Functional Analysis Of Hc WRKY17 And HcWRKY26 Response To Salinity And Drought Stress During Seedling Stage Of Kenaf(Hibiscus Cannabinus)

High salinity and drought are the two main abiotic stresses,which severely inhibit the productivity of crops.The signal transduction and the molecular regulatory system will be disorganized when the plants cope with these stresses.However,the transcription factors play an essential role in plant signaling networks.For one hand,they may act as regulatory factors involved in the cross-talk between different signaling pathways to adjust metabolic processes;on the other hand,they may actively sense the upstream signals and then regulate the expression of downstream genes to help the plants adapt to adverse conditions.WRKY proteins which comprise one of the largest transcription factor families regulate a number of developmental processes and stress responses.However,the studies about WRKYs is being concentrated on many model plants responding to biotic stresses,the roles of them in abiotic stresses are poorly understood,particularly in non-model plants.Kenaf(Hibiscus cannabinus L.)is an environmental-friendly fiber crop,can survive in the salinity and drought conditions,but there's no report about WRKY proteins studies in kenaf.Moreover,a great number of studies indicate that the housekeeping genes cannot stably express when plant responds to adverse stresses.Thus,we firstly validated and evaluated the expression stability of reference genes in kenaf and jute under the abiotic stresses,then isolated and validated 28 WRKY genes in kenaf,and analysed the expression profiles of HcWRKYs in response to different abiotic stresses and hormonal stimulus,and obtained two HcWRKY genes(HcWRKY17 and HcWRKY26)that can be actively induced by salinity and drought.Finally,the functions of the two HcWRKYs were further investigated.The main results are listed as follows:1.The stably expressed reference genes in kenaf and jute under different abiotic stressesGene expression analysis revealed that many reference genes cannot express stably under the stress conditions.Via qRT-PCR and soft analysis,a set of suitable reference genes were selected in kenaf and jute.The best reference genes were ACT7 and RAN in jute treated with salinity and biotic stressses;DnaJ and UBC were the best references in jute under drought stress;UBI and TUB were the best for different jute tissues.While the best reference genes were PP2A and PTB in kenaf under salinity,drought and cold stresses;ACT4 and PP2A were the best choices for kenaf under different hormonal stimulus;EF1?,PTB and 18S rRNA were the optimum for different kenaf tissues.The results can provide researchers with appropriate reference genes for qRT-PCR in kenaf and jute,and will facilitate gene expression study under those conditions.2.Expression of HcWRKYs were regulated by multiple abiotic stresses and signaling factorsqRT-PCR was performed to investigate the expression pattern of 28 HcWRKYs across different tissues,various abiotic stresses and hormones signaling.The results revealed that nearly half of the HcWRKYs showed no significant organ/tissue related differences,but some were clearly spatial differences.For example,Hc WRKY13,Hc WRKY8 and Hc WRKY6 were expressed at high levels in root,stem,leaf and phloem;HcWRKY5 and HcWRKY27were highly expressed in stem and phloem;while HcWRKY24 and HcWRKY25 were highly expressed in root and HcWRKY9,HcWRKY14 and HcWRKY18 expression were particularly high in leaves.In addition,in order to obtain insights into the potential roles of 28 HcWRKY genes in stress associated gene expression,5-leaf-old seedlings were exposed to salt,drought and cold stress and different hormones.qRT-PCR results revealed that five Hc WRKY genes could be up-regulated by salt,drought and cold stresses(HcWRKY15,HcWRKYl 7,Hc WRKY19,HcWRKY23,HcWRKY26),two HcWRKY gents could be up-regulated by ABA stimulus(HcWRKY8,HcWRKY 15),nine HcWRKY genes up-regulated by JA(HcWRKY3,HcWRKY8,HcWRKY13,HcWRKY15,HcWRKY17,HcWRKY19,HcWRKY20,HcWRKY22,HcWRKY26),two by SA(HcWRKYM,HcWRKY24)and two by ET(HcWRKY13,HcWRKY22).In general,HcWRKY gene expression was influenced by a broad range of abiotic stress and hormonal signals,indicating that they may play a key role in signal transduction in plant resistance regulation.3.HcWRKY 17 regulated osmotic stress responses andABA-dependent stomatal movementGene expression analysis revealed that HcWRKY17 was actively responsive to salinity and drought stresses,compared with other abiotic treatments.HcWRKY17 protein played a role in the cell nucleus,and overexpression of HcWRKY17 in Arabidopsis enhanced salinity and drought tolerance,owing to the lower MDA content,water loss rate and ionic conductivity rate.Overespression of HcWRKY17 conferred reduced seed germination and early seedling growth under salinity stress,and decreased sensitivity to ABA during germination and seedling growth,the phenotypes resembling those by lack of ABF4 and/or ABI5.Gene expression analysis revealed that ABI1 and ABI2 were up-regulated by HcWRKY17,whereas ABI5 and ABF4,the downstream genes of ABI1 and ABI2,which were positive regulator of ABA signaling,was down-regulation in the HcWRKY17ox lines.This was consistent with the observed ABA-insensitive phenotypes in seed germination and seedling growth.Moreover,it was found that overexpression of HcWRKY17 caused a reduction of stomatal density and a decreased water loss rate,and stomatal closure was more sensitive to ABA than that in the wildtype.It is probable that the reduction in the number of stomata leads to reduced transpiration during the salinity and drought period.These data suggested that the salinity and drought tolerance phenotypes exhibited by the HcWRKY17ox lines may also be attributable to the stomatal closure under stress conditions.The results also suggested that there could be different signaling pathways for the ABA-mediated effects on germination or seedling growth and ABA-induced stomatal closure.4.HcWRKY26 acted as a negative regulator to respond to salinity and drought stressesAlthough HcWRKY26 also actively responded to salinity and drought treatments,the overexpression of HcWRKY26 resulted in sensitivity to salinity and drought stresses and insensitivity to ABA treatment.Seven stress-related genes were moderately up-regulated in HcWRKY26-transgenic plants.ABI1 and ABI2 are two negative regulators of ABA responses in Arabidopsis,the up-regulation of gene expression would lead to reduced ABA sensitivity and decreased salinity and drought tolerance.Otherwise,STZ is a ZPT2-related protein,acting as a transcriptional repressor that down-regulates the transactvation of transcription factors,and overexpression of STZ resulted in growth retardation.Although the up-regulated of DREB2A and down-regulation of STZ in this study,the transgenic plants showed more sensitive to salinity and drought stresses,indicating that HcWRKY26 function as a negative regulator to respond to abiotic stresses.