial involvement in stomata closure
by Nagata C., Miwa C., Tanaka N., Kato M., Suito M., Tsuchihira A., Sato Y., Segami S., Maeshima M. (2016)
The Ca2+-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca2+-calmodulin complex as well as free Ca2+. Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown.
We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1–GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring.
Fluorescence intensity of PCaP1–GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper.
In stomatal guard cells, PCaP1–GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure.
These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.
Fig. 5: Stomatal development defects in hdg2 mutants and higher-order mutants in closely relatedHD-ZIP IV genes. (A) Stomatal index (SI; white bars), meristemoid index (MI; grey bars) and stomatal-lineage index (SLI; black bars) of 10-day-old abaxial cotyledons from wild-type (wt) and three independent hdg2 T-DNA insertion alleles: hdg2-2, hdg2-3 and hdg2-4. SLI is defined here as the sum of SI and MI. See supplementary material Fig. S1 for RT-PCR analysis. Data are means (n=8); error bars indicate s.e.m. *P<0.0005; **P<0.0001 (two-tailed Student’s t-test of each hdg2 allele against wild type). Tukey’s HSD did not reveal statistical difference of SI, MI and SLI among three hdg2 alleles. (B) SI of 10-day-old abaxial cotyledons from wild type, atml1, pdf2, hdg2, hdg2 atml1 and hdg2 pdf2. hdg2-2 and atml1-3 alleles were used for the analysis. Data are means (n=8); error bars indicate s.e.m. Total numbers of stomata counted: 680 (wild type), 639 (atml1), 673 (pdf2), 630 (hdg2), 550 (hdg2 atml1) and 620 (hdg2 pdf2). (C) MI of the six genotypes described above. Data are means; error bars indicate s.e.m. Total numbers of meristemoids counted: 25 (wild type), 34 (atml1), 35 (pdf2), 93 (hdg2), 243 (hdg2 atml1) and 94 (hdg2 pdf2). (D) SLI of the six genotypes described above. Data are mean; error bars indicate s.e.m. Total numbers of cells counted: 2516 (wild type), 2542 (atml1), 2661 (pdf2), 2786 (hdg2), 3331 (hdg2 atml1) and 2661 (hdg2 pdf2). For B,C, genotypes with non-significant phenotypes were grouped together with a letter (Tukey’s HSD test after one-way ANOVA). For D, only one genotype was significantly different from others (Tukey’s HSD test; P<0.01). (E-J) Representative DIC images of 10-day-old abaxial cotyledons from wild type (E), hdg2 (F), atml1(G), pdf2 (H), hdg2 atml1 (I) and hdg2 pdf2 (J). Asterisks indicate meristemoids. Images were taken under the same magnification. Scale bar: 100 μm. (K-N) Aberrant stomatal complexes found in hdg2. (K) Arrested stomatal precursor after extensive asymmetric amplifying divisions from 30-day-old cotyledon. (L) Confocal image of a stomatal complex with a single GC from 10-day-old abaxial cotyledon. (M,N) Singular GCs from 30-day-old cotyledon. Asterisks indicate singular GCs; + indicates arrested stomatal precursor. Scale bars: in L, 20 μm; in K,M,N, 25 μm.
Summary
The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration.
Advances have been made in our understanding of stomatal patterning and differentiation, and a set of ‘master regulatory’ transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis.
Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages.
Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata.
A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2.
Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta.
Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.
PLANT STOMATA ENCYCLOPEDIA 
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