Functional Analysis Of Populus GATA Gene In Photosynthesis And Vegetative Growth

Populus is one of the important tree species in North China.It is an important part of renewable biological resources,and forest carbon sinks.There are many factors affecting the rapid growth of Populus,such as photosynthesis,the absorption and utilization of nutrients.Photosynthesis is one of the most important physiological processes that determine the accumulation of plant biomass.Nitrogen is an essential nutrient for plant growth,and the nitrogen content of the soil also affects the photosynthetic efficiency of plants.To meet the needs of human beings and the society for wood resources,we studied the mechanism of rapid growth of trees from the physiological and molecular levels.It is significant to improve the quality,quantity of forest products,and the development of forestry.In this study,we compared the different growth of Populus from the physiological research.Then,based on the gene expression profiles of Populus in different growth rates,we found GATA transcription factor genes related to photosynthesis and nitrogen nutrition.The roles of GATA genes in the regulation of plant photosynthesis and nitrogen response were researched by bioinformatics analysis,gene overexpression,and CRISPR/Cas9 gene editing.The major results were summarized as follows:(1)Three different genotypes of NE19([P.nigra ×(P.deltoides × P.nigra)]),DN2(P.deltoides ×P.nigra),and R270(P.deltoides × P.nigra)were studied.We compared their growth rates.The results showed that NE19 plants grown faster than R270 and DN2 in leaf area,chlorophyll content,height and diameter.The effect of nitrogen nutrition on growth of NE19 was studied.We found that the nitrogen deficiency inhibited photosynthesis of NE19 plants,decreased the chlorophyll content,leaf number,and biomass seriously.(2)In this study,the GATA domain was used as a probe,and 39 genes with GATA domain were retrieved from the published genome.We described the chromosome location,replication,structure of the 39 genes and the homologous genes in Arabidopsis.The phylogenetic tree was constructed using the neighbor-jointing method,and the 39 genes were divided into four subfamilies(Ⅰ,Ⅱ,Ⅲ,Ⅳ),and each subfamily was analyzed.Through the analysis of cis-elements in the promoter of the gene,it was found that there were abundant signal components in the subfamily I of GATA,which could be inferred that the genes involved in the subfamily Ⅰ were associated with the light response.There were many stress responsive elements in the subfamily Ⅲ,which could respond to other signals such as high/low temperature and drought.Compared with the annual herbaceous plants,the members of the GATA subfamily Ⅲ of Populus showed more adaptive characteristics.Then we carried out different nitrogen treatments on NE19 plants,and analyzed the expression of GATA family members in different tissues of roots,stems and leaves by qPCR.We found that the gene expression of GATA transcription factor was varied in the same subfamily.These results indicated that GATA family members had different roles in nitrogen metabolism.(3)We firstly cloned GATA transcription factor family member PdGNC from NE19.This gene was highly expressions in mature leaves.At present,by means of CRISPR/Cas9 technique,we could acquire the Populus mutation,for the research of gene function of woody plants.Using CRISPR/Cas9 technology,we constructed the knock-out vector of the GNC gene,and successfully edited the site-directed mutation.At the same time,the construction of over expression vector of PdGNC gene was constructed,and used for transformation of Populus and Arabidopsis.(4)In the model plant Arabidopsis thaliana,oxPdGNC plants and complementary line gnc/PdGNC were obtained by the method of inflorescence impregnation.From 4-week-old plants,we could find the chlorophyll content,chloroplast number and chloroplast area of oxPdGNC line were 26.12%,22%,and 15.87%higher than wild type,respectively.PdGNC promoted longer primary roots and larger leaf areas.Root length and leaf area of oxPdGNC was 1.3-and 2.3-fold higher than wild type,respectively.The photosynthetic rate in transgenic LN lines was 42.17%higher than in the wild type through modification of the chlorophyll fluorescence parameters Fv/F0,Fv/Fm,qP,NPQ.Overexpression of PdGNC also had pronounced effects on chloroplast ultrastructure by increasing the number of grana and thylakoids.In conclusion,PdGNC not only promoted the synthesis of chlorophyll and the efficiency of light energy conversion,but also exhibited resistance to environmental damage,improved nitrogen utilization efficiency and plant growth.(5)In the woody Populus clone 717,we obtained the overexpression of oxPdGNC and CRISPR/Cas9 mediated GNC mutant crispr-GNC transgenic plants through Agrobacterium transformation of leaf.Leaves of crispr-GNC plants were yellowish green.The chlorophyll content of oxPdGNC was 25-30%higher than WT.The photosynthetic rate of oxPdGNC was increased by 20-28%than WT.Moreover,PdGNC also influences the microstructure of chloroplasts,which is like the overexpression of this gene in Arabidopsis thaliana.In Populus,PdGNC gene was associated with the fast growth and affected the vascular system development.Distinguished from Arabidopsis,the growth of crispr-GNC was significantly growth delayed.Plant height of oxPdGNC was～25%,220%higher than that of WT and crispr-GNC.Furthermore,the lignin content in stems of crispr-GNC plants was earlier than that of wild Populus clone 717 and oxPdGNC plants.PdGNC could influence the expression of downstream genes,as glucose transport gene(PMT and STP)and electron transport genes(CYP76C4,BBE14,and BBE17).We infer that PdGNC gene regulated plant growth by influencing the carbon distribution in plants.In conclusion,these studies indicated that GATA gene of Populus not only affected the growth of plants,but also responded to environmental factors.At present,there is not any study on woody plants research a member of the GATA family.We through analysis of expression system evolution of Populus GATA transcription factor family,PdGNC gene was cloned and researched in Arabidopsis and Populus.We revealed the PdGNC gene in woody plants and herbaceous plants in similarities and different points.This provides a basic study of biological functions of GATA genes for Populus or other woody plants.