Signaling and reducing the accumulation of reactive oxygen species [67]. OsWNK9 plays a function in ABAmediated anxiety tolerance mechanisms [68]. The GO functional GSK-3 MedChemExpress enrichment analysis of DEGs showed that protein phosphorylation and protein serine/threonine kinase activity have been high enriched at eight DAH, 12 DAH andXie et al. BMC Plant Biol(2021) 21:Web page 13 ofTable 2 DEGs encoding PKs differentially expressed in X11 vs. X7, X24 vs. X7, and X11 vs. X24 at eight DAH, 12 DAH and 16 DAHFamily 8 DAH X11 vs. X7 Up AGC CAMK CGMC CK1 FGGY SET TKL 0 5 0 0 0 1 8 Down 1 6 three 0 0 5 two X24 vs. X7 Up 0 3 0 0 0 1 2 Down 1 three 0 0 1 0 three X11 vs. X24 Up 0 3 0 0 0 three 5 Down 0 five 1 0 0 1 1 12 DAH X11 vs. X7 Up 1 2 0 0 0 1 three Down 0 four two 1 0 1 2 X24 vs. X7 Up 1 0 0 0 0 two five Down 1 five four 1 1 2 2 X11 vs. X24 Up 0 6 1 0 0 2 4 Down 0 1 0 0 0 9 two 16 DAH X11 vs. X7 Up 0 3 1 0 0 2 9 Down 0 four 0 0 0 3 3 X24 vs. X7 Up 2 2 0 1 0 1 0 Down 2 7 0 0 0 5 six X11 vs. X24 Up 1 6 0 0 0 five 11 Down two 4 1 0 0 0Numbers of DEGs encoding PKs differentially expressed with statistical significance (P-value 0.05 and |Log2foldchange(FC)| 1) are shown; Up: up-regulated; Down: down-regulatedFig. 8 Expression patterns of DEGHL encoding PKs obtained at eight DAH, 12 DAH and 16 DAH, which are shown as log2Foldchange levels. DEGHL had been differentially expressed with statistical significance (P-value 0.05 and |Log2foldchange(FC)| 1)DAH (Fig. 4A-C). Additional evaluation showed that 5 AGC , 32 CAMK, 5 CGMC, two CK1, 17 STE and 30 TKL have been differentially expressed in at the very least a single group of pairwise comparison (Table two). In these PKs, there had been six DEGHL at 8 DAH, 7 DEGHL at 12 DAH and four DEGHL at 16 DAH (Fig. 8). At eight DAH, 1 CAMK, 1 STE and 1 TKL were up-regulated, 1 AGC , 1 CAMK and 1 TKL had been down-regulated. At 12 DAH, 1 AGC and 1 TKL were up-regulated, two CAMK, 2 CGMC, 1 CK1 and 1 TKL have been down-regulated. At 16 DAH, 2 CAMK, 1 STE and 1 TKL have been down-regulated. The outcomes showed that the differential expression of CAMK and TKL have been located at 8 DAH, 12 DAH and 16 DAH, as well as the variety of DEGHL in these two groups have been extra than other groups. Meanwhile, TKL member (OsR498G0510476500.01) was significantlydown-regulated (Log2Foldchange -10) at three sampling dates. OsCPK21, which was involved in the response to ABA [69], was down-regulated at 12 DAH and 16 DAH. Hence, we speculated that some PKs interacted with phytohormones could possibly be critical variables inside the regulation of the caryopsis improvement and chalkiness formation. In an effort to validate the RNA-seq data, 12 DEGHL had been selected to confirm expression by real-time PCR evaluation, such as 5 DEGHL involved in starch/sucrose and protein metabolism (OsR498G0100430000.01, OsR498G0204716300.01, OsR498G0917494500.01, OsR498G0204791500.01 and PROLM9), three DEGHL encoding TFs (OsR498G0100472700.01,Validation of DEGHL by realtime PCR analysisXie et al. BMC Plant Biol(2021) 21:Web page 14 ofFig. 9 Expression patterns of 12 DEGHL confirmed by real-time PCR, which are shown as log2Foldchange levels. DEGHL had been differentially expressed with statistical significance (P-value 0.05 and |Log2foldchange(FC)| 1). Information shown as implies SD of 3 biological replicates. Asterisks indicate a substantial distinction according to a Dunnett’s test. important distinction at five level (P 0.05); substantial distinction at 1 level (P 0.01)Xie et al. BMC Plant Biol(2021) 21:Web page 15 ofOsR498G0100734500.01 and Bcl-B web WRKY76), 3 DEGHL associated with phytohormonal biosynthesis and signaling (OsYUCCA7, OsARF10 and OsSDR), 1 DEGHL e.