The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems

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 in athb8 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.     

Evolution of class III homeodomain-leucine zipper genes in streptophytes

Land plants underwent tremendous evolutionary change following the divergence of the ancestral lineage from algal relatives. Several important developmental innovations appeared as the embryophyte clade diversified, leading to the appearance of new organs and tissue types. To understand how these changes came about, we need to identify the fundamental genetic developmental programs that are responsible for growth, patterning, and differentiation and describe how these programs were modified and elaborated through time to produce novel morphologies. Class III homeodomain-leucine zipper (class III HD-Zip) genes, identified in the model plant Arabidopsis thaliana, provide good candidates for basic land plant patterning genes. We show that these genes may have evolved in a common ancestor of land plants and their algal sister group and that the gene family has diversified as land plant lineages have diversified. Phylogenetic analysis, expression data from nonflowering lineages, and evidence from Arabidopsis and other flowering plants indicate that class III HD-Zip genes acquired new functions in sporophyte apical growth, vascular patterning and differentiation, and leaf development. Modification of expression patterns that accompanied diversification of class III HD-Zip genes likely played an important role in the evolution of land plant form.     

Primary phloem-specific expression of a Zinnia elegans homeobox gene.

Some plant homeobox genes are expressed specifically in vascular cells and are assumed to function in the differentiation of specific types of vascular cells. However, homeobox genes exhibiting primary phloem-specific expression have not been reported. To elucidate the molecular mechanisms of vascular development, we undertook to isolate from Zinnia elegans primary phloem-specific homeobox genes that may function in phloem development. An HD-Zip type homeobox gene, ZeHB3, was isolated. This gene encodes a class I HD-Zip protein, and constitutes a gene subfamily with the Daucus carota gene CHB6, and Arabidopsis thaliana genes Athb-5, Athb-6, and Athb-16. In situ hybridization of 1-, 14- and 50-day-old plants demonstrated that ZeHB3 mRNA accumulation is restricted to a few cells destined to differentiate into phloem cells and to the immature phloem cells surrounding the sieve elements and companion cells. ZeHB3 protein was also localized to immature phloem cells. These findings clearly indicate that ZeHB3 is a novel homeobox gene that marks, and may function in, the early stages of phloem differentiation.     

Roles for Class III HD-Zip and KANADI genes in Arabidopsis root development

Meristems within the plant body differ in their structure and the patterns and identities of organs they produce. Despite these differences, it is becoming apparent that shoot and root apical and vascular meristems share significant gene expression patterns. Class III HD-Zip genes are required for the formation of a functional shoot apical meristem. In addition, Class III HD-Zip and KANADI genes function in patterning lateral organs and vascular bundles produced from the shoot apical and vascular meristems, respectively. We utilize both gain- and loss-of-function mutants and gene expression patterns to analyze the function of Class III HD-Zip and KANADI genes in Arabidopsis roots. Here we show that both Class III HD-Zip and KANADI genes play roles in the ontogeny of lateral roots and suggest that Class III HD-Zip gene activity is required for meristematic activity in the pericycle analogous to its requirement in the shoot apical meristem.     

Gene trapping in Arabidopsis reveals genes involved in vascular development

The procambium is made up of stem cells that give rise to various vascular cells in plants. To understand the molecular nature of procambium cells, we tried to identify genes that characterize procambium cells using Arabidopsis gene trap lines. Among 26,000 gene trap lines, we found 67 lines in which beta-glucuronidase (GUS) staining occurred along vascular tissues in cotyledons and/or adult leaves. Although four gene trap lines showed procambium-preferential GUS expression, their expression patterns differed from each other during procambium development in root tips and young rosette leaves. Genomic regions flanking the gene trap insertion points in 25 of the 67 lines were determined, including three lines showing preferential GUS staining of the procambium. The three procambium-related genes encoded PINHEAD, katanin and an unknown DUF740 domain-containing protein. We discuss procambium development based on the functions and the differential GUS staining patterns of the procambium-related genes.     

Developmental role and auxin responsiveness of Class III homeodomain leucine zipper gene family members in rice

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner.     

Evolution of the class III HD-Zip gene family in land plants

Arabidopsis class III homeodomain-leucine zipper (HD-Zip III) proteins play overlapping, distinct, and antagonistic roles in key aspects of development that have evolved during land plant evolution. To better understand this gene family's role in plant evolution and development as well as to address broader questions of how duplicated genes functionally diversify, we investigated the evolutionary history of this gene family. Phylogenetic analyses including homologs from diverse land plants indicate that a gene duplication event before the angiosperm--gymnosperm split gave rise to two gene lineages that diversified during angiosperm plant radiation. Heterologous expression of an HD-Zip III gene from the nonvascular plant moss within the Arabidopsis HD-zip III revoluta mutant modified but did not complement the phenotype. Comparison of the expression domains of flowering and nonflowering plant homologs indicate an ancestral role in vascular development and organ initiation but not in specifying organ polarity, a prominent role for angiosperm homologs.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

Expression and genome-wide analysis of the xylogen-type gene family

In higher plants, many extracellular proteins are involved in developmental processes, including cell-cell signaling and cell wall construction. Xylogen is an extracellular arabinogalactan protein (AGP) isolated from Zinnia elegans xylogenic culture medium, which promotes xylem cell differentiation. Xylogen has a unique structure, containing a non-specific lipid transfer protein (nsLTP) domain and AGP domains. We searched for xylogen-type genes in the genomes of land plants, including Arabidopsis thaliana, to further our knowledge of xylogen-type genes as functional extracellular proteins in plants. We found that many xylogen-type genes, including 13 Arabidopsis genes, comprise a gene family in land plants, including Populus trichocarpa, Vitis vinifera, Lotus japonicus, Oryza sativa, Selaginella moellendorffii and Physcomitrella patens. The genes shared an N-terminal signal peptide sequence, a distinct nsLTP domain, one or more AGP domains and a glycosylphosphatidylinositol (GPI)-anchored sequence. We analyzed transgenic plants harboring promoter::GUS (β-glucuronidase) constructs to test expression of the 13 Arabidopsis xylogen-type genes, and detected a diversity of gene family members with related expression patterns. AtXYP2 was the best candidate as the Arabidopsis counterpart of the Zinnia xylogen gene. We observed two distinct expression patterns for several genes, with some anther specific and others preferentially expressed in the endodermis/pericycle. We conclude that xylogen-type genes, which may have diverse functions, form a novel chimeric AGP gene family with a distinct nsLTP domain.