ZmSSRP1, transactivated by OPAQUE11, positively regulates starch biosynthesis in maize endosperm

Abstract

Starch synthesis is crucial for crop yield and quality. This study reveals an O11-ZmSSRP1 module of kernel starch biosynthesis in maize (Zea mays). We identify STARCH SYNTHESIS REGULATING PROTEIN 1 (ZmSSRP1) positively regulates amylose and amylopectin-dependent starch synthesis in maize endosperm. ZmSSRP1 encodes a highly conserved plastid-localized protein that is highly expressed in developing endosperm. Loss-of-function zmssrp1 mutants created by CRISPR/Cas9 exhibit much smaller starch granules and significantly reduced amylose and amylopectin synthesis, while overexpression of ZmSSRP1 causes larger starch granules and an increased amylose and amylopectin accumulation. RNA-seq analysis revealed that the expression levels of several genes involved in the synthesis of amylose and amylopectin were significantly down-regulated in the zmssrp1 mutant compared to the wild-type. Furthermore, we found that OPAQUE11 (O11), a core transcription factor essential for maize endosperm development and nutrient metabolism, transactivates the expression of ZmSSRP1 by binding to its promoter, and functions upstream of ZmSSRP1 in the regulatory pathway governing starch synthesis in the maize endosperm. The present work demonstrates that the O11-ZmSSRP1 module positively regulates starch synthesis in maize kernels, potentially paving the way for future genetic improvements of maize quality.

Keywords: O11; ZmSSRP1; maize endosperm; starch biosynthesis.

Figure 1

Phylogenetic analyses and expression pattern of ZmSSRP1. (a) ZmSSRP1 contains four low‐complexity regions and a CBM48 domain. The ZmSSRP1 protein contains 541 amino acids (a.a) with four different lengths of low‐complexity regions (The blue box) and one CBM48 domain (The orange box). (b) A neighbour‐joining tree of ZmSSRP1 and its homologues in different plant species. The numbers on the branch node refer to the bootstrap value of the phylogenetic tree. The tree was constructed using MEGA and bootstrapped with 1000 bootstrap replicates. The proteins are named according to their gene/EST names or NCBI accession numbers. Protein IDs are listed after abbreviated species names. The full species names are as follows: Triticum aestivum, Hordeum vulgare, Oryza sativa, Setaria viridis, Setaria italica, Zea mays, Sorghum bicolor, Glycine max, Arabidopsis thaliana, Manihot esculenta. The scale bar corresponds to 0.2 estimated amino acid substitutions per site. (c) Amino acid sequence alignment of the CBM48 domain in ZmSSRP1 and OsFLO6. The asterisks represent conservative sites. (d) Relative expression levels of ZmSSRP1 in developing embryo and endosperm. Total RNA was extracted from different tissues as indicated or from different developing stages of the embryo and endosperm. ZmActin was used as an internal control for gene expression normalization from the RT‐qPCR. DAP, day after pollination. Values are means ± SD (n = 3). Statistical significance was determined by Student's t‐test (P < 0.01).

Figure 2

Generation and characterization of the zmssrp1 mutants and ZmSSRP1‐overexpressing lines. (a) Construction of the zmssrp1‐1 and zmssrp1‐2 mutants by CRISPR/Cas9‐mediated genome editing. Upper panel, schematic diagram of the ZmSSRP1 gene. Black boxes and blue boxes represent the introns and exons, respectively. The position of the CRISPR/Cas9 target site in exons is indicated. Lower panel, alignment of the CRISPR/Cas9 target sequences from the WT and the zmssrp1‐1 and zmssrp1‐2 mutants. The ‘T’ insertion is indicated in blue, and the deletion is indicated by dashes. (b) Relative expression levels of ZmSSRP1 in KN5585, zmssrp1‐1, zmssrp1‐2 mutants and ZmSSRP1‐overexpressing lines (ZmSSRP1‐OE#1 and ZmSSRP1‐OE#2). Total RNA was extracted from kernels. ZmActin was used as an internal control for gene expression normalization from the RT‐qPCR. Values are means ± SD (n = 3 biological replicates). Statistical significance was determined by Student's t‐test (P < 0.01). (c) Mature ear of KN5585, zmssrp1‐1, zmssrp1‐2 mutants and ZmSSRP1‐overexpressing lines (ZmSSRP1‐OE#1 and ZmSSRP1‐OE#2). (d–g) Kernel length (d), kernel width (e), Kernel thickness (f), 100‐grain weight of different materials as indicated. Data are presented as mean ± SD (n = 3 biological replicates). Different letters indicate statistically significant differences for the indicated values, as determined by one‐way ANOVA followed by Tukey's LSD test (P < 0.01).

Figure 3

ZmSSRP1 positively regulates starch synthesis‐related genes in maize kernel. (a) Volcano plot showing DEGs (FC >1.5 and FDR < 0.05) in the zmssrp1‐1 mutant compared with KN5585 determined by RNA‐seq analysis. Up‐ and down‐regulated DEGs are displayed as red and blue dots, respectively. 9 down‐regulated genes involved in starch synthesis are highlighted. 21 DAP kernels were sampled, and 3 biological replicates were used for the analysis. (b) GO term analyses of upregulated and down‐regulated DEGs in the zmssrp1‐1 mutant. (c) The heatmap showing the expression patterns of the representative starch synthesis‐related genes in the zmssrp1‐1 mutant and KN5585. The values indicate the relative gene expression in the zmssrp1‐1 mutant and KN5585. (d) RT‐qPCR analyses of the transcript levels of 6 representative genes (ZmUPG2.3, ZmAGPS2, ZmSus1, ZmDOF36, ZmABI19 and ZmGBSS1b) involved in amylose and amylopectin synthesis. Total RNA was extracted from 21‐DAP endosperm. The expression levels were normalized to that of ZmActin. Values are means ± SD (n = 3). Statistical significance was determined by Student's t‐test (P < 0.01).

Figure 4

ZmSSRP1 positively regulates starch biosynthesis in maize endosperm. (a) Scanning electron microscopy of the central regions of the mature endosperm of KN5585, zmssrp1‐1, zmssrp1‐2 mutants and ZmSSRP1‐overexpressing lines kernel. Bars = 5 μm. (b) Transmission electron microscopy of the central regions of the mature endosperm of KN5585, zmssrp1‐1 mutants and ZmSSRP1‐overexpressing lines kernel. Bars = 5 μm. (c–h) Starch content (c), amylose content (d), amylopectin content (e), crude protein content (f), reducing sugar content (g) and soluble sugar content (h) in mature kernels of different materials as indicated. Data are presented as mean ± SD (n = 3 biological replicates). Different letters indicate statistically significant differences for the indicated values, as determined by one‐way ANOVA followed by Tukey's LSD test (P < 0.01).

Figure 5

O11 binds to the promoters of the ZmSSRP1 gene and activates its transcription. (a) The E‐box in the promoter region of ZmSSRP1. (b) Binding of O11 to the promoters of ZmSSRP1 is shown by yeast one‐hybrid assays. O11 was constructed in the AD‐Rec vector and ZmSSRP1 (about −2000 bp to −1 bp of the start codon) promoter was constructed in the pAbAi vector. Yeast cells co‐expressing pAbAi‐p53pro and AD‐Rec‐P53 were used as the positive control. pAbAi‐ZmSSRP1pro and AD‐Rec were used as the negative control. (c) Schematic diagrams of the effector and reporter constructs used in the dual‐luciferase assays. 35S, CaMV 35S promoter; REN, Renilla luciferase gene; LUC, firefly luciferase gene. (d, e) Transcriptional activation of ZmSSRP1 expression by O11 in maize protoplasts (d) and tobacco leaves (e). The ZmSSRP1 promoter drove the LUC gene. Relative luciferase activities (LUC/REN) were measured by co‐transfection with different effector and reporter plasmid combinations. The empty 62SK vector was used as the negative control. Different letters indicate statistically significant differences for the indicated values, as determined by a Student's t‐test (P < 0.01). (f) Relative expression levels of ZmSSRP1 in maize developing kernels. Total RNA was extracted from different development stages of kernels as indicated. ZmActin was used as an internal control for gene expression normalization from the RT‐qPCR. Values are means ± SD (n = 3). Statistical significance was determined by Student's t‐test (P < 0.01).

Figure 6

Phenotypes of the o11 and zmssrp1‐1 single mutants, and the o11 × zmssrp1‐1 double mutants. (a) Scanning electron microscopy of the central regions of the mature endosperm of KN5585, o11 mutant, W22, zmssrp1‐1 mutant, W22 × KN5585 and o11 × zmssrp1‐1 double mutant kernel. Bars = 10 μm. (b–f) Starch content (b), amylose content (c), amylopectin content (d), crude protein content (e) and reducing sugar content (f) in mature kernels of different materials as indicated. Data are presented as mean ± SD (n = 3 biological replicates). Different letters indicate statistically significant differences for the indicated values, as determined by one‐way ANOVA followed by Tukey's LSD test (P < 0.01).