Study On The Mechanism Underlying The Regulation Of Low-Light Tolerance By RGA1 In Rice

Light is one of the major environmental factors affecting the growth,development,yield,and quality formation of rice.Due to the limitations posed by climatic conditions,the production of rice in some areas often faces the challenge of insufficient light.In recent years,frequent persistent cloudy and rainy weather due to climate anomalies and the"global dimming"phenomenon caused by annual decreases in radiation levels due to atmospheric pollution have resulted in severe global crop yield losses.Insufficient light has become one of the major environmental stresses that threaten rice production.Thus,breeding low-light tolerant varieties and developing cultivation techniques for stress tolerance are of great significance for stabilizing food security.Currently,there are few studies on low-light stress in rice,and the relationship between carbohydrate metabolism and transport,as well as energy production,utilization and allocation and the formation of yield and quality under low-light and the related regulatory mechanisms remain to be eluciated.In this study,a low-light intolerant RGA1 mutant（d1）screened through previously experiments,and its wild type（Zhonghua 11,WT）and overexpressed transgenic plant（OE-1）were used as materials to investigate the mechanism of RGA1 regulation of low-light tolerance in rice,using a combination of an artificial climate chamber and field shading treatments.On this basis,the effects of different nitrogen fertilizer levels on rice yield and rice quality formation under low-light were investigated.The main results are as follows:1.RGA1 enhances low-light tolerance in rice by improving energy production efficiency.The survival rate of WT,d1,OE-1 and OE-2 plants decreased by 48.6%,88.6%,28.6%and 31.3%,respectively,after low-light treatment during the nutritional growth period of rice.Low-light stress markedly decreased the net photosynthetic rate and respiration rate of rice leaves,and the difference in net photosynthetic rate between lines was small,but the respiration rate of d1 leaves was markedly higher than that of WT.Under low-light,the activity of mitochondrial electron transport chain complex I and complex V in d1 leaves significantly decreased,but the content of alternative oxidase（AOX）significantly increased,resulting in a significantly lower ATP production efficiency than in WT,indicating that ATP deficiency is the main factor leading to poor low-light tolerance.Based on transcriptomic and lipidomic analyses,carbohydrate,lipid and energy metabolic pathways associated with ATP production are all involved in regulating low-light tolerance.Compared with WT,the expression of genes related to sucrose metabolism and glycolysis pathway was more down-regulated in d1 leaves under low-light conditions.The phosphatidic acid（PA）can be transformed into other glycerophospholipids in WT leaves under low-light.However,the glycerolipid and fatty acid degradation pathways of d1 were significantly up-regulated,contributing to the over-metabolism of glycerophospholipids such as PA to diglyceride,resulting in damage to mitochondrial membrane structure.Further research indicates that,acid invertase（INV）,INV activator（sucrose,Suc）,or phospholipase inhibitor（aristolochic acid,AAs）could improve the low-light tolerance of rice, especially d1,while sucrose metabolism inhibitor（acarbose,Aca）reduced the low-light tolerance of rice.Thus,RGA1 mainly promotes sucrose metabolism and prevents over-metabolism of glycerophospholipids such as PA under low-light, thereby increasing the efficiency of ATP production and thus enhancing low-light tolerance in rice.2.RGA1 alleviates the inhibition of pollen tube elongation in pistil by low-light through improving carbohydrate and energy metabolism and allocation.Low-light stress during anthesis significantly reduced spikelet fertility of rice,with 44.7%,91.6%and 14.1%reduction in spikelet fertility in WT,d1 and OE-1,respectively.And hindered pollen tube elongation was the main reason for the significant reduction in spikelet fertility.Under low-light,the activities of INV,sucrose synthase（SUS）and mitochondrial electron transport chain complexes,as well as the relative expression levels of sugar transporters（SUTs and SWEETs）,SUSs,INVs,cell wall invertases（CINs）and sucrose non-fermenting-1-related kinase 1（Sn RK1A and Sn RK1B）in OE-1 pistils significantly increased,but significantly decreased in d1.The ATP and ATPase contents in pistils of OE-1 were significantly increased under low-light treatment compared with the control,but significantly decreased in d1.In addition,INV and ATPase activators（Suc and Na₂SO₃）improved energy status in pistils and enhanced spikelet fertility under low-light,whereas the ATPase inhibitor Na₂VO₄ exacerbated spikelet abortion.In summary,RGA1 can alleviate the inhibition of pollen tube elongation and avoid the spikelet abortion by promoting sucrose transport and metabolism and improving ATP production,utilization and allocation in the pistil under low-light stress.3.RGA1 can alleviate the inhibition of rice yield and quality formation by low-light stress during the filling stage.The effect of low-light stress on rice yield and quality during the filling stage varied depending on the stage and rice line.Low-light treatment at the MS stage（7-14d after full heading）had the greatest effect on yield and appearance quality（chalky grain rate and chalkiness degree）,followed by the ES stage（0-7d after full heading）and LS stage（14-21d after full heading）,respectively.Compared with WT and OE-1,low-light treatment at MS stage significantly reduced the nonstructural carbohydrate content,ADPG pyrophosphorylase（ADPGsae）and starch synthase（SS）activities,and the relative expression of SUT1 and SUT2 in d1 grains.The contents of gibberellin（GA₃）,auxin（IAA）and zeatin（ZR）in WT and OE-1 grains increased more than that in d1 under low-light treatment.Thus,RGA1 can improve the source-sink relationship by regulating the contents of GA₃,IAA and ZR hormones to promote assimilate translocation to the grains,thus reducing the inhibition of rice yield and quality by low-light stress.4.A moderate increase in N fertilizer application is beneficial to reduce yield loss of rice caused by low-light at anthesis stage.Shading treatment significantly reduced spikelet fertility,and hindered pollen tube elongation was an important factor leading to spikelet abortion.Under shading treatment,the ratio of pollen tubes entering the ovules and the spikelet fertility were significantly increased in WT and OE-1 under moderate-nitrogen（MN）treatment compared with low-nitrogen（LN） treatment,while high-nitrogen（HN）treatment reduced the spikelet fertility and yield.Except for LN,other nitrogen treatments had little effect on spikelet fertility and yield of d1 under shading treatment,and the difference between treatments is not significant.Compared with LN treatment,MN treatment under shading treatment caused significant increases in INV,SUS,ATP and ATPase contents in spikelet of WT and OE-1.In conclusion,moderate increase in nitrogen level can promote sucrose metabolism and maintain energy balance as a way to prevent the inhibition of pollen tube elongation and spikelet fertility formation by low-light stress.5.An increase in N fertilizer application is beneficial to alleviate the inhibition of rice yield and quality formation by low-light stress during the filling stage.Under polyethylene film（PT）treatment,MN treatment increased the yield,panicle dry matter weight and N harvest index and reduced chalky grain rate and chalkiness degree of WT and OE-1 compared with LN,but the opposite trend was observed under HN treatment.Under black shade net（BT）treatment,the yield,panicle dry matter weight and nitrogen harvest index of WT and OE-1 increased with increasing nitrogen levels,and HN reduced chalky grain rate but increased chalkiness degree.Except for LN,nitrogen treatments had little effect on rice yield and quality of d1 under low-light conditions.In summary,the reasonable increase of nitrogen fertilization was beneficial to the formation of rice yield and quality under the two low-light treatments.