Transactivation of Sus1 and Sus2 by Opaque2 is an essential supplement to sucrose synthase-mediated endosperm filling in maize

Abstract

The endosperm-specific transcription factor Opaque2 (O2) acts as a central regulator for endosperm filling, but its functions have not been fully defined. Regular o2 mutants exhibit a non-vitreous phenotype, so we used its vitreous variety Quality Protein Maize to create EMS-mutagenesis mutants for screening o2 enhancers (oen). A mutant (oen1) restored non-vitreousness and produced a large cavity in the seed due to severely depleted endosperm filling. When oen1 was introgressed into inbred W64A with a normal O2 gene, the seeds appeared vitreous but had a shrunken crown. oen1 was determined to encode Shrunken1 (Sh1), a sucrose synthase (SUS, EC 2.4.1.13). Maize contains three SUS-encoding genes (Sh1, Sus1, and Sus2) with Sh1 contributing predominantly to the endosperm. We determined SUS activity and found a major and minor reduction in oen1 and o2, respectively. In o2;oen1-1, SUS activity was further decreased. We found all Sus gene promoters contain at least one O2 binding element that can be specifically recognized and be transactivated by O2. Sus1 and Sus2 promoters had a much stronger O2 transactivation than Sh1, consistent with their transcript reduction in o2 endosperm. Although sus1 and sus2 alone or in combination had no perceptible phenotype, either of them could dramatically enhance seed opacity and cavity in sh1, indicating that transactivation of Sus1 and Sus2 by O2 supplements SUS-mediated endosperm filling in maize. Our findings demonstrate that O2 transcriptionally regulates the metabolic source entry for protein and starch synthesis during endosperm filling.

Keywords: Sh1; Sus1; Sus2; O2; Quality Protein Paize; endosperm; starch; storage protein.

Figure 1

Phenotypes and biochemical analysis of WT, o2, oen1‐1 and o2;oen1‐1 in the W64A background. (a) Ear and kernel phenotypes. Top panel, ear phenotypes; middle panel, kernel vitreousness as observed on the light box. A small portion of vitreous endosperm with light translucency in o2 seeds is indicated by arrow; bottom panel, kernel transverse sections. Arrowhead and asterisk indicate the vitreous and starchy endosperm, respectively. Scale bars, 2 mm. (b) The 100‐kernel weight of WT, o2, oen1‐1 and o2;oen1‐1. (c) Starch content of WT, o2, oen1‐1 and o2;oen1‐1. (d) Protein content of WT, o2, oen1‐1 and o2;oen1‐1. (e) Levels of soluble sugars of WT, o2, oen1‐1 and o2;oen1‐1. The data represent the mean and standard deviation (SD) of independent triplicate (d) or quadruplicate (c and e) or quintuplicate (c) measurements. g, gram. The single and double asterisks represent significant difference (Student's t‐test, P < 0.05) and extremely significant difference (Student's t‐test, P < 0.01) compared to WT, respectively.

Figure 2

Map‐based cloning and genetic complementation test of oen1‐1. (a) Location of oen1 in the 0–28 Mb region of chromosome 9, as determined by the mapping‐by‐sequencing strategy. The graph shows that SNP positions (x‐axis) and delta SNP‐index values (y‐axis, blue points) depicting the difference in SNP‐Index values between the High and Low bulks (shrunken and WT bulks). The red line is average delta SNP‐index, the purple line is delta SNP‐index 95% confidence intervals, and the orange line is delta SNP‐index 99% confidence intervals. (b) Gene structure of Zm00001d045042 (Oen1) and the mutant sites of oen1‐1 and oen1‐2. (c) RT‐PCR detection of Oen1 transcripts in 12‐DAP endosperms of WT and oen1‐1. (d) Immunoblotting analysis of Sh1 in 12‐DAP endosperms of WT o2, oen1‐1 and o2;oen1‐1. Actin was used as an internal control.

Figure 3

Total SUS activity and transcript levels of the three Sus genes in the developing endosperms of WT, o2, oen1‐1, and o2;oen1‐1. (a) The total SUS activity in the developing endosperms of WT, o2, oen1‐1, and o2;oen1‐1 from 8 to 24 DAP. The data at each time point represent the mean ± SD of six measurements except for 8 DAP (quadruplicate measurements). The production of 1 μg of sucrose in 1 min per gram of tissue is defined as one unit of SUS activity (μg/min/g). (b) Amino acid sequence identity of the three SUS enzymes. (c) Transcript levels of the three Sus genes in WT and o2 endosperms at 12 DAP. All expression levels are normalized to that of Ubiquitin. The data represent the mean ± SD of triplicate measurements. The single and double asterisks represent significant difference (Student's t‐test, P < 0.05) and extremely significant difference (Student's t‐test, P < 0.01) compared to WT, respectively.

Figure 4

Transactivation assay and EMSA of the Sus gene promoters with O2 and O2‐bZIP proteins. (a) Dual‐luciferase assays of the promoters of Sh1, Sus1 and Sus2 with O2 in Arabidopsis protoplasts. REN, Renilla luciferase; LUC, firefly luciferase. LUC/REN, ratio of LUC activity to REN activity. The data represent the means ± SDs of three or more independent replicates. The single and double asterisks represent significant difference (Student's t‐test, P < 0.05) and extremely significant difference (Student's t‐test, P < 0.01) compared to WT, respectively. (b) EMSA of ACGT‐containing probes in the three Sus promoters with His‐O2 and His‐O2‐bZIP proteins. Comp, competing probes not labelled with biotin; Mp, mutated probes with a mutation in the corresponding ACGT core element. The promoter and the probe sequences of the three Sus genes are in Appendix S1.

Figure 5

Phenotypes of WT and single, double and triple mutants of the three sus genes. (a) Ears phenotypes of all genotypes of the three sus genes. (b) The total SUS activity of all genotypes from 12‐DAP developing endosperms. The data at each time point represent the mean ± SD of six measurements. The production of 1 μg of sucrose in 1 min per gram of tissue is defined as one unit of SUS activity (μg/min/g). The single and double asterisks represent significant difference (Student's t‐test, P < 0.05) and extremely significant difference (Student's t‐test, P < 0.01) compared to WT or oen1‐1, respectively. (c) Starch content of all genotypes in 100 mg dry endosperm flour. The data represent the mean ± SD of quadruplicate measurements. The double asterisks represent significant difference (Student's t‐test, P < 0.05) compared to WT.

Figure 6

A proposed model for the transcriptional regulation of the three Sus genes by O2.