Dynamic dimer formation is an elaborate means of modulating transcription factor activities in diverse cellular processes. The basic helix-loop-helix (bHLH) transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1), for example, plays a role in plant photomorphogenesis by forming non-DNA binding heterodimers with PHYTOCHROME-INTERACTING FACTORS (PIFs). Recent studies have shown that a small HLH protein KIDARI (KDR) negatively regulates the HFR1 activity in the process. However, molecular mechanisms underlying the KDR control of the HFR1 activity are unknown. Here, we demonstrate that KDR attenuates the HFR1 activity by competitively forming nonfunctional heterodimers, causing liberation of PIF4 from the transcriptionally inactive HFR1-PIF4 complex. Accordingly, the photomorphogenic hypocotyl growth of the HFR1-overexpres-sing plants can be suppressed by KDR coexpression, as observed in the HFR1-deficient hfr1-201 mutant. These results indicate that the PIF4 activity is modulated through a double layer of competitive inhibition by HFR1 and KDR, which could in turn ensure fine-tuning of the PIF4 activity under fluctuating light conditions.
Arabidopsis; competitive inhibition; KIDARI (KDR); light signaling; LONG HYPOCOTYL IN FAR-RED 1 (HFR1); PHYTOCHROME-INTERACTING FACTOR (PIF)
Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata.
We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary.
Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.
LC-MS/MS; Nectar protein; Nectarin; Nicotiana attenuata
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.
Alternative splicing; Arabidopsis; Cold stress; Peptide interference (PEPi); Small interfering peptide (siPEP); Splicing factor; Transcription factor
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.
alternative splicing; Arabidopsis; CCA1; circadian clock; cold acclimation; freezing tolerance
A plant’s endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata.
Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed.
We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.
Circadian clock; Flowering time; NaLHY; NaTOC1; NaZTL; Nicotiana attenuata; Protein interaction
Floral transition is coordinately regulated by both endogenous and exogenous cues to ensure reproductive success under fluctuating environmental conditions. Abiotic stress conditions, including drought and high salinity, also have considerable influence on this developmental process. However, the signaling components and molecular mechanisms underlying the regulation of floral transition by environmental factors have not yet been defined. In this work, we show that the Arabidopsis BROTHER OF FT AND TFL1 (BFT) gene, which encodes a member of the FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, regulates floral transition under conditions of high salinity. The BFT gene was transcriptionally induced by high salinity in an abscisic acid (ABA)-dependent manner. Transgenic plants overexpressing the BFT gene (35S:BFT) and BFT-deficient mutant (bft-2) plants were phenotypically indistinguishable from Col-0 plants in seed germination and seedling growth under high salinity. In contrast, although the floral transition was delayed significantly in Col-0 plants under high salinity, that of the bft-2 mutant was not affected by high salinity. We also observed that expression of the APETALA1 (AP1) gene was suppressed to a lesser degree in the bft-2 mutant than in Col-0 plants. Taken together, our observations suggest that BFT mediates salt stress-responsive flowering, providing an adaptive strategy that ensures reproductive success under unfavorable stress conditions.
abscisic acid; Arabidopsis; BFT; flowering; salt stress
Plants possess versatile strategies that permit efficient use of limited nutrient resources during senescing process. This metabolic adjustment is critical for prevention of diverse cellular damage and thus for reproductive success and offspring production, particularly under environmental stress conditions. However, it is largely unknown how age-dependent resistance to cellular damages is established and how it is influenced by environmental stress signals during senescing process. We found that the VNI2 (VND-INTERACTING 2) transcription factor, which belongs to the NAC (NAM/ATAF1, 2/CUC2) transcription factor family, plays a role in the age-dependent induction of stress resistance. The VNI2 transcription factor is transcriptionally induced during senescing process and regulates COR/RD genes by binding directly to their promoters. The COR/RD proteins play a role in the protection from diverse cellular damages during senescing process. Notably, the transcriptional activation activity of VNI2 is further elevated under high salinity. These results indicate that plants increase environmental stress resistance by inducing the VNI2 gene to assure their reproductive success, supporting signaling crosstalk between stress resistance response and senescing process.
abscisic acid; arabidopsis; COR/RD; salt stress; senescence; VNI2
Plants have evolved diverse adaptive strategies to cope with drought or water deficit conditions, such as stomatal closure, maintenance of root growth and water uptake, and biosynthesis of osmoprotectants. Accumulation of cuticular waxes also contributes to drought resistance. However, it is still unclear how cuticular wax biosynthesis is regulated in response to drought and how it is associated with plant responses to drought at the molecular level. The abscisic acid (ABA)-inducible MYB96 transcription factor plays a role in drought resistance. Notably, it also regulates cuticular wax biosynthesis by binding directly to the promoters of genes encoding fatty acid elongating enzymes, such as KCS, KCR and ECR that constitute a rate-limiting step in cuticular wax biosynthesis. In the myb96-1D mutant that constitutively express the MYB96 gene, many of genes involved in cuticular wax biosynthesis are upregulated and accordingly, cuticular wax accumulation is greatly elevated. In contrast, cuticular wax accumulation is reduced in the myb96-1 mutant, linking drought with cuticular wax biosynthesis. It is evident that the MYB96 transcription factor incorporates drought stress signals into a gene regulatory network that modulates cuticular wax biosynthesis under drought stress conditions, providing a first molecular mechanism by which cuticular wax biosynthesis contributes to drought resistance and protection from pathogenic and mechanical damages as well.
abscisic acid; alkane biosynthesis; arabidopsis; cuticular wax; drought; MYB96
Lateral root formation is a critical agronomic trait in plant architecture that determines crop productivity and environmental stress adaptability. It is therefore tightly regulated both by intrinsic developmental cues, such as abscisic acid (ABA) and auxin, and by diverse environmental growth conditions, including water deficit and high salinity in the soil. We have recently reported that an Arabidopsis R2R3-type MYB transcription factor, MYB96, regulates lateral root meristem activation under drought conditions via an ABA-auxin signaling crosstalk. In this signaling scheme, the MYB96-mediated ABA signals are incorporated into an auxin signaling pathway that involves a subset of GH3 gene encoding auxin-conjugating enzymes. The MYB96-overexpressing, activation tagging mutant, which is featured by having dwarfed growth and reduced lateral root formation, exhibits an enhanced drought resistance. In the mutant, expression of the GH3 genes was significantly elevated, which is consistent with the reduced lateral root formation. In contrast, the MYB96-deficient knockout mutant produced more lateral roots and was more susceptible to drought stress. Our observations strongly support that MYB96 is a molecular link that integrates ABA and auxin signals in modulating auxin homeostasis during lateral root development, particularly under water deficit conditions. It is also envisioned that the MYB96-mediated signals are related with pathogen resistance response, which is also profoundly affected by water content in plant cells.
arabidopsis; abscisic acid; auxin homeostasis; lateral root; MYB; GH3; drought
The wild grass species Brachypodium distachyon (Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.
A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST) databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR) using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (UBC18) was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (Ubi4 and Ubi10) was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (SamDC) ranked was most stable in plants grown under various environmental stresses.
This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.
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.