A novel transcription factor OsMYB73 affects grain size and chalkiness by regulating endosperm storage substances' accumulation-mediated auxin biosynthesis signalling pathway in rice

Abstract

Enhanced grain yield and quality traits are everlasting breeding goals. It is therefore of great significance to uncover more genetic resources associated with these two important agronomic traits. Plant MYB family transcription factors play important regulatory roles in diverse biological processes. However, studies on genetic functions of MYB in rice yield and quality are rarely to be reported. Here, we investigated a nucleus-localized transcription factor OsMYB73 which is preferentially expressed in the early developing pericarp and endosperm. We generated targeted mutagenesis of OsMYB73 in rice, and the mutants had longer grains with obvious white-belly chalky endosperm appearance phenotype. The mutants displayed various changes in starch physicochemical characteristics and lipid components. Transcriptome sequencing analysis showed that OsMYB73 was chiefly involved in cell wall development and starch metabolism. OsMYB73 mutation affects the expression of genes related to grain size, starch and lipid biosynthesis and auxin biosynthesis. Moreover, inactivation of OsMYB73 triggers broad changes in secondary metabolites. We speculate that rice OsMYB73 and OsNF-YB1 play synergistic pivotal role in simultaneously as transcription activators to regulate grain filling and storage compounds accumulation to affect endosperm development and grain chalkiness through binding OsISA2, OsLTPL36 and OsYUC11. The study provides important germplasm resources and theoretical basis for genetic improvement of rice yield and quality. In addition, we enriches the potential biological functions of rice MYB family transcription factors.

Keywords: OsMYB73; auxin biosynthesis; endosperm starch and lipid biosynthesis; grain filling and endosperm development; grain size and chalkiness; yield and quality.

Figure 1

Rice OsMYB73 gene expression at various tissues, histochemical GUS staining and subcellular localization of OsMYB73 protein. (a) Rice OsMYB73 gene expression at various tissues. R, root; S, steam; L, leaf; LS, leaf sheath; P, panicle; 3D, 5D, 6D, 9D, 12D, 15D, 18D means 3, 5, 6, 9, 12, 15, 18 days after fertilization. Expression level at root in the wild‐type was set as reference value of 1. Data are mean ± SD (n = 3). (b) Histochemical GUS staining. Scale bars are 1.0 cm in B. (1): root; (2): steam; (3, 4): leaf; (5): leaf sheath; (6): branch; (7): floret; (8, 9): spikelet; (10, 11): Developing seed and transection of 5 days after fertilization; (12, 13): Developing seed and transection of 10 days after fertilization; (14, 15): Mature brown rice and transection. (c) Subcellular localization in rice protoplasts, bars are 2 μm.

Figure 2

CRISPR/Cas9 mediated target mutagenesis of OsMYB73 and genotype and phenotype identification. (a) The gene structure of OsMYB73 in rice; (b) target site selection and genotype identification; (c) milled rice phenotype identification in T₁ generation (CK: ZH11), scale bar is 1.5 cm; (d) dehusked rice phenotype identification in T₁ generation (CK: HZ), scale bar is 1.0 cm; (e) grain length between wild‐type and mutants, scale bar are 1.0 and 1.5 cm; (f) grain filling rate between wild‐type and mutants; (g) grain length between wild‐type and mutant; (h) Grain width between wild‐type and mutants; (i) 1000‐grain weight between wild‐type and mutants; (j) tillers between wild‐type and mutants; (k) plant height between wild‐type and mutants.

Figure 3

Grain starch physicochemical characteristics comparison of ZH11 and cr‐myb73 in T₁ generation. (a) Total starch content; (b) amylose content; (c) total protein content; (d) total soluble sugar content; (e) total lipid content; (f) gel consistency; (g) starch solubility in 1.7% KOH solution, scale bars are 1.0 cm; (h) chain length distributions of amylopectin in ZH11 and cr‐myb73; (i) pasting properties rapid visco analyser (RVA) of endosperm starch of ZH11 and cr‐myb73. FV, final viscosity; HV, hold through viscosity; PV, peak viscosity. The viscosity value at each temperature is the average of three replicates. The green line indicates the temperature changes during the measurements. Asterisks indicate statistical significance, as determined by a Student's t‐test (*P < 0.05, **P < 0.01).

Figure 4

Yeast two‐hybrid and yeast one‐hybrid assays of rice OsMYB73. (a) Yeast two‐hybrid self‐activating phenomenon assay (scale bar is 2.0 cm); (b) yeast one‐hybrid assay showing OsMYB73 binding OsISA2 and OsLTPL36 promoters; (c) yeast two‐hybrid assay showing the interaction between OsMYB73 and OsNF‐YB1, OsLTPL36 and OsYUC11 proteins in yeast cells; (d) bimolecular fluorescence complementation assay showing the interaction between OsMYB73 and OsNF‐YB1 proteins in tobacco leaf cells; (e) luciferase complementation imaging (LCI) assay showing that OsMYB73‐nLuc and OsNF‐YB1‐cLuc interact to form a functional LUC protein in Nicotiana benthamiana leaf cells. The combinations of nLuc and cLuc, nLuc and OsNF‐YB1‐cLuc and OsMYB73‐nLuc and cLuc were used as negative controls. (f) Analysis of the relationships between OsMYB73 and OsNF‐YB1, OsMYB73 and OsISA2, OsMYB73 and OsLTPL36, OsMYB73 and OsYUC11 through luciferase reporter assay in the rice protoplasts. (g) EMSA of OsMYB73 binding to OsISA2, OsNF‐YB1, OsYUC11 and OsLTPL36 promoters motifs.

Figure 5

CRISPR/Cas9 mediated target mutagenesis of rice OsMYB73, OsNF‐YB1, OsISA2, OsLTPL36, OsMYB73 + OsNF‐YB1, OsMYB73 + OsISA2 and OsMYB73 + OsLTPL36 mutants grains phenotypic evaluation. Bar, 1.0 cm. Lowercase letters indicate statistically significant differences at P < 0.05 by one‐way ANOVA test with Tukey correction.

Figure 6

Overexpression of rice OsMYB73 causes smaller and chalky grains. (a, b) Morphology of hulled (a) and dehulled (b) grains of Zhonghua11 (ZH11) and OsMYB73 overexpression lines. Bars are 1.0 cm. (c) Panicle architecture of ZH11 and OsMYB73 overexpression lines. Bar, 10 cm. (d) qRT‐PCR analysis of OsMYB73 in ZH11 and transgenic lines endosperm at 5 DAF. (e) Seed setting rate of ZH11 and OsMYB73 overexpression lines. (f) Grain yield per plant of ZH11 and OsMYB73 overexpression lines. (g–i) Statistical analysis of grain length (g), grain width (h), grain thickness (i) of ZH11 and OsMYB73 overexpression lines. (j) 1000‐grain weight of ZH11 and OsMYB73 overexpression lines. Values are means ± SD, n = 3. *P < 0.05; **P < 0.01, determined by Student's t‐test.

FIGURE 7

A schematic model of pleiotropic biological metabolic pathways of rice OsMYB73. OsMYB73 may interact with nuclear factor OsNF‐YB1 and bind to OsISA2, OsLTPL36 and OsYUC11 to regulate endosperm storage substances accumulation to affect rice grain quality.