Molecular Mechanism Of PtPEPCK1 In Maintaining Carbon And Nitrogen Balance In Poplar

Phosphoenolpyruvate carboxykinase kinase(PEPCK;EC 4.1.1.49)is a key enzyme at the plant's major basal metabolic pathway and participates directly or indirectly in biological processes.It plays an important role in plant metabolism.In C4 plants,PEPCK plays an important role in carbon dioxide concentration,and its overexpression and RNAi inhibitory expression in the original C3 plant will definitely affect the flow of carbon and nitrogen,and thus affect the proportion of carbon and nitrogen distribution.In this study,genetic engineering,transcriptomics and metabolomics were used to study the function of PtPEPCK1 in 84K poplar.This study is of great significance for understanding the mechanism of carbon sequestration and the regulation of carbon and nitrogen balance.It provides a theoretical reference for further revealing the molecular mechanism of carbon and nitrogen key enzyme gene metabolism.The main research outcomes and results of this paper are as follows:(1)Tissue-specific expression analysis using RT-PCR,qRT-PCR,and Western blotting techniques showed that PtPEPCK1 was expressed in roots,stems,and leaves of Poplars.There were significant differences in expression levels,with the highest expression in leaves and stems while the expression in roots was significantly low.At the protein level,Western blotting showed that PtPEPCKl was expressed in the leaf and stem tissues but expression was not detected in the roots.(2)Using Gateway system,the overexpression and RNAi vectors of PtPEPCK1 gene were constructed,and the genetic transformation of 84K poplar was carried out by Agrobacterium tumefaciens-mediated method.Using molecular analysis methods,a total of 12 and 30 positive strains were obtained through overexpression of PtPEPCK1 by "light-induced"and "dark-induced" transgenes methods,respectively.Several undifferentiated callus transformants were obtained.A total of 11 positive RNAi transgenic lines were obtained by"light-induced" transgenesis.Twenty-five RNAi lines were obtained through "dark-induced"transgene method.Several undifferentiated callus lines were obtained and saved as backup experimental materials.(3)The phenotypic observations including:plant height at 20d,30d,60d,and 90d of both overexpressed and suppressed Poplars were analyzed.The results showed that the transgenic plants did not show diametrically opposite phenotypic growth characteristics.The parameters such as growth rate,tree height,and diameter were lower than those of wild-type plants.The growth and development of over-expressed strains was most clearly inhibited.The phenotypic growth rate of RNAi-inhibited lines was similarly slow,but to a lesser degree than overexpressed lines.(4)The Immunohistochemistry(IHC)results showed that PtPEPCKl was expressed both in spongy tissue and palisade tissues of leaf mesophyll cells.Strong positive expression characteristics were observed in spongy tissues of mesophyll cells while brownish-yellow particles in the nucleus depicted negative expression.(5)Using co-immunoprecipitation technology combined with mass spectrometry analysis,Score and Coverage values were used as selection criteria to further clarification of the interaction between protein networks and PtPEPCKl.The results showed that the top 10 interacting proteins fell under the following major categories:(a)carbon dioxide enrichment mechanism-related enzymes;(b)gluconeogenesis intermediates;(c)ATP synthase;(d)chaperones.(6)Based on the Illumina platform,transcriptome sequencing was performed on overexpressed,inhibited and wild-type poplar leaves.The results showed that there was a convergence effect on the overall difference in the transcriptional level of PtPEPCK1 gene,and there were significant differences in local carbon and nitrogen metabolism.The major changes in the pathways involved were mainly in pyruvate metabolism,TCA cycle,gluconeogenesis,amino acid biosynthesis,pyrimidine metabolism,biosynthesis of secondary metabolites,and metabolic pathways in the plant's immune system.Overexpression and RNAi inhibition of expression of PtPEPCKl perturbs the core metabolic pathway.Overexpression of PtPEPCKl led to abnormal pyruvate metabolism,carbon flow from pyruvate into the acetyl-CoA metabolic pathway,acetyl-CoA carbon influx into the TCA cycle,and pyruvate Acetyl-coenzyme A,citric acid,and alpha-ketoglutaric acid are all precursors of glutamic acid biosynthesis,which promotes further flow of carbon into the Glu-Orn cycle,leading to an overall upregulation of the ornithine cycle and its precursors(5-49 times).RNAi inhibition of PtPEPCK1 expression caused a compensatory response from malic acid to oxaloacetate,succinic acid to fumaric acid.These substances are precursors of aspartic acid synthesis,enabling plants to open the malate-aspartate shuttle and ammonia recycling.PtPEPCK1 showed obvious changes in amino acids biosynthesis and pyrimidine metabolism pathway.Transformation of PtPEPCK1 gene caused change in amino acids biosynthesis pathway in 3 main ways:(a)Changes in acidic family of amino acids biosynthesis pathway induced by the citric acid cycle;(b)Changes in the biosynthetic pathway of fatty acid family of amino acids caused by glycolysis;(c)Changes in the biosynthetic pathway of aromatic amino acids in the pentose phosphate metabolic pathway.Overexpression of PtPEPCK1 led to more dramatic changes in the biosynthetic pathway of amino acids with more upregulated genes in the pyrimidine metabolic pathway.(7)The metabolomic analysis of the transgenic plants showed that the metabolites of the all transgenic plants showed similar changes,and the differential metabolites were mainly concentrated in the carbon,nitrogen,and organic acid metabolic pathways.Both carbon metabolism and organic acid metabolism were decreased,and the overexpression samples showed a large decrease.Amino acids,modified amino acids,and small peptides were all elevated in transgenic plants,and the increase in overexpression lines was greater.(8)The transcriptome data and metabolome data of the transgenic plants were analyzed by association analysis.The results showed that the PtPEPCKl gene had a low degree of association with carbon metabolism,while nitrogen metabolism,amino acid biosynthesis,and associated branching were relatively high.