Analysis of expression characteristics and identification of interaction proteins of transcription factor BnaABI5 in Brassica napus.

Rapeseed is one important oil crop in China. However, its planting benefit is frequently affected by environmental stresses such as drought in the northwest region of China. The abscisic acid(ABA) signaling pathway plays an important role in plant abiotic stress response and tolerance, and ABFs/AREBs(ABA-responsive element binding factors/ABA-responsive element binding proteins) are the core transcription factors that regulate the expression of ABA-responsive genes. To dissect the key transcription factors mediated abiotic stress, we mainly characterized abscisic acid insensitive 5(BnaABI5) in rapeseed, including its subcellular localization, expression pattern in response to various stress and tissue-specific expression analysis, transcriptional activity analysis as well as interaction screening with BnaMPKs(mitogen-activated protein kinases). Our results showed that the BnaABI5-GFP fusion protein was localized in the nucleus, and its transcript level is induced by drought stress and was mainly expressed in the roots of rapeseed. Furthermore, BnaABI5 showed transcriptional activation activity through a yeast transactivation assay and it also activated the promoter activity of EM6 target gene in the transient expression system in tobacco leaves. Moreover, BnaABI5 interacted with BnaMPK6 and BnaMPK13 through BiFC and Y2H analysis. This study preliminarily explored the expression characteristics of transcription factor BnaABI5 and its interaction with BnaMPKs, which might help us for further understanding the function of BnaABI5.

Rapeseed PP2C37 Interacts with PYR/PYL Abscisic Acid Receptors and Negatively Regulates Drought Tolerance.

Global water deficit is a severe abiotic stress threatening the yielding and quality of crops. Abscisic acid (ABA) is a phytohormone that mediates drought tolerance. Protein kinases and phosphatases function as molecular switches in eukaryotes. Protein phosphatases type 2C (PP2Cs) are a major family that play essential roles in ABA signaling and stress responses. However, the role and underlying mechanism of PP2C in rapeseed (Brassica napus L.) mediating drought response has not been reported yet. Here, we characterized a PP2C family member, BnaPP2C37, and its expression level was highly induced by ABA and dehydration treatments. It negatively regulates drought tolerance in rapeseed. We further identified that BnaPP2C37 interacted with multiple PYR/PYL receptors and a drought regulator BnaCPK5 (calcium-dependent protein kinase 5) through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Specifically, BnaPYL1 and BnaPYL9 repress BnaPP2C37 phosphatase activity. Moreover, the pull-down assay and phosphatase assays show BnaPP2C37 interacts with BnaCPK5 to dephosphorylate BnaCPK5 and its downstream BnaABF3. Furthermore, a dual-luciferase assay revealed BnaPP2C37 transcript level was enhanced by BnaABF3 and BnaABF4, forming a negative feedback regulation to ABA response. In summary, we identified that BnaPP2C37 functions negatively in drought tolerance of rapeseed, and its phosphatase activity is repressed by BnaPYL1/9 whereas its transcriptional level is upregulated by BnaABF3/4.

Rapeseed NAM transcription factor positively regulates leaf senescence via controlling senescence-associated gene expression.

Leaf senescence is one of the most visible forms of programmed cell death in plants. It can be a seasonal adaptation in trees or the final stage in crops ensuring efficient translocation of nutrients to seeds. Along with developmental cues, various environmental factors could also trigger the onset of senescence through transcriptional cascades. Rapeseed (Brassica napus L.) is an important oil crop with its yielding affected by significant falling leaves as a result of leaf senescence, compared to many other crops. Therefore, a better understanding of leaf senescence and developing strategies controlling the progress of leaf senescence in rapeseed is necessary for warranting vegetable oil security. Here we functionally characterized the gene BnaNAM encoding No Apical Meristem (NAM) homologue to identify transcriptional regulation of leaf senescence in rapeseed. A combination of transient and stable expression techniques revealed overexpression of BnaNAM induced ROS production and leaf chlorosis. Quantitative evaluation of up-regulated genes in BnaNAM overexpression lines identified genes related to ROS production (RbohD, RbohF), proteases (ßVPE, γVPE, SAG12, SAG15), chlorophyll catabolism (PaO, PPH) and nucleic acid degradation (BFN1) as the putative downstream targets. A dual luciferase-based transcriptional activation assay of selected promoters further confirmed BnaNAM mediated transactivation of promoters of the downstream genes. Finally, an electrophoretic mobility shift assay further confirmed direct binding of BnaNAM to promoters of ßVPE, γVPE, SAG12, SAG15 and BFN1. Our results therefore demonstrate a novel role of BnaNAM in leaf senescence.

Calcium-dependent Protein Kinase 5 (CPK5) positively modulates drought tolerance through phosphorylating ABA-Responsive Element Binding Factors in oilseed rape (Brassica napus L.).

Drought is an environmental stress that causes severe crop loss. Drought stress can induce abscisic acid (ABA) accumulation and cytoplasmic calcium oscillation. Calcium-dependent protein kinases (CPKs) constitute a group of Ser/Thr protein kinases decoding calcium signals. However, the function and molecular mechanisms of most CPKs in oilseed rape (Brassica napus) remain unknown. Here, we report the functional characterization of BnaCPK5 in drought stress tolerance. BnaCPK5 belongs to Group I of the CPK family and was localized at the plasma membrane and nuclei. Overexpression of BnaCPK5 enhanced drought stress tolerance compared with the control. A screening of interacting proteins identified that BnaCPK5 interacted strongly with two ABA-Responsive Element Binding Factors (ABF/AREBs), BnaABF3 and BnaABF4. BnaCPK5 was shown to phosphorylate both BnaABF3 and BnaABF4 in a kinase assay. Further, it was found that the phosphorylation of BnaABF3 and BnaABF4 by BnaCPK5 increased their transcriptional activities against the famous drought stress marker gene, Responsive to Dehydration (RD) 29B and protein stability. Taken together, these data demonstrate that BnaCPK5 acts as a positive regulator of drought tolerance by, at least in part, phosphorylating two core ABA-signaling components to modulate Late-Embryogenesis Abundant (LEA)-like RD29B expression.