Photosynthetic Performance Of Aquaporin Knockout Lines In Oryza Sativa

Rice is one of the essential food crops in China.With the population increasing and cultivated land area reducing,it is urgent to improve rice yield per unit of cultivated area to ensure food security.Since the dry matter accumulated by photosynthesis supplies the biomass basis for crop yields,improving leaf photosynthetic rate is considered a significant way to promote crop yield potential.In general,CO₂ assimilated during photosynthesis is mainly from atmosphere.CO₂ diffusing from the atmosphere into chloroplasts is affected by serial resistances,and mesophyll resistance accounts for about half of the total resistance.CO₂ transmembrane transport is believed to be mediated by plasma membrane intrinsic proteins（PIPs）,localizing on the cell membrane and chloroplast membrane.Recent studies proposed that PIPs also involved in short-term environment response of mesophyll conductance.However,in rice,PIPs constitute 11 homologous proteins,and how each homologous protein influence mesophyll conductance and regulates its response to the environment is still unclear.Therefore,we constructed 11 single-gene knockout mutants with rice cultivar Nipponbare background by CRISPR/Cas9 technology in the present study and conducted two experiments.Firstly,in the growth chamber,the effects of OsPIP on the photosynthetic feature and plant growth performance in rice were identified on the vegetative and reproductive growth stage.At the same time,a field experiment was carried out to clarify the impacts of single aquaporin gene knock-out on rice yield;Secondly,to reveal the roles of OsPIPs in regulating environmental responses of mesophyll conductance,I compared the differences in photosynthetic characteristics and growth performance between knockout lines and wild-type under different growth conditions by planting plant materials in Hainan and Wuhan.In Wuhan,the comparisons were also applied to plants grown in outdoor and in the artificially controlled environment in the growth chamber.The main results were as follow.（1）The effects of OsPIP knockout on plant agronomic traits varied with the growth stage in field conditions.At 20 days after transplanting,only the Ospip1;1 line was statistically significant.However,with growth period prolong,the significance difference between Ospip1;1 and wild-type in tiller number disappeared.After transplanting 35 days,tiller numbers of Ospip2;2,Ospip2;3,Ospip2;4,Ospip2;6 and Ospip2;8 were significantly less than wild-type.Similarly,plant heights of Ospip2;1,Ospip2;3 and Ospip2;4 were significantly higher than wild-type at 20 days after transplanting,but at 35 days and 41 days after transplanting,the mutants with significant difference in plant height from wild-type were Ospip1;1,Ospip2;4 and Ospip2;5.Meanwhile,compared to other mutants,Ospip2;5had highest plant height while Ospip1;1 had maintained the lowest plant height.Moreover,under field conditions,the yields of Ospip1;1,Ospip1;3,Ospip2;1,Ospip2;2,Ospip2;4and OsPIP2;7 were significantly reduced.In these mutants,Ospip1;1 had the lowest yield,with an average of 4.55 t ha-1,which was 33.7 % lower than wild-type.From the perspective of yield components,the significant reduction in yield were mainly related to the decrease of 1000-grain weight and spikelet number per panicle.（2）Among the 11 knockout lines,only OsPIP1;2,OsPIP1;3,OsPIP2;1 and OsPIP2;8knock-out had significant effect on leaf mesophyll conductance.Compared with the wildtype,the mesophyll conductance of Ospip1;2 and Ospip2;8 decreased by 15.1% and 13.7%respectively on the vegetative growth stage which result in a significant decrease in photosynthetic rate.However,on the reproductive growth stage,no significant differences were observed in the mesophyll conductance between wild-type and Ospip1;2,as well as Ospip2;8.At this stage,the mesophyll conductance of Ospip1;3 and Ospip2;1 decreased by 14.2% and 23.6% respectively,and the photosynthetic rate was also significantly lower than that wild-type.In addition,the stomatal conductance of Ospip1;2,Ospip1;3 and Ospip2;1 was also significantly lower than the wild-type on the reproductive growth stage,and the stomatal conductance of Ospip2;1 was the lowest among all knockout lines,which was 44.6% lower than wild-type.Therefore,different OsPIP had different effects on leaf photosynthetic physiology,and the difference in mesophyll conductance between OsPIP knockout mutants and wild-type was related to plant growth stages.Ospip1;1,Ospip1;2and Ospip2;1（3）In Hainan,the photosynthetic rate and mesophyll conductance of knockout lines were not significantly different from wild-type,but the significant difference in stomatal conductance was observed.Ospip1;2 had a higher stomatal conductance than wild-type while Ospip1;2 had a lower stomatal conductance.In contrast to Hainan,except for Ospip2;1,the photosynthetic rate and mesophyll conductance of Ospip1;1 and Ospip1;2were significantly lower than wild-type in Wuhan,especially Ospip1;1,whose photosynthetic rate and mesophyll conductance were lowest in all mutants.In addition,when compared the photosynthetic characteristics of plants under artificially controlled environment and outdoor natural environmental conditions,the effects of OsPIP on plant photosynthetic physiology also showed significant differences.Under artificially controlled environmental conditions,the knockout of OsPIP2;1 significantly reduced the stomatal conductance and mesophyll conductance,which in turn affected the photosynthetic rate.However,under natural environmental conditions,the knockout of OsPIP2;1 had no significant effect on CO₂ transport efficiency,but the mesophyll conductance and photosynthetic rate of Ospip1;1 were significantly reduced.In summary,the effect of OsPIP on leaf photosynthetic characteristics is regulated by the plant growth environment.In conclusion,OsPIP1;2,OsPIP1;3,OsPIP2;1 and OsPIP2;8 can regulate the CO₂ transport efficiency in rice leaves,and this regulation is affected by plant growth stage and growth environment.Meanwhile,compared with wild-type,OsPIP1;3,OsPIP2;1knockout also significantly reduced rice yield.This suggests that OsPIP1;3 and OsPIP2;1may affect photosynthetic rate by regulating CO₂ transport efficiency in leaves,thereby influence yield.