Increasing sucrose uptake capacity of wheat grains stimulates storage protein synthesis

Abstract

Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations.

Figure 1.

Characterization of HvSUT1-overexpressing wheat lines. A, Independent lines HOSUT10 (T4 generation), -11, and -20 (T2 generation) were analyzed for transgene integration by DNA gel-blot analysis. WT, Wild type. B, Quantitative real-time PCR analysis of HOSUT caryopses for transgene expression. Values are means of three biological replicates ± sd. C, Total nitrogen (N) and thousand grain weight of mature HOSUT grains (greenhouse grown). n = 5 to 10 ± sd. Significant differences according to t test are as follows: ^aP < 0.05, ^bP < 0.01. W, Wild type.

Figure 2.

Suc uptake and TaSUT gene expression. A and B, ¹⁴C label incorporation was measured after 10 min of labeling with [¹⁴C]Suc of immature grains of wild-type Certo and lines HOSUT10 (A) and HOSUT20 (B) at 19 DAF. Thirty-minute preincubation was done with the covalent protein modifier diethylpyrocarbonate (DEPC), known to inhibit proton-coupled symport. Significant differences according to t test are as follows: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. C, Quantitative RT-PCR of gene expression of endogenous wheat Suc transporters in HOSUT10 and wild-type endosperm. Values shown are means of at least three replicates ± sd.

Figure 3.

Growth parameters of HOSUT10 grains. A, Suc concentration in caryopses ± sd (n = 5). B, Fresh weight accumulation (n = 100). C, Dry weight accumulation (n = 100). D, ABA concentration (pmol g⁻¹ fresh weight) ± sd (n = 5). Significant differences according to t test are as follows: ^aP < 0.05, ^bP < 0.01.

Figure 4.

Ratios of gene expression, HOSUT10:wild type. Gene expression was determined by quantitative real-time PCR at 10 stages of development with three biological replicates. Data were extracted from Supplemental Table S2 and are log₂ transformed. A, Storage protein and storage-associated genes. B, Genes revealing a cluster similar to that presented in A. C and D, Genes revealing a cluster inverse to that presented in B. For full description of genes, see Supplemental Table S2. Identifier numbers refer to the Affymetrix GeneChip.

Figure 5.

Independent component (IC) analysis based on the median centered and log₁₀-transformed response ratios. The graph shows that deviations of metabolite levels during grain development occur in the early phase DAF 8 to 10 (arrow in B) and the late phase (26 DAF), indicating a continued development in transgenic grains at day 26 (arrow in A). The size of each circle indicates its developmental stage: smallest circle, 6 DAF; largest circle, 26 DAF. WT, Wild type.

Figure 6.

Ratios of amino acid levels, HOSUT10:wild type. Amino acids were determined by GC-MS, and data were extracted from Supplemental Table S3. A, Cluster with amino acid levels lower at 8 to 12 DAF and higher at later stages, 22 to 26 DAF. B, Cluster with amino acid levels lower at 8 to 12 DAF and lower at later stages, 22 to 26 DAF.

Figure 7.

Summary of changed transcript and metabolite levels in HOSUT10 caryopses. Data are derived from Supplemental Tables S2 (transcripts) and S3 (metabolites). Color code is as follows: red, down-regulated; green, up-regulated in HOSUT10 caryopses with respect to the wild type.