Construction Of GOC Photorespiratory Bypass In Solanum Tuberosum And Its Impact On Photosynthetic Efficiency And Yield

Photorespiratory is also referred to as the C2 cycle and,in which light-dependent uptake of O₂coupled with CO₂release occurs.Photorespiratory fluxes can consume about 25-30%of photosynthates in C3 plants under normal environmental conditions,and even higher up to 50%under environmental stresses such as under hot and dry growth conditions,since these stresses may futher stimulate photorespiration.Potato is an important crop that can be eaten as either staple food or vegetable in the world.This study aims to introduce the three enzymes,i.e.Os GLO,Kat E/Os CATC and OsOxO,into potato chloroplasts through Cre/lox P recombinant technology.In GOC/（GC）O bypass,part of the glycolate produced by photorespiration is directly metabolized to CO₂in chloroplasts,which may increase the concentration of CO₂around Rubisco and form a CO₂concentrating mechanism（CCMs）similar to C4 plants,so as to finally obtain engineered potato plants with high photosynthetic efficiency and yiled.The results were achieved as follows:1.Phenotype and functional analysis of engineered potato with OsOxO3 expressed in chloroplasts（called cOxO below）OsOxO is a key enzyme in downstream of the GOC/（GC）O pathway,in order to verify whether OsOxO enzyme plays a functional role in potato chloroplasts,OsOxO were introduced into potato chloroplasts（called cOxO below）and the phenotype of cOxO transgenic plants was analyzed.The results showed that cOxO potato plants showed various negative phenotypes,such as dwarf growth,smaller but thicker and greener leaves.Further physiological analyses showed that OxO activity was increased while oxalate content was markedly reduced by 38.9-55%in the cOxO transgenic plants.These cOxO transgenic plants showed reduced photosynthetic rate,stomatal conductance,intercellular CO₂concentration and soluble protein.The above results indicated that the introduced OsOxO has played a functional role in potato chloroplasts,likely because the enzyme catalyzed oxalate degradation,and finally affected the growth and development of the cOxO plants.2.Construction of the vectors expressing either（GC）O or GOC bypass and genetic transformation into potatoTwo bypasses were designed and contructed,i.e.,the GOC bypass that expresses glycolate oxidase（Os GLO）,oxalate oxidase（OsOxO）and catalase（Kat E）and the（GC）O bypass that expresses a fused protein with glycolate oxidase and catalase linked（Os GLO3*Os CATC）and OsOxO3.In both bypasses,Os GLO catalyzed part of the photorespiratory glycolate to glyoxylate,further to oxalate,then the oxalate was finally decomposed into CO₂as catalyzed by OsOxO within potato chloroplasts.In such manner,CO₂concentrations around Rubisco are increased and a CO₂concentrating mechanism is introduced,similar to that of C4 plants.The catalase was used to remove the H₂O₂produced during glycolate and oxalate oxidation.The multiple gene expressing vectors,i.e.,（GC）O-pBIA13 and GOC-pBIA13 were then constructed,in which Os GLO3 was promoted by p XNOS（agrobacterium cochinine synthase promoter）,（Os GLO3*Os CATC）and Kat E were promoted by p35S（Ca MV 35S promoter）,and OsOxO3 was promoted by p E35S（Ca MV 35S enhanced promoter）.（GC）O-pBIA13 or GOC-pBIA13 vectors was transformed into potato by LBA4404agrobacterium,and GOC and（GC）O lines were finally obtained through screening processes and analysis on molecular and enzyme activities.Further analyses have showed that the target proteins,i.e.Os GLO3,（Os GLO3*Os CATC）,Kat E and OsOxO3,were all successfully located into potato chloroplasts by rbc S chloroplast transit peptide.3.The GOC bypass significantly inhibited photorespiration and increased photosynthetic efficiencyUnder normal atmospheric circumstances,the photorespiratory rate was decreased by6.59-12.01%with significance in GOC transgenic potato plants,while the net photosynthetic rate was increased by 7.02%to 11.68%.Meanwhile,GOC potato had higher intercellular CO₂concentration,maximum net photosynthetic rate at light saturation point（Amax）and saturation point（LSP）,carboxylation efficiency（CE）and maximum carboxylation efficiency（Vcmax）and lower CO₂compensation point（Γ*）.These results indicated that the introduced GOC bypass effectively inhibited photorespiration and improved the photosynthetic characteristics in GOC potato,through forming a CO₂concentrationing mechanism,like in C4 plants.4.The GOC bypass significantly improved growth and agronomic performance of potatoGrowth and agronomic perfermance of the GOC transgenic potato were tested for two consecutive years by greenhouse pot experiments,and it was found that the plant height,stem diameter,biomass and yield of the GOC transgenic plants were all steadily increased.Compared with WT,the plant height and stem diameter of the GOC transgenic plants increased by 1.62-2.77%and 3.69-3.77%,respectively,the aboveground fresh/dry mass of per transgenic plants was increased by（9.65-22.18%）/（5.9-10.4%）,and the fresh/dry weight of tuber per transgenic plants significantly increased by（10.34-11.14%）/（11.8-14.7%）,the number of potato per transgenic plants steadily increased by 3-5.In order to further confirm the effect and practical significance of GOC bypass,we tested GOC transgenic potato under field conditions in Guangzhou,and the results showed that the increase of biomass and yield of GOC potato was similar between field and greenhouse pot experiments.Compared with WT,the aboveground fresh/dry mass per transgenic plants was increased by（10.83-12.59%）/（7.97-8.72%）,and the fresh/dry weight of tuber per transgenic plants significantly increased by（11.78-12.69%）/（11.68-12.27%）.We further tested GOC transgenic potato under field conditions in China Northern Area,where longer sunshine time and stronger light intensity usually occur.The results showed that the plant height,stem diameter,biomass and yield of the GOC transgenic plants all increased much more significantly.The plant height,stem diameter and leaf area per GOC transgenic plants increased by 5.04-5.05%,8.5-10.58%and 21.1-38.2%,respectively,the aboveground fresh/dry mass of per transgenic plants was increased by（4.6-18.5%）/（6.5-19.9%）,and the fresh/dry weight of tuber per transgenic plants increased by（31-46.7%）/（30.8-53.1%）,the number of potato per transgenic plants steadily increased by 4-7.These results indicated that the GOC bypass could effectively promote the growth of GOC transgenic plants and,correspondingly,biomass and yield,especially under high light conditions.5.Phenotype and charateristics of（GC）O potatoAnalyses on the obtained（GC）O potato showed that the linker between Os CATC and Os GLO3 protein was broken.Compared with WT,the activity of GLO was increased by9.62-14.3%,while the activity of CAT was only increased by 2.03-8.32%.The net photosynthetic rate,stomatal conductance,intercellular CO₂concentration and Fv/Fm value of（GC）O plants were not different from those of WT under normal atmospheric circumstances.The above results indicates that the introduced（GC）O bypass failed to work effectively to divert the photorespiratory glycolate,inhibit photorespiration and increase photosynthesis,likely due to breakage of the linker and or the low increases of the introduced enzymes.