| Rice is the most important food crops in the world.High yield is the most effective safeguard for food safty and agricultural sustainable development.Energetic activities primarily emerge from the solar energy,among of which green plants play an important role,because of the energy conversion apparatus-chloroplasts.The wlp2-1,wlp2-2 mutants were derived from the japonica rice variety Zhonghua 11 by EMS chemically.Map-based cloning of WLP2,showed that it encodes a pfk B-type carbohydrate kinase family protein named Fructokinase-like 1（OsFLN1）,and the defect can even cause albinos lethal during seedlings stage under higher temperature condition.Here,we showed our main results in this study.Our results showed that wlp2（white leaves and white panicles 2）exhibits two allelic mutants（wlp2-1,wlp2-2）,which were both sensitive to higher temperature even albinos lethal during seedlings stage.Interestingly,the two allelic mutants sensitive to temperature with different extent:wlp2-1exhibits white-leaf stripe in low temperature（12 h of light at 22°C/12 h of darkness at 22°C）and albino lethal in high temperature(12 h of light at 35°C/12 h of darkness at 35°C,while wlp2-2 was dull to low temperature until high temperature that are also albino lethal through early seedling stage.Both the mutants show white-striped basal leaves and white panicles with differently sensitive to temperature under Hangzhou field conditions in 2013.Taken together with the mainly agronomic characteristics of wild-type and wlp2-1,wlp2-2 mutants at maturity in the two years,we deduced that wlp2 mutants show susceptible to higher temperature during all life cycle even effect the biological yield.Besides,wlp2-1showed more severe mutational phenomenon than wlp2-2.the WT and wlp2-2 chloroplasts ultrastructures developed with well lamellar structures,normally stacked stroma and thylakoid membranes.While wlp2-1 appeared less well-structured chloroplast with little developmental lamellar structure.Under the 35°C condition,both of wlp2 mutants showed devastating chloroplast structure with scarcely lamellar structures and an amount of osmiophilic plastoglobuli production.Similarly,the wlp2-1 displayed barely well chloroplast structure,while wlp2-2 showed less-well chloroplast with thylakoid membrane and less lamellar structures under Hangzhou field condition.At the same time,net photosynthetic rate of the flag leaves were detected significantly different between the wlp2 and WT plants（Figure 2.L）.conclusively,the wlp2 mutants show defect in photosynthesis synthesis,chlorophyll accumulation and impaired chloroplast biogenesis during the life cycle in rice.A genetic analysis was carried out to identify the white-striped leaf and white panicle phenomenon was controlled by a single recessive locus in nucleus.By a BC1F2 population of the wlp2-1 mutant and Nanjing 11,we localized the WLP2 gene to a 206.5 kb physical segment on chromosome 1 defined by the insertion/deletion polymorphism markers Indel 10 and Indel 20.With the targeting region,there were 35 open reading frames（ORFs）.A genomic sequence analysis of the ORF21 showed that the only sequence substitution between the WT and wlp2 mutants:wlp2-1 was a base change（G→A）at the position 674bp from the ATG starting codon;wlp2-2 also played a base substitution（C→T）at the position 674bp from the ATG starting codon.Complementation assay showed that the ORF21（LOCOs01g63220）indisputably corresponds to WLP2.Protein sequence alignment and phylogenetic analysis were surveyed to predict the function of WLP2,which showed that the pfkB-type domain was highly conserved among different species.AtFLN1 involves in the PEP complex to regulate the plastid genes transcription process and chloroplast development in Arabidopsis and mustard.We investigated the expression patterns of WLP2 during the flowering period by RT-PCR in wild-type plants and GUS chemical staining of WLP2 transgenic plants,our results showed that WLP2was constitutively transcribed in the leaf,young panicles,leaf sheath,culm,and roots,and expressed more actively in flag leaves and young panicles.Simultaneously,we found that WLP2 expression level was significantly increased in L4 section（P4）.It has been reported that activation of photosynthetic apparatus begins during the P4 stage.Our results suggest may indicate that WLP2 is connected to PEP activity to involve in the photosynthesis and chloroplast development with an established plastid genetic system.OsFLN1 was localized to the chloroplast in rice.Besides,WLP2 likely associate with the chloroplast biogenesis by light regulation.Taken together,plastid-associated genes,Chlorophyll biosynthesis,and photosynthesis and chloroplasts biogenesis genes in wlp2 mutants expressed more active with higher or lower levels may take account for more serious albino defective phenotype under higher heat condition.It has been reported that AtFLN1 homologous to WLP2 is associated with TAC complex to activate the PEP to regulate the PEP-mediated plastid genes transcription process,further to control the chloroplast biogenesis in Arabidopsis.To investigate the mechanism of regulation in rice,yeast two-hybrid assay and bimolecular fluorescence complementation（BiFC）assay were performed to seek the interacting proteins.Luckily,we found WLP2 interact with a thioredoxin z（OsTrx Z）,which also has been identified to be an important component of TAC complex through redox regulation.Besides,we found WLP2 only containing pfkB domain can not interact with OsTrx Z,which concordant with the results that AtFLN1 connects with Trx Z through a conserved double Cys motif.Meanwhile,we found OsTrx Z can also interact with another pfkB-type family carbohydrate kinase protein,fructokinase-like2（Os FLN2）,but OsFLN2 did not interact with WLP2.All above results imply that OsTrx Z interacts with WLP2 and OsFLN2 to combine the TAC complex to participate in the regulation of PEP-dependent plastid genes transcription course.During our research,we found the wlp2 mutants were sensitive to higher temperature conditions,and showed spontaneous heat-stress-dependent death formation with amount hydrogen peroxide production.Besides,the expression levels of many nucleus genes associated with stress response,photosynthesis,and chlorophyll were distinctively different from the wild type plants in seedlings by RNA-seqs,which may implicate the retrograde signals accommodation between the chloroplast and nucleus. |
