W-box Cis-elements Research Articles

MdWRKY45 contributes to resistance against Botryosphaeria dothidea in apple

Malus domestica “Fuji” is the predominant apple cultivar grown around the world, but it is highly susceptible to apple ring rot caused by Botryosphaeria dothidea. WRKY transcription factors (TFs) play key roles in multiple physiological responses to plant defense. We have previously reported the involvement of several MdWRKY TFs in plant defense against. In this study, we identified a new MdWRKY TF, MdWRKY45, which is induced by B. dothidea infection. MdWRKY45 was localized in the nucleus and exhibited transcriptional activity. Over-expression of MdWRKY45 enhanced the resistance of transgenic apple calli and fruits to B. dothidea infection. Conversely, suppression of MdWRKY45 decreased the disease resistance of apple calli and fruits. Further transcriptome profiling and expression analysis showed that various genes regulating the release of salicylic acid (SA), jasmonic acid (JA), and reactive oxygen species (ROS) were either up-regulated or down-regulated in MdWRKY45-RNAi transgenic apple calli compared with wild-type (WT). During B. dothidea infection, MdWRKY45 modulated the levels of SA and JA, as well as ROS burst. Moreover, MdWRKY45 could bind to the W-box cis-element in the promoters of MdPR1, MdJAZ1, and MdRbohF genes, thereby regulating their expression. In conclusion, MdWRKY45 primarily confers resistance to B. dothidea infection through pathways involving SA, JA, and ROS burst defense.

Transcriptional Profiling of BpWRKY49 Reveals Its Role as a Master Regulator in Stress Signaling Pathways in Birch (Betula platyphylla)

The WRKY family of transcription factors (TFs) is one of the most diverse families in plants, playing crucial roles in various plant growth and stress response processes. Asian white birch (Betula platyphylla) is a globally distributed tree species that holds ecological, medical, and economic significance. However, the regulatory mechanisms of WRKY TFs in birch remain poorly understood. Herein, we cloned and characterized the BpWRKY49 gene from birch. Through bioinformatics analyses, we revealed the potential involvement of BpWRKY49 in both biotic and abiotic stress responses. In addition, BpWRKY49 was found to be localized in the nucleus and exhibited transcriptional activity in yeast. Transactivation assays further confirmed that BpWRKY49 exhibited transcriptional activity at its C-terminal end. Notably, our binding specificity assays demonstrated the specific interaction of BpWRKY49 with the W-box cis element in vitro. Furthermore, tissue-specific expression analysis demonstrated that BpWRKY49 exhibited the highest expression level in the roots. Real-time quantitative PCR (RT-qPCR) analysis of birch plants subjected to salt and drought treatments revealed that BpWRKY49 displayed significant 30-fold and 10-fold upregulations under salt and drought stress conditions, respectively. DAP-seq analysis of BpWRKY49 identified a total of 21,832 peaks, with 3477 occurring in the promoter region of genes. Gene ontology (GO) enrichment analysis highlighted prominent terms related to defense against biotic stress, followed by terms associated with abiotic stress and development. Y1H assays of three genes provided evidence for the binding ability of BpWRKY49 to the promoters of BpPUB21, BpBTL15, and BpHIP47 in vitro. Collectively, our findings strongly suggest that BpWRKY49 possesses diverse functions and may activate multiple genes to contribute to various biological processes, including salt stress tolerance, in birch.

Ethylene enhances resistance to cucumber green mottle mosaic virus via the ClWRKY70-ClACO5 module in watermelon plants.

Ethylene (ET) is involved in plant responses to viral infection. However, its molecular mechanisms and regulatory network remain largely unknown. In the present study, we report that cucumber green mottle mosaic virus (CGMMV) in watermelon (Citrullus lanatus) triggers ET production by inducing the expression of ClACO5, a key gene of the ET biosynthesis pathway through transcriptome data analysis and gene function validation. The knock-down of ClACO5 expression through virus-induced gene silencing in watermelon and overexpressing ClACO5 in transgenic Nicotiana benthamiana indicated that ClACO5 positively regulates CGMMV resistance and ET biosynthesis. The salicylic acid-responsive transcription factor gene ClWRKY70 shares a similar expression pattern with ClACO5. We demonstrate that ClWRKY70 directly binds to the W-box cis-element in the ClACO5 promoter and enhances its transcription. In addition, ClWRKY70 enhances plant responses to CGMMV infection by regulating ClACO5 expression in watermelon. Our results demonstrate that the ClWRKY70-ClACO5 module positively regulates resistance to CGMMV infection in watermelon, shedding new light on the molecular basis of ET accumulation in watermelon in response to CGMMV infection.

Multifaceted roles of WRKY transcription factors in abiotic stress and flavonoid biosynthesis.

Increasing biotic and abiotic stresses are seriously impeding the growth and yield of staple crops and threatening global food security. As one of the largest classes of regulators in vascular plants, WRKY transcription factors play critical roles governing flavonoid biosynthesis during stress responses. By binding major W-box cis-elements (TGACCA/T) in target promoters, WRKYs modulate diverse signaling pathways. In this review, we optimized existing WRKY phylogenetic trees by incorporating additional plant species with WRKY proteins implicated in stress tolerance and flavonoid regulation. Based on the improved frameworks and documented results, we aim to deduce unifying themes of distinct WRKY subfamilies governing specific stress responses and flavonoid metabolism. These analyses will generate experimentally testable hypotheses regarding the putative functions of uncharacterized WRKY homologs in tuning flavonoid accumulation to enhance stress resilience.

Arabidopsis transcription factor WRKY45 confers cadmium tolerance via activating PCS1 and PCS2 expression

Cadmium (Cd) has long been recognized as toxic pollutant to crops worldwide. The biosynthesis of glutathione-dependent phytochelatin (PC) plays crucial roles in the detoxification of Cd in plants. However, its regulatory mechanism remains elusive. Here, we revealed that Arabidopsis transcription factor WRKY45 confers Cd tolerance via promoting the expression of PC synthesis-related genes PCS1 and PCS2, respectively. Firstly, we found that Cd stress induces the transcript levels of WRKY45 and its protein abundance. Accordingly, in contrast to wild type Col-0, the increased sensitivity to Cd is observed in wrky45 mutant, while overexpressing WRKY45 plants are more tolerant to Cd. Secondly, quantitative real-time PCR revealed that the expression of AtPCS1 and AtPCS2 is stimulated in overexpressing WRKY45 plants, but decreased in wrky45 mutant. Thirdly, WRKY45 promotes the expression of PCS1 and PCS2, electrophoresis mobility shift assay analysis uncovered that WRKY45 directly binds to the W-box cis-element of PCS2 promoter. Lastly, the overexpression of WRKY45 in Col-0 leads to more accumulation of PCs in Arabidopsis, and the overexpression of PCS1 or PCS2 in wrky45 mutant plants rescues the phenotypes induced by Cd stress. In conclusion, our results show that AtWRKY45 positively regulates Cd tolerance in Arabidopsis via activating PCS1 and PCS2 expression.

MdWRKY39 negatively regulates apple phosphorus-deficiency tolerance by inhibiting MdPHT1;7 expression

WRKY transcription factors have been reported to participate extensively in plant responses to abiotic stress. Phosphorus deficiency can limit apple plant growth and bud differentiation resulting in reduced yield; however, the response of WRKY transcription factors to phosphorus deficiency in apple plants has rarely been studied. In this study, we identified a MdWRKY39 gene in apple and investigated its role in phosphorus-deficiency tolerance. The MdWRKY39 protein fused to green fluorescent protein was localized to the nucleus and its expression was repressed by low phosphorus supply. Overexpression of MdWRKY39 reduced apple plant phosphorus content and root hair density. In vitro and in vivo experiments indicated that MdWRKY39 down-regulates MdPHT1;7 transcription by directly binding to its W-box cis-elements. More specifically, MdWRKY39 binds to the TGAC motif of the W-box element in the MdPHT1;7 promoter. Our results showed that MdWRKY39 directly targets MdPHT1;7, thereby negatively regulating phosphorus-deficiency tolerance in apple plants.

The CRK5 and WRKY53 Are Conditional Regulators of Senescence and Stomatal Conductance in Arabidopsis.

In Arabidopsis thaliana, cysteine-rich receptor-like kinases (CRKs) constitute a large group of membrane-localized proteins which perceive external stimuli and transduce the signal into the cell. Previous reports based on their loss-of-function phenotypes and expression profile support their role in many developmental and stress-responsive pathways. Our study revealed that one member of this family, CRK5, acts as a negative regulator of leaf aging. Enrichment of the CRK5 promoter region in W-box cis-elements demonstrated that WRKY transcription factors control it. We observed significantly enhanced WRKY53 expression in crk5 and reversion of its early-senescence phenotype in the crk5 wrky53 line, suggesting a negative feedback loop between these proteins antagonistically regulating chlorophyll a and b contents. Yeast-two hybrid assay showed further that CRK5 interacts with several proteins involved in response to water deprivation or calcium signaling, while gas exchange analysis revealed a positive effect of CRK5 on water use efficiency. Consistent with that, the crk5 plants showed disturbed foliar temperature, stomatal conductance, transpiration, and increased susceptibility to osmotic stress. These traits were fully or partially reverted to wild-type phenotype in crk5 wrky53 double mutant. Obtained results suggest that WRKY53 and CRK5 are antagonistic regulators of chlorophyll synthesis/degradation, senescence, and stomatal conductance.

The transcription factor TaGAMYB modulates tapetum and pollen development of TGMS wheat YanZhan 4110S via the gibberellin signaling

Male reproductive development in higher plants experienced a series of complex biological processes, which can be regulated by Gibberellins (GA). The transcriptional factor GAMYB is a crucial component of GA signaling in anther development. However, the mechanism of GAMYB in wheat male reproduction is less understood. Here, we found that the thermo-sensitive genic male sterilitywheat line YanZhan 4110S displayed delayed tapetum programmed cell death and pollen abortive under the hot temperature stress. Combined with RNA-Sequencing data analysis, TaGAMYB associated with fertility conversion was isolated, which was located in the nucleus and highly expressed in fertility anthers. The silencing of TaGAMYB in wheat displayed fertility decline, defects in tapetum, pollen and exine formation, where the abortion characteristics were the same as YanZhan 4110S. In addition, either hot temperature or GA3 treatment in YanZhan 4110S caused the downregulation of TaGAMYB at binucleate stage and trinucleate stage, as well as fertility decrease. Further, the transcription factor TaWRKY2 significantly changed under GA3-treatment and directly interacted with the TaGAMYB promoter by W-box cis-element. Therefore, we suggested that TaGAMYB may be essential for anther development and male fertility, and GA3 activates TaGAMYB by TaWRKY2 to regulate fertility in wheat.

Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis.

OscWRKY1 from Ocimum sanctum positively regulates phenylpropanoid pathway genes and rosmarinic acid content. OscWRKY1 overexpression promotes resistance against bacterial pathogen in Arabidopsis. WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TF's is not well characterized. In the present study, we have characterized an OscWRKY1 gene from Ocimum sanctum which shows induced expression by methyl jasmonate (MeJA), salicylic acid (SA), and wounding. The recombinant OscWRKY1 protein binds to the DIG-labeled (Digoxigenin) W-box cis-element TTGAC[C/T] and activates the LacZ reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have been identified to contain the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. Furthermore, the metabolite analysis of OscWRKY1 silenced plants showed reduced rosmarinic acid content. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes leading to the alteration of rosmarinic acid content and enhances the resistance against bacterial pathogen in Arabidopsis.

Dioscorea composita WRKY3 positively regulates salt-stress tolerance in transgenic Arabidopsis thaliana

Dioscorea composita (D. composita) is a perennial dioecious herb with strong biotic and abiotic stress tolerance. However, what roles WRKY transcription factors might play in regulating abiotic stress responses in this medicinal plant is unknown. Here, we isolated DcWRKY3 from D. composita and analyzed its role in stress tolerance. DcWRKY3 is a group I WRKY transcription factor that localized to the nucleus and specifically bound to the W-box cis-elements, but lacked transcriptional activation activity in yeast cells. The expression of DcWRKY3 was strongly affected by salt stress. The heterologous expression of DcWRKY3 strongly enhanced the seed germination rate and root length of Arabidopsis thaliana under salt stress. The DcWRKY3-expressing transgenic lines (DcWRKY3-OEs) also showed higher proline content and antioxidant enzyme activity but lower malondialdehyde and reactive oxygen (ROS) levels compared with the wild type. Moreover, these plants showed upregulated expression of genes related to the salt-stress response and ROS clearance. These findings indicate that DcWRKY3 plays a positive role in the salt-stress response by improving the ROS scavenging ability and maintaining the balance of osmotic pressure in plants. Further studies showed that DcWRKY3 binds to the promoter of AtP5CS1, but not AtSOD and AtRD22, suggesting that DcWRKY3 improves salt tolerance in plants by directly or indirectly regulating the expression of downstream genes. This functional characterization of DcWRKY3 provides new insight into the molecular mechanism underlying the response of D. composita to salt stress.

A novel SAPK10-WRKY87-ABF1 biological pathway synergistically enhance abiotic stress tolerance in transgenic rice (Oryza sativa)

WRKY transcription factors play a role in a variety of biological processes. Several studies have revealed that abiotic stress regulates the transcription of a large number of WRKY genes. In this study, we report the identification of a novel ‘SAPK10-WRKY87-ABF1’ biological pathway, through which they harmoniously enhance drought and salinity tolerance. We generated OsWRKY87-overexpressing transgenic rice and found that the transgenic seedlings exhibited significantly improved drought and salinity stress tolerance. Subcellular localization in rice seedling protoplast revealed that OsWRKY87-GFP fusion protein mostly accumulated in the nucleus, suggesting that OsWRKY87 is a nucleus-localized protein, in line with the predicted function of OsWRKY87 as a transcription factor. In vivo interaction between SAPK10 and WRKY87 was demonstrated by Yeast two-hybrid-assay. In addition, phosphorylation assays showed that SAPK10 exhibits autophosphorylation activity on the 177th serine, enabling it to phosphorylate WRKY87. OsWRKY87 functioned as a transcriptional initiator, according to a yeast one-hybrid assay and a luciferase assay. Remarkably, gel mobility shift assay showed that phosphorylated WRKY87 enhances its DNA-binding ability to the W-box cis-element of ABF1 promoter and activated its transcription, thereby elevating the ABF1 transcription and improving drought and salinity tolerance. Overall, our findings revealed a novel ‘SAPK10- WRKY87-ABF1’ module, which synergistically interacts to improve drought and salt tolerance in rice (Oryza sativa).

The long noncoding RNA MdLNC499 bridges MdWRKY1 and MdERF109 function to regulate early-stage light-induced anthocyanin accumulation in apple fruit

Anthocyanin pigments contribute to plant coloration and are valuable sources of antioxidants in the human diet as components of fruits and vegetables. Their production is known to be induced by light in apple fruit (Malus domestica); however, the underlying molecular mechanism responsible for early-stage light-induced anthocyanin biosynthesis remains unclear. Here, we identified an ethylene response factor (ERF) protein, ERF109, involved in light-induced anthocyanin biosynthesis and found that it promotes coloration by directly binding to anthocyanin-related gene promoters. Promoter::β-glucuronidase reporter analysis and Hi-C sequencing showed that a long noncoding RNA, MdLNC499, located nearby MdERF109, induces the expression of MdERF109. A W-box cis-element in the MdLNC499 promoter was found to be regulated by a transcription factor, MdWRKY1. Transient expression in apple fruit and stable transformation of apple calli allowed us to reconstruct a MdWRKY1-MdLNC499-MdERF109 transcriptional cascade in which MdWRKY1 is activated by light to increase the transcription of MdLNC499, which in turn induces MdERF109. The MdERF109 protein induces the expression of anthocyanin-related genes and the accumulation of anthocyanins in the early stages of apple coloration. Our results provide a platform for better understanding the various regulatory mechanisms involved in light-induced apple fruit coloration.

Comparison and Characterization of a Cell Wall Invertase Promoter from Cu-Tolerant and Non-Tolerant Populations of Elsholtzia haichowensis.

Cell wall invertase (CWIN) activity and the expression of the corresponding gene were previously observed to be significantly elevated in a Cu-tolerant population of Elsholtzia haichowensis relative to a non-tolerant population under copper stress. To understand the differences in CWIN gene regulation between the two populations, their CWIN promoter β-glucuronidase (GUS) reporter vectors were constructed. GUS activity was measured in transgenic Arabidopsis in response to copper, sugar, and phytohormone treatments. Under the copper treatment, only the activity of the CWIN promoter from the Cu-tolerant population was slightly increased. Glucose and fructose significantly induced the activity of CWIN promoters from both populations. Among the phytohormone treatments, only salicylic acid induced significantly higher (p < 0.05) activity of the Cu-tolerant CWIN promoter relative to the non-tolerant promoters. Analysis of 5′-deletion constructs revealed that a 270-bp promoter fragment was required for SA induction of the promoter from the Cu-tolerant population. Comparison of this region in the two CWIN promoters revealed that it had 10 mutation sites and contained CAAT-box and W-box cis-elements in the Cu-tolerant promoter only. This work provides insights into the regulatory role of SA in CWIN gene expression and offers an explanation for differences in CWIN expression between E. haichowensis populations.

Characterization of MYB35 regulated methyl jasmonate and wound responsive Geraniol 10-hydroxylase-1 gene from Bacopa monnieri.

BmG10H-1 transcript from B. monnieri was functionally active. BmG10H-1 promoter drives GUS activity in response to MeJA and wounding. BmMYB35 regulates BmG10H-1 transcript by binding to its promoter. Geraniol 10-hydroxylase (G10H) is one of the important regulatory cytochrome P450 monooxygenase, which is involved in the biosynthesis of monoterpene alkaloids. However, G10H is not characterized at the enzymatic or at the regulatory aspect in B. monnieri. In the present study, we have identified two transcripts of BmG10H (BmG10H-1and BmG10H-2) and characterized the methyl jasmonate (MeJA) and wound responsive BmG10H-1 transcript from B. monnieri. BmG10H-1 showed induced expression after 3h of MeJA and wounding treatment in the shoot. Yeast purified recombinant BmG10H-1 protein is enzymatically active, having Vmax of 0.16µMsec-1μg-1 protein and catalyzes the hydroxylation of geraniol to 10-hydroxy geraniol. The BmG10H-1 promoter was isolated by using the genome walking method. BmG10H-1 promoter can drive GUS expression in transgenic Arabidopsis thaliana. GUS activity of MeJA and wound-treated Arabidopsis seedlings were found to be increased as compared to the control untreated seedlings, whereas no GUS activity was found in deleted MeJA responsive and W-box cis-elements. This shows that the BmG10H-1 promoter contains functional MeJA (TGACG) and wound responsive (TGACCT) cis-elements. Further, shoot specific and MeJA responsive recombinant BmMYB35 protein was purified, which binds with the MYB recognition cis-element (TGGTTA) present in the BmG10H-1 promoter and transcriptionally activates the reporter gene in yeast. In conclusion, the characterization of MeJA and wound responsive BmG10H-1 provides novel information about its transcriptional regulation by binding with MYB transcription factor in B. monnieri.

The SrWRKY71 transcription factor negatively regulates SrUGT76G1 expression in Stevia rebaudiana

SrUGT76G1 is vital for the biosynthesis of rebaudioside A, D and M in Stevia rebaudiana Bertoni; however, its transcriptional regulatory mechanism remains unknown. In this study, the 2050-bp promoter region of SrUGT76G1 was isolated by the TAIL-PCR method, and sequence analysis revealed the presence of several W-box cis-elements, which are the recognition motifs of WRKY transcription factors. Furthermore, SrWRKY71, characterized by a typical WRKY domain and a C2H2 zinc finger-like motif, was identified as a putative transcriptional regulator of SrUGT76G1. The transcript of SrWRKY71 predominantly accumulated in leaves and was present at a lower level in stems, roots and flowers. The SrWRKY71-GFP fusion protein was specifically localized to the nucleus in tobacco epidermal cells. In addition, the N and C terminal regions of SrWRKY71 contributed to its transactivation activity. Y1H and EMSA assays validated that SrWRKY71 binds directly to W-box1 and W-box2 in the proximal promoter region of SrUGT76G1. Moreover, SrWRKY71 represses the expression level of SrUGT76G1 in both tobacco leaves and stevia callus. Taken together, the data in this study represent the first identification of an essential upstream transcription factor of SrUGT76G1 and provides new insight into the regulatory network of steviol glycoside biosynthesis in Stevia rebaudiana.

MdWRKY11 Participates in Anthocyanin Accumulation in Red-Fleshed Apples by Affecting MYB Transcription Factors and the Photoresponse Factor MdHY5.

Red-fleshed apples are popular as a result of their high anthocyanin content. MdMYB10 and its homologues are known to be important regulators of anthocyanin synthesis in apple, but the roles of other transcription factors are not well-understood. Here, we explored the role of MdWRKY11 in regulating anthocyanin synthesis in apple flesh. Overexpression of MdWRKY11 in apple callus could significantly promote anthocyanin accumulation, and the expression of some MYB transcription factors and structural genes increased significantly. In binding analyses, MdWRKY11 bound to W-box cis-elements in the promoters of MdMYB10, MdMYB11, and MdUFGT. However, MdWRKY11 did not interact with MdMYB10, MdbHLH3, or MdWD40 proteins, the members of the MBW complex. Sequence analyses revealed that another W-box cis-element was present in the promoter of MdHY5 (encoding a photoresponse factor), and MdWRKY11 was able to bind to the promoter of MdHY5 and promote its activity. Our findings clarify the role of MdWRKY11 in anthocyanin synthesis in red-fleshed apple and imply that other novel genes may be involved in anthocyanin synthesis.

WRKY18 and WRKY53 Coordinate with HISTONE ACETYLTRANSFERASE1 to Regulate Rapid Responses to Sugar.

Sugars provide a source of energy; they also function as signaling molecules that regulate gene expression, affect metabolism, and alter growth in plants. Rapid responses to sugar signaling and metabolism are essential for optimal growth and fitness, but the regulatory mechanisms underlying these are largely unknown. In this study, we found that the rapid induction of sugar responses in Arabidopsis (Arabidopsis thaliana) requires the W-box cis-elements in the promoter region of GLC 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2, a well-studied sugar response marker gene. The transcription factors WRKY18 and WRKY53 directly bind to the W-Box cis-elements in the promoter region of sugar response genes and activate their expression. In addition, HISTONE ACETYLTRANSFERASE 1 (HAC1) is recruited to the WRKY18 and WRKY53 complex that resides on the promoters. In this complex, HAC1 facilitates the acetylation of histone 3 Lys 27 (H3K27ac) on the sugar-responsive genes. Taken together, our findings demonstrate a mechanism by which sugar regulates chromatin modification and gene expression, thus helping plants to adjust their growth in response to environmental changes.

TaWRKY51 promotes lateral root formation through negative regulation of ethylene biosynthesis in wheat (Triticum aestivum L.)

Common wheat (Triticum aestivum L.) is an important staple food crop worldwide. Lateral roots (LRs), as the major component of root architecture, affect water and nutrient uptake in wheat. The phytohormone ethylene is known to affect LR formation; however, the factor(s) modulating ethylene during this process have not yet been elucidated in wheat. Here we identified wheat TaWRKY51 as a key factor that functions in LR formation by modulating ethylene biosynthesis. Wheat TaWRKY51RNA interference lines (TaWRKY51-RNAi) and the homozygous mutants tawrky51-2a and tawrky51-2b all produced fewer LRs than the wild type and negative transgenic plants, whereas the TaWRKY51 overexpression lines (TaWRKY51-OE) had the opposite phenotype. Transcription analysis revealed that 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes (TaACS2, TaACS7 and TaACS8) involved in ethylene biosynthesis were downregulated in TaWRKY51-OE lines but upregulated in TaWRKY51-RNAi lines. The rate of ethylene production also decreased in TaWRKY51-OE lines but increased in TaWRKY51-RNAi lines compared with their respective negative transgenic controls. Electrophoretic mobility shift and transient expression assays revealed that TaWRKY51 inhibits the expression of ACS genes by binding to the W-box cis-element present in their promoter region. Moreover, overexpression of ACS2 or exogenous application of 1-aminocyclopropane-1-carboxylic acid reversed the phenotype of enhanced LR number in TaWRKY51-OE Arabidopsis lines, and overexpression of TaWRKY51 in the ethylene-overproducing mutant eto1-1 rescued its LR defect phenotype. In addition, genetic evidence demonstrates that TaWRKY51-regulated LR formation is also dependent on ethylene and auxin signaling pathways. Our findings reveal a molecular genetic mechanism by which a WRKY gene coordinates ethylene production and LR formation in wheat.

Vascular preferential activity of the Pennisetum purpureum cinnamyl alcohol dehydrogenase promoter in transgenic tobacco plants

Little is known about the cross talk between the lignin biosynthesis gene promoters and the regulatory proteins that modulate molecular signaling and respond to various stresses. In this study, we characterized the promoter region of the lignin biosynthesis pathway cinnamyl alcohol dehydrogenase (CAD) gene in elephant grass, Pennisetum purpureum. Quantification of the transcript levels of the PpCAD promoter revealed it is preferentially expressed in vascular tissue, especially xylem. Histochemical and fluorometric assays confirmed the vascular-preferential expression of the PpCAD promoter, as the highest β-glucuronidase (GUS) activity was found in the basal stem in transgenic tobacco plants expressing a 1154-bp PpCAD promoter-GUS fusion construct. Moreover, 5′-deleted PpCAD promoter analyses showed that the 1154-bp PpCAD promoter fragment had the highest transcriptional activity, whereas the 2054-bp fragment had multifarious inducible activity responding to gibberellin (GA), methyl jasmonate (MeJA), abscisic acid (ABA), and wounding. The regions from −248 to −243 bp and −1416 to −1411 bp contained W-box cis-elements, which were detected by electrophoretic mobility shift assay (EMSA). The binding effects of the GA-responsive elements (from −561 to −555 bp and −1077 to −1071 bp), MeJA-responsive element (from −1146 to −1142 bp), and the ABA-responsive cis-element (from −1879 to −1874 bp) were also validated by EMSA. Based on our results, we suggest that lignin deposition associated with PpCAD promoter activity adapts to the environment through molecular signaling involving GA, MeJA, and ABA.

An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF.

Overexpression of BnaWGR1 causes ROS accumulation and promotes leaf senescence. BnaWGR1 binds to promoters of RbohD and RbohF and regulates their expression. Manipulation of leaf senescence process affects agricultural traits of crop plants, including biomass, seed yield and stress resistance. Since delayed leaf senescence usually enhances tolerance to multiple stresses, we analyzed the function of specific MAPK-WRKY cascades in abiotic and biotic stress tolerance as well as leaf senescence in oilseed rape (Brassica napus L.), one of the important oil crops. In the present study, we showed that expression of one WRKY gene from oilseed rape, BnaWGR1, induced an accumulation of reactive oxygen species (ROS), cell death and precocious leaf senescence both in Nicotiana benthamiana and transgenic Arabidopsis (Arabidopsis thaliana). BnaWGR1 regulates the transcription of two genes encoding key enzymes implicated in production of ROS, that is, respiratory burst oxidase homolog (Rboh) D and RbohF. A dual-luciferase reporter assay confirmed the transcriptional regulation of RbohD and RbohF by BnaWGR1. In vitro electrophoresis mobility shift assay (EMSA) showed that BnaWGR1 could bind to W-box cis-elements within promoters of RbohD and RbohF. Moreover, RbohD and RbohF were significantly upregulated in transgenic Arabidopsis overexpressing BnaWGR1. In summary, these results suggest that BnaWGR1 could positively regulate leaf senescence through regulating the expression of RbohD and RbohF genes.