Effects Of Free-air Temperature Increase On Indica Rice Grain Protein Content In A Double Rice Cropping System And Its Mechanisms

The average global surface temperature continues to rise due to the massive emissions of anthropogenic greenhouse gases.Rice yield and quality can be significantly affected by climate warming.Proteins are the most important nutrients in rice grains.The contents of rice grain proteins not only determine rice nutritional quality,but are also closely related to rice processing,appearance,cooking,and eating qualities.Identifying the responses of rice grain protein contents to climate warming in double rice cropping systems are essential to improving rice quality in the warming future.Therefore,we conducted a field warming experiment from 2021 to 2022 by using a free-air temperature increase facility with conventional high-yielding（early rice:Zhongjiazao17;late rice:Tianyouhuazhan）and superior-quality palatable（early rice:Xiangzaoxian 45;late rice:Wanxiangyouhuazhan）indica rice varieties to investigate the effects of warming on the rice grain protein content and relevant physiological and ecological mechanisms in a double rice cropping system in southern China.Meanwhile,a pot experiment subjected to ¹⁵N-labeled urea application was conducted to clarify sources of rice nitrogen（N）uptake and N use efficiency in response to warming.Furthermore,transcriptomic analysis was performed to reveal the molecular mechanisms of protein biosynthesis and metabolism in rice grains as affected by warming.The main results were as follows:（1）The free-air temperature increase system resulted in an increase in daily average temperature of 1.9°C and 2.0°C in the canopy of early and late rice,respectively.For early rice,warming significantly increased effective panicles and filled-grain percentage,while decreased spikelet number and grain weight,which led to non-significant impact on grain yield.However,warming significantly decreased late rice yield（-6.3%）,primarily due to the reduction in grain weight.Experimental warming significantly increased N uptake（+9.4%）of early rice but did not significantly influence that of late rice.（2）Responses of crude protein content and protein component contents in the grain to warming were consistent between early and late rice,and between conventional high-yielding and superior-quality palatable rice cultivars.Warming significantly increased crude protein content in brown rice（+10.0%and+7.5%,respectively）and milled rice（+11.1%and+7.6%,respectively）of both early and late rice.Under the warming treatment,the albumin contents in the brown rice of both early and late rice were significantly reduced but the contents of globulin,prolamin,and glutelin showed an increasing trend.In the milled rice,the contents of the four protein components all tended to increase in response to warming.（3）Results from the ¹⁵N-tracer experiment showed that warming significantly increased N uptake derived from both fertilizer and soil of early rice,increased N uptake derived from soil but reduced that from fertilizer of late rice.N recovery rate and N retention rate was significantly increased and reduced by warming,respectively,in the early rice season,while cases were opposite in the late rice season.Under the warming treatment,N loss rate was markedly increased in both early and late rice seasons.（4）The free-air temperature increase system resulted in an increase in daily average temperature of 1.3°C and 1.2°C in the soil during the early and late rice growing seasons,respectively.Warming significantly improved root activity at the heading stage of early and late rice and concentrations of ammonium N and nitrate N in the soil,which benefited rice N uptake from soil.Besides,warming had significant influences on key enzyme activities related to protein synthesis in rice grains during the grain-filling period for both early and late rice.The protease activity tended to decrease,while activities of glutamate synthase,glutamine synthase,glutamate aminotransferase,and glutathione aminotransferase all tended to increase in rice grains under the warming condition,which promoted synthesis and accumulation of rice grain protein.（5）Transcriptomic analysis showed that regulation of rice grain protein biosynthesis pathways as affected by warming were different between early and late rice.For early rice,the differentially expressed genes（DEGs）of the two cultivars under warming were both related to“response to reactive oxygen species”,“cellular response to heat”,and“chaperone-mediated protein folding”.Warming activated“protein processing in the endoplasmic reticulum”pathway in the two cultivars for early rice.For late rice,DEGs of the two varieties were different in the functional enrichments but were both involved in amino acid metabolisms.Above all,free-air temperature increase（about 2.0℃）did not affect early rice yield but significantly decreased late rice yield when ambient daily mean temperatures were24.9°C and 28.4°C during early and late rice growing seasons,respectively.Warming increased the contents of crude protein and protein components in the grains of double rice,for both the high-yielding and superior-quality cultivars.For early rice,on the one hand,warming accelerated the net N mineralization rate of soil and improved the activity of rice roots,thus promoting rice N uptake.On the other hand,warming increased the activities of key enzymes involved in protein synthesis in rice grains and induced the up-regulated expressions of heat shock protein genes,promoting protein synthesis and processing,which consequently resulted in the increase in protein contents.For late rice,the N uptake was not significantly affected by warming,though the net N mineralization rate of soil and root activity were enhanced.However,warming promoted the activities of protein synthesis enzymes and amino metabolisms in rice grains,suggesting that the increase in rice grain protein content was dominantly attributed to enhanced protein synthesis processes by warming rather than the increased N uptake.However,the specific synthesis pathways of the four kinds of protein components in rice grains and the regulation of warming on these pathways remain unclear and need further study.