Physiological Responses Of Pyrus Betulaefolia To Low Potassium And Analysis Of Its Potassium Uptake Key Genes

Potassium(K)is one of the essential macronutrients for plant growth and development,widely involved in a series of plant physiological and biochemical processes.Pear has a great demand on potassium and K plays a decisive role in fruit yield and quality.K in soil was unevenly distributed.Soil of pear orchards varies in its K+content(19?547 mg/kg)and ability to supply K+to plants.Potassium deficiency occurs from time to time.Pyrus betulaefolia is widely used as a rootstock for many pears.Rootstock plays important roles in improving K use efficiency of pear scion by enhancing root K uptake.HAK/KUP/KT K+transporter gene family and Shaker K+channel gene family are important for root K+uptake,but these two families have not been reported in the pear.Therefore,identifying and analyzing the HAK/KUP/KT and Shaker gene families in P.betulaefolia,mining key genes involved in root K+uptake,analyzing their expression patterns,investigating the signal transduction and regulation mode of roots in response to low K could provide important clues to clarifying the mechnisms of pear tolerant to low K+environment.In this study,P.betulaefolia seedlings were grown in solution culture method under two potassium levels for 3 mM K+(CK)and 0.1 mM K+(LK)to study the effect of K+deficiency on physiological response characteristics and root morphology.We identified HAK/KUP/KT and Shaker gene family and analysed their expression patterns.Roots of P.betulaefolia seedlings with 3 mM K+(CK)and 0.1 mM K+(LK)were harvested at 6 h and 15 d to analyse their transcriptomes by Illumina RNA-seq.Heterogeneous system was used to detect function of PbHAK12.The main results are listed as follows:1.Under K deficiency,the dry weight of P.betulaefolia seedlings was reduced with increased stress time.Compared to control,the total root length,total root surface area,total volume and number of root tips decreased by 1 8.1%,25.9%,14.7%,and 26.0%under low K stress(21 d),respectively.The net photosynthetic rate(Pn),stomatal conductance(Gs)and intercellular carbon dioxide(Ci)concentrations of seedling leaves decreased by 21.6%,23.7%and 21.6%under low K stress(21 d),respectively.Potassium content of root and shoot respectively decreased by 23.9%and 18.6%at low K stress(21 d).Anatomic structures of P.betulaefolia seedlings were changed by K deficiency:the upper and lower epidermis of leaves ruptured and cavitation increased;the volume of vascular tissues and pith parenchyma in stem and petiole reduced.MDA content and SOD activity in seedling leaves increased,indicating the degree of membrane lipid peroxidation increased.2.We identified 21 HAK/KUP/KTs(named as PbHAK1-21)from pear genome and they were grouped into five major clusters with eight subclades,along with HAK/KUP/KTs for Arabidopsis and rice according to phylogenetic analysis,but Cluster ? was lost in pear.Chromosome localization and gene duplication showed that whole-genome duplication/segmental duplication greatly contributed to the expansion of HAK/KUP/KT family in pear.Comparative analysis of orthologous genes between pear and Arabidopsis showed that PbHAK12-14 and PbHAK17 are orthologous to AtHAK5.Cis-element analysis suggested that PbHAKs could respond to enriched environment stimulation including various stresses and hormones.The expression patterns of PbHAKs showed PbHAK12 and PbHAK17 are preferentially expressed in roots and they were rapidly up-regulated under K+deficiency.Interestingy,PbHAK12 and PbHAK17 were not involved in fruit development,but many genes from Cluster ? and ? were expressed during fruit development,especially at 50 DAB and 80 DAB.3.We identified 9 Shaker genes from pear genome and they were grouped into five major clusters,along with Shakers fom Arabidopsis and rice based on phylogenetic analysis.The differentiation of each group comes after the bryophytes and group V is most conserved during evolution.Chromosome localization and colinear analysis showed that whole-genome duplication/segmental duplication greatly contributed to the expansion of Shaker family in pear.Calculation of Ka/Ks between the paralogous pairs IN PEAR indicated that all of them have evolved under positive selection.Various phytohormone-and stress-responsive cis-elements were present in promoter regions of PbShakers.Gene expression levels measured by RNA-Seq and qRT-PCR indicated that PbAKT1.l and PbKC1.2 might regulate root K+absorption at K+deficiency.PbAKT1.2 might play crucial roles in pollen development and pollen tube growth.4.We analyzed the transcriptomic profiles of roots of P.betulaefolia in response to low-K+stress.The roots of P.betulaefolia seedlings with or without low-K+treatment were harvested after 6 h and 15 d.Different response mechanisms of roots to K+deficiency for short-(6 h)and long-term(15 d)stress were observed.We identified a total of 1820(1330 up-regulated and 490 down-regulated)and 1843(629 up-regulated and 1214 down-regulated)differentially expressed genes responding to short-and long-term low-K stress,respectively.448 genes were coregulated in both short-and long-term low-K stress,compared with those under control.Many nutrient transporters,transcription factors,kinases,oxidative stress-related genes and genes in Ca+and hormone signaling pathways displayed different expression in response to short-and long-term low-K stress.Based on the analysis of transmembrane transportation-related genes responsive to low K+,different regulatory mechanism of the K+acquisition system were detected at short-and long-term low-K stress.PbHAK12 rapidly responded to short-term low-K stress,while PbHAK17 and PbAKT1.1 played important roles in long-term low K stress.RCI3(Pbr034488.2)-RAP2.11(Pbr007699.1)-HAK5(PbHAK17),SYP121(Pbr035718.1/Pbr03572 7.1)-KC1(PbKC1.2)-AKT1(PbAKT1)and CBL-CIPK23(Pbr042907.1)-AKT1(PbAKT1)pathway regulatory network might function at long-term low-K stress.These results showed P.betulaefolia,as perennial woody plant,has evolved complex regulatory mechanisms to adapt to K deficiency,especially long-term low-K stress.5.Based on RNA-seq data and expression analysis,PbHAK12 was selected as target gne.Yeast mutant was used to detect the function of PbHAK12 in heterologous system and PbHAK12 could restore K+absorption of yeast mutant R5421.Subcellular localization showed PbHAK12 was on the plasma membrane of Arabidopsis protoplast.PbHAK12 was transformed into Micro-Tom tomato and obtained 13 transgenic plants.Compared with WT lines,K+content,net photosynthetic rate and chlorophyll content of leaves of transgenic plant lines Z1,Z6 and Z12 were increased,but further research is still needed.Taken together,low K stress inhibited the growth of P.betulaefolia seedlings and led to adaptive changes for root architecture.Moreover,there are 21 HAK/KUP/KT and 9 Shaker genes identified in pear genome,respectively.RNA-seq data showed that PbHAK12,PbHAK1 7 and PbAKT1.1 played crucial roles in root K+absorption of P.betulaefolia under low K.Among them,PbHAK12 was located on plasma membrane and could restore K+absorption of yeast mutant,indicating its role in regulating K+absorption under low K.