Knockout of SlSBPASE Suppresses Carbon Assimilation and Alters Nitrogen Metabolism in Tomato Plants

Abstract

Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme in the Calvin⁻Benson cycle and has been documented to be important in carbon assimilation, growth and stress tolerance in plants. However, information on the impact of SBPase on carbon assimilation and nitrogen metabolism in tomato plants (Solanum lycopersicum) is rather limited. In the present study, we investigated the role of SBPase in carbon assimilation and nitrogen metabolism in tomato plants by knocking out SBPase gene SlSBPASE using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology. Compared with wild-type plants, slsbpase mutant plants displayed severe growth retardation. Further analyses showed that knockout of SlSBPASE led to a substantial reduction in SBPase activity and as a consequence, ribulose-1,5-bisphosphate (RuBP) regeneration and carbon assimilation rate were dramatically inhibited in slsbpase mutant plants. It was further observed that much lower levels of sucrose and starch were accumulated in slsbpase mutant plants than their wild-type counterparts during the photoperiod. Intriguingly, mutation in SlSBPASE altered nitrogen metabolism as demonstrated by changes in levels of protein and amino acids and activities of nitrogen metabolic enzymes. Collectively, our data suggest that SlSBPASE is required for optimal growth, carbon assimilation and nitrogen metabolism in tomato plants.

Keywords: CRISPR/Cas9; SBPase; carbon assimilation; nitrogen metabolism; tomato.

Figure 1

Schematic diagram of SlSBPASE gene structure, vector structure and CRSIPR/Cas9-induced mutagenesis. (A) Schematic diagram of SlSBPASE gene structure and target sequence. ATG, start codon; TGA, stop codon; sgRNA, single guide RNA; PAM, protospacer adjacent motif; AGG in red, PAM sequence. (B) Schematic diagram of the vector used in this study. LB, left border of T-DNA (Transfer DNA); RB, right border of T-DNA; U6, Arobidopsis U6 promoter; gRNA, guide RNA; e35S, enhanced 35S promoter; Cas9, optimized Cas9; NOS (nopaline synthase) Ter, NOS terminator; 35S, CaMV (Cauliflower mosaic virus) 35S promoter; HPT (hygromycin phosphotransferase), hygromycin selection marker; PolyA Ter, PolyA terminator. (C) Representative genotypes of slsbpase mutants. WT, wild type. Target sequences are in blue.

Figure 2

Characterization of slsbpase mutant. (A) Phenotypes of slsbpase mutant plants (slsbpase) and wild-type plants (WT). (B) Plant height of wild-type and mutant plants. (C) Leaf chlorosis phenotype of slsbpase mutant. (D) Leaf chlorophyll content of wild-type and mutant plants. The values presented are means ± SDs (standard deviation, n = 3). Asterisks indicate significant difference at ** p < 0.01 between slsbpase mutant plants and wild-type plants.

Figure 3

Changes of SBPase activity (A), RuBP regeneration (B) and CO₂ assimilation rates (C) in slsbpase mutant plants. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between slsbpase mutant plants and wild-type plants.

Figure 4

Diurnal carbohydrate accumulation in wild-type and slsbpase mutant plants. Levels of sucrose (A,B) and starch (C,D) were determined in leaves of wild-type and slsbpase mutant plants in the light period (end of day) and at the end of the night. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between slsbpase mutant plants and wild-type plants.

Figure 5

Night respiration rates in wild-type tomato plants and slsbpase mutant plants. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 between slsbpase mutant plants and wild-type plants.

Figure 6

Changes in levels of total protein and amino acids as affected by mutation of SlSBPASE in tomato plants. (A) Protein level. (B) Amino acids level. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 and * p < 0.05 between slsbpase mutant plants and wild-type plants.

Figure 7

Activities of metabolic enzymes involved in nitrogen metabolism in wild-type plants and slsbpase mutant plants. (A) NR. (B) GS. (C) GOGAT. (D) GDH. The values presented are means ± SDs (n = 3). Asterisks indicate significant difference at ** p < 0.01 and * p < 0.05 between slsbpase mutant plants and wild-type plants.