Arabidopsis Transcription Research Articles

DNA recognition by Arabidopsis transcription factors ABI3 and NGA1

B3 transcription factors constitute a large plant-specific protein superfamily, which plays a central role in plant life. Family members are characterized by the presence of B3 DNA-binding domains (DBDs). To date, only a few B3 DBDs were structurally characterized; therefore, the DNA recognition mechanism of other family members remains to be elucidated. Here, we analyze DNA recognition mechanism of two structurally uncharacterized B3 transcription factors, ABI3 and NGA1. Guided by the structure of the DNA-bound B3 domain of Arabidopsis transcriptional repressor VAL1, we have performed mutational analysis of the ABI3 B3 domain. We demonstrate that both VAL1-B3 and ABI3-B3 recognize the Sph/RY DNA sequence 5'-TGCATG-3' via a conserved set of base-specific contacts. We have also solved a 1.8 Å apo-structure of NGA1-B3, DBD of Arabidopsis transcription factor NGA1. We show that NGA1-B3, like the structurally related RAV1-B3 domain, is specific for the 5'-CACCTG-3' DNA sequence, albeit tolerates single base pair substitutions at the 5'-terminal half of the recognition site. Employing distance-dependent fluorophore quenching, we show that NGA1-B3 binds the asymmetric recognition site in a defined orientation, with the 'N-arm' and 'C-arm' structural elements interacting with the 5'- and 3'-terminal nucleotides of the 5'-CACCTG-3' sequence, respectively. Mutational analysis guided by the model of DNA-bound NGA1-B3 helped us identify NGA1-B3 residues involved in base-specific and DNA backbone contacts, providing new insights into the mechanism of DNA recognition by plant B3 domains of RAV and REM families. DATABASES: RCSB Protein Data Bank, accession number 5OS9.

Arabidopsis Transcription Factor MYB102 Increases Plant Susceptibility to Aphids by Substantial Activation of Ethylene Biosynthesis.

Induction of ethylene biosynthesis by aphids increases the susceptibility of several plant species to aphids. Recent studies have indicated that some MYB transcription factors regulate the phloem-based defense against aphid infestation by modulating ethylene (ET) signaling. Arabidopsis MYB102 has previously been shown to be induced by wound signaling and regulate defense response against chewing insects. However, it remains unclear whether Arabidopsis MYB102 takes part in the defense response of plants to aphids. Here, we investigated the function of MYB102 in the response of Arabidopsis to aphid infestation. Arabidopsis MYB102 was primarily expressed in vascular tissues, and its transcription was remarkably induced by green peach aphids (GPA; Myzus persicae). The results of RNA-Sequencing revealed that overexpression of MYB102 in Arabidopsis promoted ET biosynthesis by upregulation of some 1-aminocyclopropane-1-carboxylate synthase (ACS) genes, which are rate-limiting enzymes of the ET-synthetic pathway. Enhanced ET levels led to reduced Arabidopsis resistance to GPA. Furthermore, dominant suppression of MYB102 inhibited aphid-induced increase of ET levels in Arabidopsis. In agreement with a negative regulatory role for ET in aphid defense responses, the MYB102-overexpressing lines were more susceptible to GPA than wild-type (WT) plants. Overexpression of MYB102 in Arabidopsis obviously repressed aphid-induced callose deposition. Conversely, overexpression of MYB102 failed to increase aphid susceptibility in both the ET-insensitive mutants and plants treated with inhibitors of ET signaling pathways, demonstrating that the ET was critical for promoting aphid performance conferred by overexpression of MYB102. Collectively, our findings indicate that the Arabidopsis MYB102 increases host susceptibility to GPA through the ET-dependent signaling pathways.

A Dual Repeat Cis-Element Determines Expression of GERANYL DIPHOSPHATE SYNTHASE for Monoterpene Production in Phalaenopsis Orchids.

Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs) cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.

Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis.

SummaryThe flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5‐like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5‐like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1‐amino‐cyclopropane‐1‐carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation–quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5‐like TFs in the regulation of ethylene‐mediated petal development and growth.

H2A.Z-containing nucleosomes are evicted to activate AtMYB44 transcription in response to salt stress

Transcripts of the Arabidopsis transcription factor gene, AtMYB44, accumulate rapidly to mediate a tolerance mechanism in response to salt stress. The AtMYB44 promoter is activated by salt stress, as illustrated in AtMYB44pro::GUS transgenic plants. Chromatin immunoprecipitation (ChIP) assays revealed that RNA polymerases were enriched on the AtMYB44 gene, especially on TSS-proximal regions, and nucleosome density was markedly reduced in the AtMYB44 gene-body region in response to salt stress. In addition, H2A.Z occupation was significantly decreased at the AtMYB44 promoter, transcription start site (TSS), and gene-body regions. Histone modifications including histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 and H4 acetylation (H3ac and H4ac) were not affected under the same stress conditions. We found a decrease in the number of AtMYB44 proteins bound to their own gene promoters in response to salt stress. These results suggest that salt stress induces the eviction of H2A.Z-containing nucleosomes from the AtMYB44 promoter region, which may weaken its affinity for binding AtMYB44 protein that acts as a repressor for AtMYB44 gene transcription under salt stress-free conditions.

Characterization and transcriptional regulation of chlorophyll b reductase gene NON-YELLOW COLORING 1 associated with leaf senescence in perennial ryegrass (Lolium perenne L.)

Chlorophyll (Chl) degradation leads to leaf senescence and adversely affects biomass production of forage grasses and aesthetic appearance of turfgrasses. The objectives of this study were to characterize the function of a Chl catabolic gene, NON-YELLOW COLORING 1 (LpNYC1), and to understand its transcriptional regulatory pattern involved in leaf senescence in perennial ryegrass (Lolium perenne L.). The LpNYC1 was initially cloned using the RACE-PCR method. Its encoded protein was localized in chloroplasts and its expression pattern was correlated with the progression of leaf senescence. LpNYC1 shared the same biological function with Arabidopsis NYC1, as overexpression of its encoding gene accelerated chlorophyll degradation in Nicotiana benthamiana and rescued the stay-green phenotype of the Arabidopsis nyc1 null mutant. A yeast one-hybrid cDNA library was prepared from senescent leaves of perennial ryegrass. Using the LpNYC1 promoter as bait, five putative transcription factors upstream of LpNYC1 were pooled out using this method, among which three (LpABI5, LpABF3, and LpEIN3) were orthologous to Arabidopsis transcription factors involved in the ABA and ethylene signaling pathways. The expression of LpNYC1 was highly inducible by ABA and ethephon (ethylene releasing reagent) but was suppressed by treatment with AVG (ethylene biosynthesis inhibitor). Furthermore, LpABI5, LpABF3, LpEIN3 directly activated the expression of LpNYC1 by binding to its promoter. The current result laid the groundwork for future in-depth analysis of the molecular regulation of LpNYC1 and Chl metabolism during leaf senescence in perennial grass species.

UVR8 Interacts with BES1 and BIM1 to Regulate Transcription and Photomorphogenesis in Arabidopsis

UV-B light (UV-B radiation) is known to inhibit plant growth, but the mechanism is not well understood. UVR8 (UV RESISTANCE LOCUS 8) is a UV-B light photoreceptor that mediates UV-B light responses in plants. We report here that UV-B inhibits plant growth by repressing plant steroid hormone brassinosteroid (BR)-promoted plant growth. UVR8 physically interacts with the functional dephosphorylated BES1 (BRI1-EMS-SUPPRESSOR1) and BIM1 (BES1-INTERACTING MYC-LIKE 1) transcription factors that mediate BR-regulated gene expression and plant growth to inhibit their activities. Genome-wide gene expression analysis defined a BES1-dependent UV-B-regulated transcriptome, which is enriched with genes involved in cell elongation and plant growth. We further showed that UV-B-activated and nucleus-localized UVR8 inhibited the DNA-bindingactivities of BES1/BIM1 to directly regulate transcription of growth-related genes. Our results therefore establish that UVR8-BES1/BIM1 interaction represents an early photoreceptor signaling mechanism in plants and serves as an important module integrating light and BR signaling.

UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in Arabidopsis

UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UVB) radiation photoreceptor that mediates light responses in plants. How plant UVR8 acts in response to UVB light is not well understood. Here, we report the identification and characterization of the Arabidopsis WRKY DNA-BINDING PROTEIN 36 (WRKY36) protein. WRKY36 interacts with UVR8 in yeast and Arabidopsis cells and it promotes hypocotyl elongation by inhibiting HY5 transcription. Inhibition of hypocotyl elongation under UVB requires the inhibition of WRKY36. WRKY36 binds to the W-box motif of the HY5 promoter to inhibit its transcription, while nuclear localized UVR8 directly interacts with WRKY36 to inhibit WRKY36-DNA binding both in vitro and in vivo, leading to the release of inhibition of HY5 transcription. These results indicate that WRKY36 is a negative regulator of HY5 and that UVB represses WRKY36 via UVR8 to promote the transcription of HY5 and photomorphogenesis. The UVR8-WRKY36 interaction in the nucleus represents a novel mechanism of early UVR8 signal transduction in Arabidopsis.

Transcriptome profile of NO-induced Arabidopsis transcription factor genes suggests their putative regulatory role in multiple biological processes

TFs are important proteins regulating plant responses during environmental stresses. These insults typically induce changes in cellular redox tone driven in part by promoting the production of reactive nitrogen species (RNS). The main source of these RNS is nitric oxide (NO), which serves as a signalling molecule, eliciting defence and resistance responses. To understand how these signalling molecules regulate key biological processes, we performed a large scale S-nitrosocysteine (CySNO)-mediated RNA-seq analysis. The DEGs were analysed to identify potential regulatory TFs. We found a total of 673 (up- and down-regulated) TFs representing a broad range of TF families. GO-enrichment and MapMan analysis suggests that more than 98% of TFs were mapped to the Arabidopsis thaliana genome and classified into pathways like hormone signalling, protein degradation, development, biotic and abiotic stress, etc. A functional analysis of three randomly selected TFs, DDF1, RAP2.6, and AtMYB48 identified a regulatory role in plant growth and immunity. Loss-of-function mutations within DDF1 and RAP2.6 showed compromised basal defence and effector triggered immunity, suggesting their positive role in two major plant defence systems. Together, these results imply an important data representing NO-responsive TFs that will help in exploring the core mechanisms involved in biological processes in plants.

Construction of Arabidopsis Transcription Factor ORFeome Collections and Identification of Protein-DNA Interactions by High-Throughput Yeast One-Hybrid Screens.

Identification of transcription factor (TF)-promoter interactions is key to understanding the basic molecular underpinnings of gene regulation. The complexity of gene regulation, however, is driven by the combined function of several TFs recruited to the promoter region, which often confounds the discovery of transcriptional regulatory mechanisms. Genome sequencing enabled the construction of TF-specific ORFeome clone collections that can be used to study TF function with unprecedented coverage. Among the recently developed methods, gene-centered yeast one-hybrid (Y1H) screens performed with these ORFeome collections provide a simple and reliable strategy to identify TF-promoter interactions. Here, we describe high-throughput cloning protocols used to generate a gold standard TF ORFeome collection for the model organism Arabidopsis thaliana. Furthermore, we outline the protocol to build a daughter clone collection suitable for the Y1H assay and a high-throughput Y1H screening procedure that enables rapid assessment of thousands TF-promoter interactions using a robotic platform. These protocols can be universally adopted to build ORFeome libraries and thus expand the usage of gene-centered Y1H screens or other alternative strategies for discovery and characterization of TF functions.

Generation of Inducible Transgenic Lines of Arabidopsis Transcription Factors Regulated by MicroRNAs.

Transcription factors play key regulatory roles in all the life processes across kingdoms. In plants, the genome of a typical model species such as Arabidopsis thaliana encodes over 1500 transcription factors that regulate the expression dynamics of all the genes in time and space. Therefore, studying their function by analyzing the loss and gain-of-function lines is of prime importance in basic plant biology and its agricultural application. However, the current approach of knocking out genes often causes embryonic lethal phenotype, while inactivating one or two members of a redundant gene family yields little phenotypic changes, thereby making the functional analysis a technically challenging task. In such cases, inducible knock-down or overexpression of transcription factors appears to be a more effective approach. Restricting the transcription factors in the cytoplasm by fusing them with animal glucocorticoid/estrogen receptors (GR/ER) and then re-localizing them to the nucleus by external application of animal hormone analogues has been a useful method of gene function analysis in the model plants. In this chapter, we describe the recent advancements in the GR and ER expression systems and their use in analyzing the function of transcription factors in Arabidopsis.

KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis

Germ-line specification is essential for sexual reproduction. In the ovules of most flowering plants, only a single hypodermal cell enlarges and differentiates into a megaspore mother cell (MMC), the founder cell of the female germ-line lineage. The molecular mechanisms restricting MMC specification to a single cell remain elusive. We show that the Arabidopsis transcription factor WRKY28 is exclusively expressed in hypodermal somatic cells surrounding the MMC and is required to repress these cells from acquiring MMC-like cell identity. In this process, the SWR1 chromatin remodeling complex mediates the incorporation of the histone variant H2A.Z at the WRKY28 locus. Moreover, the cytochrome P450 gene KLU, expressed in inner integument primordia, non-cell-autonomously promotes WRKY28 expression through H2A.Z deposition at WRKY28. Taken together, our findings show how somatic cells in ovule primordia cooperatively use chromatin remodeling to restrict germ-line cell specification to a single cell.

The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes.

Agrobacterium-mediated genetic transformation not only represents a technology of choice to genetically manipulate plants, but it also serves as a model system to study mechanisms employed by invading pathogens to counter the myriad defenses mounted against them by the host cell. Here, we uncover a new layer of plant defenses that is targeted by A. tumefaciens to facilitate infection. We show that the Agrobacterium F-box effector VirF, which is exported into the host cell, recognizes an Arabidopsis transcription factor VFP4 and targets it for proteasomal degradation. We hypothesize that VFP4 resists Agrobacterium infection and that the bacterium utilizes its VirF effector to degrade VFP4 and thereby mitigate the VFP4-based defense. Indeed, loss-of-function mutations in VFP4 resulted in differential expression of numerous biotic stress-response genes, suggesting that one of the functions of VFP4 is to control a spectrum of plant defenses, including those against Agrobacterium tumefaciens. We identified one such gene, ATL31, known to mediate resistance to bacterial pathogens. ATL31 was transcriptionally repressed in VFP4 loss-of-function plants and activated in VFP4 gain-of-function plants. Gain-of-function lines of VFP4 and ATL31 exhibited recalcitrance to Agrobacterium tumorigenicity, suggesting that A. tumefaciens may utilize the host ubiquitin/proteasome system to destabilize transcriptional regulators of the host disease response machinery.

From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress

Plants have established different mechanisms to cope with environmental fluctuations and accordingly fine‐tune their growth and development through the regulation of complex molecular networks. It is largely unknown how the network architectures change and what the key regulators in stress responses and plant growth are. Here, we investigated a complex, highly interconnected network of 20 Arabidopsis transcription factors (TFs) at the basis of leaf growth inhibition upon mild osmotic stress. We tracked the dynamic behavior of the stress‐responsive TFs over time, showing the rapid induction following stress treatment, specifically in growing leaves. The connections between the TFs were uncovered using inducible overexpression lines and were validated with transient expression assays. This study resulted in the identification of a core network, composed of ERF6, ERF8, ERF9, ERF59, and ERF98, which is responsible for most transcriptional connections. The analyses highlight the biological function of this core network in environmental adaptation and its redundancy. Finally, a phenotypic analysis of loss‐of‐function and gain‐of‐function lines of the transcription factors established multiple connections between the stress‐responsive network and leaf growth.

A compendium of transcription factor and Transcriptionally active protein coding gene families in cowpea (Vigna unguiculata L.)

BackgroundCowpea (Vigna unguiculata (L.) Walp.) is the most important food and forage legume in the semi-arid tropics of sub-Saharan Africa where approximately 80% of worldwide production takes place primarily on low-input, subsistence farm sites. Among the major goals of cowpea breeding and improvement programs are the rapid manipulation of agronomic traits for seed size and quality and improved resistance to abiotic and biotic stresses to enhance productivity. Knowing the suite of transcription factors (TFs) and transcriptionally active proteins (TAPs) that control various critical plant cellular processes would contribute tremendously to these improvement aims.ResultsWe used a computational approach that employed three different predictive pipelines to data mine the cowpea genome and identified over 4400 genes representing 136 different TF and TAP families. We compare the information content of cowpea to two evolutionarily close species common bean (Phaseolus vulgaris), and soybean (Glycine max) to gauge the relative informational content. Our data indicate that correcting for genome size cowpea has fewer TF and TAP genes than common bean (4408 / 5291) and soybean (4408/ 11,065). Members of the GROWTH-REGULATING FACTOR (GRF) and Auxin/indole-3-acetic acid (Aux/IAA) gene families appear to be over-represented in the genome relative to common bean and soybean, whereas members of the MADS (Minichromosome maintenance deficient 1 (MCM1), AGAMOUS, DEFICIENS, and serum response factor (SRF)) and C2C2-YABBY appear to be under-represented. Analysis of the AP2-EREBP APETALA2-Ethylene Responsive Element Binding Protein (AP2-EREBP), NAC (NAM (no apical meristem), ATAF1, 2 (Arabidopsis transcription activation factor), CUC (cup-shaped cotyledon)), and WRKY families, known to be important in defense signaling, revealed changes and phylogenetic rearrangements relative to common bean and soybean that suggest these groups may have evolved different functions.ConclusionsThe availability of detailed information on the coding capacity of the cowpea genome and in particular the various TF and TAP gene families will facilitate future comparative analysis and development of strategies for controlling growth, differentiation, and abiotic and biotic stress resistances of cowpea.

Three stress-responsive NAC transcription factors from Populus euphratica differentially regulate salt and drought tolerance in transgenic plants.

Stress-responsive NAM, Arabidopsis transcription activation factor 1/2 (ATAF1/2) and CUC2 (SNAC) genes are being used to alter stress tolerance in Arabidopsis or grasses through genetic engineering. However, limited reports are available about the functional characteristics of SNAC in trees. In this study, three putative NAC proteins were identified from Populus euphratica. PeNAC034 and PeNAC045 were classified into the ATAF subgroup and PeNAC036 into the ANAC072 subgroup. These three SNAC transcription factors were localized in the nucleus and contained the transcription activation domain in their C-terminal. Under drought and salt stresses, PeNAC036 was strongly induced in the whole plant, but PeNAC034 was significantly suppressed in the roots and stems, and PeNAC045 was inhibited in the roots. PeNAC036 overexpression in Arabidopsis wild-type (WT) (OEPeNAC036) and PeNAC036 complementation in mutant anac072 (anac072/PeNAC036) lines increased tolerance to salt and drought, whereas PeNAC034 overexpression in WT (OEPeNAC034) and PeNAC034 complementation in mutant ataf1 (ataf1/PeNAC034) lines enhanced salt and drought sensitivity. After drought and salt treatments, the expression levels of COR47, RD29B, ERD11, RD22 and DREB2A were upregulated in OEPeNAC036 and anac072/PeNAC036 lines, but were downregulated in OEPeNAC034 and ataf1/PeNAC034 plants. Compared with WT and Vector lines, PeNAC045 overexpression in poplar WT (OEPeNAC045) led to a significant decrease in the net photosynthesis rate, stomatal conductance and transpiration rate under salinity and drought conditions. These results suggest that P. euphratica can adapt to the environment of high salinity and drought, which may be related to the differential expression patterns of SNAC genes.

Positive regulation of AMS by TDF1 and the formation of a TDF1-AMS complex are required for anther development in Arabidopsis thaliana.

Tapetum development and pollen production are regulated by a complex transcriptional network that consists of a group of tapetum-specific Arabidopsis transcription factors (TFs). Among these TFs, DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) encodes an R2R3 MYB factor, and ABORTED MICROSPORE (AMS) encodes a basic helix-loop-helix (bHLH) factor. However, knowledge regarding the regulatory role of TDF1 in anther development remains limited. Here, we discovered that TDF1 directly regulates AMS via an AACCT cis-element. We found the precocious AMS transcript and absence of AMS protein in ams-/- gpTDF1:AMS-FLAG lines, suggesting the timing of the TDF1-regulated AMS expression is a prerequisite for AMS functioning. We found that TDF1 interacts with AMS. Additionally, the TDF1-AMS complex additively promotes the expression of AMS-regulated genes, suggesting that TDF1 and AMS regulate the downstream genes through a feed-forward loop. EPXB5, encoding a beta-expansin family protein, is another direct target of TDF1, and it is highly expressed in the tapetum and pollen grains. The TDF1-AMS complex acts in concert to activate EXPB5 expression through a feed-forward loop. The identification of the regulatory pathway between TDF1 and AMS provides an interlocked feed-forward loop circuit that precisely regulates the transcriptional cascades that support anther development.

Interaction of Arabidopsis TGA3 and WRKY53 transcription factors on Cestrum yellow leaf curling virus (CmYLCV) promoter mediates salicylic acid-dependent gene expression in planta.

This paper highlighted a salicylic acid-inducible Caulimoviral promoter fragment from Cestrum yellow leaf curling virus (CmYLCV). Interaction of Arabidopsis transcription factors TGA3 and WRKY53 on CmYLCV promoter resulted in the enhancement of the promoter activity via NPR1-dependent salicylic acid signaling. Several transcriptional promoters isolated from plant-infecting Caulimoviruses are being presently used worldwide as efficient tools for plant gene expression. The CmYLCV promoter has been isolated from the Cestrum yellow leaf curling virus (Caulimoviruses) and characterized more than 12years ago; also we have earlier reported a near-constitutive, pathogen-inducible CmYLCV promoter fragment (-329 to +137 from transcription start site; TSS) that enhances stronger (3×) expression than the previously reported fragments; all these fragments are highly efficient in monocot and dicot plants (Sahoo et al. Planta 240: 855-875, 2014). Here, we have shown that the full-length CmYLCV promoter fragment (-729 to +137 from TSS) is salicylic acid (SA) inducible. In this context, we have performed an in-depth study to elucidate the factors responsible for SA-inducibility of the CmYLCV promoter. We found that the as-1 1 and W-box1 elements (located at -649 and -640 from the TSS) of the CmYLCV promoter are required for SA-induced activation by recruiting Arabidopsis TGA3 and WRKY53 transcription factors. Consequently, as a nascent observation, we established the physical interaction between TGA3 and WYKY53; also demonstrated that the N-terminal domain of TGA3 is sufficient for the interaction with the full-length WRKY53. Such interaction synergistically activates the CmYLCV promoter activity in planta. Further, we found that activation of the CmYLCV promoter by SA through TGA3 and WRKY53 interaction depends on NPR1. Finally, the findings presented here provide strong support for the direct regulatory roles of TGA3 and WRKY53 in the SA and NPR1-dependent activation of a Caulimoviral promoter (CmYLCV).

Intergenic transformation of AtMYB44 confers drought stress tolerance in rice seedlings

We transformed rice (Oryza sativa L. Japonica cv. Ilmi) calli with the Arabidopsis transcription factor gene AtMYB44 using Agrobacterium-mediated transformation. The T-DNA construct to be transformed contained tflA cDNA (encoding a toxoflavin lyase) as a selectable marker. Since toxoflavin is a photosensitizing phytotoxin, transgenic plantlets were selected based on their capacity for root development on medium containing this toxin in the light. Homozygous lines were selected by determining the segregation patterns, expression levels, and copy numbers of AtMYB44. Intergenic genomic locations of the inserted T-DNA in the three transgenic lines were confirmed by adaptor-ligation polymerase chain reaction and analysis using FSTVAL ( http://bioinfo.mju.ac.kr/fstval/ ), an open-access web tool used to localize the flanking sequences of the transgene. Drought tolerance of young seedlings of the transgenic lines was determined based on the recovery of wilted leaves by re-watering after 3 days of water deprivation in a 105-well (35W × 35L × 45D mm/per well) plate. The three transgenic lines showed average survival rates of 80.4, 93.5, and 72.6%, respectively, whereas wild-type plants failed to recover after re-watering. Thus, the transgenic rice plants exhibited significantly enhanced tolerance to drought stress, as was shown previously in AtMYB44-overexpressing transgenic Arabidopsis and soybean. These results suggest that AtMYB44 activates a drought tolerance mechanism that is conserved in both monocotyledonous and dicotyledonous plants.

MicroRNA319-regulated TCPs interact with FBHs and PFT1 to activate CO transcription and control flowering time in Arabidopsis.

The transcription factor CONSTANS (CO) is a central component that promotes Arabidopsis flowering under long-day conditions (LDs). Here, we show that the microRNA319-regulated TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors promote photoperiodic flowering through binding to the CO promoter and activating its transcription. Meanwhile, these TCPs directly interact with the flowering activators FLOWERING BHLH (FBHs), but not the flowering repressors CYCLING DOF FACTORs (CDFs), to additively activate CO expression. Furthermore, both the TCPs and FBHs physically interact with the flowering time regulator PHYTOCHROME AND FLOWERING TIME 1 (PFT1) to facilitate CO transcription. Our findings provide evidence that a set of transcriptional activators act directly and additively at the CO promoter to promote CO transcription, and establish a molecular mechanism underlying the regulation of photoperiodic flowering time in Arabidopsis.