High nighttime temperature (HNT)-responsive MicroRNA profiles in developing caryopses and flag leaves of 13 rice varieties

• MicroRNAs (miRNAs) regulate various biological processes, including grain development but their role in high nighttime temperature (HNT)-induced grain chalkiness in rice is unknown • 138 small RNA-seq libraries were generated from the developing caryopses (R6-stage) and flag leaves from a panel of 13 rice varieties with varying chalkiness under HNT. • A total of 787 distinct miRNAs (including 5 novel miRNAs) belonging to 299 miRNA families were identified in these two tissues of rice. • Comparative analyses revealed differential regulation of specific miRNAs (Osa-miR1432-5p, Osa-miR1861 etc.) in one phenotype (low chalky phenotype) relative to the other (high chalky phenotype), suggesting potential role of miRNA in regulating chalki phenotype under HNT. • Four degradome libraries were generated from caryopses and flag leaves, which confirmed validation of 171 targets in these two tissues. : Higher ambient temperatures during the cropping season, especially during nighttime, reduces rice grain quality by increasing chalkiness, primarily defined by altered starch metabolism. MicroRNAs (miRNAs) regulate various biological processes, including grain development and maturation. However, their role in high nighttime temperature (HNT)-induced grain chalkiness in rice is unknown. To address this phenomenon, an attempt was made to identify HNT-responsive miRNAs in developing caryopsis and flag leaves. The miRNA expression levels were investigated between several high chalky (HC) and low chalky (LC) varieties to identify potential microRNAs involved in impacting grain chalkiness traits. : 138 small RNA-seq libraries were generated from the R6-stage caryopses and flag leaves from a panel of 13 rice varieties with varying chalkiness under HNT. Genetic variation among rice varieties for miRNAs expression was found. A comparison of miRNAs for differential regulation patterns between genetically closely related HC and LC varieties revealed specific miRNAs with consistently higher expression levels in one phenotype relative to the other. Notably, some of these miRNAs have predicted or confirmed effects in regulating grain quality and HNT responses. Furthermore, a degradome analysis of mRNA in caryopsis and flag leaf tissues revealed several novel miRNA-mRNA target pairs with some targets possibly involved in rice starch metabolism. : This study revealed a number of differentially regulated miRNAs with potential roles towards alterations of grain chalkiness in rice under HNT stress. These miRNAs are good potential candidates for future analyses and investigation regarding their effects on improving rice grain quality under HNT-stress conditions in field settings.