Regulation Mechanisms Of NO₃^- Transport Mediated By SLAC/SLAH In Pear

Nitrogen,identified as an indispensable element in multiple biological processes of plants,not only participates in the growth and development stages as nutrients,but also acts as a signaling molecule to regulate the morphogenesis of plants.Nitrate nitrogen is the primary source for plants to absorb and utilize nitrogen,and the regulation mechanisms of absorption,transport and metabolism of it have been specifically studied in multiple plants.For fruit trees,the level of nitrogen supply significantly affects various physiology indexes including fresh weight,dry weight,leaf area and growth rate.Applying nitrogen to fruit trees based on scientific evidences not only enriches the content of carbon,but also extends the duration of growing development period by promoting the sink activity of fruits.It can also enhance the activity of the fruit bank and prolong the growth and development of the fruit.In addition,employing of scientific amount nitrogen fertilizers can offer crucial assistance for increasing the fruit yields and fortifying the fruit qualities.Nevertheless,excess of nitrogen supply brings negative effects on the growing and development as well as other significant functions of fruit plants.Applying nitrogen fertilizers in excessive amount leads to the massive accumulation of nitrogen,which causes eutrophication of soil.The vegetative growth of the fruit trees is significantly promoted by excessively applying nitrogen,which results in the disruption of the C/N ratio balance,and then inhibiting the reproductive growth.Consequently,it is of great scientific significance and practical value to penetrate the understandings for the absorption and utilization of nitrogen in fruit trees and related regulation mechanisms.The physiological characteristics and molecular regulation mechanism of nitrogen absorption and utilization of Pyrus L.,which is the third largest yield fruit in China,are remaining highly unexplored.In this study,the effects of different concentrations of nitrate nitrogen on the growth and development of pear trees,as well as the regulation mechanism of pear seedlings absorbing,transporting and assimilating nitrates are analyzed;the functional characteristics and regulation mechanisms of S-type Anion Channel(SLAC/SLAH)are characterized.Taken them together,we offer a theoretical basis for improving the nitrogen use efficiency of pear trees,and provide genetic resources for molecular breeding of pear with efficient nitrogen utilization.The main results are as follows:1.The growing development of the pear(Pyrus betulifolia Bunge)seedlings as well as their root-morphological traits and leaf phenotype are proved to be responsive to the changes of nitrogen in this study.To analyze the responding patterns of the plant to nitrogen of different concentrations,the pear seedlings are treated by nitrogen with five different concentrations.The results presented that the deficiency and the excess of the nitrogen have impacts on the morphogenesis of the roots and the phenotypic symptoms of the leaves,and they both can inhibit the growing development of the plant.Under the condition of nitrogen deficiency,the treated pear seedlings were observed with leaf etiolation and stimulation of root elongation,as well as decrease in nitrate reductase activity and chlorophyll content.Pear seedlings treated with excess nitrogen,nevertheless,had presented phenotypes of leaf coking and suppression of root elongation.Additionally,it is verified that the changes of nitrogen concentration impact the nitrogen contents as well as the absorption of mineral nutrients in plant tissues.Taken together,the physiological characteristics of pear seedlings responding to nitrogen provide a good reference standard for the diagnosis of nitrogen supply in pear,and lay a foundation for studying the improvement of nitrogen utilization rate of pear.2.To gain a further understanding for the molecular mechanism in pear(Pyrus bretschneideri Rehd.)responsible for the regulation of nitrate transport and assimilation,RNA-seq was performed on samples with different nitrogen treatments.In analyzing the data acquired from the RNA-seq,after starvation treatment and re-supply of nitrate,the 10,273 genes of differential expressions were obtained and annotated to 49 GO terms,which can be classified into 45 clusters having co-expression trends and identified to be involved in 18 KEGG-defined significantly overrepresented pathways.Identified by the KEGG pathways,15 genes of differential expressions including 1 NRT gene,2 NR genes,1 NiR gene,2 GDH genes,6 GS genes and 3 GOGAT genes are related to the nitrogen metabolism,and they significantly differentially expressed in response to nitrate starvation and a nitrate re-supply treatment.Furthermore,449 transcription factors belonging to 35 different families were identified during the nitrate treatments.The expression patterns of 14 randomly selected genes of differentially expressed were verified by qRT-PCR.Taken together,these results and data provide valuable resources for investigating the genetics of the nitrogen metabolic pathways and improving nitrogen utilization efficiency in pear.3.More than playing irreplaceable roles in anion transport pathways of plant(nitrate,chloride,etc.),S-type anion channels also function significantly in abiotic stress responses and hormone signaling of plants.In Rosaceae species,a total of 21 SLAC/SLAH genes were identified,which can be classified into three subfamilies based on the structural characteristics and phylogenetic analysis.Transcriptome data indicated that PbrSLAC/SLAH genes were located in all issues of the pear.As the transient expression and subcellular localization experiments demonstrate,PbrSLAC/SLAH genes are located on the plasma membrane in Arabidopsis protoplasts cells.Additionally,functional complementation experiments showed that PbrSLAH2/3-1 successfully alleviated the ammonium salt toxicity phenotype in Arabidopsis slah3-3 mutant.These results provide valuable information that help to gain further understandings of the evolution,expression and functions of the SLAC/SLAH gene family in higher plants.4.Calcium-dependent protein kinase(CPK),characterized as a calcium ion sensor,plays crucial role in stimulating phosphorylation of SLAC/SLAH proteins by interaction.A total of 43 PbrCPKs were identified from pear genome and divided into 4 subfamilies according to the phylogenetic analysis.Duplication mode analysis showed that genome-wide replication(WGD)or dispersed replication was the main driving force in the expansion of this family.By carrying out the Yeast two-hybrid and bimolecular fluorescence complementation assays,it is confirmed that PbrSLAH2/3-1 interacts directly with PbrCPK32.When PbrSLAH2/3-1 was co-expressed with either the Arabidopsis calcium-dependent protein kinase(CPK)21 or PbrCPK32 in Xenopus oocytes,yellow fluorescence was emitted from the oocytes,and the typical anion currents were recorded in the presence of extracellular NO3-.In addition,the electrophysiological results showed that PbrSLAH2/3-1 was more permeable to NO3-than Cl-.Accumulating these evidences,we suggest that PbrSLAH2/3-1 crossing-talk with PbrCPK32 probably participate in transporting of nitrate nutrition in pear.