| The development and maturation of rice pollen determine the seeds setting and yield,and starch accumulation is a key process of rice pollen maturation.However,the regulation mechanism for pollen starch accumulation is still unclear.L-type lectin receptor-like protein kinases are a class of protein receptors that can recognize the ligands and have phosphorylation activity.In plants,these kinases have been reported to participate in a variety of biological processes,such as disease resistance and defense response,regulation of development,and abiotic stress response.Previous studies have shown that L-type lectin receptor-like protein kinases are also involved in the regulation of rice pollen development.Therefore,it is of great significance to study the functions of L-type lectin receptor-like protein kinase for further understanding the regulatory network of rice pollen development.In this study,we identified a rice male-sterile mutant（abnormal pollen 1,ap1）and conducted a series of phenotypic analysis,gene cloning and functional study.The biochemical functions of AP1 was further studied using the transcriptomic approach,phosphoproteomic analysis and protein-interaction assays.This study demonstrated that AP1 interacts with and phosphorylates OsUGP2（uridine diphosphate glucose pyrophosphorylase,UGPase,a key factor of pollen starch accumulation during pollen maturation）to improve its enzymatic activity,and thus promoting starch accumulation during pollen maturation.The main results are as follows:1.The anther of ap1 mutant was pale yellow,and was slightly smaller than that of wild-type.Then FDA and I2-KI staining results showed that the pollen grains of ap1 were inactive and sterile.And microscopic observations indicated that the pollen grains of ap1were hollow and lacking of typical inclusions and exhibited extremely low starch accumulation level.The observations of meiosis in pollen mother cells showed that the ap1mutation did not significantly affect the meiotic processes.The results of semi-thin sections,transmission electron microscopy and scanning electron microscopy indicated that the dysfunction of AP1 not only led to the defect of pollen content accumulation but also affected the development of pollen intine and aperture.2.A C/T mutated SNP was identified in LOC＿Os02g26160 on the long arm of chromosome 2 using Mut Map re-sequencing analysis.This SNP induced a premature stop codon and was co-segregated with the male-sterile phenotype.These results suggested this SNP is the causal mutation.Gene annotation of public database showed that AP1/LOC＿Os02g26160 encodes an L-type lectin receptor-like kinase（lec RLK）.Further using the CRISPR/Cas9 genome editing technology we have obtained several AP1 knockout mutant lines in the background of Nipponbare.The homozygous CRISPR/Cas9 knockout（cr）mutants are male-sterile due to lack of pollen inclusions,mimicking the phenotype of the ap1 mutant.Then,we used these ap1/cr mutants for functional complementation analysis,and found that the positive transgenic plants of both mutants showed wild-type male fertility restorations.These results confirmed that AP1 is the key gene in controlling rice pollen fertility.3.The results of q RT-PCR showed that the expression level of AP1 was higher in spikelets with anther at developmental stages 8-9 and 11-12 than in other tissues.GUS histochemical staining and in situ RNA hybridization assays showed that AP1 was preferentially expressed in microspores of anther at developmental stage 11.On the other hand,subcellular localization analysis showed that AP1 protein was located to the plasma membrane.4.The transcriptomic analysis showed that numerous genes involved in sugar signaling/metabolism were down-regulated in the ap1 mutant,which supported the notion that AP1is involved in regulating pollen starch accumulation.Further in vitro analysis showed that AP1 protein has phosphorylation function.The phosphoproteomic analysis showed that the phosphorylation levels of several proteins in the sucrose/starch metabolic pathway were also down-regulated in the ap1 mutant,including OsUGP2.Since no significant difference in the transcription level of OsUGP2 was found between the wild-type and the ap1 mutant,it is reasonable to speculate that the down-regulated phosphorylation level of OsUGP2 was a direct consequence of the ap1 mutation.5.The q RT-PCR and subcellular localization analysis showed that OsUGP2 and AP1 have the characteristics of space-time overlapping.Furthermore,using Yeast Two Hybrids（Y2H）,Bi-molecular Fluorescent Complimentary（Bi-FC）,Luciferase Complementation Imaging（LCI）and GST pull-down assays,we demonstrated that AP1 directly interacts with OsUGP2 through its kinase domain,indicating that the kinase domain of AP1 is necessary for protein interaction.6.The in vitro phosphorylation and phosphoproteomic analysis showed that AP1 could directly phosphorylate the serine at sites 151 and 413 of OsUGP2,while the ap1 mutant protein showed no phosphorylation activity.We further constructed two mutated versions of OsUGP2 protein,namely OsUGP2DD and OsUGP2AA.The former could mimic the continuous phosphorylation of OsUGP2 protein,while the latter is phosphonegative(phosphorylation of OsUGP2AA by AP1 decreased significantly).These results indicate that the kinase domain of AP1 is essential for phosphorylation,and the serine 151 and 413of OsUGP2 are two key phosphorylation sites.In vitro enzyme activity assays showed that AP1 could promote the enzymatic activity of OsUGP2,while the ap1 mutant protein had no significant effect on the enzymatic activity of OsUGP2.The enzymatic activity of OsUGP2AA was lower than that of OsUGP2DD,and could not be increased by incubation with AP1.Further,the transient expression of these proteins in tobacco leaves and the determination of starch content also showed the similar results.These in vitro and in vivo results indicate that AP1 can promote the enzymatic activity of OsUGP2 through phosphorylation modification,and thus affecting pollen starch synthesis.In conclusion,we cloned a rice pollen fertility controlling gene AP1,which encodes an L-type lectin receptor-like protein kinase.AP1 interacts with and phosphorylates OsUGP2,and promotes the enzyme activity of OsUGP2 to regulate the synthesis and accumulation of starch during rice pollen maturation. |
