Sucrose phosphate synthase (SPS), sucrose synthase (SUS) and their products in the leaves of Miscanthus × giganteus and Zea mays at low temperature

Abstract

The changes in the expression of key sugar metabolism enzymes (SPS and SUS), sucrose content and arrangement of chloroplast starch may play a significant role in the cold response in M. giganteus and maize plants. To understand the mechanism of the chilling-response of two closely-related C₄ plants, we investigated the changes in the expression of sucrose phosphate synthase (SPS) and sucrose synthase (SUS) as well as changes in their potential products: sucrose, cellulose and starch in the leaves of Miscanthus × giganteus and Zea mays. Low temperature (12-14 °C) increased SPS content in Miscanthus (MG) and chilling-sensitive maize line (Zm-S), but not in chilling-tolerant one (Zm-T). In Zm-S line, chilling also caused the higher intensity of labelling of SPS in the cytoplasm of mesophyll cells, as demonstrated by electron microscopy. SUS labelling was also increased by cold stress only in MG plants what was observed in the secondary wall between mesophyll and bundle sheath cells, as well as in the vacuoles of companion cells. Cold led to a marked increase in total starch grain area in the chloroplasts of Zm-S line. In turn, Fourier transform infrared spectroscopy (FTIR) showed a slight shift in the cellulose band position, which may indicate the formation of more compact cellulose arrangement in Zm-T maize line. In conclusion, this work presents new findings supporting diversified cold-response, not only between two C₄ plant species but also within one species of maize.

Keywords: Cellulose; Immunolocalization; Starch grains; Sucrose; Sugar metabolism.

Fig. 1

The relative expression of a sucrose phosphate synthase (SPS) and b sucrose synthase 1 (SUS1) in leaves of the control (white bars) and chilled (grey bars) plants of Miscanthus × giganteus (MG), chilling-tolerant (Zm-T) and chilling-sensitive maize line (Zm-S). Representative immunoblots are reported. The normalization was performed relative to actin. a For SPS, there is a significant effect of genotype [ANOVA, F (2;12) = 68.73; P < 0.0001], treatment [ANOVA, F (1;12) = 90.83; P < 0.0001] and of the interaction of genotype: treatment [ANOVA, F (2;12) = 5.14; P = 0.024]. b For SUS1, there is a significant effect of genotype [ANOVA, F (2;12) = 18.39; P = 0.0002], treatment [ANOVA, F (1;12) = 17.10; P = 0.0014], yet there is no significant effect of the interaction of genotype: treatment [ANOVA, F (2;12) = 2.73; P = 0.11]. Protein lysates separated during SDS-PAGE electrophoresis were obtained by pooling the leaf material from at least 6 plants for each treatment from three independent experiments (n = 3). Bars represent the means ± SD, asterisks indicating a significant effect of chilling (Tukey’s HSD test): ***P ≤ 0.001

Fig. 2

Examples of localization of sucrose phosphate synthase (SPS) in the leaves of Miscanthus × giganteus, MG, chilling-tolerant, Zm-T and chilling-sensitive maize line, Zm-S. Control (not-chilled plants) of MG (a), Zm-T line (d) and Zm-S maize line (g). Chilled-treated plants of MG (b), Zm-T line (e) and Zm-S maize line (h). Single gold particles (arrowheads) and the clusters of colloidal gold grains (arrows, more than three particles) visualizing anti-SPS antibody were mainly observed in the cytoplasm of mesophyll and bundle sheath cells, including areas near plasmodesmata (a, g, h). Note: more intense labelling in the cytoplasm of mesophyll cells of chilled plants of Zm-S maize line (h) compared to the control one (g). No signal was detected in the negative control, where the incubation with primary antibodies was omitted, in Miscanthus (c) and maize leaves (f, i). MS, mesophyll; BS, bundle sheath; VP, vascular parenchyma; ch, chloroplast; CW, cell wall; cyt, cytoplasm; ER, endoplasmic reticulum; Pd, plasmodesmata. Scale bar = 500 nm

Fig. 3

Localization of synthase sucrose 1 (SUS1) in the leaves of Miscanthus × giganteus, MG, chilling-tolerant, Zm-T and chilling-sensitive maize line, Zm-S. Control (not-chilled plants) of MG (a, b), Zm-T line (e, f) and Zm-S maize line (i, j). Chilled-treated plants of MG (c, d), Zm-T line (g, h) and Zm-S maize line (k, l). Single gold particles (arrowheads) and the clusters of colloidal gold grains (arrows, more than three particles) visualizing anti-SUS1 antibody were mainly observed along the plasma membrane in all types of cells tested (a-l), in the secondary cell wall (b-g, i, k), including areas near plasmodesmata (b, c, f, g), at endoplasmic reticulum (a, d, j), as well as in the vacuole (d) and cytoplasm (l). Note: more intense labelling in chilled plants of MG compared to the control one, in the secondary cell wall of mesophyll cells (b vs. c) and vacuole of companion cells (a vs. d). Negative control for Miscanthus (m) and maize leaves (n, o). MS, mesophyll; BS, bundle sheath; VP, vascular parenchyma; CC companion cell; SE, sieve element; ch, chloroplast; CW, cell wall; cyt, cytoplasm; ER, endoplasmic reticulum; pm, plasma membrane; Pd, plasmodesmata; V, vacuole. Scale bar = 500 nm

Fig. 4

The level of sucrose analyzed by GC in the leaves of the control (white bars) and chilled (grey bars) plants of Miscanthus × giganteus (MG), chilling-tolerant (Zm-T) and chilling-sensitive maize line (Zm-S). The sucrose content is expressed as % leaf dry weight (DW). The significant effect of genotype [ANOVA, F (2;12) = 79.03; P < 0.0001], treatment [ANOVA, F (1;12) = 66.89; P < 0.0001] and effect of the interaction of genotype: treatment were noted [ANOVA, F (2;30) = 14.76; P < 0.0001]. Results represent six independent extractions (n = 6) with 80% (v/v) ethanol. Bars represent the means ± SD, asterisks indicating a significant effect of chilling (Tukey’s HSD test): ***P ≤ 0.001

Fig. 5

Absorbance of infrared spectra (means ± 95% confidence intervals) of cell wall isolated from the 3^rd leaves in the control (red/.co) and chilled (blue/.ch) plants of Miscanthus × giganteus (MG), chilling-tolerant (Zm-T) and chilling-sensitive maize line (Zm-S). The spectra are given for the region between c. 667 and 673.5 cm⁻¹ wavelength assigned to the OH out-of-plane bending vibrations in cellulose. The significant genotype effect for the peak position [ANOVA, (F (2;29) = 13.571; P < 0.01)], and the significant genotype: treatment interaction (F (2;29) = 4.009; P = 0.029) were noted. The confidence intervals from Tukey.HSD post-hoc test for estimated difference of means between the maximum peak position of chilled and control plants were as follows: MG [95% CIs (− 0.2739, 0.3772), P = 0.99], Zm-T [95% CIs (− 0.0055, 0.6455), P = 0.056] and Zm-S [95% CIs (− 0.2295, 0.4534), P = 0.91]. Positions of mean maximum absorbances are marked with arrows. The spectra are shifted in absorbance for clarity

Fig. 6

Ultrastructure of mesophyll chloroplasts with starch grains arrangement in the control (a, c, e) and chilled (b, d, f) plants of Miscanthus × giganteus (MG), chilling-tolerant (Zm-T) and chilling-sensitive maize line (Zm-S). In the chilled leaves of Zm-S line (f) more starch grains are observed than in the control plants of this maize line (e). Ch, chloroplast; CW, cell wall; S, starch grain. Scale bar = 1 µm

Fig. 7

Ultrastructure of bundle sheath chloroplasts with starch grains arrangement in the control (a, c, e) and chilled (b, d, f) plants of Miscanthus × giganteus (MG), chilling-tolerant (Zm-T) and chilling-sensitive maize line (Zm-S). Note the extensive starch grains filling in the chloroplast of chilled plants of Zm-S maize line (f). Ch, chloroplast; CW, cell wall; S, starch grain. Scale bar = 1 µm

Fig. 8

Total starch grain area in the chloroplasts of mesophyll (a) and bundle sheath cells (b) in the control (white bars) and chilled (grey bars) plants of Miscanthus × giganteus (MG), chilling-tolerant (Zm-T) and chilling-sensitive maize line (Zm-S). Values are expressed as % of total area of starch grain in the total area of chloroplast. a Significant effect of genotype [ANOVA, F (2;102) = 73.45; P < 0.0001], treatment [ANOVA, F (1;102) = 80.78; P < 0.0001] and of the interaction of genotype: treatment [ANOVA, F (2;102) = 54.89; P < 0.001]. b Significant effect of genotype [ANOVA, F (2;102) = 153.35; P < 0.0001], treatment [ANOVA, F (1;102) = 61.27; P < 0.0001] and of the interaction of genotype: treatment [ANOVA, F (2;102) = 31.41; P < 0.0001]. For each treatment, the total area of starch grain in the chloroplasts was measured from at least thirty Kranz mesophyll or bundle sheath cells from six plants with three independent experiments (n = 18). Bars represent the means ± SD; asterisks indicating a significant effect of chilling (Tukey’s HSD test): *P ≤ 0.05; ***P ≤ 0.001