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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background

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.

Results

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.

Conclusions

We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

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.

Background

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.

Results

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.

Conclusion

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.

Summary

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.

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.