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1.  HOS1-mediated activation of FLC via chromatin remodeling under cold stress 
Plant Signaling & Behavior  2013;8(12):e27342.
The Arabidopsis E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) has been shown to act as a negative regulator of cold responses by degrading the INDUCER OF CBF EXPRESSION 1 (ICE1) transcription factor through the ubiquitin/proteasome pathway. Notably, loss-of-function hos1 mutants exhibit early flowering, and the transcript level of the floral repressor FLOWERING LOCUS C (FLC) is downregulated in the mutants. However, it is largely unknown how HOS1 regulates FLC transcription. We found that HOS1 activates FLC transcription by inhibiting the activity of histone deacetylase 6 (HDA6) under cold stress. Cold temperatures induce the binding of HOS1 to FLC chromatin in an FVE-dependent manner. Cold-activated HOS1 promotes the dissociation of HDA6 from FLC chromatin, and the cold effects disappear in both hos1 and fve mutants. It is therefore clear that HOS1 regulates FLC transcription via chromatin remodeling, providing new insights into the signaling crosstalks between cold response and flowering time control.
PMCID: PMC4091338  PMID: 24390058
Arabidopsis; chromatin remodeling; cold stress; FLC; flowering time; FVE; HDA6; HOS1
2.  CCA1 alternative splicing as a way of linking the circadian clock to temperature response in Arabidopsis 
Plant Signaling & Behavior  2012;7(9):1194-1196.
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.
PMCID: PMC3489659  PMID: 22899064
alternative splicing; Arabidopsis; CCA1; circadian clock; cold acclimation; freezing tolerance
3.  Regulation of reactive oxygen species generation under drought conditions in Arabidopsis 
Plant Signaling & Behavior  2012;7(6):599-601.
Reactive oxygen species (ROS) are produced when plants are exposed to environmental stresses, such as drought and heat conditions. Oxidative stress imposed by ROS under drought conditions profoundly affects plant growth and development. However, ROS production and scavenging mechanisms under adverse environmental conditions are largely unknown. We have recently reported that a NAM/ATAF1/2/CUC2 (NAC) transcription factor NTL4 is required for generation of ROS under drought conditions in Arabidopsis. 35S:4ΔC transgenic plants overexpressing a truncated NTL4 form (4ΔC) lacking the C‑terminal transmembrane (TM) motif were hypersensitive to drought stress, and ROS accumulated to a high level in the transgenic plants. In contrast, NTL4-deficient ntl4 mutants were less sensitive to drought stress and contained reduced levels of ROS. Furthermore, the plasma membrane-associated NTL4 transcription factor is proteolytically activated by treatments with drought and abscisic acid (ABA) and nuclear-localized, where it induces expression of NADPH oxidase genes involved in ROS biosynthesis. Notably, the 35S:4ΔC transgenic plants showed accelerated leaf senescence and cell death under drought conditions. Taken together, these observations indicate that NTL4 regulation of ROS generation underlies the drought-induced leaf senescence.
PMCID: PMC3442848  PMID: 22580707
ABA; Arabidopsis; Drought stress; NAM/ATAF1/2/CUC2 (NAC); ROS; leaf senescence
4.  Signaling linkage between environmental stress resistance and leaf senescence in Arabidopsis 
Plant Signaling & Behavior  2011;6(10):1564-1566.
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.
PMCID: PMC3256385  PMID: 21921691
abscisic acid; arabidopsis; COR/RD; salt stress; senescence; VNI2
5.  Auxin modulation of salt stress signaling in Arabidopsis seed germination 
Plant Signaling & Behavior  2011;6(8):1198-1200.
Seed germination is an elaborate developmental process that is regulated through intricate signaling networks integrating diverse environmental cues into endogenous hormonal signaling pathways. Accumulating evidence in recent years supports the role of auxin in seed germination. Whereas the roles of gibberellic acid (GA) and abscisic acid (ABA) in the germination process have been studied extensively, how auxin modulates seed germination is largely unknown. We found that a membrane-bound NAC transcription factor NTM2 mediates the signaling crosstalk between auxin and salt stress via the IAA30 gene during seed germination in Arabidopsis. Germination of the NTM2-deficient ntm2-1 mutant seeds exhibited enhanced resistance to high salinity. However, the salt resistance was reduced in the ntm2-1 mutant overexpressing the IAA30 gene, which was induced by high salinity in a NTM2-dependent manner. Exogenous auxin treatment further suppressed the reduced germination rate of control seeds under high salinity. In contrast, the auxin effects disappeared in the ntm2-1 mutant. These observations indicate that NTM2 is a molecular link that incorporates auxin signal into salt stress signaling during seed germination, providing a role of auxin in modulating seed germination under high salinity.
PMCID: PMC3260721  PMID: 21757997
Arabidopsis; auxin; IAA30; NTM2; high salinity; MTF; seed germination
6.  Cuticular wax biosynthesis as a way of inducing drought resistance 
Plant Signaling & Behavior  2011;6(7):1043-1045.
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.
PMCID: PMC3257791  PMID: 21617381
abscisic acid; alkane biosynthesis; arabidopsis; cuticular wax; drought; MYB96
7.  Modulation of reactive oxygen species by salicylic acid in arabidopsis seed germination under high salinity 
Plant Signaling & Behavior  2010;5(12):1534-1536.
Potential roles of salicylic acid (SA) on seed germination have been explored in many plant species. However, it is still controversial how SA regulates seed germination, mainly because the results have been somewhat variable, depending on plant genotypes used and experimental conditions employed. We found that SA promotes seed germination under high salinity in Arabidopsis. Seed germination of the sid2 mutant, which has a defect in SA biosynthesis, is hypersensitive to high salinity, but the inhibitory effects are reduced in the presence of physiological concentrations of SA. Abiotic stresses, including high salinity, impose oxidative stress on plants. Endogenous contents of H2O2 are higher in the sid2 mutant seeds. However, exogenous application of SA reduces endogenous level of reactive oxygen species (ROS), indicating that SA is involved in plant responses to ROS-mediated damage under abiotic stress conditions. Gibberellic acid (GA), a plant hormone closely associated with seed germination, also reverses the inhibitory effects of high salinity on seed germination and seedling establishment. Under high salinity, GA stimulates SA biosynthesis by inducing the SID2 gene. Notably, SA also induces genes encoding GA biosynthetic enzymes. These observations indicate that SA promotes seed germination under high salinity by modulating antioxidant activity through signaling crosstalks with GA.
PMCID: PMC3115096  PMID: 21150285
arabidopsis; gibberellic acid; reactive oxygen species; salicylic acid; salt stress; seed germination
8.  Auxin homeostasis during lateral root development under drought condition 
Plant Signaling & Behavior  2009;4(10):1002-1004.
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.
PMCID: PMC2801374  PMID: 19826230
arabidopsis; abscisic acid; auxin homeostasis; lateral root; MYB; GH3; drought
9.  Gibberellic acid-mediated salt signaling in seed germination 
Plant Signaling & Behavior  2008;3(10):877-879.
Seed germination initiates the postembryonic development of plants, which determines successful seedling establishment and plant propagation. It is therefore tightly regulated by diverse environmental conditions, including high salinity and drought, as well as by intrinsic developmental programs, among which gibberellic acid (GA) is best understood. Regulatory roles of GA in seed germination have been extensively studied. It is also known that high salinity inhibits germination by repressing genes encoding GA biosynthetic enzymes. However, it is still unclear how salt signals are coordinately incorporated into the GA signaling pathway at the molecular level. We recently demonstrated that a membrane-bound NAC transcription factor, NTL8, mediates salt signaling, primarily through a RGL2-independent GA pathway, in regulating seed germination. High salinity promotes NTL8 transcription and proteolytic activation of NTL8. Notably, the NTL8-mediated salt signaling is independent of abscisic acid (ABA). These observations indicate that membrane-mediated transcription control is an important component of salt signaling during seed germination.
PMCID: PMC2634403  PMID: 19704528
Arabidopsis; gibberellic acid (GA); salt stress; membrane-bound transcription factor; NAC; NTL8; RGL2
10.  Small interfering peptides as a novel way of transcriptional control 
Plant Signaling & Behavior  2008;3(9):615-617.
Transcription factors are key components of transcriptional regulatory networks governing virtually all aspects of plant growth and developmental processes. Their activities are regulated at various steps, including gene transcription, posttranscriptional mRNA metabolism, posttranslational modifications, nucleocytoplasmic transport, and controlled proteolytic cleavage of membrane-anchored, dormant forms. Dynamic protein dimerization also plays a critical role in this process. An exquisite regulatory scheme has recently been proposed to modulate the action of transcription factors. Small peptides possessing a protein dimerization motif but lacking the DNA-binding motif form nonfunctional heterodimers with a group of specific TFs, inhibiting their transcriptional activation activities. Extensive searches for small proteins that have a similar structural organization in the databases revealed that small peptide-mediated transcription control is not an exceptional case but would be a regulatory mechanism occurring widespread in the Arabidopsis genome.
PMCID: PMC2634540  PMID: 19513250
Arabidopsis; flowering time; HD-ZIP III; homodimer; transcription factor; ZPR
11.  Membrane-Mediated Salt Stress Signaling in Flowering Time Control 
Plant Signaling & Behavior  2007;2(6):517-518.
More than 10% of the plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factors have been predicted to have alpha-helical transmembrane (TM) domain in their C-terminal regions, among which at least three members have been proven to be membrane-associated and play a role in cell cycle control and stress responses. These observations suggest that membrane-mediated regulation would be an important molecular mechanism mediating rapid transcriptional responses to internal and external stimuli in plants. Recently, we showed that a salt-responsive NTL (NTM1-Like's) transcription factor NTL8 is localized primarily in plasma membranes as dormant form and subsequently processed into transcriptionally active, nuclear form. Overexpression of an active NTL8 form exhibited delayed flowering as well as reduced growth with small curled leaves. Consistent with this, expression of FLOWERING LOCUS T (FT) and its downstream genes was significantly reduced in the transgenic plants. Furthermore, FT was notably repressed by high salt. These results indicate that NTL8 mediates salt-responsive flowering via FT in Arabidopsis and that membrane-mediated transcription regulation underlies the salt signaling in mediating flowering initiation.
PMCID: PMC2634355  PMID: 19704545
Arabidopsis; flowering time; flowering locus T (FT); membrane-bound transcription factor; NAC; salt stress
12.  Auxin Homeostasis in Plant Stress Adaptation Response 
Plant Signaling & Behavior  2007;2(4):306-307.
Auxin plays a wide range of regulatory roles in diverse aspects of plant growth and developmental processes through a complex network of signaling interactions. In the May issue of Journal of Biological Chemistry, we have demonstrated that auxin homeostasis directly links growth regulation with stress adaptation responses through interactions with salicylic acid (SA) and abscisic acid (ABA) signals. In this signaling network, the endogenous auxin content is coordinately regulated through negative feedback by a group of auxin-inducible GH3 genes that encode auxin-conjugating enzymes. The Arabidopsis mutant wes1-D overexpressing a GH3 gene WES1 exhibits typical auxin-deficient traits, such as reduced growth and leaf curling, but is resistant to both biotic and abiotic stresses. In addition, various stress-regulated genes, including pathogenesis- related protein genes (PRs) and C-repeat/dehydration responsive element binding factor genes (CBFs), are up-regulated in the mutant. Consistent with these observations, WES1 is activated by pathogenic infections and abiotic stresses as well as by exogenous SA and ABA. We therefore propose that the WES1-mediated growth suppression would underlie the commonly observed symptoms of infected or stressed plants and provide a mechanism for auxin action in the fitness costs of induced resistance in plants.
PMCID: PMC2634159  PMID: 19704630
Arabidopsis; abscisic acid; auxin homeostasis; GH3; light; salicylic acid; stress adaptation
13.  Membrane Regulation of Cytokinin-Mediated Cell Division in Arabidopsis 
Plant Signaling & Behavior  2007;2(1):15-16.
Controlled proteolytic activation of membrane-bound transcription factors (MTFs) is an efficient adaptation strategy that ensures prompt transcriptional responses to intrinsic and environmental changes in eukaryotes. The proteolytic processing liberates active transcription factors from the membranes, which subsequently enter the nucleus and turn on downstream target genes. In the December issue of Plant Cell, we have demonstrated that an Arabidopsis membrane-bound NAC transcription factor, designated NTM1, is activated by proteolytic cleavage through regulated intramembrane proteolysis (RIP). The transcriptionally active NTM1 form induces a subset of CDK inhibitor genes (KRPs), resulting in reduced cell division. We have also shown that cytokinins regulate NTM1 activity by modulating its protein stability via an ubiquitin (Ub)-mediated protein degradation pathway, defining a unique molecular scheme by which cytokinins regulate cell division. It is thus envisioned that both positive and negative signaling components would be required for tight control of cell cycling by cytokinins. In this addendum, we propose a working hypothesis in which environmental stresses affect cell division by regulating NTM1 expression or NTM1 processing step.
PMCID: PMC2633889  PMID: 19704799
Arabidopsis; cell division; cytokinins; membrane-bound transcription factor; NAC; regulated intramembrane proteolysis
14.  Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis 
BMC Plant Biology  2014;14:136.
The 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.
We 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.
Taken 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.
PMCID: PMC4035800  PMID: 24885185
Arabidopsis thaliana; Circadian clock; Transcription factor; Alternative splicing; Nonsense-mediated decay (NMD); Environmental stress
15.  A Competitive Peptide Inhibitor KIDARI Negatively Regulates HFR1 by Forming Nonfunctional Heterodimers in Arabidopsis Photomorphogenesis 
Molecules and Cells  2012;35(1):25-31.
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.
PMCID: PMC3887847  PMID: 23224238
Arabidopsis; competitive inhibition; KIDARI (KDR); light signaling; LONG HYPOCOTYL IN FAR-RED 1 (HFR1); PHYTOCHROME-INTERACTING FACTOR (PIF)
16.  Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon 
BMC Plant Biology  2014;14:15.
Adverse environmental conditions severely influence various aspects of plant growth and developmental processes, causing worldwide reduction of crop yields. The C-repeat binding factors (CBFs) are critical transcription factors constituting the gene regulatory network that mediates the acclimation process to low temperatures. They regulate a large number of cold-responsive genes, including COLD-REGULATED (COR) genes, via the CBF-COR regulon. Recent studies have shown that the CBF transcription factors also play a role in plant responses to drought and salt stresses. Putative CBF gene homologues and their downstream genes are also present in the genome of Brachypodium distachyon, which is perceived as a monocot model in recent years. However, they have not been functionally characterized at the molecular level.
Three CBF genes that are responsive to cold were identified from Brachypodium, designated BdCBF1, BdCBF2, and BdCBF3, and they were functionally characterized by molecular biological and transgenic approaches in Brachypodium and Arabidopsis thaliana. Our results demonstrate that the BdCBF genes contribute to the tolerance response of Brachypodium to cold, drought, and salt stresses by regulating downstream targets, such as DEHYDRIN5.1 (Dhn5.1) and COR genes. The BdCBF genes are induced under the environmental stress conditions. The BdCBF proteins possess transcriptional activation activity and bind directly to the promoters of the target genes. Transgenic Brachypodium plants overexpressing the BdCBF genes exhibited enhanced resistance to drought and salt stresses as well as low temperatures, and accordingly endogenous contents of proline and soluble sugars were significantly elevated in the transgenic plants. The BdCBF transcription factors are also functional in the heterologous system Arabidopsis. Transgenic Arabidopsis plants overexpressing the BdCBF genes were also tolerant to freezing, drought, and salt stresses, and a set of stress-responsive genes was upregulated in the transgenic Arabidopsis plants.
Taken together, our results strongly support that the BdCBF transcription factors are key regulators of cold stress responses in Brachypodium and the CBF-mediated cold stress signaling pathway is conserved in this plant species. We believe that this study would confer great impact on stress biology in monocot species and could be applied to engineer abiotic stress tolerance of bioenergy grass species.
PMCID: PMC3898008  PMID: 24405987
Brachypodium distachyon; C-repeat binding factor (CBF); COLD-REGULATED (COR); Abiotic stress tolerance; Arabidopsis thaliana
17.  Natural variation in floral nectar proteins of two Nicotiana attenuata accessions 
BMC Plant Biology  2013;13:101.
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.
PMCID: PMC3728157  PMID: 23848992
LC-MS/MS; Nectar protein; Nectarin; Nicotiana attenuata
18.  Alternative splicing of transcription factors in plant responses to low temperature stress: mechanisms and functions 
Planta  2013;237(6):1415-1424.
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.
PMCID: PMC3664756  PMID: 23624977
Alternative splicing; Arabidopsis; Cold stress; Peptide interference (PEPi); Small interfering peptide (siPEP); Splicing factor; Transcription factor
19.  The Floral Repressor BROTHER OF FT AND TFL1 (BFT) Modulates Flowering Initiation under High Salinity in Arabidopsis 
Molecules and Cells  2011;32(3):295-303.
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.
PMCID: PMC3887636  PMID: 21809215
abscisic acid; Arabidopsis; BFT; flowering; salt stress
20.  Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata 
BMC Plant Biology  2012;12:172.
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.
PMCID: PMC3489836  PMID: 23006446
Circadian clock; Flowering time; NaLHY; NaTOC1; NaZTL; Nicotiana attenuata; Protein interaction
21.  Activation of a Mitochondrial ATPase Gene Induces Abnormal Seed Development in Arabidopsis 
Molecules and Cells  2011;31(4):361-369.
The ATPases associated with various cellular activities (AAA) proteins are widespread in living organisms. Some of the AAA-type ATPases possess metalloprotease activities. Other members constitute the 26S proteasome complexes. In recent years, a few AAA members have been implicated in vesicle-mediated secretion, membrane fusion, cellular organelle biogenesis, and hypersensitive responses (HR) in plants. However, the physiological roles and biochemical activities of plant AAA proteins have not yet been defined at the molecular level, and regulatory mechanisms underlying their functions are largely unknown. In this study, we showed that overexpression of an Arabidopsis gene encoding a mitochondrial AAA protein, ATPase-in-Seed-Development (ASD), induces morphological and anatomical defects in seed maturation. The ASD gene is expressed at a high level during the seed maturation process and in mature seeds but is repressed rapidly in germinating seeds. Transgenic plants overexpressing the ASD gene are morphologically normal. However, seed formation is severely disrupted in the transgenic plants. The ASD gene is induced by abiotic stresses, such as low temperatures and high salinity, in an abscisic acid (ABA)- dependent manner. The ASD protein possesses ATPase activity and is localized into the mitochondria. Our observations suggest that ASD may play a role in seed maturation by influencing mitochondrial function under abiotic stress.
PMCID: PMC3933970  PMID: 21359673
AAA-type ATPase; abiotic stress; abscisic acid; Arabidopsis; seed development
22.  Nuclear Import and DNA Binding of the ZHD5 Transcription Factor Is Modulated by a Competitive Peptide Inhibitor in Arabidopsis* 
The Journal of Biological Chemistry  2010;286(2):1659-1668.
Competitive inhibition of transcription factors by small proteins is an intriguing component of gene regulatory networks in both animals and plants. The small interfering proteins possess limited sequence homologies to specific transcription factors but lack one or more protein motifs required for transcription factor activities. They interfere with the activities of transcription factors, such as DNA binding and transcriptional activation, by forming nonfunctional heterodimers. A potential example is the Arabidopsis MIF1 (mini zinc finger 1) protein consisting of 101 residues. It has a zinc finger domain but lacks other protein motifs normally present in transcription factors. In this work, we show that MIF1 and its functional homologues physically interact with a group of zinc finger homeodomain (ZHD) transcription factors, such as ZHD5, that regulate floral architecture and leaf development. Gel mobility shift assays revealed that MIF1 blocks the DNA binding activity of ZHD5 homodimers by competitively forming MIF1-ZHD5 heterodimers. Accordingly, the transcriptional activation activity of ZHD5 was significantly suppressed by MIF1 coexpressed transiently in Arabidopsis protoplasts. Notably, MIF1 also prevents ZHD5 from nuclear localization. Although ZHD5 was localized exclusively in the nucleus, it was scattered throughout the cytoplasm when MIF1 was coexpressed. Transgenic plants overexpressing the ZHD5 gene (35S:ZHD5) exhibited accelerated growth with larger leaves. Consistent with the negative regulation of ZHD5 by MIF1, the 35S:ZHD5 phenotypes were diminished by MIF1 coexpression. These observations indicate that MIF1 regulates the ZHD5 activities in a dual step manner: nuclear import and DNA binding.
PMCID: PMC3020774  PMID: 21059647
Arabidopsis; DNA-binding Protein; Gene Expression; Plant; Transcription Factors; Zinc Finger
23.  Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR 
BMC Plant Biology  2008;8:112.
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.
PMCID: PMC2588586  PMID: 18992143
24.  microRNA-directed cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems 
Class 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.
PMCID: PMC1382282  PMID: 15773855
Arabidopsis; ATHB15; HD-ZIP; microRNA; mRNA cleavage; vascular development
25.  Exploring membrane-associated NAC transcription factors in Arabidopsis: implications for membrane biology in genome regulation 
Nucleic Acids Research  2006;35(1):203-213.
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.
PMCID: PMC1802569  PMID: 17158162

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