| Grain filling efficiency in inferior grains is lower than superior grains in many cereal crops,especially in rice cultivar with large panicle and numerous spikelets.Low grain filling efficiency in inferior spikelet has become one of the limiting factors for higher yield.In our previous study,we found moderate soil drying after anthesis significantly promoted grain filling of inferior spikelet and increased yield.However,our understanding of the molecular mechanism of grain filling difference between inferior spikelet and superior spikelet is extremely poor.Based on the previous research,this study not only revealed the physiological mechanism of moderate soil drying regulating NSC remobilization in straw and grain filling in inferior spikelets of rice,but also identified OsABA8ox2 which is the key gene to regulate ABA content and difference of grain filling efficiency between superior and inferior grains using RNA-seq and reversed genetics.Moreover,miRNA and synergistic interaction between ABA and IAA resulted from moderate soil drying promotes grain filling of inferior spikelet in rice.The main findings are as follows:(1)Moderate soil drying promoted starch hydrolysis by increasing α-amylase and β-amylase activity,and accelerated transport from stem to grain.In grains,sucrose was conversed into starch by increasing the activity of starch synthase and sucrose synthase,thus accelerated the accumulation of starch in inferior grains,which thus promoted grain filling of inferior spikelet,increased weight and promotes the yield.(2)RNA-seq analysis showed that signal pathways such as phenylpropanoid biosynthesis,plant-pathogen interaction and protein processing in endoplasmic reticulum were involved in moderate soil drying promoted inferior grain filling.More importantly,plant hormone signaling,starch and sucrose metabolism signaling pathways were also involved in this process.(3)OsABA8ox2 is the key gene to regulate ABA content and difference of grain filling efficiency in superior and inferior grains.Knockout of OsABA8ox2 by CRISPR/Cas9 technology can significantly increase the ABA content and grain filling.Grain weight of inferior grain was increased by 7.09%-20.26% and the yield per plant was promoted by 6.71%-12.46%,seed setting rate was also increased by 10.26%-12.53% in fall seeding.(4)Moderate soil drying and knockout of OsABA8ox2 gene can elevate ABA content in inferior grains,which thus up-regulated the expression of IAA biosynthesis and promoted IAA signal transduction by down-regulating the auxin responsive genes.In addition,several TPP but not TPS genes were down-regulated in inferior spikelet of both MD treatment and OsABA8ox2 knockout mutant,resulting in lower trehalose content and might lead to accumulation of T6P,which thus increased the expression of OsTAR2,promoted IAA biosynthesis.Taken together,our results suggested that a synergistic interaction of ABA mediated accumulation of IAA promotes grain filling of inferior spikelet under moderate soil drying.(5)miRNA was involved in moderate soil drying promotes grain filling of inferior grains by small RNA sequencing analysis,target genes for differential miRNA were predicted using bioinformatics websites.GO analysis shown that the pathway binding,catalytic activity,metabolic process,transcription regulator activity,carbohydrate metabolism and lipid metabolism etc.were enriched.Moderate soil drying promoted amylase activity through miR1861-OsSBDCP1-OsSSIIIa pathway,resulting in enhanced inferior grain filling.miR1432 was also decreased by moderate soil drying which targets on OsACOT gene,resulting in higher fatty acid metabolism and modulating ABA signaling and auxin transport.miR397-OsLAC was also involved in the process of moderate soil drying promoted grain filling,in which BRs may play a key role.Taken together,our results suggested that a synergistic interaction of ABA-mediated accumulation of IAA promotes grain filling of inferior spikelet under moderate soil drying,and miRNAs also play critical role in this process. |
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