Yeast One-hybrid Analysis Research Articles

OsWRKY5 Promotes Rice Leaf Senescence via Senescence-Associated NAC and Abscisic Acid Biosynthesis Pathway.

The onset of leaf senescence is triggered by external cues and internal factors such as phytohormones and signaling pathways involving transcription factors (TFs). Abscisic acid (ABA) strongly induces senescence and endogenous ABA levels are finely tuned by many senescence-associated TFs. Here, we report on the regulatory function of the senescence-induced TF OsWRKY5 TF in rice (Oryza sativa). OsWRKY5 expression was rapidly upregulated in senescing leaves, especially in yellowing sectors initiated by aging or dark treatment. A T-DNA insertion activation-tagged OsWRKY5-overexpressing mutant (termed oswrky5-D) promoted leaf senescence under natural and dark-induced senescence (DIS) conditions. By contrast, a T-DNA insertion oswrky5-knockdown mutant (termed oswrky5) retained leaf greenness during DIS. Reverse-transcription quantitative PCR (RT-qPCR) showed that OsWRKY5 upregulates the expression of genes controlling chlorophyll degradation and leaf senescence. Furthermore, RT-qPCR and yeast one-hybrid analysis demonstrated that OsWRKY5 indirectly upregulates the expression of senescence-associated NAM/ATAF1/2/CUC2 (NAC) genes including OsNAP and OsNAC2. Precocious leaf yellowing in the oswrky5-D mutant might be caused by elevated endogenous ABA concentrations resulting from upregulated expression of ABA biosynthesis genes OsNCED3, OsNCED4, and OsNCED5, indicating that OsWRKY is a positive regulator of ABA biosynthesis during leaf senescence. Furthermore, OsWRKY5 expression was suppressed by ABA treatment. Taken together, OsWRKY5 is a positive regulator of leaf senescence that upregulates senescence-induced NAC, ABA biosynthesis, and chlorophyll degradation genes.

Transcriptional regulation of Arabidopsis copper amine oxidase ζ (CuAOζ) in indole-3-butyric acid-induced lateral root development

Spatiotemporal regulation of reactive oxygen species (ROS) is fine-tuned at multiple levels, including transcriptional regulation. Arabidopsis copper amine oxidase ζ (CuAOζ) is localized in peroxisomes, and the CuAOζ-derived ROS is required for indole-3-butyric acid (IBA)-induced lateral root (LR) development. Here, we showed that both GATA2 and NIGT1.2 transcription factors interacted with the 5′ untranslated region (5′ UTR) of CuAOζ, using enhanced yeast one-hybrid analysis. NIGT1.2 was highly expressed in root tips and was upregulated by IBA. Phenotypic analysis, localization, and genetic studies demonstrate that NIGT1.2 positively regulated IBA-induced CuAOζ expression, ROS generation, and LR development. Furthermore, another transcription factor, UPBEAT1 (UPB1), regulated the basal expression of CuAOζ in roots, suggesting a putative functional regulation of CuAOζ in cellular proliferation and differentiation in the root. Together, our findings indicate that transcriptional regulation of peroxisomal CuAOζ plays an important role in determining CuAOζ spatiotemporal activity and ROS homeostasis within a developing root.

Abnormal tapetum development in hermaphrodites of an androdioecious tree, Tapiscia sinensis.

Tapiscia sinensis Oliv. (Tapisciaceae) has been proven to be a functional androdioecious species with both male and hermaphroditic individuals, and the pollen viability of males is far higher than that of hermaphrodites. To better understand the causes of the low pollen viability in hermaphroditic flowers, different stages of anther development were observed. We found that hermaphroditic flowers exhibit abnormal tapetum development, resulting in low pollen viability. To clarify the underlying molecular mechanism of abnormal tapetum development in hermaphrodites, quantitative real-time PCR analyses were performed. The results revealed that the expression levels of an important transcription factor for tapetum development and function, T. sinensis DYSFUNCTIONAL TAPETUM1 (TsDYT1), and its potential downstream regulatory genes T. sinensis DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1 (TsTDF1), T. sinensis ABORTED MICROSPORE (TsAMS) and T. sinensis MALE STERILITY 1 (TsMS1) were all significantly downregulated in hermaphrodites compared with males at some key stages of anther development. The amino acid sequence similarity, expression pattern, gene structure and subcellular localization of these genes were analyzed, and the results indicated functional conservation between T. sinensis and homologues in Arabidopsis thaliana. Next, rapid amplification of cDNA end and thermal asymmetric interlaced PCR were employed to clone the full-length cDNA and promoter sequences of these genes, respectively. In addition, results of yeast two-hybrid analysis showed that TsDYT1 can form heterodimers with TsAMS, and yeast one-hybrid analysis demonstrated that TsDYT1 directly binds to the promoter regions of TsTDF1 and TsMS1. TsTDF1 can directly regulate expression of TsAMS, suggesting that a functionally conserved pathway exists between A. thaliana and T. sinensis to regulate tapetum development. In conclusion, the results suggest that abnormal expression of core transcription factors for tapetum development, including TsDYT1, TsTDF1, TsAMS and TsMS1, plays an important role in the abnormal development of the tapetum in T. sinensis hermaphrodites. Furthermore, a hermaphroditic tapetum with abnormal function causes the low pollen viability of hermaphroditic trees. Our results provide new insight into our understanding of the underlying mechanism of why pollen viability is much higher in males than hermaphrodites of the androdioecious tree T. sinensis.

Reaumuria trigyna transcription factor RtWRKY23 enhances salt stress tolerance and delays flowering in plants

Reaumuria trigyna (Reaumuria Linn genus, family Tamaricaceae), an endangered dicotyledonous shrub with the features of a recretohalophyte, is endemic to the Eastern Alxa–Western Ordos area of China. Based on R. trigyna transcriptome data and expression pattern analysis of RtWRKYs, RtWRKY23, a Group II WRKY transcription factor, was isolated from R. trigyna cDNA. RtWRKY23 was mainly expressed in the stem and was induced by salt, drought, cold, ultraviolet radiation, and ABA treatments, but suppressed by heat treatment. Overexpression of RtWRKY23 in Arabidopsis increased chlorophyll content, root length, and fresh weight of the transgenic lines under salt stress. Real-time quantitative PCR (qPCR) analysis and yeast one-hybrid analysis demonstrated that RtWRKY23 protein directly or indirectly modulated the expression levels of downstream genes, including stress-related genes AtPOD, AtPOD22, AtPOD23, AtP5CS1, AtP5CS2, and AtPRODH2, and reproductive development-related genes AtMAF5, AtHAT1, and AtANT. RtWRKY23 transgenic Arabidopsis had higher proline content, peroxidase activity, and superoxide anion clearance rate, and lower H2O2 and malondialdehyde content than WT plants under salt stress conditions. Moreover, RtWRKY23 transgenic Arabidopsis exhibited later flowering and shorter pods, but little change in seed yield, compared with WT plants under salt stress. Our study demonstrated that RtWRKY23 not only enhanced salt stress tolerance through maintaining the ROS and osmotic balances in plants, but also participated in the regulation of flowering under salt stress.

A R2R3-type MYB transcription factor gene from soybean, GmMYB12, is involved in flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis

The R2R3-type MYB transcription factors have been shown to increase flavonoids accumulation by regulating the expression of key enzyme genes related to flavonoid biosynthesis pathway. However, the roles and underlying mechanisms of the soybean GmMYB12 gene in regulation of flavonoids accumulation and tolerance to abiotic stresses are rarely known. In the present study, the GmMYB12 gene was isolated and its function was characterized. Sequence and yeast one-hybrid analyses showed that GmMYB12 contained two MYB domains and belonged to R2R3-MYB protein with transactivation activity. Subcellular localization analysis in onion epidermal cells indicated that GmMYB12 was localized to the nucleus. Overexpression of GmMYB12 increased the production of downstream flavonoids and the expression of related genes in the flavonoid biosynthesis pathway. It also improved resistance to salt and drought stresses during seed germination, root development, and growing stage. Further component and enzymatic analyses showed significant increases of proline content, pyrroline-5-carboxylate synthase (P5CS), superoxide dismutase (SOD), and peroxidase (POD) activities, as well as significant reduction of H2O2 and malonaldehyde (MDA) content under salt and drought stresses in transgenic plants. Meanwhile, the expression level of AtP5CS, AtSOD and AtPOD genes was up-regulated against salt and drought stresses. Together, our finding indicated that changing the expression level of GmMYB12 in plants alters the accumulation of flavonoids and regulates plantlet tolerance to abiotic stress by regulating osmotic balance, protecting membrane integrity and maintaining ROS homeostasis. The GmMYB12 gene has the potential to be used to increase the content of valuable flavonoids and improve the tolerance to abiotic stresses in plants.

BZR1 Mediates Brassinosteroid-Induced Autophagy and Nitrogen Starvation in Tomato.

Autophagy, an innate cellular destructive mechanism, plays crucial roles in plant development and responses to stress. Autophagy is known to be stimulated or suppressed by multiple molecular processes, but the role of phytohormone signaling in autophagy is unclear. Here, we demonstrate that the transcripts of autophagy-related genes (ATGs) and the formation of autophagosomes are triggered by enhanced levels of brassinosteroid (BR). Furthermore, the BR-activated transcription factor brassinazole-resistant1 (BZR1), a positive regulator of the BR signaling pathway, is involved in BR-induced autophagy. Treatment with BR enhanced the formation of autophagosomes and the transcripts of ATGs in BZR1-overexpressing plants, while the effects of BR were compromised in BZR1-silenced plants. Yeast one-hybrid analysis and chromatin immunoprecipitation coupled with quantitative polymerase chain reaction revealed that BZR1 bound to the promoters of ATG2 and ATG6 The BR-induced formation of autophagosomes decreased in ATG2- and ATG6-silenced plants. Moreover, exogenous application of BR enhanced chlorophyll content and autophagosome formation and decreased the accumulation of ubiquitinated proteins under nitrogen starvation. Leaf chlorosis and chlorophyll degradation were exacerbated in BZR1-silenced plants and the BR biosynthetic mutant d^im but were alleviated in BZR1- and BZR1-1D-overexpressing plants under nitrogen starvation. Meanwhile, nitrogen starvation-induced expression of ATGs and autophagosome formation were compromised in both BZR1-silenced and d^im plants but were increased in BZR1- and BZR1-1D-overexpressing plants. Taken together, our results suggest that BZR1-dependent BR signaling up-regulates the expression of ATGs and autophagosome formation, which plays a critical role in the plant response to nitrogen starvation in tomato (Solanum lycopersicum).

Methyl jasmonate enhances apple’ cold tolerance through the JAZ–MYC2 pathway

Improving the cold resistance of plants is important, because their growth and development are negatively affected by cold stress. In this study, exogenous applications of methyl jasmonate could enhance the cold resistance of ‘Orin’ apple (Malus × domestica) calli by increasing the expression levels of the cold-signal response genes MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3. In addition, yeast two-hybrid and pull-down assays demonstrated that MdCIbHLH1 interacts with MdJAZ1/4 and MdMYC2 in vitro and in vivo. Protein degradation experiments demonstrated that the stability of the MdJAZ1/4 proteins were affected by the application of exogenous methyl jasmonate, which was followed by their degradation by the 26S proteasome. MdJAZ1/4 act as repressors, binding MdMYC2 in the jasmonate-signaling pathway. The overexpression of MdMYC2 in ‘Orin’ calli increased the expression levels of MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3, resulting in an increased freeze tolerance. Furthermore, the overexpression of MdJAZ1 or MdJAZ4 in transgenic red-fleshed apple calli weakened the promotive effect of MdMYC2 on cold tolerance. Yeast one-hybrid and chromatin immunoprecipitation-PCR analyses showed that MdMYC2 could bind the G-box element found in the MdCBF1 promoter. Thus, jasmonate may function as a critical upstream signal in the ICE–CBF/DREB1 pathway to positively regulate apple freeze tolerance.

Phosphorylation of E-box binding USF-1 by PI3K/AKT enhances its transcriptional activation of the WBP2 oncogene in breast cancer cells.

WW domain binding protein 2 (WBP2), a transcriptional coactivator, plays a vital role in breast tumorigenesis. It positively regulates estrogen receptor, Hippo, and Wnt pathways, which subsequently enhance the transcription of downstream target genes contributing to cancer. Understanding the regulation of the expression and activity of WBP2 oncoprotein has implication in cancer therapy. We have previously reported that WBP2 is regulated at the post-translational and post-transcriptional levels. However, its regulation at the transcriptional level is not known. In this study, the minimal promoter region of WBP2 that is critical for its transcription was identified. The E-box motif in the WBP2 promoter was demonstrated to be essential for its transcription. The E-box binding protein upstream stimulatory factor 1 (USF-1) was discovered to be a key transcription factor for WBP2 by yeast one-hybrid analysis and was validated through reporter and chromatin immunoprecipitation assays and tandem mass spectrometry, which also suggested that USF-1 acts by regulating a network of genes, in addition to WBP2, associated with cell movement, proliferation, cell-cycle, and survival cellular processes. USF-1 is overexpressed in majority of the breast cancer cell lines and tissues tested, and has profound effects on cancer cell proliferation. USF-1-mediated transcription of WBP2 was demonstrated to be inducible by insulin, which led to AKT-mediated phosphorylation of USF-1 that modulated its ability to bind to the WBP2 promoter and activate its transcription. This study sheds new light onto the regulation of the WBP2 oncogene at the transcriptional level by a novel oncogenic transcription factor, USF-1. USF-1 is a potential drug target for treatment of WBP2-positive breast cancer.-Ramos, A., Miow, Q. H., Liang, X., Lin, Q. S., Putti, T. C., Lim, Y. P. Phosphorylation of E-box binding USF-1 by PI3K/AKT enhances its transcriptional activation of the WBP2 oncogene in breast cancer cells.

The global regulator of pathogenesis PnCon7 positively regulates Tox3 effector gene expression through direct interaction in the wheat pathogen Parastagonospora nodorum.

To investigate effector gene regulation in the wheat pathogenic fungus Parastagonospora nodorum, the promoter and expression of Tox3 was characterised through a series of complementary approaches. Promoter deletion and DNase I footprinting experiments identified a 25 bp region in the Tox3 promoter as being required for transcription. Subsequent yeast one-hybrid analysis using the DNA sequence as bait identified that interacting partner as the C2H2 zinc finger transcription factor PnCon7, a putative master regulator of pathogenesis. Silencing of PnCon7 resulted in the down-regulation of Tox3 demonstrating that the transcription factor has a positive regulatory role on gene expression. Analysis of Tox3 expression in the PnCon7 silenced strains revealed a strong correlation with PnCon7 transcript levels, supportive of a direct regulatory role. Subsequent pathogenicity assays using PnCon7-silenced isolates revealed that the transcription factor was required for Tox3-mediated disease. The expression of two other necrotrophic effectors (ToxA and Tox1) was also affected but in a non-dose dependent manner suggesting that the regulatory role of PnCon7 on these genes was indirect. Collectively, these data have advanced our fundamental understanding of the Con7 master regulator of pathogenesis by demonstrating its positive regulatory role on the Tox3 effector in P. nodorum through direct interaction.

Isolation and functional characterization of a floral repressor, BcFLC2, from Pak-choi (Brassica rapa ssp. chinensis).

BcFLC2 functioned as a repressor of flowering by directly regulating BcTEM1, BcMAF2, BcSOC1 and BcSPL15 in Pak-choi. FLOWERING LOCUS C (FLC) plays an important role in regulating flowering time. Here, we functionally described an FLC homologous gene, BcFLC2, that negatively regulated flowering in Pak-choi (Brassica rapa ssp. chinensis). The sequence comparison to Arabidopsis FLC showed that BcFLC2 also had a MADS-box domain at the N terminus. BcFLC2 was highly expressed in the leaves, roots, stems and stamens, and its expression was repressed by vernalization in Pak-choi. Interestingly, BcFLC2 expression exhibited a small peak at 2weeks of vernalization treatment, suggesting that BcFLC2 may be involved in preventing premature flowering under short-term cold exposure in Pak-choi, which is different from the AtFLC expression pattern. Overexpression of BcFLC2 in Arabidopsis caused late flowering, while silencing of BcFLC2 in Pak-choi caused early flowering. BcFLC2 localized to the cell nucleus and functioned as a transcription factor. Yeast one-hybrid analysis revealed that BcFLC2 could bind to the promoters of Pak-choi Tempranillo 1 (BcTEM1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BcSOC1), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (BcSPL15) and MADS AFFECTING FLOWERING 2 (BcMAF2). Taken together, the present results suggested that BcFLC2 played a key role in flowering regulation as a negative regulator by controlling BcTEM1, BcMAF2, BcSOC1 and BcSPL15 expression.

Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis.

Arbuscular mycorrhizal (AM) fungi are soil-borne fungi belonging to the ancient phylum Glomeromycota and are important symbionts of the arbuscular mycorrhiza, enhancing plant nutrient acquisition and resistance to various abiotic stresses. In contrast to their significant physiological implications, the molecular basis involved is poorly understood, largely due to their obligate biotrophism and complicated genetics. Here, we identify and characterize three genes termed Fm201, Ri14-3-3 and RiBMH2 that encode 14-3-3-like proteins in the AM fungi Funneliformis mosseae and Rhizophagus irregularis, respectively. The transcriptional levels of Fm201, Ri14-3-3 and RiBMH2 are strongly induced in the pre-symbiotic and symbiotic phases, including germinating spores, intraradical hyphae- and arbuscules-enriched roots. To functionally characterize the Fm201, Ri14-3-3 and RiBMH2 genes, we took advantage of a yeast heterologous system owing to the lack of AM fungal transformation systems. Our data suggest that all three genes can restore the lethal Saccharomyces cerevisiae bmh1 bmh2 double mutant on galactose-containing media. Importantly, yeast one-hybrid analysis suggests that the transcription factor RiMsn2 is able to recognize the STRE (CCCCT/AGGGG) element present in the promoter region of Fm201 gene. More importantly, Host-Induced Gene Silencing of both Ri14-3-3 and RiBMH2 in Rhizophagus irregularis impairs the arbuscule formation in AM symbiosis and inhibits the expression of symbiotic PT4 and MST2 genes from plant and fungal partners, respectively. We further subjected the AM fungus-Medicago truncatula association system to drought or salinity stress. Accordingly, the expression profiles in both mycorrhizal roots and extraradical hyphae reveal that these three 14-3-3-like genes are involved in response to drought or salinity stress. Collectively, our results provide new insights into molecular functions of the AM fungal 14-3-3 proteins in abiotic stress responses and arbuscule formation during AM symbiosis.

CsATAF1 Positively Regulates Drought Stress Tolerance by an ABA-Dependent Pathway and by Promoting ROS Scavenging in Cucumber.

The NAC transcription factors play vital roles in responding to drought stress in plants; however, the molecular mechanisms remain largely unknown in cucumber. Suppression of CsATAF1 via RNA interference (RNAi) weakened drought stress tolerance in cucumber due to a higher water loss rate in leaves, a higher level of hydrogen peroxide (H2O2) and superoxide radicals (O2·-), increased malondialdehyde (MDA) content, lower Fv/Fm ratios and lower antioxidant enzyme activity. The analysis of root length and stomatal apertures showed that CsATAF1-RNAi cucumber plants were less responsive to ABA. In contrast, CsATAF1-overexpression (OE) plants showed increased drought stress tolerance and sensitivity to ABA. Quantitative PCR (qPCR) analysis showed that expression of several stress-responsive genes was significantly up-regulated in CsATAF1-OE transformants and down-regulated in CsATAF1-RNAi transformants. CsABI5, CsCu-ZnSOD and CsDREB2C were verified as direct target genes of CsATAF1. Yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) further substantiated that CsATAF1 bound to the promoters of CsABI5, CsCu-ZnSOD and CsDREB2C. Transient expression in tobacco leaves and cucumber protoplasts showed that CsATAF1 directly up-regulated the expression of CsABI5, CsCu-ZnSOD and CsDREB2C. Our results demonstrated that CsATAF1 functioned as a positive regulator in response to drought stress by an ABA-dependent pathway and decreasing reactive oxygen species (ROS) accumulation in cucumber.

Overexpression of a New Zinc Finger Protein Transcription Factor OsCTZFP8 Improves Cold Tolerance in Rice.

Cold stress is one of the most important abiotic stresses in rice. C2H2 zinc finger proteins play important roles in response to abiotic stresses in plants. In the present study, we isolated and functionally characterized a new C2H2 zinc finger protein transcription factor OsCTZFP8 in rice. OsCTZFP8 encodes a C2H2 zinc finger protein, which contains a typical zinc finger motif, as well as a potential nuclear localization signal (NLS) and a leucine-rich region (L-box). Expression of OsCTZFP8 was differentially induced by several abiotic stresses and was strongly induced by cold stress. Subcellular localization assay and yeast one-hybrid analysis revealed that OsCTZFP8 was a nuclear protein and has transactivation activity. To characterize the function of OsCTZFP8 in rice, the full-length cDNA of OsCTZFP8 was isolated and transgenic rice with overexpression of OsCTZFP8 driven by the maize ubiquitin promoter was generated using Agrobacterium-mediated transformation. Among 46 independent transgenic lines, 6 single-copy homozygous overexpressing lines were selected by Southern blot analysis and Basta resistance segregation assay in both T1 and T2 generations. Transgenic rice overexpressing OsCTZFP8 exhibited cold tolerant phenotypes with significantly higher pollen fertilities and seed setting rates than nontransgenic control plants. In addition, yield per plant of OsCTZFP8-expressing lines was significantly (p < 0.01) higher than that of nontransgenic control plants under cold treatments. These results demonstrate that OsCTZFP8 was a C2H2 zinc finger transcription factor that plays an important role in cold tolerance in rice.

Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars

Anthocyanins are secondary metabolites in land plants that contribute to the colors of leaves and flowers, and are nutritionally valuable components of the human diet. The DFR gene plays an important role in the anthocyanin biosynthetic pathway. In this study, we investigated the regulation of DFR expression and in different Malus crabapple cultivars that show distinct patterns of leaf coloration, and how it influences leaf anthocyanin accumulation and coloration. Specifically, we studied the ever-red leaved cultivar ‘Royalty’, the ever-green leaved cultivar ‘Flame’ and the spring-red leaved cultivar ‘Radiant’. RT-PCR analysis showed that the expression of McDFR1 correlated with the expression of a MYB transcription factor, McMYB10, and with anthocyanin accumulation. We isolated five McDFR1 promoter fragments from the three cultivars and identified four different fragments (F1–4) that were present either in several cultivars, or only in one. Yeast one-hybrid and electrophoretic mobility shift assay analyses showed that McMYB10 could bind to all the McDFR1 promoters, except McDFR1-Ra2. The F1, F2 and F3 fragments did not affect McMYB10 binding to the McDFR1 promoters; however, we found evidence that the F4 fragment suppressed binding, and that the MYBGAHV amino-acid sequence maybe an important cis-element for McMYB10 protein binding. This information has potential value for strategies to modify plant color through genetic transformation.

TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis.

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

Litchi Fruit LcNAC1 is a Target of LcMYC2 and Regulator of Fruit Senescence Through its Interaction with LcWRKY1.

Senescence is a key factor resulting in deterioration of non-climacteric fruit. NAC transcription factors are important regulators in plant development and abiotic stress responses, yet little information regarding the role of NACs in regulating non-climacteric fruit senescence is available. In this study, we cloned 13 NAC genes from litchi (Litchi chinensis) fruit, and analyzed subcellular localization and expression profiles of these genes during post-harvest natural and low-temperature-delayed senescence. Of the 13 NAC genes, expression of LcNAC1 was up-regulated in the pericarp and pulp as senescence progressed, and was significantly higher in senescence-delayed fruit than that in naturally senescent fruit. LcNAC1 was induced by exogenous ABA and hydrogen peroxide. Yeast one-hybrid analysis and transient dual-luciferase reporter assay showed that LcNAC1 was positively regulated by the LcMYC2 transcription factor. LcNAC1 activated the expression of LcAOX1a, a gene associated with reactive oxygen species regulation and energy metabolism, whereas LcWRKY1 repressed LcAOX1a expression. In addition, LcNAC1 interacted with LcWRKY1 in vitro and in vivo. These results indicated that LcNAC1 and LcWRKY1 form a complex to regulate the expression of LcAOX1a antagonistically. Taken together, the results reveal a hierarchical and co-ordinated regulatory network in senescence of harvested litchi fruit.

Rice PcG gene OsEMF2b controls seed dormancy and seedling growth by regulating the expression of OsVP1

The induction and release of seed dormancy are a precisely regulated process that influences seed germination. ABA promotes seed dormancy but suppresses seed germination and seedling growth. However, how chromatin and epigenetic mechanisms regulate the expression of ABA related genes during these processes remains unclear. Polycomb gene OsEMF2b was required for regulation of seed dormancy and seedling growth by dynamically activating and repressing ABA signal response genes. Downregulation of OsEMF2b led to vivipary and decreased expression level of OsVP1, which was involved in ABA signal pathway in seed dormancy. While, the seedlings with downregulation of OsEMF2b exhibited hyper-sensitive response to ABA and the expression of OsVP1 is upregulated. Yeast one-hybrid assay and ChIP analyses proved that OsEMF2b could bind to the promoter of OsVP1 directly and affected H3K27me3 enrichments of OsVP1 in seedling. Interestingly, both H3K27me3 and H3K4me3 enrichments of OsVP1 were changed with OsEMF2b mis-expression in seed and seedling. We proposed that OsEMF2b may play a pivotal role in seed dormancy and seedling growth by regulating the expression of OsVP1 indirectly or directly.

Ethylene response factor AtERF72 negatively regulates Arabidopsis thaliana response to iron deficiency

Ethylene regulates the plant's response to stress caused by iron (Fe) deficiency. However, specific roles of ERF proteins in response to Fe deficiency remain poorly understood. Here, we investigated the role of ERF72 in response to iron deficiency in Arabidopsis thaliana.In this study, the levels of the ethylene response factor AtERF72 increased in leaves and roots induced under the iron deficient conditions. erf72 mutant plants showed increased growth compared to wild type (WT) when grown in iron deficient medium for 5 d. erf72 mutants had increased root H+ velocity and the ferric reductase activity, and increase in the expression of the iron deficiency response genes iron-regulated transporter 1 (IRT1) and H+-ATPase (HA2) levels in iron deficient conditions. Compared to WT plants, erf72 mutants retained healthy chloroplast structure with significantly higher Fe and Mg content, and decreased chlorophyll degradation gene pheophorbide a oxygenase (PAO) and chlorophyllase (CLH1) expression when grown in iron deficient media. Yeast one-hybrid analysis showed that ERF72 could directly bind to the promoter regions of iron deficiency responses genes IRT1, HA2 and CLH1. Based on our results, we suggest that ethylene released from plants under iron deficiency stress can activate the expression of ERF72, which responds to iron deficiency in the negative regulation.

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.

Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.

The transcription factor SlDof22 involved in ascorbate accumulation and salinity stress in tomato

Ascorbic acid (AsA) is an important antioxidant and its biosynthesis in plants has extensively been investigated. However, the key regulatory factors controlling the accumulation of AsA remain elusive. Here we report that tomato SlDof22, a member of the Dof family, negatively regulated AsA accumulation in tomato. RNA interference (RNAi) of SlDof22 in transgenic lines induced AsA levels, and affected the expression of genes in the D-mannose/L-galactose pathway and AsA recycling. In addition, SlSOS1 was significantly down-regulated in SlDof22 RNAi plants which resulted in reduced tolerance to salt stress. We further found that SlDof22 could bind to the promoter sequence of SlSOS1 gene by yeast one-hybrid analysis. Taken together, our data suggested that the Dof transcription factor SIDof22 involved in ascorbate accumulation and salt stress response in tomato.