Responses to Hypoxia and Endoplasmic Reticulum Stress Discriminate the Development of Vitreous and Floury Endosperms of Conventional Maize (Zea mays) Inbred Lines

Abstract

Major nutritional and agronomical issues relating to maize (Zea mays) grains depend on the vitreousness/hardness of its endosperm. To identify the corresponding molecular and cellular mechanisms, most studies have been conducted on opaque/floury mutants, and recently on Quality Protein Maize, a reversion of an opaque2 mutation by modifier genes. These mutant lines are far from conventional maize crops. Therefore, a dent and a flint inbred line were chosen for analysis of the transcriptome, amino acid, and sugar metabolites of developing central and peripheral endosperm that is, the forthcoming floury and vitreous regions of mature seeds, respectively. The results suggested that the formation of endosperm vitreousness is clearly associated with significant differences in the responses of the endosperm to hypoxia and endoplasmic reticulum stress. This occurs through a coordinated regulation of energy metabolism and storage protein (i.e., zein) biosynthesis during the grain-filling period. Indeed, genes involved in the glycolysis and tricarboxylic acid cycle are up-regulated in the periphery, while genes involved in alanine, sorbitol, and fermentative metabolisms are up-regulated in the endosperm center. This spatial metabolic regulation allows the production of ATP needed for the significant zein synthesis that occurs at the endosperm periphery; this finding agrees with the zein-decreasing gradient previously observed from the sub-aleurone layer to the endosperm center. The massive synthesis of proteins transiting through endoplasmic reticulum elicits the unfolded protein responses, as indicated by the splicing of bZip60 transcription factor. This splicing is relatively higher at the center of the endosperm than at its periphery. The biological responses associated with this developmental stress, which control the starch/protein balance, leading ultimately to the formation of the vitreous and floury regions of mature endosperm, are discussed.

Keywords: endosperm; hypoxia; maize; transcriptome; unfolded protein response (UPR); vitreousness.

Figure 1

MEP and MEC transcriptome pattern. (A) Biological process GO categories significantly enriched in genes only up-regulated in MEC and MEP in almost a tested condition. (B) Annotated genes related to stress differentially expressed between MEC and MEP at 15 DAP and 20 DAP in flint and dent maize.

Figure 2

Transcriptome patterns of zein genes. Log₂ ratio between MEP (V) and MEC (F) for genes specifying zeins at 15 and 20 DAP in flint and dent inbred lines. Genes ID and log₂ ratio are listed in Table S3.

Figure 3

Spatiotemporal evolution of UPR during endosperm development. (A) bzip60 (GRMZM2G025812) splicing was detected by RT-PCR in MEP and MEC at 15 and 20 DAP. Ubi: RT-PCR of ubiquitin (GRMZM2G109977). Arrow: spliced mRNA. Relative expression of bzip60 (B), unspliced bzip60 (C) and spliced bzip60 (D) measured by qRT-PCR in MEP and MEC at 15 and 20 DAP. Relative expression level was normalized to ubiquitin (GRMZM2G109977). Asterisks indicate significant difference (paired t-test; ^*p < 0.05; ^**p < 0.01).

Figure 4

Metabolite profiling of MEP and MEC. Soluble sugars (A) and free amino acids (B) of MEP and MEC in dent and flint maize at 30 DAP. Asterisks indicate significant differences (paired t-test; p < 0.05).

Figure 5

Metabolic switch between MEP and MEC. Transcriptome pattern of genes involved in glycolysis, fermentation and TCA cycle in MEP (V) and MEC (F) at 15 and 20 DAP in flint and dent inbred lines. Right box lines represent MEP/MEC log₂ expression ratio. Left box lines represent 20/15 DAP log₂ expression ratio. For multigenic families, the results represent the mean of all genes. Genes ID and log₂ ratio of all genes are listed in Table S3. Blue lines, amino acid synthesis pathway; PFK, phosphofructokinase; PFP, pyrophosphate-fructose-6-phosphate-1-phosphotransferase; ALD, fructose-bisphosphate aldolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; PPDK, pyruvate orthophosphate dikinase; PK, pyruvate kinase; PDH, pyruvate; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase; ALA-T, alanine aminotransferase; CTS, citrate synthase; ACO, aconitase; IDH, isocitrate dehydrogenase; OGDC, oxoglutarate dehydrogenase complex; SCS, succinyl-CoA synthetase; SDH, succinate dehydrogenase; FUM, fumarate hydratase; MDH, malate dehydrogenase; ME, malic enzyme; GOGAT, glutamate synthase; GAD, glutamate decarboxylase; GABA-T, γ-aminobutyric acid trans-aminase; SSADH, succinate-semialdehyde dehydrogenase.