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. 2013 May;140(9):1924-35.
doi: 10.1242/dev.090209. Epub 2013 Mar 20.

Arabidopsis Homeodomain-Leucine Zipper IV Proteins Promote Stomatal Development and Ectopically Induce Stomata Beyond the Epidermis

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Free PMC article

Arabidopsis Homeodomain-Leucine Zipper IV Proteins Promote Stomatal Development and Ectopically Induce Stomata Beyond the Epidermis

Kylee M Peterson et al. Development. .
Free PMC article

Abstract

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.

Figures

Fig. 1.
Fig. 1.
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.
Fig. 2.
Fig. 2.
Ectopic overexpression of HDG2 confers ectopic stomata within mesophyll tissues and formation of multiple epidermal layers. (A-D) In situ hybridization analysis of HDG2 expression in 10-day-old wild-type (A,B) and HDG2-OX (C,D) seedlings treated with HDG2 antisense (A,C) or sense (B,D) probes. In wild type, endogenous HDG2 shows stomatal-lineage expression (A, arrowheads). Inset shows an enlarged image. Scale bars: 40 μm. (E-H) Histological cross-sections of plastic-embedded 10-day-old cotyledons from wild-type (E) and transgenic plants overexpressing HDG2 (HDG2-OX), which are CaMV35S::HDG2 (F-H). In HDG2-OX, ectopic differentiation of stomata in internal tissues (arrowheads) is evident. Bottom insets indicate enlarged images. Multiple epidermal layers (asterisks) occasionally formed. Images are taken under the same magnification. Scale bars: 100 μm. (I-K) Live sections of HDG2-OX cotyledons expressing mature guard cell marker E994 (I) and stomatal-lineage marker TMMpro::GUS-GFP (J,K). GFP is detected in ectopic internal stomata (I, arrowhead) and internal stomatal-lineage cells (J,K, arrowheads). A mature ectopic internal stoma is also evident in J (cyan arrowhead). Scale bars: 20 μm. (L) Optical section of mPS-PI stained mesophyll layer from HDG2-OX cotyledon expressing SPCHpro::GUS (arrowhead).
Fig. 3.
Fig. 3.
Ectopic overexpression of HDG2 induces some epidermal reporters. (A,B) Histological cross-sections of emerging rosette leaves from 10-day-old wild-type (wt: A) and HDG2-OX seedlings expressing AtML1pro::GUS (B). Ectopic GUS expression (asterisks) can be seen in B. Scale bars: 100 μm. (C-F) In situ hybridization analysis of FDH expression in 10-day-old wild-type (C,D) and HDG2-OX (E,F) seedlings treated with HDG2 antisense (C,E) or sense (D,F) probes. Scale bars: 40 μm. (G) At5g17710pro::nuc-3xVENUS in wild-type rosette leaf abaxial epidermis showing specific signals in large pavement cells. Scale bar: 50 μm. (H,I) Confocal optical sections (xy, xz and yz planes) of 10-day-old HDG2-OX cotyledon expressing At5g17710pro::nuc-3xVenus, without (H) or with (I) HDG2 induction. Asterisk, GFP in subepidermal mesophyll nucleus. Arrowheads, GFP epidermal nuclei (co-stained with propidium iodide). Scale bars: 50 μm.
Fig. 4.
Fig. 4.
Optical serial sectioning through intact cotyledons by mPS-PI staining reveals ectopic internal stomata. (A) Cartoon of a cotyledon indicating locations of each tissue: (i) adaxial epidermis; (ii) palisade mesophyll; (iii) mid-vein region; (iv) spongy mesophyll; (v) abaxial epidermis. Stomata are found in both adaxial and abaxial epidermis in Arabidopsis. (B) mPS-PI stained optical serial sections of locations (i-v) in wild-type (top); induced ectopic HDG2 overexpression (middle: HDG2-OX) and induced ectopic AtML1 overexpression (bottom: AtML1-OX). Images were taken under the same magnification. Scale bar: 20 μm. See supplementary material Movies 1-3 for z-stack 3D sections.
Fig. 5.
Fig. 5.
Stomatal development defects in hdg2 mutants and higher-order mutants in closely related HD-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.
Fig. 6.
Fig. 6.
HDG2 is a transcriptional activator that can bind to both L1 box and HAHR1 box. (A) The HDG2 gene, from translational initiation codon to termination codon. Boxes indicate exons; lines indicate introns; light-gray boxes indicate homeodomain; dark-gray boxes indicate START domain, which also contains a transcriptional activation domain of maize OCL1 (black boxes). HDG2-SRDX has additional 12 amino acids (LDLDLELRLGFA) for forced gene repression. (B) Sequence similarity of HDG2 with AtML1, PDF2 and OCL1 activation domain region. (C) Transcriptional autoactivation of reporter lacZ gene in yeast by HDG2. Addition of SRDX reduces autoactivation. SCRM and MUTE, a known transcriptional activator and repressor, respectively (Kanaoka et al., 2008), were used as positive and negative controls. (D) Induced overexpression of HDG2-SRDX confers an epidermal phenotype with delayed stomatal precursors, resulting in expanded SLGC-like cells (brackets). Shown are 10-day-old cotyledon abaxial epidermis of wild type (left) and esHDG2-SRDX (right) expressing TMMpro::GUS (top) and EPF2pro::erGFP (bottom). Scale bars: 50 μm. (E) Yeast one-hybrid analysis for activation of lacZ reporter possessing 5′ upstream L1-A-box (white bars), L1-T-box (gray bars) or L1-m-box (black bars) by HDG2, AtML1 or PDF2. SCRM, a known E-box binder (Chinnusamy et al., 2003), was used as a negative control. Bars represent mean values of triplicates. Error bars indicate s.e.m. (F) Yeast one-hybrid analysis for activation of lacZ reporter possessing 5′ upstream HAHR1-A-box (white bars), HAHR1-T-box (gray bars) or HAHR1-m-box (black bars), respectively, by HDG2, AtML1 or PDF2. SCRM was used as a negative control. Bars represent mean value of triplicates. Error bars indicate s.e.m.
Fig. 7.
Fig. 7.
HDG2 transactivates TMM and MUTE promoters in planta. (A) Effector and reporter constructs used. The exact locations of L1 boxes are indicated. (B) Relative luciferase activity in N. benthamiana transiently transformed with 35S::HDG2 and the indicated reporters. Luciferase activities were measured 7 days after infiltration. Relative luciferase activities are shown by firefly luciferase (LUC) activities normalized to renilla luciferase (REN) activities. Bars indicate mean; error bars indicate s.e.m. (n=4).

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