Ectopic Expression In Arabidopsis Research Articles

JrATHB-12 mediates JrMYB113 and JrMYB27 to control the different types of red walnut1

Red walnut has broad market prospects because it is richer in anthocyanin than ordinary walnut. However, the anthocyanin biosynthetic mechanism of red walnut is still unknown. We studied two types of red walnut, called red walnut 1 (R1), with a red pericarp and seed coat, and red walnut 2 (R2), with a red seed coat only. R1 mostly contained cyanidin-3-O-galactoside, while R2 contained a certain amount of each of cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-glucoside. The LDOX-2 (LOC109007163) and LDOX-3 (LOC109010746), encoding genes of leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS), were preliminarily appraised as crucial genes for anthocyanin biosynthesis in R1 and R2, respectively. The MYB differential genes analysis showed that MYB27 and MYB113 are specifically expressed in the red parts of R1 and R2, respectively, and are regarded as candidate regulatory genes. Ectopic expression in Arabidopsis and transient injection in walnut showed that both MYB27 and MYB113, located in the nucleus, promoted anthocyanin accumulation, MYB27 promoted the LDOX-2 expression, and MYB113 promoted the expressions of LDOX-3 and UAGT-3. Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind the LDOX-2 promoter, while MYB113 could bind the promoters of LDOX-3 and UAGT-3. In addition, we also identified an HD-Zip transcription factor ATHB-12, which is specifically expressed in the pericarp. After silencing the expression of ATHB-12, the R2 pericarp turned red, and the MYB113 expression increased. Further experiments showed that ATHB-12 could specifically interact with MYB113 and bind to its promoter. This suggests that MYB27 controls R1 coloration by regulating LDOX-2, while MYB113 controls R2 coloration by regulating LDOX-3 and UAGT-3, but the ATHB-12 can specifically bind and inhibit the MYB113 of R2 pericarp so that it becomes unpigmented. This study reveals the anthocyanin biosynthesis mechanisms in different types of red walnut and provides a scientific basis for the selection and breeding of red walnut varieties.

PgWRKY44, a pearl millet WRKY transcription factor-Calmodulin module, enhances salt and drought stress resilience in transgenic plants

WRKY transcription factors (TFs) regulate signal transduction pathways during stress response and can also modulate the activity of downstream genes through binding to their cognate W-box elements [(T) TGAC(C/T)]. Previous studies have identified and in-silico characterized WRKY family members in millets. However, their functional elucidation and molecular mechanism in millets remain vastly unexplored. In this study, a pearl millet WRKY TF (PgWRKY44) belonging to Group IId was characterized, and its ectopic expression in Arabidopsis was found to be positively regulating abiotic stress tolerance in transgenic plants through ABA-mediated signalling. Also, reduced accumulation of reactive oxygen species (ROS) and up-regulation of stress-related genes confirmed improved defense systems of transgenic plants upon abiotic stress treatments. Functional network analysis and expression data indicated towards co-regulation of multiprotein bridging factor (MBF1C), HSFs, and calmodulin (CAM) members with PgWRKY44 in response to osmotic stress. Yeast one-hybrid also confirmed W-box-dependent binding of PgWRKY44. These findings enriched our understanding of the PgWRKY44 functions in pearl millet and exhibited its potential application in developing climate-resilient crop plants.

Identification and characterization of the karrikins signaling gene SsSMAX1 in Sapium sebiferum

SUPPRESSOR OF MAX2 LIKE 1 (SMAX1) is a member of the SUPPRESSOR of MAX2 1‑LIKE family of genes and is known as a target protein of KARRIKIN INSENSITIVE2 (KAI2)-MORE AXILLARY BRANCHES2 (MAX2), which mediates karrikin signaling in Arabidopsis. SMAX1 plays a significant role in seed germination, hypocotyl elongation, and root hair development in Arabidopsis. SMAX1 has not yet been identified and characterized in woody plants. This study identified and characterized SsSMAX1 in Sapium sebiferum and found that SsSMAX1 was highly expressed in the seed, hypocotyl, and root tips of S. sebiferum. SsSMAX1 was functionally characterized by ectopic expression in Arabidopsis. SsSMAX1 overexpression lines of Arabidopsis showed significantly delayed seed germination and produced seedlings with longer hypocotyl and roots than wild-type and Atsmax1 functional mutants. SsSMAX1 overexpression lines of Arabidopsis also had broader and longer leaves and petioles than wild-type and Atsmax1, suggesting that SsSMAX1 is functionally conserved. This study characterizes the SMAX1 gene in a woody and commercially valuable bioenergy plant, Sapium sebiferum. The results of this study are beneficial to future research on the molecular biology of woody plants.

Cloning, Identification, and Functional Analysis of the Chalcone Isomerase Gene from Astragalus sinicus.

Astragalus sinicus is an important winter-growing cover crop. It is widely utilized, not only as a cover crop for its benefits in fertilizing the soil but also as a landscape ground cover plant. Anthocyanins are involved in the pigmentation of plants in leaves and flowers, which is a crucial characteristic trait for A. sinicus. The formation of anthocyanins depends significantly on the enzyme chalcone isomerase (CHI). However, research on the CHI gene of A. sinicus remains unexplored. The rapid amplification of cDNA ends (RACE) approach was used in this research to clone the CHI sequence from A. sinicus (AsiCHI). The expression profiles of the AsiCHI gene in multiple tissues of A. sinicus were subsequently examined by qRT-PCR (Quantitative Real-Time PCR). Furthermore, the function of the AsiCHI was identified by the performance of ectopic expression in Arabidopsis (Arabidopsis thaliana). The outcomes revealed that the full-length cDNA of the AsiCHI gene (GeneBank: OQ870547) measured 972 bp in length and included an open reading frame of 660 bp. The encoded protein contains 219 amino acids with a molecular weight of 24.14 kDa and a theoretical isoelectric point of 5.11. In addition, the remarkable similarity between the AsiCHI protein and the CHI proteins of other Astragalus species was demonstrated by the sequence alignment and phylogenetic analysis. Moreover, the highest expression level of AsiCHI was observed in leaves and showed a positive correlation with anthocyanin content. The functional analysis further revealed that the overexpression of AsiCHI enhanced the anthocyanidin accumulation in the transgenic lines. This study provided a better understanding of AsiCHI and elucidated its role in anthocyanin production.

Genome-Wide Identification of LBD Genes in Foxtail Millet (Setaria italica) and Functional Characterization of SiLBD21.

Plant-specific lateral organ boundaries domain (LBD) proteins play important roles in plant growth and development. Foxtail millet (Setaria italica) is one new C4 model crop. However, the functions of foxtail millet LBD genes are unknown. In this study, a genome-wide identification of foxtail millet LBD genes and a systematical analysis were conducted. A total of 33 SiLBD genes were identified. They are unevenly distributed on nine chromosomes. Among these SiLBD genes, six segmental duplication pairs were detected. The thirty-three encoded SiLBD proteins could be classified into two classes and seven clades. Members in the same clade have similar gene structure and motif composition. Forty-seven kinds of cis-elements were found in the putative promoters, and they are related to development/growth, hormone, and abiotic stress response, respectively. Meanwhile, the expression pattern was investigated. Most SiLBD genes are expressed in different tissues, while several genes are mainly expressed in one or two kinds of tissues. In addition, most SiLBD genes respond to different abiotic stresses. Furthermore, the function of SiLBD21, which is mainly expressed in roots, was characterized by ectopic expression in Arabidopsis and rice. Compared to controls, transgenic plants generated shorter primary roots and more lateral roots, indicating the function of SiLBD21 in root development. Overall, our study laid the foundation for further functional elucidation of SiLBD genes.

Dual functions of PsmiR172b-PsTOE3 module in dormancy release and flowering in tree peony (Paeonia suffruticosa).

MicroRNAs (miRNAs) are non-coding RNAs that interact with target genes and are involved in many physiological processes in plants. miR172-AP2 mainly plays a role in the regulation of flowering time and floral organ differentiation. Bud dormancy release is necessary for forcing culture of tree peony in winter, but the mechanism of dormancy regulation is unclear. In this study, we found that a miR172 family member, PsmiR172b, was downregulated during chilling-induced bud dormancy release in tree peony, exhibiting a trend opposite to that of PsTOE3. RNA ligase-mediated (RLM) 5'-RACE (rapid amplification of cDNA ends) confirmed that miR172b targeted PsTOE3, and the cleavage site was between bases 12 (T) and 13 (C) within the complementary site to miR172b. The functions of miR172b and PsTOE3 were detected by virus-induced gene silencing (VIGS) and their overexpression in tree peony buds. PsmiR172b negatively regulated bud dormancy release, but PsTOE3 promoted bud dormancy release, and the genes associated with bud dormancy release, including PsEBB1, PsEBB3, PsCYCD, and PsBG6, were upregulated. Further analysis indicated that PsTOE3 directly regulated PsEBB1 by binding to its promoter, and the specific binding site was a C-repeat (ACCGAC). Ectopic expression in Arabidopsis revealed that the PsmiR172b-PsTOE3 module displayed conservative function in regulating flowering. In conclusion, our results provided a novel insight into the functions of PsmiR172-PsTOE3 and possible molecular mechanism underlying bud dormancy release in tree peony.

Fine mapping and cloning of a novel BrSCC1 gene for seed coat color in Brassica rapa L.

A novel BrSCC1 gene for seed coat color was fine mapped within a 41.1-kb interval on chromosome A03 in Brassica rapa and functionally validated by ectopic expression analysis. Yellow seed is a valuable breeding trait that can be potentiality applied for improving seed quality and oil productivity in oilseed Brassica crops. However, only few genes for yellow seed have been identified in B. rapa. We previously identified a minor quantitative trait locus (QTL), qSC3.1, for seed coat color on chromosome A03 in B. rapa. In order to isolate the seed coat color gene, a brown-seeded chromosome segment substitution line, CSSL-38, harboring the qSC3.1, was selected and crossed with the yellow-seeded recurrent parent, a rapid cycling inbred line of B. rapa (RcBr), to construct the secondary F2 population. Metabolite identification suggested that seed coat coloration in CSSL-38 was independent of proanthocyanidins (PAs) accumulation. Genetic analysis revealed that yellow seed was controlled by a single recessive gene, Seed Coat Color 1 (BrSCC1). Utilizing bulked segregant analysis (BSA)-seq and secondary F2 and F2:3 recombinants analysis, BrSCC1 was fine mapped within a 41.1-kb interval. By integrating gene expression profiling, genome sequence comparison, metabolite analysis, and functional validation through ectopic expression in Arabidopsis, the BraA03g040800.3C gene was confirmed to be BrSCC1, which positively correlated with the seed coat coloration. Our study provides a novel gene resource for the genetic improvement of yellow seeds in oilseed B. rapa.

CsERF003, CsMYB7 and CsMYB102 promote cuticular wax accumulation by upregulating CsKCS2 at fruit ripening in Citrus sinensis

Cuticular wax is a hydrophobic protective layer covering the surface of citrus fruit, which plays an important role in preventing fruit water loss and improving fruit appearance quality. Our previous study has demonstrated that CsKCS2 is highly expressed at fruit ripening and involved in fruit cuticular wax synthesis in Citrus sinensis. However, the upstream regulators of CsKCS2 and the transcription factors regulating fruit cuticular wax synthesis at ripening remain unclear. Here, we performed a gene co-expression analysis with the published transcriptomic data of five developmental stages of Citrus sinensis fruit. The results showed that the gene expression trend of the blue module was synchronized with the accumulation pattern of fruit cuticular wax during fruit development and ripening. Further dual luciferase assay suggested that the promoter of CsKCS2 could be activated by CsERF003, CsMYB7 and CsMYB102 in the blue module. Moreover, ectopic expression in Arabidopsis revealed that CsERF003, CsMYB7 and CsMYB102 could increase cuticular wax content in leaves by upregulating the expression of wax-related genes (such as KCS, CER1, CER3 and CER4). Our results provide new insights into the transcriptional regulation of fruit cuticular wax synthesis, and important information for the mining of regulatory factors involved in cuticular wax synthesis in Citrus sinensis fruit.

Inhibition profile of trifludimoxazin towards PPO2 target site mutations.

Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides. Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations. Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Physiological and transcriptomic analysis uncovers salinity stress mechanisms in a facultative crassulacean acid metabolism plant Dendrobium officinale.

Dendrobium officinale is a precious medicinal Chinese herb that employs facultative crassulacean acid metabolism (CAM) and has a high degree of abiotic stress tolerance, but the molecular mechanism underlying the response of this orchid to abiotic stresses is poorly understood. In this study, we analyzed the root microstructure of D. officinale plantlets and verified the presence of chloroplasts by transmission electron microscopy. To obtain a more comprehensive overview of the molecular mechanism underlying their tolerance to abiotic stress, we performed whole‐transcriptome sequencing of the roots of 10-month-old plantlets exposed to salt (NaCl) treatment in a time‐course experiment (0, 4 and 12 h). The total of 7376 differentially expressed genes that were identified were grouped into three clusters (P < 0.05). Metabolic pathway analysis revealed that the expression of genes related to hormone (such as auxins, cytokinins, abscisic acid, ethylene and jasmonic acid) biosynthesis and response, as well as the expression of genes related to photosynthesis, amino acid and flavonoid metabolism, and the SOS pathway, were either up- or down-regulated after salt treatment. Additionally, we identified an up-regulated WRKY transcription factor, DoWRKY69, whose ectopic expression in Arabidopsis promoted seed germination under salt tress. Collectively, our findings provide a greater understanding of the salt stress response mechanisms in the roots of a facultative CAM plant. A number of candidate genes that were discovered may help plants to cope with salt stress when introduced via genetic engineering.

Improvement of plant tolerance to drought stress by cotton tubby-like protein 30 through stomatal movement regulation

IntroductionCotton is a vital industrial crop that is gradually shifting to planting in arid areas. However, tubby-like proteins (TULPs) involved in plant response to various stresses are rarely reported in cotton. The present study exhibited that GhTULP30 transcription in cotton was induced by drought stress. ObjectiveThe present study demonstrated the improvement of plant tolerance to drought stress by GhTULP30 through regulation of stomatal movement. MethodsGhTULP30 response to drought and salt stress was preliminarily confirmed by qRT-PCR and yeast stress experiments. Ectopic expression in Arabidopsis and endogenous gene silencing in cotton were used to determine stomatal movement. Yeast two-hybrid and spilt-luciferase were used to screen the interacting proteins. ResultsEctopic expression of GhTULP30 in yeast markedly improved yeast cell tolerance to salt and drought. Overexpression of GhTULP30 made Arabidopsis seeds more resistant to drought and salt stress during seed germination and increased the stomata closing speed of the plant under drought stress conditions. Silencing of GhTULP30 in cotton by virus-induced gene silencing (VIGS) technology slowed down the closure speed of stomata under drought stress and decreased the length and width of the stomata. The trypan blue and diaminobenzidine staining exhibited the severity of leaf cell necrosis of GhTULP30-silenced plants. Additionally, the contents of proline, malondialdehyde, and catalase of GhTULP30-silenced plants exhibited significant variations, with obvious leaf wilting. Protein interaction experiments exhibited the interaction of GhTULP30 with GhSKP1B and GhXERICO. ConclusionGhTULP30 participates in plant response to drought stress. The present study provides a reference and direction for further exploration of TULP functions in cotton plants.

Cloning, characterization, and ectopic expression in Arabidopsis to determine the function of FvSPL1 gene from woodland strawberry (Fragaria vesca)

BACKGROUND: SQUAMOSA promoter binding protein-like (SPL) proteins are a class of plant-specific transcription factors that play important roles in plant development. However, the majority of SPL genes in strawberry are functionally uncharacterized. OBJECTIVE: To understand the biological functions and elucidate the molecular regulatory mechanisms of SPL genes in strawberry development. METHODS: The FvSPL1 gene from woodland strawberry was cloned and the phylogenetic tree was analyzed using bioinformatics methods. Subcellular localization, transcriptional activity, DNA binding ability and regulatory mechanisms of FvSPL1 were analyzed through biochemical and genetic approaches. RESULTS: Phylogenetic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that FvSPL1 clustered in the same group as those of the miR156 target site located at the 3′-untranslated region. Further biochemical analysis indicated that FvSPL1 was exclusively localized in the nucleus. Electrophoretic mobility shift assay demonstrated that FvSPL1 could specifically recognize the GTAC motif. Transcriptional activity analysis showed that FvSPL1 is a transcriptional activator that could activate the expression of the FvAP1 gene. Finally, transgenic Arabidopsis overexpressing the FvSPL1 gene exhibited significantly early flowering. Taken together, our study indicated that FvSPL1, similar to its orthologs in Arabidopsis, mainly functions in regulating plant flowering. CONCLUSIONS: These findings provide insight into the mechanism of flowering in strawberry, and contribute to the understanding of strawberry flowering time manipulation which will facilitate the molecular breeding of strawberries.

Cold induced genes (CIGs) regulate flower development and dormancy in Prunus avium L.

The flower buds continue to develop during the whole winter in tree fruit species, which is affected by environmental factors and hormones. However, little is known about the molecular mechanism of flower development during dormancy phase of sweet cherry in response to light, temperature and ABA. Therefore, we identified two cold induced gene (CIG) PavCIG1 and PavCIG2 from sweet cherry, which were closely to PpCBF and PyDREB from Prunus persica and Prunus yedoensis by using phylogenetic analysis, suggesting conserved functions with these evolutionarily closer DREB subfamily genes. Subcellular localization analysis indicated that, PavCIG1 and PavCIG2 were both localized in the nucleus. The seasonal expression levels of PavCIG1 and PavCIG2 were higher at the stage of endodormancy in winter, and induced by low temperature. Ectopic expression of PavCIG1 and PavCIG2 resulted in a delayed flowering in Arabidopsis. Furthermore, PavCIG2 increased light-responsive gene PavHY5 transcriptional activity by binding to its promoter, meanwhile, PavHY5-mediated positive feedback regulated PavCIG2. Moreover, ABA-responsive protein PavABI5-like could also increase transcriptional activity of PavCIG and PavCIG2. In addition, PavCIG and PavCIG2 target gene PavCAL-like was involved in floral initiation, demonstrated by ectopic expression in Arabidopsis. These findings provide evidences to better understand the molecular mechanism of CIG-mediated flower development and dormancy in fruit species, including sweet cherry.

A Meloidogyne incognita C-type lectin effector targets plant catalases to promote parasitism.

Root-knot nematodes, Meloidogyne spp., secrete effectors to modulate plant immune responses and establish a parasitic relationship with host plants. However, the functions and plant targets of C-type lectin (CTL)-like effectors of Meloidogyne incognita remain unknown. Here, we characterized a CTL-like effector of M.incognita, MiCTL1a, and identified its target and role in nematode parasitism. In situ hybridization demonstrated the expression of MiCTL1 in the subventral glands; and in planta, immunolocalization showed its secretion during M.incognita parasitism. Virus-induced gene silencing of the MiCTL1 reduced the infection ability of M.incognita in Nicotiana benthamiana. The ectopic expression in Arabidopsis not only increased susceptibility to M.incognita but also promoted root growth. Yeast two-hybrid and co-immunoprecipitation assays revealed that MiCTL1a interacts with Arabidopsis catalases, which play essential roles in hydrogen peroxide homeostasis. Knockout or overexpression of catalases showed either increased or reduced susceptibility to M.incognita, respectively. Moreover, MiCTL1a not only reduced catalase activity invitro and in planta but also modulated stress-related gene expressions in Arabidopsis. Our data suggest that MiCTL1a interacts with plant catalases and interferes with catalase activity, allowing M.incognita to establish a parasitic relationship with its host by fine-tuning responses mediated by reactive oxygen species.

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.)

Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

Overexpression of an Antisense RNA of Maize Receptor-Like Kinase Gene ZmRLK7 Enlarges the Organ and Seed Size of Transgenic Arabidopsis Plants.

Leucine-rich repeat (LRR)-receptor-like protein kinases (LRR-RLKs) play vital roles in plant growth, development, and responses to environmental stresses. In this study, a new LRR-RLK gene, ZmRLK7, was isolated from maize, and its function within plant development was investigated through ectopic expression in Arabidopsis. The spatial expression pattern analysis reveals that ZmRLK7 is highly expressed in embryos prior to programmed cell death (PCD) of starchy endosperm tissues, and its encoded protein has been localized to both plasm and nuclear membranes subcellularly. Overexpression of sense ZmRLK7 reduced the plant height, organ size (e.g., petals, silique, and seeds), and 1000-seed weight in transgenic lines, while the antisense transgene enlarged these traits. Cytological analysis suggested that ZmRLK7 negatively regulates petal size through restricting both cell expansion and proliferation. In addition, abnormal epidermal cell structure was observed, and the stomata number decreased obviously in sense ZmRLK7 transgenic lines with a lower stomatal index than that in the wild type. Quantitative RT-PCR analysis indicated that transcript levels of genes that are involved in the brassinosteroid and ERACTA signaling pathways were coordinately altered, which could partially explain the phenotypic variation. Moreover, overexpression of antisense ZmRLK7 substantially rescued the Arabidopsis bak1-3 mutant phenotype. All these results together suggest that ZmRLK7 can serve as an important regulator in regulating plant architecture and organ size formation. This work will provide insight into the function of ZmRLK7 in maize.

Brassica napus COL transcription factor BnCOL2 negatively affects the tolerance of transgenic Arabidopsis to drought stress

Rapeseed (Brassica napus) is an important oil crop in the world but shows poor stress resistance. Extreme weather events, such as frequent droughts, have severely reduced rapeseed production. In recent decades, transcription factors, which are responsive to diverse stresses, have been used to explore the stress tolerance of plants by transgenic methods. In this study, we functionally characterized a member of the CONSTANS-Like transcription factor family, BnCONSTANS-LIKE2 (BnCOL2), by ectopic expression in Arabidopsis. BnCOL2 showed transcriptional activation activity and was localized in the nucleus. Tissue expression analysis showed that BnCOL2 was mainly expressed in cotyledons and leaves of rapeseed seedlings. Expression patterns indicated that BnCOL2 was induced by NaCl, PEG-6000, and abscisic acid (ABA). Overexpression of BnCOL2 in Arabidopsis resulted in mannitol- and ABA-sensitive phenotypes compared with the wild-type (WT) Arabidopsis phenotype. BnCOL2-overexpression lines exhibited significantly reduced drought stress tolerance, as illustrated by lower survival percentage and leaf temperature compared with those of the WT under drought stress. The water loss rate was remarkably reduced in BnCOL2-overexpression lines in comparison to the WT. Transcription of nine stress-responsive genes, such as AtRD29A and AtCOR27, in BnCOL2-overexpressing lines changed significantly under drought stress compared with those of the WT. The results indicate that BnCOL2 may regulate plant tolerance to drought stress by regulating expression of ABA-responsive and drought stress-related genes.

A Rapeseed WRKY Transcription Factor Phosphorylated by CPK Modulates Cell Death and Leaf Senescence by Regulating the Expression of ROS and SA-Synthesis-Related Genes

Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence. Here, we identified a novel WRKY transcription factor gene WSR1 (WRKY regulating SA and ROS 1) in Brassica napus (rapeseed) in promoting SA and ROS production, which eventually led to leaf senescence thereafter. Its expression increased in senescing leaves. Ca2+-dependent protein kinase (CPK) 5 and -6 interacted with and phosphorylated BnaWSR1. Overexpression of phosphomimic BnaWSR1 (BnaWSR1ca) in rapeseed protoplasts elicited ROS production and cell death while its ectopic expression in Arabidopsis enhanced SA and ROS levels and, hence, accelerated leaf senescence. Furthermore, BnaWSR1ca activated the expression of Isochorismate Synthase 1 (ICS1), Respiratory Burst Oxidase Homologue (Rboh) D, and Senescence-Associated Gene 14 (SAG14). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated that BnaWSR1ca directly bound to promoter regions of ICS1, RbohD, and SAG14. These data have identified a CPK-WSR1 module that integrates SA and ROS to control cell death and leaf senescence.

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

Background“Bairihua”, a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development.ResultsSPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in “Bairihua” and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in “Bairihua”. Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time.ConclusionsOur results implied a novel pathway that CbuSPL9 regulated flowering development, but not flowering transition, with the participation of CbuHMGA. Further investments need to be done to verify the details of this pathway.

HD-ZIP I Transcription Factor (PtHB13) Negatively Regulates Citrus Flowering through Binding to FLOWERING LOCUS C Promoter.

For floral induction in adult citrus, low temperature is one of the most important environmental factors. FLOWERING LOCUS C (FLC) plays a very important role in low-temperature-induced Arabidopsis flowering by repressed FLC expression under exposure to prolonged low-temperature conditions. However, little is known about the FLC regulation mechanism in perennial woody plants such as citrus. In this study, the functions of citrus FLC homolog (PtFLC) were investigated by ectopic expression in Arabidopsis. Transcription factor of homeodomain leucine zipper I (HD-ZIP I) as an upstream regulator of PtFLC was identified by yeast one-hybrid screen to regulate its transcription. The HD-ZIP I transcription factor was highly homologous to Arabidopsis ATHB13 and thus was named PtHB13. Ectopically expressed PtHB13 inhibited flowering in transgenic Arabidopsis. Furthermore, the expression of PtFLC and PtHB13 showed a seasonal change during the floral induction period and was also affected by low temperature. Thus, we propose that PtHB13 binds to PtFLC promoter to regulate its activity during the citrus floral induction process.