Study On The Physiological And Molecular Mechanism Of Carbon Enrichment Promoting Tomato Growth

CO₂ is the main raw material of plant photosynthesis and one of the main factors that affects plant growth in greenhouses.Using the captured CO₂ as a gas fertilizer in protected cultivation to increase photosynthesis rate and transform more CO₂ to organic matter is one of the important ways to achieve high yield and optimal product quality.Tomato（Solanum Lycopersicon L.）is one of vegetables in Solanaceae family that is widely cultivated in China and especially the largest vegetable area in protected cultivation in the north.At present,the completion of tomato genome sequencing and the enrichment of gene annotation information provide favorable conditions for the research on photosynthesis and carbohydrate metabolism of tomato and other vegetables in the Solanaceae family.Some reports about vegetable growth,physiological characteristics and related gene functions under CO₂ enrichment environment were published,but the molecular mechanism is still unknown.Therefore,it is important to study the response mechanism of tomato under CO₂ enrichment for the production and breeding of Solanaceae vegetables.In this study,four large fruit type tomatoes varieties ‘Jindun’,‘Zhongyan 868’,‘Hongzhuanshi’ and‘Meiguohongzhixin’ were used as experimental materials.The morphological changes and photosynthetic characteristics of tomato under natural environment and CO₂ enrichment treatment(800 ± 50 μmol·mol^-1)were compared.The yield,quality and transcriptome differential expression of sensitive varieties were analyzed and the key genes responding to CO₂ enrichment for tomato were screened by gene function annotation and metabolic pathway analysis as well.We also verified their functions by gene overexpression and knockout technology,which laid a foundation for future research.The main results were as follows:1.CO₂ enrichment promoted plant height,stem diameter,internode length of certain nodes,leaf length and width of the four tomato cultivars with large fruits,especially ’American Red Star’ and ’Red Diamond’.However,it had little effect on root shoot ratio.Under carbon enrichment,the thickness of palisade tissue and sponge tissue,the ratio of grid to sea and the overall thickness of leaf were increased and early growth rate of tomato fruit were also promoted as well.2.The photosynthetic CO₂ response curves of four tomato varieties showed that the net photosynthetic rate from high to low was ’Mei guo hong zhi xing’>’Hong zuan shi’>’Zhongyan 868’>’Jin dun’ under each CO₂ concentration gradient in the range of 400-2200 μmol·mol^-1.Among these four varieties,’Mei guo hong zhi xing’ exhibited the maximum catalytic rate of Ru Bis CO enzyme,regeneration rate of Ru Bp,and the transportation rate of triose phosphate.The growth dynamic of light intensity in response to the net photosynthetic rate is ‘Mei guo hong zhi xing’>’Zhongyan 868’>‘Jin dun’>‘Hong zuan shi’.The net photosynthetic rate of ’Red zuan shi’ and ’Mei guo hong zhi xing’ under carbon enrichment increased by74.16% and 78.52% respectively compared with the control group,and the difference was significant.Under the carbon enrichment,the Ru Bis CO enzyme and the TK enzyme activity in functional leaves of’Mei guo hong zhi xing’ increased by 12.84% and 21.16% compared with the control respectively which showed the significant difference;the FBAase and the SBPase activity increased by 9.35% and 1.59%compared with the control,but the difference was not significant.The results indicated that CO₂ enrichment is beneficial to increase the photosynthetic rate and key enzyme activities of photosynthesis,and the "Mei guo hong zhi xing" variety is the most sensitive.3.The yields of four large-fruit tomato varieties had different increase under CO₂ enrichment.’Mei guo hong zhi xing’ significantly increased by 16.93% compared with the control;The soluble sugar content increased and the titratable acid content decreased,the sugar-to-acid ratio,soluble solids and lycopene content significantly increased by 47.13%,35.25% and 72.24%,respectively compared with the control.4.A total of 28.70 Gbs of high-quality transcriptome data were obtained by high-throughput sequencing under different CO₂ concentrations.We found 208 significantly differentially expressed genes（DEGs）by comparing gene expression under different CO₂ concentrations,of which 179 and 29 were up-regulated and down-regulated,respectively.DEGs involve a variety of biological processes,cell components and molecular functions,but are mainly enriched in physiological processes such as photosystem I,photosystem II,phosphate binding in photosynthesis.Nine DEGs involved the electron transport system of photosystem I,6 DEGs involved the electron transport system of Photosystem II,3DEGs involved the electron transport of cytochrome b6 f,and 3 DEGs involved NADPH dehydrogenase activity.In addition,DEGs involved in sugar synthesis and plant hormone signal transduction was also discovered.It is predicted that these genes are directly related to the growth and physiological changes of tomato in response to CO₂ enrichment.5.Two key genes,Solyc07g043480 related to sugar metabolism（it was found in the tomato database that Solyc07g043480 is a UDP-glucosyltransferase,the gene name is GLYCOALKALOID METABOLISM17（GAME17））and Solyc01g007720 related to photosynthesis,in response to CO₂ enrichment were obtained through transcriptome analysis in this experiment.（1）We cloned GAME17 and Soly720,constructed an overexpression vector,and carried out tomato genetic transformation.After hygromycin resistance screening and PCR detection,12 OE GAME17-positive plants and 13 OE Soly720-positive plants were obtained;we constructed gene knockout vector,transformed into tomato and obtained 10Cas9-GAME17 positive plants and 10 Cas9-Soly720 positive plants after hygromycin resistance screening and PCR detection.（2）It showed that expression levels of genes in OE GAME17 and OE Soly720 transgenic tomatoes were higher than wild-type in the tender and functional leaves of tomato.（3）The net photosynthetic rate of wild type and OE Soly720 were measured by using photosynthesis related gene Soly720.The net photosynthetic rate of OE Soly720 was higher than wild type.（4）Under CO₂ enrichment environment,Ru Bis CO activities in OE Soly720 were more than wild type;Activities of other 3 types enzymes including FBAase,TK and SBPase in OE Soly720 were all > wild type.