Plants constantly monitor environmental fluctuations to optimize their growth and metabolism. One example is adaptive growth occurring in response to biotic and abiotic stresses. Here, we demonstrate that GH3-mediated auxin homeostasis is an essential constituent of the complex network of auxin actions that regulates stress adaptation responses in Arabidopsis. Endogenous auxin pool is regulated, at least in part, through negative feedback by a group of auxin-inducible GH3 genes encoding auxin-conjugating enzymes. An Arabidopsis mutant, wes1-D, in which a GH3 gene WES1 is activated by nearby insertion of the 35 S enhancer, exhibited auxin-deficient traits, including reduced growth and altered leaf shape. Interestingly, WES1 is also induced by various stress conditions as well as by salicylic acid and abscisic acid. Accordingly, wes1-D was resistant to both biotic and abiotic stresses, and stress-responsive genes, such as pathogenesis-related genes and CBF genes, were upregulated in this mutant. In contrast, a T-DNA insertional mutant showed reduced stress resistance. We therefore propose that GH3-mediated growth suppression directs reallocation of metabolic resources to resistance establishment and represents the fitness costs of induced resistance.

Controlled release of membrane-tethered, dormant precursors is an intriguing activation mechanism that regulates diverse cellular functions in eukaryotes. An exquisite example is the proteolytic activation of membrane-bound transcription factors. The proteolytic cleavage liberates active transcription factors from the membranes that can enter the nucleus and evokes rapid transcriptional responses to incoming stimuli. Here, we show that a membrane-bound NAC (for NAM, ATAF1/2, CUC2) transcription factor, designated NTM1 (for NAC with transmembrane motif1), is activated by proteolytic cleavage through regulated intramembrane proteolysis and mediates cytokinin signaling during cell division in Arabidopsis thaliana. Cell proliferation was greatly reduced in an Arabidopsis mutant with retarded growth and serrated leaves in which a transcriptionally active NTM1 form was constitutively expressed. Accordingly, a subset of cyclin-dependent kinase (CDK) inhibitor genes (the KIP-related proteins) was induced in this mutant with a significant reduction in histone H4 gene expression and in CDK activity. Consistent with a role for NTM1 in cell cycling, a Ds element insertional mutant was morphologically normal but displayed enhanced hypocotyl growth with accelerated cell division. Interestingly, cytokinins were found to regulate NTM1 activity by controlling its stability. These results indicate that the membrane-mediated activation of NTM1 defines a molecular mechanism by which cytokinin signaling is tightly regulated during cell cycling.

SummaryClass III homeodomain-leucine zipper proteins regulate critical aspects of plant development, including lateral organ polarity, apical and lateral meristem formation, and vascular development. ATHB15, a member of this transcription factor family, is exclusively expressed in vascular tissues. Recently, a microRNA (miRNA) binding sequence has been identified in ATHB15 mRNA, suggesting that a molecular mechanism governed by miRNA binding may direct vascular development through ATHB15. Here, we show that miR166-mediated ATHB15 mRNA cleavage is a principal mechanism for the regulation of vascular development. In a gain-of-function MIR166a mutant, the decreased transcript level of ATHB15 was accompanied by an altered vascular system with expanded xylem tissue and interfascicular region, indicative of accelerated vascular cell differentiation from cambial/procambial cells. A similar phenotype was observed in Arabidopsis plants with reduced ATHB15 expression but reversed in transgenic plants overexpressing an miR166-resistant ATHB15. ATHB15 mRNA cleavage occurred in standard wheat germ extracts and in Arabidopsis and was mediated by miR166 in Nicotiana benthamiana cells. miR166-assisted ATHB15 repression is likely to be a conserved mechanism that regulates vascular development in all vascular plants.

Controlled proteolytic cleavage of membrane-associated transcription factors (MTFs) is an intriguing activation strategy that ensures rapid transcriptional responses to incoming stimuli. Several MTFs are known to regulate diverse cellular functions in prokaryotes, yeast, and animals. In Arabidopsis, a few NAC MTFs mediate either cytokinin signaling during cell division or endoplasmic reticulum (ER) stress responses. Through genome-wide analysis, it was found that at least 13 members of the NAC family in Arabidopsis contain strong α-helical transmembrane motifs (TMs) in their C-terminal regions and are predicted to be membrane-associated. Interestingly, most of the putative NAC MTF genes are up-regulated by stress conditions, suggesting that they may be involved in stress responses. Notably, transgenic studies revealed that membrane release is essential for the function of NAC MTFs. Transgenic plants overexpressing partial-size NAC constructs devoid of the TMs, but not those overexpressing full-size constructs, showed distinct phenotypic changes, including dwarfed growth and delayed flowering. The rice genome also contains more than six NAC MTFs. Furthermore, the presence of numerous MTFs is predicted in the whole transcription factors in plants. We thus propose that proteolytic activation of MTFs is a genome-wide mechanism regulating plant genomes.

Posttranscriptional RNA metabolism plays versatile roles in the regulation of gene expression during eukaryotic growth and development. It is mediated by a group of RNA binding proteins with distinct conserved motifs. In this study, an Arabidopsis (Arabidopsis thaliana) gene, designated FLK, was identified and shown to encode a putative RNA binding protein with K homology motifs. A mutant in which FLK was inactivated by T-DNA insertion exhibited a severe late flowering phenotype both in long and short days. The late flowering phenotype was reversed by gibberellin and vernalization treatments. The FLOWERING LOCUS C (FLC) transcription was greatly upregulated, whereas those of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 decreased in the mutant. These observations demonstrate that FLK regulates the autonomous flowering pathway via FLC. It is now evident that a battery of different RNA binding proteins are involved in the posttranscriptional regulation of flowering time in Arabidopsis.