Function Analysis Of DEAD-box RNA Helicase CmRH56 Regulating Morphogenesis Of Suckers And Drought Stress Tolerance In Chrysanthemum

Chrysanthemum has long cultivation and culture history,and is an important flower using for ornament,food,tea and medicines.As ornamental crop,chrysanthemum is widely used for cut flowers,potted flowers,landscape and environment beautification.Plants have evolved various of intrinsic tolerance strategies to acclimate to ambient adversity in long-term natural selection,such as drought stress.The incidence of chryssanthemum suckers are the key factors reflecting plant to acclimate to ambient adversity,asexual reproduction,and regeneration ability of plant.The type of suckers can be changed by environmental factors.But the study of the relationship between the number and type of chrysanthemum suckers and drought stress tolerance has not reported yet.This study aims to explore the adaptive relationship between chrysanthemum suckers incidence and drought stress tolerance,looking forward to understand the adaptation mechanism of drought tolerance in chrysanthemum,provide new ideas for drought tolerance breeding in chrysanthemum.In this study,we used landscaping chrysanthemum ’Fall Color’ as experimental material.We isolated a DEAD-box RNA helicase gene CmRH56 in chrysanthemum,examined the expression pattern of CmRH56 in different organs of chrysanthemum,as well as its expression pattern induced by drought stress and ABA.We obtained CmRH56 overexpression and RNAi lines by agrobcterium-mediated transformation,and observed sucker incidence and drought stress tolerance of WT and CmRH56 transgenic chrysanthemum.We explored molecular mechanism of CmRH56 involved in chrysanthemum sucker occurrence and drought stress tolerance by RNA-seq.Our results demonstrated that:1)We obtained a 1675 bp CmRH56 gene from chrysanthemum.It coded 428 amino acid,the deduced amino acid sequence contains nine conserved motifs of DExD/H-box RNA helicase.According to the specific amino acid sequence of motif Ⅱ,it belongs to DEAD-box RNA helicase subfamily.The protein of CmRH56 located in the nucleus.The gene of CmRH56 was reduced by drought stress and ABA.CmRH56 is expressed in all the tested tissues of chrysanthemum.While in juvenile phase of chrysanthemum the highest expression is existed in root,during adult phase the highest expression is existed in suckers,suggesting that the expression of CmRH56 can be changed along with the growth and development.2)Promoter of CnRH56 contains cis-acting element about drought stress,cold stress,ABA and GA,indicating that CmRH56 may play roles in these regulation pathway.CmRH56 overexpression lines can increase the number of suckers,and have early germination.while CmRH56-RNAi lines show less suckers and delayed germination.3)Drought stress tolerance of WT and CmRH56 transgenic chrysanthemum found that overexpression of CmRH56 reduced drought stress tolerance,CmRH56-RNAi lines show increased drought stress tolerance.The number of suckers is reduced by drought stress at different degrees,and result in suckers from bud-type into root-type.Rehydration can recovered the number of suckers and bud-type suckers in WT and CmRH56 overexpression,but root-type suckers in RNAi lines.It suggests that the number and type change of suckers in RNAi lines are morphological basis for enhanced drought tolerance.4)RNA-seq results showed that 6 DREBs and 1 GA2ox were up-regulated in CmRH56-RNAi lines.Further experimental results showed that the expression of DREBs and GA2ox were significantly increased by drought stress,and DREBs were decreased after rehydration,but GA2ox did not changed significantly.GA treatment could significantly increase the number of bud-type suckers in WT and RNAi lines,suggesting that CmRH56-RNAi lines increased drought tolerance may be related to participate and mediate ABA-independent induced transcription factor DREB pathway,and the GA may participate in the suckers germination and morphological changes.5)The correlation analysis of drought stress tolerance and suckers incidence in 22 chrysanthemum cultivars revealed that there was a slight negative correlation between the number of suckers and drought tolerance:the variety with more suckers are not conducive to drought tolerance of the plants,but it was conducive to the recovery after drought stress,the plants could resume growth through regeneration of suckers.In conclusion,CmRH56 regulates the number and morphology of suckers through GA biosynthesis pathway under drought stress in Chrysanthemum,and regulates the expression of DREBs in ABA-independent signal pathway to enhance the tolerance of drought stress in Chrysanthemum.In addition,we confirmed the number of suckers was slight negative correlation with drought tolerance.