Water stress combined with sulfur deficiency in pea affects yield components but mitigates the effect of deficiency on seed globulin composition

Abstract

Water stress and sulfur (S) deficiency are two constraints increasingly faced by crops due to climate change and low-input agricultural practices. To investigate their interaction in the grain legume pea (Pisum sativum), sulfate was depleted at the mid-vegetative stage and a moderate 9-d water stress period was imposed during the early reproductive phase. The combination of the stresses impeded reproductive processes in a synergistic manner, reducing seed weight and seed number, and inducing seed abortion, which highlighted the paramount importance of sulfur for maintaining seed yield components under water stress. On the other hand, the moderate water stress mitigated the negative effect of sulfur deficiency on the accumulation of S-rich globulins (11S) in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustment of the ratio of S-poor (7S) to 11S globulins. Transcriptome analysis of developing seeds at the end of the combined stress period indicated that similar biological processes were regulated in response to sulfur deficiency and to the combined stress, but that the extent of the transcriptional regulation was greater under sulfur deficiency. Seeds from plants subjected to the combined stresses showed a specific up-regulation of a set of transcription factor and SUMO ligase genes, indicating the establishment of unique regulatory processes when sulfur deficiency is combined with water stress.

Keywords: Pisum sativum; Abiotic stresses; drought; nutrient partitioning; seed quality; seed transcriptomics, storage proteins; sulfur.

Fig. 1.

Experimental design for studying the interaction between water stress and S deficiency in pea (cv. ‘Caméor’). (A) Developmental stages at which stresses were imposed. Control, plants were well watered under non-limiting S conditions; S–, plants were deprived of S from the mid-vegetative stage until harvest; WS, plants were subjected to water stress from flowering of the 2nd or 3rd reproductive node for 9 d, then re-watered for recovery; S–WS, plants were subjected to a combination of the two stresses. Developing seeds were collected at the end of the water-stress period for transcriptome analyses, and other plants were harvested at maturity for phenotyping and analyses of seed composition. (B) Leaf water potential measured at the end of the water-stress period. Data are means (±SE), n=4 plants. Different letters indicate significant differences as determined by ANOVA followed by a SNK test (P<0.1). (C) Schematic representation of how mature seeds were collected. For each plant, seeds from each individual flowering node (dots on the left diagram) were collected at maturity and pooled into four groups (G1–G4). G1 seeds corresponded to flowering nodes that flowered before the beginning of the water stress period; G2 seeds corresponded to the flower that opened on the day that water stress was imposed; G3 and G4 seeds corresponded to nodes that flowered during the water-stress period. G1–G3 seeds had passed the final stage of seed abortion (FSSA) 3 d after the end of the water-stress period, while G4 seeds had not. Note that no G4 seeds developed on the double-stressed plants.

Fig. 2.

Effects of water stress combined with S deficiency on protein composition of mature pea seeds from seed groups G1–G4. (A) Protein profiles of mature seeds from plants with or without stresses. Representative one-dimensional electrophoresis (1-DE) protein patterns for mature control seeds from G1–G4 are shown on the left. The 27 individual bands that were detected and quantified in all the seed groups are shown. Protein annotation refers to major proteins in each band, according to the Normalized Weighted Spectra counts (Supplementary Table S1). Six protein bands corresponded to 7S globulins (vicilins and convicilin, in green) and five to 11S globulins (legumins, in orange). One 1-DE gel illustrating qualitative changes in the accumulation of the 7S and 11S globulins between the different treatments is shown on the right for two biological replicates of G1 (G1-1, G1-2) and G3 (G3-1, G3-2). (B) Quantitative variations in response to stresses. Significant changes in the relative abundance of the protein bands are shown for each group of seeds compared to the control. The ratio of values for stressed plants versus control plants is indicated for each group. Note that no G4 seeds could be harvested for S–WS plants. The 7S/11S ratio corresponds to the relative quantity of 7S divided by that of 11S. The colors indicate whether the ratio decreased (grey scale) or increased (red scale) when values were significantly different to that of control seeds, as determined by ANOVA followed by a SNK test (P<0.05, n=4 plants). The percentage of amino acids in the sequence of storage-proteins is indicated (S-AA, sulfur amino acids, methionine and cysteine). S–, S deficiency alone; WS, water stress alone; S–WS, combined stresses.

Fig. 3.

Effects of water stress combined with S deficiency on the accumulation and distribution of N, C, and S in different pea plant compartments at maturity. (A) Sulfur (S), nitrogen (N), and carbon (C) contents (%), and N/S and C/N ratios, in each compartment. (B–D) Absolute quantity (mg) of S, N, and C in each compartment. (E–G) Proportions of N, C, and S in the dry biomass (% dry weight) of each compartment. Data are means (±SE). Bold and colored values (A), different letters (B–D), or arrows (E–G) indicate significant differences as determined by ANOVA followed by a SNK test (P<0.05, n=4 plants per condition). S–, Sulfur deficiency alone; WS, water stress alone; S–WS, combined stresses. Data are shown for seeds pooled from G1–G4.

Fig. 4.

Effect of water stress combined with S deficiency on the transcriptome of pea seeds from group 2 (G2) at 9 d post-pollination. (A) Venn diagrams showing the numbers of up- and down-regulated genes in response to stresses compared to the control. (B) GO term enrichment analysis for the differentially expressed genes in response to stresses. Terms in the ‘Biological Process’ category are shown for genes up- and down-regulated in response to the different stresses. For each term, the number of genes present in the genome (‘annotated’), the number of genes present in the gene lists (‘significant’), and the associated P-value (Fisher’s test, threshold of P<0.001) are given. S–, S deficiency alone; WS, water stress alone; S–WS, combined stress.

Fig. 5.

Effects of water stress combined with S deficiency on the expression of genes related to S and N transport and metabolism in pea. For each gene, the fold-change in expression under stress conditions compared to the control is given. The values are color-coded from low expression in blue to high expression in red, and values and enzymes in bold indicate significant differences between stress and control conditions. S–, S deficiency alone; WS, water stress alone; S–WS, combined stresses. Gene annotation refers to the closest Arabidopsis thaliana homolog (Supplementary Table S3). For each gene, the mean expression level under control conditions (expressed in counts) is given in square brackets. Enzymes: APK, APS kinase; APR, APS reductase; ATPS, ATP sulfurylase; CBL, cystathionine β-lyase; CBS, cystathionine β-synthase; CGS, cystathionine γ-synthase; γ-ECS, γ-glutamylcysteine synthase; GGT, γ-glutamyl transferase; GOGAT, glutamate synthase; GS, glutamine synthetase; GSHS, glutathione synthetase; GSHR, glutathione reductase; GPX, glutathione peroxydase; GST, glutathione S-transferase; HMT; homocysteine-S-methyltransferase; MMT, S-adenosylmethionine methyltransferase; MS, methionine synthase; NiR, nitrite reductase; NR, nitrate reductase; OAS-TL, OAS thiol-lyase; PIP, 3´(2´),5´-bisphosphate nucleotidase; SAHase, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine synthetase; SAT, serine acetyltransferase; SiR, sulfite reductase; SO, sulfite oxidase. Metabolites: AAP, amino acid permease; AdoMet, S-adenosylmethionine; APS, adenosine 5´-phosphosulfate; ASN, asparagine; ASP, aspartate; Cys, cysteine; Cyst, cystathionine; Gln, glutamine; Glu, glutamate; γ-GluCys, γ-glutamylcysteine; γ-CysGly, γ-cysteinylglycine; GSH, glutathione; GSSG, glutathione disulfide; Hcy, homocysteine; Met, methionine; OAS, O-acetylserine; PAPS, 3´-phoshoadenosine 5´-phosphosulfate; SAHC, S-adenosylhomocysteine; SMM, S-methylmethionine. Transporters: AAP, amino acid permeases; NPF, nitrate peptide transporter family; SULTR, sulfate transporters (green shading indicates N transport and metabolism).

Fig. 6.

Summary of the effects of water stress with or without S deficiency on yield components, nutrient partitioning, and seed nutrient and protein composition at harvest in pea. (A) Summary for water-stress conditions. (B) Summary for sulfur-deficient conditions. (C) Summary for combined stress condition. The colors correspond to the ratios of values for stressed plants versus control plants, and indicate whether the ratio was significantly decreased (blue) or increased (red). S–, S deficiency; WS, water stress; S–WS, combined stresses.