The PGS1 Basic Helix-Loop-Helix (bHLH) Protein Regulates Fl3 To Impact Seed Growth And Grain Yield In Cereals

Common wheat is one of the three major food crops.Improving its yield is very important to solve the problem of food shortage.Basic helix-loop-helix(bHLH)transcription factors(TFs)are widely distributed in eukaryotic organisms.They play critical roles in regulating the expression of developmental genes in cereals.To find important transcription factors specifically expressed in seeds,225 bHLH predicted from wheat using genomic,and RNA sequencing data were subjected to identification,classification,conserved motif characterization,chromosomal distribution determination,and expression pattern analysis.The molecular function and regulatory mechanism of one of the key bHLH transcription factors specifically expressed in seeds were identified.1.One basic region,two helix regions,and one loop region were conserved in wheat bHLH TFs.The bHLH proteins could be separated into four categories based on sequence profiles in their basic regions.Neighbor-joining-based phylogenetic analysis of conserved bHLH domains from wheat,Arabidopsis,and rice identified 26 subfamilies of bHLH TFs,23 were found in wheat.One hundred and nine wheat bHLH genes had homologs in Arabidopsis(54)and rice(55).Seven tissue-specific bHLH TF clusters were identified according to their expression patterns in the endosperm,aleurone,seedlings,heading-stage spikes,flag leaves,shoots,and roots.Expression levels of six endosperm-specifically expressed TFs measured by q PCR and RNA-seq showed a good correlation between the two methods.2.We identified the bHLH protein specifically expressed in the seeds at wheat’s 5–20 days post-anthesis.It was ectopically overexpressed(OE)in wheat and rice,leading to increased grain weight(up to 17.24% in wheat and 13.40% in rice lines)and grain size.Carbohydrate and total protein levels also increased.Therefore,Ta PGS1(T.aestivum Positive Regulator of Grain Size 1).We also investigated plant height,ear length,effective panicle number,and grain number per panicle and found no significant differences in these phenotypes between Ts PGS1 overexpressed lines and wild type.Scanning electron microscopy results of endosperm indicated that the starch granules in the endosperm of Ta PGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix.Furthermore,experimental results demonstrate that Ta PGS1 was bound directly to the E-box motif at the promoter of a PLATZ transcription factor gene and positively regulated its expression in wheat and rice,respectively.The genes are homologous to Zm Fl3,a PLATZ transcription factor gene that regulates endosperm development in maize,so they were named Ta Fl3 and Os Fl3,respectively.In rice,the Os Fl3 CRISPR/Cas9 knockout lines showed reduced average thousand-grain weight,grain width,and grain length in rice.The endosperm structure is loose and porous,which is precisely opposite to the phenotype of Ta PGS1 overexpressed lines.This study provides insight into understanding the structure divergence,evolutionary features,expression profiles,and potential function of bHLH and PLATZ in wheat.Our results reveal that Ta PGS1 functions as a valuable trait-associated gene for improving cereal grain yield.