Mechanism Of Drought Response Cooperatively Regulated By A Long Intergenic Noncoding RNA DALIR And HLP2 In Arabidopsis Thaliana

Drought is currently one of the most severely affected climates by global temperature changes,and it is also one of the main factors limiting agricultural production and development.Solving the problem of crop drought tolerance is conducive to ensuring the sustainable agricultural development and food security.Understanding how plants respond to drought stress is helpful to guide the cultivation of new varieties with high quality and drought tolerance,and provide theoretical basis for reducing the adverse effects of drought disasters in agriculture.Plants have developed many adaptations at the morphological,physiological,cellular and molecular levels to cope with the serious effects of drought.Studies have shown that long non-coding RNA plays an important role in plant drought resistance.We previous identified a linc RNA,DALIR(drought associated linc RNA),which is involved in the plant drought response.dalir mutant leaves have faster water loss rate and lower tolerance to drought.It was further found that DALIR could positively regulate the expression of drought-related gene NAC055,but the regulatory mechanism remained unclear.In this study,a glycine-rich RNA binding protein HLP2 interacting with DALIR was screened by yeast three-hybrid assay,physiological and biochemical methods are used to further clarify alternative polyadenylation of NAC055 co-regulated by DALIR and HLP2.The main results are as follows:1.DALIR interacts with HLP2 in the nucleusThe yeast three hybrid experiment was used to screen the yeast library of Arabidopsis thaliana RNA binding proteins previously created in our laboratory,and the interaction between the RNA binding protein HLP2 and DALIR was screened and identified.The RNA pull-down experiment also confirmed the interaction between HLP2 and DALIR in vitro.Previous studies had shown that DALIR was mainly enriched in the nucleus,and HLP2 was also localized in the nucleus.Moreover,the interaction between DALIR and HLP2 in the nucleus of tobacco was demonstrated by using the Trimolecular Fluorescence Complementation Experiment(Tri FC).RNA immunoprecipitation(RIP)assay also demonstrated the interaction of DALIR and HLP2 in plant in vivo.2.HLP2 negatively regulates plant response to drought stressFunctional deletion mutant of HLP2 that terminated translation prematurely due to base deletion via gene editing technology CRISPR/Cas9 was constructed.The PEG simulated drought plate experiment found that the hlp2 mutant was insensitive to PEG,compared to the wild type.Similarly,in the soil drought experiment,the mutant of the hlp2 enhanced plants drought resistance.In addition,the decrease in stomatal aperture and the slow rate of water loss in detached leaves of the hlp2 mutant were contributed to the enhanced drought resistance.The PEG sensitivity and stomatal aperture phenotypes were consistent with those of the wild type when HLP2 was inserted into the hlp2 deletion mutant.These results suggested that HLP2 was a negative regulator of drought regulatory responses in A thaliana.3.HLP2 and DALIR cooperatively regulate alternative polyadenylation of NAC055qRT-PCR detection found that the expression of NAC055,a positive regulator involved in drought stress,was lower in the dalir mutant than in the wild type,while the expression of NAC055 was higher in the hlp2 mutant.Additionally,NAC055 has two poly(A)sites: the proximal poly(A)site and the distal poly(A)site.Different poly(A)sites can be selected to form two transcripts of different lengths.APA analysis showed that the proportion of NAC055 short transcripts decreased and the proportion of long transcripts increased in the hlp2 mutant.We constructed the NAC055 long and short transcript complementation line and found thant only NAC055 long transcript complementation line and Col-0 exhibited comparable stomatal indices.4.HLP2 interacts with PABNsThe PABNs protein family includes PABN1,PABN2,and PABN3.Previous studies had shown that they all interacted with DALIR in vivo and in vitro.The interaction of HLP2 with PABN1,PABN2,and PABN3 in yeast cells was demonstrated by yeast two hybrid assays.In summary,DALIR and HLP2 interact in the nucleus,they cooperatively regulate both expression level and alternative polyadenylation of NAC055,thereby affecting the drought stress response in A thaliana.The research results of this topic not only enrich the molecular mechanism of regulating gene expression by long non-coding RNA and interacting proteins,but also provide theoretical guidance for using molecular biological methods to improve plant varieties and improve crop drought resistance.