Evolutionary Engineering Study Of C₄ Photosynthetic Pathway

The production of organic matter by photosynthesis in green plants is an important way of producing global primary products.Compared with C₃plants,C₄plants have the advantages of low CO₂compensation points,high water and nitrogen fertilizer utilization,and high yield.The C₄photosynthetic pathway evolved from the C₃photosynthetic pathway,and the driving force for the evolution of the C₄photosynthetic pathway was proposed to be low CO₂.Introducing C₄photosynthetic pathway into C₃crops may bring a second green revolution to human society.Although our understanding of the comprehensive traits of C₄is gradually deepening,cultivating the prototype of C₄rice is still a huge challenge.Here we propose a novel plant photosynthetic pathway transformation strategy,which combines genetic engineering and evolution engineering.First,genetic engineering is used to increase some C₄enzymes in C₃plants,and artificially construct low CO₂mutant cumulative lines.The transgenic lines are continuously cultivated for many generations in low CO₂,and it is expected that the plants will turn to C₄at multiple levels.Previously,our lab introduced the key gene PEPC of C₄photosynthetic pathway into C₃plant Arabidopsis thaliana,and we got the line with high PEPC enzyme expression.Since the C₄pathway is considered to be an evolutionary strategy for plants to adapt to the new environment in a certain stress environment,and low CO₂concentration is an external driving force for the evolution of the C₄photosynthetic pathway.Organisms can maintain a constant growth balance in the adaptation of new environments and existing conditions,but stress-induced mutations can break this balance,increase the mutation rate of plants,and higher mutation rates contribute to plant adaptation in stressful environments.More beneficial mutations occur during the adaptive evolution of organisms,which is conducive to the evolution of plants.We grew the highest expressing PEPC overexpression lines oe PEPC under normal and low CO₂conditions and performed the physiological measurements of each generation,including PEPC enzyme activity,plant size,leaf area,flower size and chlorophyll content.Then the transcriptome sequencing of the eighth generation continuously grown under low CO₂was performed to analyze the changes of different lines at the transcriptome level,and to explore how low concentration of CO₂stress affects the transcriptome changes of multi-generational accumulation lines and gene expression influences.Genomic resequencing of the eighth generation plants was performed to track the genetic changes associated with low CO₂stress,and then to study the evolutionary trajectory of the C₄photosynthetic pathway.1.Effect of low CO₂concentration on the growth status of overexpressing PEPC Arabidopsis thaliana and wild type Arabidopsis thaliana.Under the condition of CO₂concentration of 100ppm,Arabidopsis thaliana plants grew short and grew slowly.PEPC activity,leaf area and flower length and width decreased.However,in the low CO₂environment,overexpression of PEPC Arabidopsis thaliana has higher specific activity,higher chlorophyll a and chlorophyll b,and larger leaf area and flower area than wild type Arabidopsis thaliana.2.Analysis of transcriptome data of wild-type and overexpressing PEPC Arabidopsis under low CO₂concentration.According to the analysis of sequencing data,plants have gene differential expression in photosynthesis,chlorophyll content,floral organ development,stress response etc,and are basically consistent with the observed phenotype.Moreover,the expression of the PEPC line over these lines was up-regulated compared with the wild-type line,which indicated that the over-expressed PEPC line had enhanced photosynthesis under low CO₂environment,and the degree of stress was more severe,resulting in more mutations.Adapt to the stress environment and promote the evolution of plants.3.Genomic sequencing data analysis of wild type and overexpressing PEPC Arabidopsis.According to the analysis of sequencing data,the multi-generation growth of Arabidopsis under low CO₂stress increased the frequency of mutation,and the overexpression of PEPC Arabidopsis thaliana increased the frequency of mutation more than wild-type Arabidopsis thaliana.In this study,created low-CO₂stress induced Arabidopsis mutation accumulation lines,and compared the growth of overexpressing PEPC Arabidopsis thaliana and wild-type Arabidopsis under low CO₂stress.We also observed big difference in gene expression of the 8th generation between wild-type and oe PEPC and the whole genome seqeunce data showed,overexpression of PEPC accumulated more mutations.It provides a new idea for the transformation of C₄genetic engineering.