Homeodomain-leucine zipper (HD-ZIP) proteins are transcription factors present only in the plant kingdom. They have a homeodomain (HD), which is a conserved 60-amino-acid motif for DNA binding, and a leucine zipper motif, which mediates their homo- and hetero-dimerization. The class-IV HD-ZIP (HD-ZIP IV) proteins are characterized by an internal loop in the middle of the leucine zipper motif, which is thus called a zipper-loop-zipper (ZLZ) domain (Schrick et al., 2004). They also contain a steroidogenic acute regulatory protein-related lipid transfer (START) domain and a START-adjacent (SAD) domain in the C terminus. The HD-ZIP IV family in Arabidopsis thaliana consists of 16 genes: GLABRA2 (GL2), ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1), ANTHOCYANINLESS 2 (ANL2), PROTODERMAL FACTOR 2 (PDF2) and HOMEODOMAIN GLABROUS 1 (HDG1)–HDG12, among which HDG6 is identical to the previously identified FWA (Nakamura et al., 2006). Expression analyses with promoter-GUS gene fusions have revealed that ATML1, PDF2, HDG2, HDG5, HDG11 and HDG12 are expressed predominantly in the epidermal layer of shoot meristems and organs (Nakamura et al., 2006), suggesting that many members of the HD-ZIP IV family regulate gene expression in the epidermis. However, only four single knock-out mutants are known to show morphological alterations. The gl2 mutant is defective in trichome and seed mucilage formation, and has ectopic root hairs in non-hair cell files (Rerie et al., 1994; Di Cristina et al., 1996). In anl2, anthocyanin accumulation is reduced in the shoot, and several extra cells are formed between cortical and epidermal layers of the root (Kubo et al., 1999; Kubo and Hayashi, 2011). The hdg11 mutant has trichomes with increased branching, and the phenotype is enhanced in hdg11 hdg12 double mutants (Nakamura et al., 2006), whereas hdg2 has trichomes with smooth cell walls (Marks et al., 2009). ATML1 and PDF2, which are closely similar in sequence to each other, are both expressed specifically in the outermost cell layer (L1) of shoot apical meristems (Lu et al., 1996; Sessions et al., 1999; Abe et al., 2003). While each T-DNA insertion mutant of ATML1 and PDF2 shows the wild-type phenotype, the atml1-1 pdf2-1 double mutant has severe defects in the differentiation of shoot epidermal cells, indicating that they play redundant but critical roles in the formation of the shoot epidermis (Abe et al., 2003). So far, however, there has been no further information on double mutants within the family that display abnormal phenotypes, except for pdf2-1 hdg3-1 and atml-1 hdg3-1, which show slight defects in cotyledon development (Nakamura et al., 2006), and the precise functions of HD-ZIP IV members and functional redundancy among them remain largely to be elucidated.
There are also accumulating reports of epidermis-related functions of HD-ZIP IV genes in other plants. In Zea mays (maize), OUTER CELL LAYER 4 (OCL4) has been suggested to regulate trichome patterning and anther development (Vernoud et al., 2009), whereas OCL1 may be involved in lipid metabolism and cuticle biosynthesis (Javelle et al., 2010). The cotton GbML1 regulates fiber development (Zhang et al., 2010). In Solanum lycopersicum (tomato), Woolly (Wo), the closest homolog of Arabidopsis PDF2, is essential for embryo development, and its dominant allele is known to exhibit the woolly trichome phenotype (Yang et al., 2011). However, these genes represent just a part of the HD-ZIP IV family, and we are still far from a comprehensive understanding of the HD-ZIP IV family.
The epidermis is organized in a continuous monolayer of cells that covers the plant body. Epidermal cells in shoot organs are exclusively derived from L1, the outermost layer of shoot apical meristems, which continues to undergo anticlinal cell division (Barton and Poethig, 1993). Shoot epidermis plays a critical role in organ separation and defense responses against drought or pathogens, as well as in the integrity of organs. In flowers, the Arabidopsis cytochrome P450 KLUH (KLU) expressed in the epidermis stimulates organ growth non-cell-autonomously, and may be one of the major players in the coordination of the final size of floral organs (Anastasiou et al., 2007; Eriksson et al., 2010). When floral organ identity genes in Arabidopsis such as AGAMOUS (AG), SEPALLATA 3 (SEP3), APETALA 3 (AP3) and PISTILLATA (PI) are expressed specifically in the L1, the flowers show similar modifications to those with constitutive expression of the respective genes, suggesting that the differentiation and maturation of floral organs can be partially directed from the L1 cells (Urbanus et al., 2010). Although AP3 expressed only in the epidermis is not sufficient for full restoration of floral organ identity in ap3 mutants, it acts non-autonomously to recover the overall shape of petals, confirming the important contribution of the epidermis to organ development (Jenik and Irish, 2001; Urbanus et al., 2010).
To examine further the function of the HD-ZIP IV genes in Arabidopsis, we generated their double mutants and found that the combinations of pdf2-1 with mutant alleles of HDG1, HDG2, HDG5 or HDG12 resulted in abnormal floral organ formation, suggesting the importance of the interplay in the epidermis between PDF2 and these HD-ZIP IV proteins during flower development.