Function,Signaling Mechanism And Stress Response Of The CRYPTOCHROME 1a And CRYPTOCHROME 1b In Sweet Sorghum

Blue light receptors cryptochromes was found in Arabidopsis at first,regulate a series of blue light responses,including inhibition of hypocotyl/stems elongation,cotyledon expansion,accumulation of anthocyanin,photoperiodic flowering,stomatal opening and so on.It was subsequently found in a variety of higher plants.But the molecular mechanism of CRY-mediated blue light signal transduction has not been well characterized in the crops except a detailed study on soybean.In particular,there is a lack of in-depth research on how CRYs can participate in regulating ABA signal transduction.Sweet sorghum has high photosynthetic efficiency,high biological yield and strong resistance,and it is becoming more and more important as sugar,fodder and energy crops.This study mainly discussed the tissue specific expression patterns of sweet sorghum Sb CRY1 a and Sb CRY1 b,the expression pattern of circadian rhythm and the subcellular localization of protein.The conservatism of the function and mechanism of signal transduction of Sb CRY1 a and Sb CRY1 b was studied in transgenic Arabidopsis.In addition,Sb CRY1 a and Sb CRY1 b invoved in ABA and salt stress response,regulated stress genes expression.The main results are as follows:1.The primary protein structure of sweet sorghum cryptochromes and its phylogenetic relationship to other cryptochromesThe amino acid sequences alignment of cryptochromes indicates Sb CRY1 a and Sb CRY1 b showed higher sequence similarity to At CRY1,including the conservative N-terminal PHR domain and DAS domain of C-terminal domain.And all the 13 amino acids predicted to interact with FAD in At CRY1 are conserved in Sb CRY1 a and Sb CRY1 b,the TGYP and LLDAD motifs which are involved in formingFAD-binding domain and FAD-binding pocket of cryptochromes were found in Sb CRY1 a and Sb CRY1 b.Six of seven amino acid residues known to interact with MTHF,the light-harvesting cofactor of At CRY1,are conserved in Sb CRY1 a with the exception of histidine at position 54,which was replaced by tyrosine.Based on phylogenetic analysis result,21 diverse species CRYs evolved in different branches clearly between dicotyledonous and monocotyledonous species.And Sb CRY1 a and Sb CRY1 b were grouped in monocot CRY1 clade and showed the highest homology with Zm CRY1 and Os CRY1 b.The results indicted the predicated Sb CRY1 a and Sb CRY1 b are sweet sorghum blue light receptors.2.The tissue-specific,light-responsive and diurnal-regulated expression of sweet sorghum CRY1q PCR(Real-time Quantitative PCR)results indicated Sb CRY1 a and Sb CRY1 b transcripts were leaf-specific,less expression in other tissues and the expression model of Sb CRY1 a and Sb CRY1 b were coincident and showed a clear diurnal regulation no matter in LD or SD condition.But the protein level of Sb CRY1 a and Sb CRY1 b exhibited no obvious oscillation.The results demonstrated a transcriptional or posttranscriptional regulation to Sb CRY1 a and Sb CRY1 b exists.3.Sb CRY1 a and Sb CRY1 b mediate de-etiolation in a blue light–specific manner in ArabidopsisTo investigate the function of Sb CRY1 a and Sb CRY1 b in plants,overexpression transgenic Arabidopsis in Col4,cry1 and cry1cry2 mutant background were obtained through floral dipping.The transgenic lines were analyzed in different light condition,the results showed Sb CRY1a/Col4 and Sb CRY1b/Col4 were hypersensitive to blue light and inhibited hypocotyl elongation in response to white and blue light,however,hypocotyl length has no obvious difference in dark and red light.Long hypocotyl phenotype of cry1 and cry1cry2 mutant was completely rescued in Sb CRY1a/cry1 and Sb CRY1b/cry1cry2 lines.Those results demonstrated Sb CRY1 a and Sb CRY1 b exerted the same function as Arabidopsis cryptochromes in mediating blue light stimulation of de-etiolation in plants.4.Sb CRY1 b regulated photoperiodic flowering rather than Sb CRY1 a in LDcondition.The transgenic lines of Sb CRY1 a and Sb CRY1 b photoperiodic flowering analysis result indicted Sb CRY1b/cry1cry2 rescued the late-flowering phenotype of the cry1cry2 mutant in long day condition,however,transgenic plants expressing Sb CRY1a-Myc also showed accelerated flowering in Arabidopsis of cry1cry2 mutant background.The flowering time of four genotypes was no difference in short day condition.Sb CRY1 b promotes flowering by stimulating m RNA expression of the FLOWERING LOCUS T(FT).The results suggested Sb CRY1 b and Arabidopsis CRY2 have a similar mode of action in regulating flowering time.5.Sb CRY1 a and Sb CRY1 b interacts with At SPA1 and At COP1 in response to blue light and mediates blue-light-induced HY5 accumulation in ArabidopsisYeast two-hybrid result indicated Sb CRY1 a and Sb CRY1 b interacted with At SPA1 and At COP1 in a blue light-dependent manner.The stability of HY5 was analyzed in Col4,cry1cry2 mutant,Sb CRY1a/cry1,Sb CRY1b/cry1cry2 in response to blue light,the results showed blue light induces more obvious increase in the abundance of the Myc HY5 protein in Sb CRY1a/cry1 and Sb CRY1b/cry1cry2 than that in wild type,cry1 or cry1cry2.The results demonstrated the mechanism of Sb CRY1a-mediated genes expression and photomorphogenesis is similar to that mediated by At CRY1.6.Overexpression of Sb COP1 results in a longer hypocotyl in the transgenic Arabidopsis and Sb COP1 directly interacts with Sb HY5Overexpression transgenic Arabidopsis of Sb COP1 was obtained through floral dipping.Sb COP1 overexpression lines exhibited longer hypocotyls than Col4 in continuous blue light.And yeast two-hybrid result indicted Sb HY5 interacted with At COP1 and Sb COP1.Furthermore,blue light significantly induced Sb HY5 accumulation in the transgenic Arabidopsis.Those results demonstrated Sb HY5 probably functions as a target protein of At COP1 to regulate light response and the mechanism of photomorphogenic development regulated by COP1 interaction with and regulation of HY5 is probably conserved in sweet sorghum,a monocot plant.7.Sb CRY1 a and Sb CRY1 b interact with Sb COP1 in blue light-depend mannerThe liquid assay of yeast two-hybrid indicated Sb CRY1 a and Sb CRY1 b interactedwith Sb COP1 instead of Sb SPA1 in response to blue light through C-terminal domain of Sb CRY1 a and Sb CRY1 b.The blue light-dependent interaction was also proved by Bi FC and co-immunoprecipitation,suggesting the interaction between CRY1 and COP1 was conservative in monocotyledon and dicotyledon.8.Sb CRY1 a interacts with Sb COP1 to suppress the interaction of Sb COP1 with Sb HY5 in response to blue lightSb CRY1a-Sb COP1 and Sb COP1-Sb HY5 interaction have been proved.In order to understand the relationship among Sb CRY1,Sb COP1 and Sb HY5,Co-IP assay was performed by HEK293 T cells.The result displayed the amount of Sb HY5-Myc coprecipitated by Sb COP1-HA was similar regardless of blue-light irradiation in the absence of Sb CRY1 a,however,a relatively lower level of Sb HY5-Myc was coprecipitated by Sb COP1a-HA in the presence of Sb CRY1 a and the amount of Sb CRY1a-Myc coprecipitated by Sb COP1-HA increased in the cells treated with blue light.The result demonstrated Sb CRY1-Sb COP1 interaction suppressed Sb COP1-Sb HY5 interaction in response to blue light,suggesting photoexcited CRY1 regulated HY5 protein accumulation in response to blue light was conserved in sweet sorghum.9.Overexpression of Sb CRY1 a and Sb CRY1 b causes susceptibility to ABA and salinity in the transgenic ArabidopsisTransgenic seeds of Sb CRY1 a and Sb CRY1 b,cry1 mutant,Col4 germination rate statistical results displayed,Sb CRY1 a and Sb CRY1 b were more sensitive to ABA,germination rates of Sb CRY1 a and Sb CRY1 b were obviously lower than wild type and cry1 mutant and the elongation of primary root was severely inhibited by ABA.In addition,the transgenic seedlings of Sb CRY1 a and Sb CRY1 b were lower tolerance to salinity.The results indicted Sb CRY1 a and Sb CRY1 b were involved in abiotic stress,more sensitive to ABA and salinity.10.Sb CRY1 a and Sb CRY1 b regulate ABA-related genes expression The expression of the following ABA-responsive genes Sn RK2.2,Sn RK2.3,ABF1,ABF2,ABF3,ABI3,ABI4,ABI5,EM6 were tested,the results displayed Sn RK2.2,Sn RK2.3,ABF1,ABI3,ABI4,ABI5,EM6 transcript level increased in Sb CRY1 a and Sb CRY1 b transgenic seedlings.When treated with ABA,the expression levelincreased more significantly.Thus,it is believed that Sb CRY1 a and Sb CRY1 b are involved in ABA-inhibited seed germination and seedling growth by increasing the transcription level of ABA signal transduction related genes.