SUMMARYMYB46 functions as a transcriptional switch that turns on the genes necessary for secondary wall biosynthesis. Elucidating the transcriptional regulatory network immediately downstream of MYB46 is crucial to our understanding of the molecular and biochemical processes involved in the biosynthesis and deposition of secondary walls in plants. To gain insights into MYB46-mediated transcriptional regulation, we first established an inducible secondary wall thickening system in Arabidopsis by expressing MYB46 under the control of dexamethasone-inducible promoter. Then, we used an ATH1 GeneChip microarray and Illumina digital gene expression system to obtain a series of transcriptome profiles with regard to the induction of secondary wall development. These analyses allowed us to identify a group of transcription factors whose expression coincided with or preceded the induction of secondary wall biosynthetic genes. A transient transcriptional activation assay was used to confirm the hierarchical relationships among the transcription factors in the network. The in vivo assay showed that MYB46 transcriptionally activates downstream target transcription factors, three of which (AtC3H14, MYB52 and MYB63) were shown to be able to activate secondary wall biosynthesis genes. AtC3H14 activated the transcription of all of the secondary wall biosynthesis genes tested, suggesting that AtC3H14 may be another master regulator of secondary wall biosynthesis. The transcription factors identified here may include direct activators of secondary wall biosynthesis genes. The present study discovered novel hierarchical relationships among the transcription factors involved in the transcriptional regulation of secondary wall biosynthesis, and generated several testable hypotheses.

Summary RING (really interesting new gene) zinc-finger proteins have important regulatory roles in the development of a variety of organisms. The XERICO gene encodes a small protein (162 amino acids) with an N-terminal transmembrane domain and a RING-H2 zinc-finger motif located at the C-terminus. In silico gene-expression analysis indicated that XERICO is induced by salt and osmotic stress. Compared with wild-type (WT) Arabidopsis plants, transgenic plants overexpressing XERICO (35S::XERICO) exhibited hypersensitivity to salt and osmotic stress and exogenous abscisic acid (ABA) during germination and early seedling growth. When subjected to a drought treatment, transcriptional upregulation of a key ABA-biosynthesis gene, AtNCED3, was much faster and stronger in 35S::XERICO plants compared with WT plants. Further, upregulation of XERICO substantially increased cellular ABA levels. The adult 35S::XERICO plants, in contrast to early seedling growth, showed a marked increase in their tolerance to drought stress. Yeast two-hybrid screening indicated that XERICO interacts with an E2 ubiquitin-conjugating enzyme (AtUBC8) and ASK1-interacting F-box protein (AtTLP9), which is involved in the ABA-signaling pathway. Affymetrix GeneChip array analysis showed that the expressions of many of the genes involved in the biosynthesis of plant hormones (e.g. ethylene, brassinosteroid, gibberellic acid) were significantly changed in the 35S::XERICO plants. These results suggest that the homeostasis of various plant hormones might be altered in 35S::XERICO plants, possibly by overaccumulation of ABA.

Wood is an important raw material and environmentally cost-effective renewable source of energy. However, the molecular biology of wood formation (i.e. secondary growth) is surprisingly understudied. A novel experimental system was employed to study the molecular regulation of secondary xylem formation in Arabidopsis. First, we demonstrate that the weight carried by the stem is a primary signal for the induction of cambium differentiation and the plant hormone, auxin, is a downstream carrier of the signal for this process. We used Arabidopsis whole-transcriptome (23 K) GeneChip analysis to examine gene expression profile changes in the inflorescent stems treated for wood formation by cultural manipulation or artificial weight application. Many of the genes up-regulated in wood-forming stems had auxin responsive cis-acting elements in their promoter region, indicating auxin-mediated regulation of secondary growth. We identified 700 genes that were differentially expressed during the transition from primary growth to secondary growth. More than 40% of the genes that were up-regulated (>5×) were associated with signal transduction and transcriptional regulation. Biological significance of these regulatory genes is discussed in light of the induction and development of secondary xylem.

SummaryVascular plants evolved to have xylem that provides physical support for their growing body and serves as a conduit for water and nutrient transport. In a previous study, we used comparative-transcriptome analyses to select a group of genes that were upregulated in xylem of Arabidopsis plants undergoing secondary growth. Subsequent analyses identified a plant-specific NAC-domain transcription factor gene (ANAC012) as a candidate for genetic regulation of xylem formation. Promoter-GUS analyses showed that ANAC012 expression was preferentially localized in the (pro)cambium region of inflorescence stem and root. Using yeast transactivation analyses, we confirmed the function of ANAC012 as a transcriptional activator, and identified an activation domain in the C terminus. Ectopic overexpression of ANAC012 in Arabidopsis (35S::ANAC012 plants) dramatically suppressed secondary wall deposition in the xylary fiber and slightly increased cell-wall thickness in the xylem vessels. Cellulose compositions of the cell wall were decreased in the inflorescent stems and roots of 35S::ANAC012 plants, probably resulting from defects in xylary fiber formation. Our data suggest that ANAC012 may act as a negative regulator of secondary wall thickening in xylary fibers.