Functional Characterization Of Maize Phytochrome C In Photomorphogenesis And Flowering

Maize as the largest crop of the world is widely used for food,feedstocks and other industrial products.In the process of domestication and breeding,the planting area of maize has been continuously expanded from tropical low latitudes to temperate high latitudes with the relaxation of photoperiod sensitivity for flowering.To continuously increase maize yield by increasing maize planting density is also an important feature of modern maize breeding and cultivation.So,it is always an important goal of maize breeding to breed maize with moderate early flowering and high-density tolerance.Shade avoidance syndrome(SAS),which is triggered when plants sense competition of light from neighboring vegetation,is detrimental for maize yield production under high-density planting conditions.Previous studies have shown that the red and far-red photoreceptors phytochromes are responsible for perceiving the shading signals and triggering SAS in Arabidopsis and phytochromes are also participating in the regulation of flowering time.However,their roles in maize are less clear.The functions of maize PHYA and PHYB in photomorphogenesis have been studied,but the studies on PHYC have not been reported in detail.In this study,we cloned ZmPHYC1 and ZmPHYC2 from maize inbred line B73;then did the bioinformatics analysis and subcellular localization analysis of ZmPHYC1/2 protein.The tissue-specific expression of ZmPHYC1 and ZmPHYC2 in maize and their transcriptional abundance that response to different photoperiodic treatments(long and short day)were detected by real-time quantitative PCR(RT-qPCR).The interaction between ZmPHYC1/2 protein and other maize phytochrome proteins were determined by biochemical test.Meanwhile,the genes of ZmPHYC1 and ZmPHYC2 were overexpressed in the model plant Arabidopsis Col-0 ecotype,phyC-2 mutant and the wild-type maize inbred line ZC01 respectively by transgenic technology to study their functions.In addition,we also used CRISPR/Cas9 genome-editing technology to knock out ZmPHYC1 and ZmPHYC2 together in maize inbred line ZC01 to detect the role of ZmPHYCs in maize photomorphogenesis,flowering and plant height.The main results are as follows1.Phylogenetic tree and expression features of ZmPHYCland ZmPHYC2.ZmPHYC1 and ZmPHYC2 were clustered on the same branch of the evolutionary tree and were most closely to rice PHYC(OsPHYC).ZmPHYC1 and ZmPHYC2 all had six functional domains including a PAS-2 domain,a cGMP-specific phosphodiesterases/adenyl cyclase/Fh1A(GAF)domain,a phytochrome(PHY)domain,two PAS domains,a His Kinase A(HisKA)domain and a Histidine kinase-like ATPases(HATPase_c)domain.The expression profiles of ZmPHYCs showing that both ZmPHYC genes were highest expressed in leaf and the expression level of ZmPHYC1 was higher than ZmPHYC2.The diurnal expression patterns of ZmPHYCs were also monitored under artificial long-day(LD)and short-day(SD)conditions.We found that ZmPHYC1 and ZmPHYC2 showed diurnal oscillation and the diurnal oscillation of ZmPHYC1 was obviously different from ZmPHYC2.Under light condition,both ZmPHYC1 and ZmPHYC2 were mainly localized in the nucleus with minor in cytoplasm2.ZmPHYC1 and ZmPHYC2 proteins can interact with themselves and other maize phytochromes.Using LCI and BiFC assay,we detected that ZmPHYC1 could interact with itself and its homologous protein ZmPHYC2.The same condition was to ZmPHYC2.At the same time,we found that ZmPHYC1 and ZmPHYC2 could also interact with ZmPHYB1 and ZmPHYB2.In the BiFC assays,the interactions were in the nucleus of N.benthamiana leaf epidermal cells3.Overexpression of ZmPHYCs rescued the hypocotyl phenotype of the Arabidopsis phyC-2 mutant.By screening the expression levels of ZmPHYC1 and ZmPHYC1 in transgenic lines(phyC-2 background),two transgenic lines were used for the following experiments.In the darkness,all of the ZmPHYCs-OE lines exhibited similar hypocotyl lengths without significant difference comparing with Arabidopsis phyC-2 mutant.But under the condition of 30 ?mol photons m-2 s-1 continuous red light(Rc),ZmPHYC1 and ZmPHYC1 could inhibit the hypocotyl elongation of transgenic lines with obvious reduced hypocotyl comparing with phyC-2 mutant4.Effects of ZmPHYC1 and ZmOHYC1 on Arabidopsis hypocotyl length under different light quality.We overexpressed ZmPHYC1 and ZmPHYC1 in the Arabidopsis wild-type(Col-0)plants.Two independent transgenic line for ZmPHYC1 and ZmPHYC1 were selected and used to perform further analysis.Under the condition of 30 ?mol photons·m-2·s-1 continuous red light(Rc)and 1 ?mol photons·m-2·s-1 continuous blue light(Bc),the hypocotyls of all transgenic lines were significantly shorter than that of Col-0.But ZmPHYCs-OE seedlings failed to suppress the hypocotyl elongation in 2.1?mol·m2·s-1 continuous Far-red light(FRc).In the meantime,all the seedlings had no apparent hypocotyl elongation differences in the dark Further,we checked the fluence rate response of all the ZmPHYC1-OE/Col and ZmPHYC2-OE/Col transgenic lines under Rc,FRc and Bc conditions.The results of fluence-rate response curves for hypocotyl growth indicated that both ZmPHYC1-OE and ZmPHYC2-OE transgenic lines were hypersensitive to Rc from low fluence rates(1.6 ?mol m-2 s-1)to high fluence rates(34 ?mol m-2 s-1).And the growth suppression of hypocotyl length was only detected at low fluence rate(1.4 ?mol m-2 s-1)of Bc.However,there was no significant difference for the hypocotyl lengths in different genotype plants at each point of FRc fluence rate5.ZmPHYC1 and ZmPHYC2 attenuated responses to simulated shade in Arabidopsis seedlings.The ZmPHYC1 and ZmPHYC2 overexpression lines(Col-0 background)exhibited obviously shorter hypocotyls compared with the Col-0 and phyC-2 under simulated shade treatment(EOD-FR treatment).When grown under normal white light(WL)conditions,the hypocotyl lengths had no significant difference between the transgenic lines and the wild-type plants.These results showed that heterologous expression of ZmPHYC1 and ZmPHYC1 could attenuate responses to simulated shade in Arabidopsis seedlings and decreased the hypocotyl lengths under shade condition.We confirmed that the expression levels of four genes which related to hypocotyls elongation and shade avoidance response,LONG HYPOCOTYL INFAR-RED1(HFR1),YUCCA2(YUC2),ATHB-2(HB-2)and PIF3-LIKE1(PIL1),were downregulated in Col-0 plants overexpressing ZmPHYC1 or ZmPHYC1 after EOD-FR treatment.These results indicated the possible pathway of ZmPHYC1 and ZmPHYC1 participating in the shade avoidance response6.ZmPHYC1 and ZmPHYC1 regulated the maize flowering time.Knockout of both ZmPHYC1 and ZmPHYC1 by CRISPR/Cas9 caused early flowering 4 to 6 days under natural LD conditions.It was worth mentioning that the flowering time of zmphyC1 zmphyC2 double mutant lines in the SD conditions did not show early-flowering.In the meantime,we investigated other relative agronomic traits containing plant height,ear height and total leaf number which found that there were no various differences in zmphyC1 zmphyC2 mutants compared with WT plants.We examined the expression patterns of maize FT gene(ZCNS)and CONZ1 in WT and zmphyCl zmphyC2 mutant under artificial LD and SD conditions.ZCN8 exhibited increased expression in zmphyC1 zmphyC2 mutant compared with WT plants for all the sampling points during LD conditions.Next,we examined the expression levels of CONZ1,a key factor of the photoperiod pathway.The CONZ1 expression level was also higher in zmphyC1 zmphyC2 mutant than in WT plants under LD condition.While under SD conditions,the expression of ZCN8 and CONZ1 were nearly the same between zmphyC1 zmphyC2 mutant and WT7.Overexpression of ZmPHYC2 reduced plant height and ear height obviously in maize The analysis of agronomic traits in the field showed that the plant height and ear height in the ZmPHYC2-OE lines were obviously reduced than WT plants but the transgenic lines overexpressing ZmPHYC2 had similar flowering time and total leaf numbers compared with WT.However,ZmPHYC1-OE lines did not have significant difference compared with WT in plant height,ear height and other agronomic traits8.Evolutionary analysis of ZmPHYC2.In previous research,we finding that at the 144th nucleotide of ZmPHYC2 coding region,there was a transversion of cytosine("C")to adenine("A"),leading early termination of ZmPHYC2.We sequenced the partial DNA sequences containing potential mutant site of ZmPHYC2 in 165 maize inbred lines and 40 teosintes.The association analyses of the mutation site with maize plant height and flowering time were performed,and no significant signals were found.The mutation site was only found in the 20 maize inbred lines but not in the teosinte.Moreover,the nucleotide diversity(?)of the region around the mutant site(800 bp accurately around the mutant site)also did not show obvious selection.In conclusion,these results provide a new idea for studying the function of ZmPHYC1 and ZmPHYC2 to breed early flowering and high-density tolerant maize cultivars.