Mi-Jeong Park

The MYB96 Transcription Factor Regulates Cuticular Wax Biosynthesis under Drought Conditions in Arabidopsis

This work provides evidence that deposition of cuticular waxes is intimately associated with plant responses to drought. The Arabidopsis MYB96 transcription factor functions as a regulator of ABA-mediated cuticular wax biosynthesis under drought conditions by binding directly to the promoters of genes encoding very-long-chain fatty acid–condensing enzymes involved in cuticular wax biosynthesis. Drought stress activates several defense responses in plants, such as stomatal closure, maintenance of root water uptake, and synthesis of osmoprotectants. Accumulating evidence suggests that deposition of cuticular waxes is also associated with plant responses to cellular dehydration. Yet, how cuticular wax biosynthesis is regulated in response to drought is unknown. We have recently reported that an Arabidopsis thaliana abscisic acid (ABA)–responsive R2R3-type MYB transcription factor, MYB96, promotes drought resistance. Here, we show that transcriptional activation of cuticular wax biosynthesis by MYB96 contributes to drought resistance. Microarray assays showed that a group of wax biosynthetic genes is upregulated in the activation-tagged myb96-1D mutant but downregulated in the MYB96-deficient myb96-1 mutant. Cuticular wax accumulation was altered accordingly in the mutants. In addition, activation of cuticular wax biosynthesis by drought and ABA requires MYB96. By contrast, biosynthesis of cutin monomers was only marginally affected in the mutants. Notably, the MYB96 protein acts as a transcriptional activator of genes encoding very-long-chain fatty acid–condensing enzymes involved in cuticular wax biosynthesis by directly binding to conserved sequence motifs present in the gene promoters. These results demonstrate that ABA-mediated MYB96 activation of cuticular wax biosynthesis serves as a drought resistance mechanism.

A Self-Regulatory Circuit of CIRCADIAN CLOCK-ASSOCIATED1 Underlies the Circadian Clock Regulation of Temperature Responses in Arabidopsis

The CCA1 MYB transcription factor is a component of the circadian clock, which plays an important role in plant responses to cold temperatures. This work shows that cold temperatures regulate CCA1 activity by modulating CCA1 alternative splicing and thus altering the molecular ratios of alternatively spliced isoforms to induce cold tolerance responses. The circadian clock synchronizes biological processes to daily cycles of light and temperature. Clock components, including CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), are also associated with cold acclimation. However, it is unknown how CCA1 activity is modulated in coordinating circadian rhythms and cold acclimation. Here, we report that self-regulation of Arabidopsis thaliana CCA1 activity by a splice variant, CCA1β, links the clock to cold acclimation. CCA1β interferes with the formation of CCA1α-CCA1α and LATE ELONGATED HYPOCOTYL (LHY)-LHY homodimers, as well as CCA1α-LHY heterodimers, by forming nonfunctional heterodimers with reduced DNA binding affinity. Accordingly, the periods of circadian rhythms were shortened in CCA1β-overexpressing transgenic plants (35S:CCA1β), as observed in the cca1 lhy double mutant. In addition, the elongated hypocotyl and leaf petiole phenotypes of CCA1α-overexpressing transgenic plants (35S:CCA1α) were repressed by CCA1β coexpression. Notably, low temperatures suppressed CCA1 alternative splicing and thus reduced CCA1β production. Consequently, whereas the 35S:CCA1α transgenic plants exhibited enhanced freezing tolerance, the 35S:CCA1β transgenic plants were sensitive to freezing, indicating that cold regulation of CCA1 alternative splicing contributes to freezing tolerance. On the basis of these findings, we propose that dynamic self-regulation of CCA1 underlies the clock regulation of temperature responses in Arabidopsis.

Controlled nuclear import of the transcription factor NTL6 reveals a cytoplasmic role of SnRK2.8 in the drought-stress response

Controlled proteolytic activation of membrane-anchored transcription factors provides an adaptation strategy that guarantees rapid transcriptional responses to abrupt environmental stresses in both animals and plants. NTL6 is a plant-specific NAC [NAM/ATAF1/2/CUC2] transcription factor that is expressed as a dormant plasma membrane-associated form in Arabidopsis. Proteolytic processing of NTL6 is triggered by abiotic stresses and ABA (abscisic acid). In the present study, we show that NTL6 is linked directly with SnRK (Snf1-related protein kinase) 2.8-mediated signalling in inducing a drought-resistance response. SnRK2.8 phosphorylates NTL6 primarily at Thr142. NTL6 phosphorylation by SnRK2.8 is required for its nuclear import. Accordingly, a mutant NTL6 protein, in which Thr142 was mutated to an alanine, was poorly phosphorylated and failed to enter the nucleus. In accordance with the role of SnRK2.8 in drought-stress signalling, transgenic plants overproducing either NTL6 or its active form 6ΔC (35S:NTL6 and 35S:6ΔC) exhibited enhanced resistance to water-deficit conditions such as those overproducing SnRK2.8 (35S:SnRK2.8). In contrast, NTL6 RNAi (RNA interference) plants were susceptible to dehydration as observed in the SnRK2.8-deficient snrk2.8-1 mutant. Furthermore, the dehydration-resistant phenotype of 35S:NTL6 transgenic plants was compromised in 35S:NTL6 X snrk2.8-1 plants. These observations indicate that SnRK2.8-mediated protein phosphorylation, in addition to a proteolytic processing event, is important for NTL6 function in inducing a drought-resistance response.

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

BackgroundThe circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level.ResultsWe systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD.ConclusionTaken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.

Alternative splicing of transcription factors in plant responses to low temperature stress: mechanisms and functions.

Transcription factors play a central role in the gene regulatory networks that mediate various aspects of plant developmental processes and responses to environmental changes. Therefore, their activities are elaborately regulated at multiple steps. In particular, accumulating evidence illustrates that post-transcriptional control of mRNA metabolism is a key molecular scheme that modulates the transcription factor activities in plant responses to temperature fluctuations. Transcription factors have a modular structure consisting of distinct protein domains essential for DNA binding, dimerization, and transcriptional regulation. Alternative splicing produces multiple proteins having different structural domain compositions from a single transcription factor gene. Recent studies have shown that alternative splicing of some transcription factor genes generates small interfering peptides (siPEPs) that negatively regulate the target transcription factors via peptide interference (PEPi), constituting self-regulatory circuits in plant cold stress response. A number of splicing factors, which are involved in RNA binding, splice site selection, and spliceosome assembly, are also affected by temperature fluctuations, supporting the close association of alternative splicing of transcription factors with plant responses to low temperatures. In this review, we summarize recent progress on the temperature-responsive alternative splicing of transcription factors in plants with emphasis on the siPEP-mediated PEPi mechanism.

CCA1 alternative splicing as a way of linking the circadian clock to temperature response in Arabidopsis

Most living organisms on the earth have the circadian clock to synchronize their biochemical processes and physiological activities with environmental changes to optimize their propagation and survival. CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) is one of the core clock components in Arabidopsis. Notably, it is also associated with cold acclimation. However, it is largely unknown how CCA1 activity is modulated by low temperatures. We found that the CCA1 activity is self-regulated by a splice variant CCA1β and the CCA1β production is modulated by low temperatures, linking the circadian clock with cold acclimation. CCA1β competitively inhibits the activities of functional CCA1α and LATE ELONGATED HYPOCOTYL (LHY) transcription factors by forming nonfunctional CCA1α-CCA1β and LHY-CCA1β heterodimers. Consequently, CCA1β-overexpressing plants (35S:CCA1β) exhibit shortened circadian periods as observed in cca1 lhy double mutants. In addition, elongated hypocotyls and petioles and delayed flowering of CCA1α-overexpressing plants (35S:CCA1α) were rescued by coexpression of CCA1β. Interestingly, low temperatures suppress CCA1 alternative splicing and thus derepress the CCA1α activity in inducing cold tolerance. These observations indicate that a cold-responsive self-regulatory circuit of CCA1 plays a role in plant responses to low temperatures.

Beyond ubiquitination: proteolytic and nonproteolytic roles of HOS1

The E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) functions as a cold signaling attenuator by degrading the INDUCER OF CBF EXPRESSION 1 transcription factor, which is a key regulator of the cold-induced transcriptome and freezing tolerance in plants. Recent studies demonstrate that HOS1 also plays nonproteolytic roles in gene expression regulation. HOS1 acts as a chromatin remodeling factor that modulates FLOWERING LOCUS C chromatin in cold regulation of flowering time. It associates with the nuclear pore complex to facilitate nucleocytoplasmic mRNA export to maintain circadian periodicity over a range of light and temperature conditions. In this review, we summarize recent advances in molecular mechanisms underlying HOS1 function during plant development in response to fluctuating environmental conditions.

Controlled turnover of CONSTANS protein by the HOS1 E3 ligase regulates floral transition at low temperatures

The timing of flowering is coordinately regulated by complex gene regulatory networks that integrate developmental and environmental cues. Light and temperature are major environmental determinants in flowering time control. Temperature signals include two major categories: ambient temperature signals and cold nonfreezing temperature signals. Notably, the effects of cold temperatures on flowering timing are profoundly differentiated, depending on the duration of cold exposure. Whereas long-term exposure to cold temperatures, designated vernalization, promotes flowering, short-term cold exposure delays flowering. Genes constituting the vernalization pathway and underlying molecular mechanisms have been extensively studied. However, how cold stress signals delay flowering is largely unknown. We have recently reported that the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1)-CONSTANS (CO) module is at least partly responsible for the daily sensing of cold stress signals in flowering time control. Intermittent cold stress triggers the degradation of CO, a central activator of photoperiodic flowering, via a ubiquitination pathway that involves the HOS1 E3 ubiquitin ligase, leading to suppression of FLOWERING LOCUS T (FT) gene and delayed flowering. It is proposed that CO serves as a molecular knot that integrates photoperiod and temperature signals into the flowering pathways, fine-tuning photoperiodic flowering under short-term temperature fluctuations.

Alternative splicing provides a proactive mechanism for the diurnal CONSTANS dynamics in Arabidopsis photoperiodic flowering

The circadian clock control of CONSTANS (CO) transcription and the light-mediated stabilization of its encoded protein coordinately adjust photoperiodic flowering by triggering rhythmic expression of the floral integrator flowering locus T (FT). Diurnal accumulation of CO is modulated sequentially by distinct E3 ubiquitin ligases, allowing peak CO to occur in the late afternoon under long days. Here we show that CO abundance is not simply targeted by E3 enzymes but is also actively self-adjusted through dynamic interactions between two CO isoforms. Alternative splicing of CO produces two protein variants, the full-size COα and the truncated COβ lacking DNA-binding affinity. Notably, COβ, which is resistant to E3 enzymes, induces the interaction of COα with CO-destabilizing E3 enzymes but inhibits the association of COα with CO-stabilizing E3 ligase. These observations demonstrate that CO plays an active role in sustaining its diurnal accumulation dynamics during Arabidopsis photoperiodic flowering.