Characterization of the Class IV Homeodomain-Leucine Zipper Gene Family in Arabidopsis

Abstract

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial β-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5′-GCATTAAATGC-3′ as the preferential binding site.

Arabidopsishomeodomain-leucine zipper IV proteins promote stomatal development and ectopically induce stomata beyond the epidermis

“…1B). This is consistent with a previous report on HDG2pro::GUS (Nakamura et al, 2006). Furthermore, the HDG2 translational reporter (HDG2pro::HDG2-GFP) accumulated in the nuclei of meristemoids and SLGCs (Fig.…”

“…Our molecular phylogenetic analysis of 16 AtHD-ZIP IV family members using Bayesian algorithms shows that, similar to the neighbor-joining analysis by Nakamura et al (Nakamura et al, 2006), HDG2 is most closely related to AtML1 and PDF2, two wellstudied L1 layer (protoderm)-specific genes (Abe et al, 2003;Lu et al, 1996) (supplementary material Fig. S1).…”

“…To address this, we investigated the hdg2 loss-of-function phenotype. A previous report on the systematic characterization of the Arabidopsis HD-ZIP IV family members did not reveal any phenotype associated with the hdg2 T-DNA knockout mutation (Nakamura et al, 2006). We therefore predicted that hdg2 phenotype, if any, would be less obvious.…”

“…Some other HD-ZIP IV members are associated with differentiation of shoot epidermal cell types. For example, Arabidopsis GLABRA2 (GL2) promotes trichome differentiation (Rerie et al, 1994), and HDG11 and HDG12 regulate trichome branching (Nakamura et al, 2006). Microarray studies of maize plants overexpressing HD-ZIP IV gene, OUTER CELL LAYER1 (OCL1) identified target genes in lipid metabolism and cuticle biosynthesis, suggesting a role in pavement cell differentiation (Javelle et al, 2010).…”

“…In Arabidopsis, two closely related genes, A. thaliana MERISTEM LAYER1 (AtML1) and PROTODERMAL FACTOR2 (PDF2), redundantly specify epidermal identity; their double loss of function results in an embryo that is lethally devoid of a protoderm (Abe et al, 2003). AtML1 and PDF2 belong to a family of plant-specific homeodomain leucine zipper class IV (HD-ZIP IV) transcription factors (Mukherjee et al, 2009), many showing preferential expression in the epidermis (Ingram et al, 2000;Javelle et al, 2011a;Nakamura et al, 2006). The Arabidopsis genome contains 16 HD-ZIP IV (AtHD-ZIP IV) genes, of which at least nine exhibit expression in leaf epidermal cells (Nakamura et al, 2006;supplementary material Fig.…”