ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected inathb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.

Homeodomain‐leucine zipper (HD‐Zip) proteins are putative transcription factors identified only in plants. Related Arabidopsis homeobox genes, isolated by virtue of sequence conservation within the helix‐3 region of the homeodomain, fall into four families based on sequence similarity. This paper reports the characterization of Athb‐10, a 747 amino acid protein belonging to the fourth HD‐ZIP family. The studies indicate that, although less conserved, the leucine zipper of Athb‐10 can functionally replace that of Athb‐2 in an in vitro DNA‐binding assay. Gene mapping experiments and sequence comparison analysis revealed that Athb‐10 corresponds to GLABRA2, a homeodomain protein involved in trichome development. The mRNA expression analysis revealed that Athb‐10/GLABRA2 is expressed not only in trichome‐bearing organs, but also in the root. The analysis of wild‐type and mutant plants showed that the Athb‐10/GLABRA2 gene expression in the aerial part of the plant and in the root is affected by mutations at the TTG locus. Morphological analysis of the gl2‐1 mutant revealed that the gene is necessary not only for local outgrowth of the trichome, but also for the regulation of root hair development in a subset of epidermal cells. Interestingly, the development of root hair cells in a position normally occupied by non‐hair cells is dependent upon the ethylene regime in which the gl2‐1 plants are grown. Sequence analysis of the gl2‐1 allele revealed that the mutant gene encodes a truncated protein that might still retain a partial activity responsible for the formation of aborted trichomes and for the ethylene‐dependent regulation of root hair formation.

We have characterized an Arabidopsis homeobox gene coding for a putative DNA binding protein that represents an early marker for vascular development. The full-length cDNA encodes a protein of 833 amino acids that we have designated Athb-8; it contains the conserved DNA binding domain that characterizes the HD-Zip family of transcription factors. RNA analysis showed that the Athb-8 gene is expressed during the vegetative and the reproductive phases of plant growth. A higher steady-state level of the Athb-8 mRNA was found in flowering stem and root. In situ mRNA analysis of Arabidopsis plants demonstrated that Athb-8 expression is restricted to the procambial cells of embryo and developing organs. Moreover, Athb-8-GUS expression was found in single parenchyma cells which are differentiating into tracheary elements in wounded tobacco transgenic plants. Finally, we showed that the auxin, indole-3-acetic acid, which is involved in vascular development and differentiation, modulates the expression of the gene. Taken together, these results suggest that Athb-8 might be a regulator of vascular development in Arabidopsis thaliana.

The homeodomain-leucine zipper (HD-Zip) class of transcription factors is unique to plants. HD-Zip proteins bind to DNA exclusively as dimers recognizing dyad symmetric sequences and act as positive or negative regulators of gene expression. On the basis of sequence homology in the HD-Zip DNA-binding domain, HD-Zip proteins have been grouped into four families (HD-Zip I–IV). Each HD-Zip family can be further divided into subfamilies containing paralogous genes that have arisen through genome duplication. Remarkably, all the members of the HD-Zip IIγ and -δ clades are regulated by light quality changes that induce in the majority of the angiosperms the shade-avoidance response, a process regulated at multiple levels by auxin. Intriguingly, it has recently emerged that, apart from their function in shade avoidance, the HD-Zip IIγ and -δ transcription factors control several auxin-regulated developmental processes, including apical embryo patterning, lateral organ polarity, and gynoecium development, in a white-light environment. This review presents recent advances in our understanding of HD-Zip II protein function in plant development, with particular emphasis on the impact of loss-of-function HD-Zip II mutations on auxin distribution and response. The review also describes evidence demonstrating that HD-Zip IIγ and -δ genes are directly and positively regulated by HD-Zip III transcription factors, primary determinants of apical shoot development, known to control the expression of several auxin biosynthesis, transport, and response genes. Finally, the interplay between HD-Zip II and III transcription factors in embryo apical patterning and organ polarity is discussed.

The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 (ACL5) and bushy and dwarf2 (BUD2) is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper (HD-ZIP) III and key auxin signaling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD-ZIP III protein Arabidopsis thaliana homeobox8 tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signaling genes such as like auxin resistant2 (LAX2) and LAX3. We propose that proper formation and differentiation of xylem depend on a balance between positive and negative feedback loops operating through HD-ZIP III genes.