Nitrogen supply influences photosynthesis establishment along the sugarcane leaf

Abstract

Nitrogen (N) is a major component of the photosynthetic apparatus and is widely used as a fertilizer in crops. However, to the best of our knowledge, the dynamic of photosynthesis establishment due to differential N supply in the bioenergy crop sugarcane has not been reported to date. To address this question, we evaluated physiological and metabolic alterations along the sugarcane leaf in two contrasting genotypes, responsive (R) and nonresponsive (NR), grown under high- and low-N conditions. We found that the N supply and the responsiveness of the genotype determined the degree of senescence, the carboxylation process mediated by phosphoenolpyruvate carboxylase (PEPcase) and differential accumulation of soluble sugars. The metabolite profiles indicated that the NR genotype had a higher respiration rate in the youngest tissues after exposure to high N. We observed elevated levels of metabolites related to photosynthesis in almost all leaf segments from the R genotype under high-N conditions, suggesting that N supply and the ability to respond to N influenced photosynthesis. Therefore, we observed that N influence on photosynthesis and other pathways is dependent on the genotype and the leaf region.

Figure 1

Schematic of segments of sugarcane leaf + 1 that were collected. B0, Base Zero; B, Base; M, Middle; and T, Tip.

Figure 2

Chlorophyll content in different segments of sugarcane leaf blade. (a) Total chlorophyll; (b) chlorophyll a; (c) chlorophyll b. 10 N and 270 N correspond to treatments of 10 and 270 mg of N per kg of sand, respectively. B0, Base “zero”; B, Base; M, Middle; T, Tip. FW, fresh weight. Data are presented as the mean ± SE. Letters indicate statistical significance using ANOVA followed by Fisher’s exact test (n = 3; P ≤ 0.05).

Figure 3

(a) Carbon isotopic discrimination (Δ¹³C) and (b) total leaf N content along sugarcane leaf blade. B0, Base “zero”; B, Base; M, Middle; T, Tip. 10 N and 270 N correspond to treatments of 10 and 270 mg of N per kg of sand, respectively. DW, dry weight. Data are presented as the mean ± SE. Letters indicate statistical significance using ANOVA followed by Fisher’s exact test (n = 3; P ≤ 0.05).

Figure 4

Enzyme activity and the level of carboxylation enzymes along the sugarcane leaf blade. (a) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO); (b) Phosphoenolpyruvate carboxylase (PEPcase). The commercial peptide sequences of RubisCO large subunit form I and PEPC1 (Agrisera, SWE) were used. The cropped gel image corresponds to a western-blot assay representative of three independent experiments. The full-length gels are presented in Supplementary Figs 8 and 9. B0, Base “zero”; B, Base; M, Middle; T, Tip. 10 N and 270 N correspond to treatments of 10 and 270 mg of N per kg of sand, respectively. Data are presented as the mean ± SE. Letters indicate statistical significance using ANOVA followed by Fisher’s exact test (n = 3; P ≤ 0.05).

Figure 5

Results of partial least square discriminant analysis (PLS-DA) in relation to metabolites detected by GC-MS. low N corresponds to the treatment of 10 mg of N per kg of sand; high N corresponds to the treatment of 270 mg of N per kg of sand; R, responsive genotype; NR, nonresponsive genotype; B0, Base “zero”; B, Base; M, Middle; T, Tip.

Figure 6

Summary plot for the over representation analysis (ORA) of metabolic pathways. The fold enrichment was calculated among all metabolites detected (67) and the metabolites present in each pathway of the customized library. The p values are colour-coded, with dark red being highly significant and white being least significant. (*) Significant pathway at the P < 0.05 level and FDR ≤ 0.1.

Figure 7

Hierarchical cluster analysis coupled with a heat map showing metabolite profiles of different segments along sugarcane leaf blade. The ratio of metabolite abundance is represented as the relative concentration in relation to the total ion count (TIC). The intensity of each metabolite was normalized to both by dividing the dry weight of the sample and by the sum of the total ion counts. To further correct for the measurement effects, values were then normalized by the median of the all measured data and log2 transformed. 10 and 270 correspond to treatments of 10 and 270 mg of N per kg of sand, respectively. R corresponds to responsive genotype while NR corresponds to nonresponsive genotype. B0, Base “zero”; B, Base; M, Middle; T, Tip. The colour key represents the relative concentration of metabolites.

Figure 8

Scheme of the sugarcane photosynthetic pathway showing the quantification of phosphate (a), pyruvate (b), malate (c) and ribose (d) along the sugarcane leaf blade. ATP, adenosine triphosphate; CA, carbonic anhydrase; MDH, malate dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate, NADP ME, NADP malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; PGA, phosphoglycerate; PKK, ribulose-5-phosphate kinase; PPDK, pyruvate Pi-dikinase; RBC, Rubisco; RUBP, Ribulose-1,5-bisphosphate; TP, triose phosphates; RBKS, ribokinase; rpiA, ribose 5-phosphate isomerase. R corresponds to the responsive genotype, and NR refers to the nonresponsive genotype. B0, Base “zero”; B, Base; M, Middle; T, Tip. Note: The ratio of metabolite abundance is represented by relative concentration in log₂ scale. Data are presented as the mean ± SE with five replications. (*) indicates values determined by the Student’s t-test to be significantly.