Identification Of Brassica Napus PHT5 Family Genes And Functional Characterization Of BNPHT5;1s Involved In Phosphate Homeostasis

Phosphorus(P)is one of the essential macronutrients for plant growth.Rapeseed is an important oil crop in China,which accounted for one-third in the planting area and seed yield in the world.Rapeseed need more P and showed more sensitive to P deficiency than other crops.The soil available P concentration in rapeseed production in China is low and the application of P fertilizer is an important solutions to keep high and stable seed yield.The vacuole is the largest P reservoir in plant and play important roles in the P homostasis in cell.In this study,PHT5 family genes in Brassica napus were identified in the whole genome,and their gene structure characteristics and transcription in response to P fluctuations in the growth medium have been investigated.Bn PHT5;1a double mutant(Bn PHT5;1a DM)and Bn PHT5;1b double mutant(Bn PHT5;1b DM)were developed by CRISPR-Cas9 to study the functions of Bn PHT5;1a and Bn PHT5;1b in regulating cellular Pi homeostasis respectively.The main results are as follows.1 The gene structure characterization of Bn PHT5 family and their expression in response to Pi fluctuationsThe Bn PHT5 family had eight homologous genes,which were divided into three subfamilies.Among them,four,two and two genes belonged to Bn PHT5;1 subfamily,Bn PHT5;2 subfamily,and Bn PHT5;3 subfamily,respectively.The protein similarity of the corresponding homologous genes on the A and C chromosomes in each subfamily of Bn PHT5 ranged from 97.4 to 99.6%.Bn PHT5;1a has 9 exons,while Bn PHT5;1b has 10 exons.Bn PHT5;1a(Bn A09PHT5;1a and Bn C09PHT5;1a)and Bn PHT5;3(Bn A01PHT5;3and Bn C01PHT5;3)were induced by Pi deficiency,while Bn PHT5;1b(Bn A09PHT5;1b and Bn Cn PHT5;1b)were on the contrary in the shoot by q-PCR analysis.The expression levels of Bn PHT5;1a and Bn PHT5;3 were gradually up-regulated with the P deficient time,and decreased rapidly after the Pi were resupplied.However,the expression levels of Bn PHT5;1b were not changed significantly with the P deficient time.These indicated that Bn PHT5;1a and Bn PHT5;3 were Pi-starvation-induced genes,and Bn PHT5;1b was not sensitive to Pi-deficiency.In addition,the gene expression patterns of the two homologous genes of Bn A09PHT5;1a and Bn C09PHT5;1a in Bn PHT5;1a subfamily and two homologous genes of Bn A09PHT5;1b and Bn Cn PHT5;1b in Bn PHT5;1b subfamily were all different in leaves at different leaf ages and different organs of rapeseed at the flowering stage,indicating that the functions of the four homologous genes of the Bn PHT5;1subfamily genes in Brassica napus were functional divergence2 The function of Bn PHT5;1b in Pi homeostasisBoth Bn A09PHT5;1b and Bn Cn PHT5;1b,which shared similar sequence,structure,Pi transport activity and tonoplast location to Arabidopsis VPT1,were induced by high P.Compared with the wild type,the Bn PHT5;1b DM had smaller shoot growth and higher Pi concentration in the shoot,which was very different from the Atpht5;1 mutant,indicating that PHT5;1 functioned a unique mechanism of cellular Pi homeostasis in B.napus.Mutation of the Bn PHT5;1b also affected the reproductive growth of rapeseed,increased P in flower organs,decreased seed yield,and changed seed trait.These results suggested that Bn PHT5;1bs were vacuolar Pi influx transporter and played an important role in maintaining cellular Pi homeostasis at reproductive stage in B.napus.3 The function of Bn PHT5;1a in Pi homeostasisBoth Bn A09PHT5;1a and Bn C09PHT5;1a are induced by Pi-deplete and also located to the tonoplast.The P concentration and distribution ratio in old leaves in Bn PHT5;1a DM were higher than that in wild type(W10),indicating that Bn PHT5;1a affected the remobilization of P from old leaves to new leaves.The lower seed germination rate of Bn PHT5;1a DM than that of Bn PHT5;1b DM was due to the lower phytic acid concentration in the seed,indicating that Bn PHT5;1a affected the accumulation of phytic acid in rapeseed seeds.Both Bn PHT5;1a DM and Bn PHT5;1b DM exhibited Pi toxicity symptoms under Pi fluctuation in the growth medium,indicating that Bn PHT5;1a also plays an important role in Pi homeostasis.At high P,the Pi concentration of Bn PHT5;1a DM was lower than that of W10 but higher than Bn PHT5;1b DM in leaves,indicating that Bn PHT5;1a was not the key gene which affect Pi accumulation.The Pi concentration in Bn PHT5;1a DM roots did not change during 0 to 12 h at low P,indicating that Bn PHT5;1a might affect Pi consumption.4.Differential protein analysis of Bn PHT5;1a DM and W10A total of 5756 proteins were identified in the leaves of Bn PHT5;1a DM and W10 treated with Pi-replete(250 μm)and Pi-deplete(0 μm)by comparative proteome analysis.Under Pi-replete conditions,388 differentially expressed proteins were identified between the Bn PHT5;1a DM and W10,and they were mainly enriched in ATP biosynthesis and metabolic processes.Under Pi-deplete conditions,263 differentially expressed proteins were identified between WT and Bn PHT5;1a DM,and they were mainly enriched in carbohydrate metabolism process,cell wall polysaccharide metabolism process and response to stress.Compared with W10,Bn PHT5;1a DM showed weaker enrichment in biological processes such as carbohydrate metabolism,cell wall polysaccharide metabolism and ATP biosynthesis and metabolism by the combination of GO and KEGG pathway analysis.Some important metabolic pathways such as oxidative phosphorylation and α-linolenic acid metabolism,were significantly inhibited in Bn PHT5;1a DM.