Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential

Abstract

To better define the still unclear role of proline (Pro) metabolism in drought resistance, we analyzed Arabidopsis (Arabidopsis thaliana) Δ(1)-pyrroline-5-carboxylate synthetase1 (p5cs1) mutants deficient in stress-induced Pro synthesis as well as proline dehydrogenase (pdh1) mutants blocked in Pro catabolism and found that both Pro synthesis and catabolism were required for optimal growth at low water potential (ψ(w)). The abscisic acid (ABA)-deficient mutant aba2-1 had similar reduction in root elongation as p5cs1 and p5cs1/aba2-1 double mutants. However, the reduced growth of aba2-1 but not p5cs1/aba2-1 could be complemented by exogenous ABA, indicating that Pro metabolism was required for ABA-mediated growth protection at low ψ(w). PDH1 maintained high expression in the root apex and shoot meristem at low ψ(w) rather than being repressed, as in the bulk of the shoot tissue. This, plus a reduced oxygen consumption and buildup of Pro in the root apex of pdh1-2, indicated that active Pro catabolism was needed to sustain growth at low ψ(w). Conversely, P5CS1 expression was most highly induced in shoot tissue. Both p5cs1-4 and pdh1-2 had a more reduced NADP/NADPH ratio than the wild type at low ψ(w). These results indicate a new model of Pro metabolism at low ψ(w) whereby Pro synthesis in the photosynthetic tissue regenerates NADP while Pro catabolism in meristematic and expanding cells is needed to sustain growth. Tissue-specific differences in Pro metabolism and function in maintaining a favorable NADP/NADPH ratio are relevant to understanding metabolic adaptations to drought and efforts to enhance drought resistance.

Figure 1.

Root elongation and fresh weight of the Col-0 wild type, aba2-1, p5cs1, and p5cs1/aba2-1 mutants. Five-day-old seedlings were transferred to either control medium (−0.25 MPa) or PEG-infused agar plates for low ψ_w (−1.2 MPa) or low ψ_w with added ABA (2 μm) or Pro (10 mm). Root elongation was measured over the next 7 d for control seedlings and 10 d for low-ψ_w-treated seedlings. Fresh weight and dry weight were quantified at the end of an experiment. A, Quantification of root elongation, fresh weight, and dry weight. Within each panel, significant differences found by two-factor ANOVA are indicated by different letters. Data shown are means ± se combined from two to four independent experiments, with each experiment including more than 30 measurements for root elongation, 12 measurements (each including five to six seedlings) for fresh weight, or three to six measurements (each including 25–35 seedlings) for dry weight. Dashed lines in each panel mark the wild-type (Col-0) value. B, Photographs of representative seedlings from each treatment taken at the end of an experiment. One seedling of each genotype is shown for the control, and two seedlings of each genotype are shown for low-ψ_w treatments.

Figure 2.

Response of root elongation at −1.2 MPa to various concentrations of exogenous Pro for the Col-0 wild type, aba2-1, p5cs1-4, and p5cs1-4/aba2-1 mutants. Five-day-old seedlings were transferred to PEG-infused agar plates (−1.2 MPa) containing Pro (0–10 mm). Root elongation was measured over the next 10 d. Data shown are means ± se combined from two independent experiments (n = 20–30). Asterisks indicate significant differences compared with the Col-0 wild type (P > 0.05).

Figure 3.

Tissue-specific expression of Pro metabolism genes in wild-type seedlings. A, Quantitative PCR analysis of gene expression. Seven-day-old seedlings were transferred to either control (−0.25 MPa) or low-ψ_w (−1.2 MPa) medium for 96 h, and samples of shoot tissue as well as three sequential sections of the root were collected (the sampling scheme is shown in the diagram in the middle of the panel). Asterisks indicate significant differences between control and low-ψ_w treatments based on results of an unpaired t test (P < 0.05). Data are shown as means ± se (n = 3–4) from one representative experiment out of two independent experiments. B, Histochemical GUS staining of T3 homozygous plants expressing PDH1_pro:GUS containing a 1.6-kb fragment of the PDH1 promoter. Control and low-ψ_w treatments were performed as described in A. Data are for one representative transgenic line out of three independent lines analyzed. Bars = 1 mm.

Figure 4.

pdh1 growth response to low ψ_w and Pro. Five-day-old seedlings were transferred to control (−0.25MPa), low-ψ_w (−1.2 MPa), or low-ψ_w plus Pro (10 mm) medium. A, Root elongation, fresh weight, and dry weight data were collected as described for Figure 1A. Within each panel, statistically significant differences detected by two-factor ANOVA and/or t test are indicated by different letters (P > 0.05). Data are shown as means ± se combined from two to four independent experiment, with each experiment including more than 15 measurements for root elongation, eight or more measurements for fresh weight, or three or more measurements for dry weight. B, Photographs of representative seedlings of the Col-0 wild type and pdh1 mutants taken at the end of an experiment. One seedling of each genotype is shown for the control, and two seedlings of each genotype are shown for the low-ψ_w treatments. C, Response of root elongation to a range of Pro concentrations from 0 to 10 mm. Data shown are means ± se combined from two independent experiments (n = 20–30). Asterisks indicate significant differences compared with the Col-0 wild type (P > 0.05).

Figure 5.

Root tip oxygen consumption rates and tissue-specific Pro contents of the Col-0 wild type and Pro metabolism mutants. A, Five-day-old seedlings were transferred to control (−0.25 MPa), low-ψ_w (−1.2 MPa), or low-ψ_w plus Pro (10 mm) medium for 96 h. Root sections (0–10 mm) were excised and immediately used for oxygen consumption rate measurement in a Clark-type electrode. Data are means ± se (n = 7–8) from two independent experiments. Asterisks indicate significant differences compared with the wild type in the same treatment detected by unpaired t test (P < 0.05). B, Free Pro content measured in shoot and 10-mm root sections after 96 h at low ψ_w (−1.2 MPa). Note that shoot data are expressed as μmol g⁻¹ fresh weight (F.W.), while root data are expressed as nmol root section⁻¹. Data are means ± se (n = 6–8) combined from two independent experiments. Asterisks mark significant differences from the wild type detected by unpaired t test (P < 0.05).

Figure 6.

Effect of Pro or GABA applied specifically to shoot or root using a split-plate assay system. A, Five-day-old seedling were transferred to low ψ_w (−1.2MPa) with the addition of 10 mm Pro to either root or shoot using split plates (the split-plate system is shown in Supplemental Fig. S5). Root elongation was then monitored over the next 10 d. For root elongation, data are means ± se (n > 40) combined from three independent experiments. Pro contents were determined in the 0- to 10-mm root section at 10 d after transfer and are means ± se (n = 3–6) combined from three independent experiments. Within each panel, different letters indicate significant differences detected by two-factor ANOVA (P < 0.05). B, Effect of 10 mm GABA applied to root or shoot on root elongation and seedling fresh weight of the wild type or p5cs1 mutants. The experimental design is the same as in A. Data are means ± se (n = 20–25). Asterisks indicate significant differences compared with the wild type detected by unpaired t test (P < 0.05).

Figure 7.

Pyridine nucleotide contents and NADP/NADPH and NAD/NADH ratios in the wild type (Col-0), p5cs1-4, and pdh1-2 after control or low-ψ_w treatment. Seven-day-old seedlings were transferred to control (−0.25 MPa) or low-ψ_w (−1.2 MPa) medium for 96 h, and samples of shoot tissue were collected for pyridine nucleotide assay. A, Assay of NADP and NADPH and calculation of NADP/NADPH ratio. NADP and NADPH contents are representative data from two independent experiments (means ± se; n = 8–12). One ratio was calculated for each independent experiment, and the NADP/NADPH ratio shown is the mean of ratios from four independent experiments. Asterisks indicate significant differences relative to the wild type detected by unpaired t test (P < 0.05). B, Assay of NAD and NADH and calculation of NAD/NADH ratio. Data and ratio calculation are as described for A. No significant differences were detected between mutants and the wild type for NAD, NADH, or NAD/NADH. FW, Fresh weight.

Figure 8.

Model of tissue-specific Pro synthesis and catabolism at low ψ_w. At low ψ_w, Pro synthesis increases in the photosynthetic tissue of the shoot (Pro source), as indicated by increased expression of both P5CS1 and P5CR. This synthesis generates NADP to maintain a more oxidized NADP/NADPH ratio. A portion of the Pro is transported to Pro sinks in the growing regions of root and shoot. In Pro sink tissues, Pro is catabolized in the mitochondria to support growth. Whether a product of Pro catabolism moves back to the shoot to continue the cycle is not known. In both the Pro source and Pro sink regions, Pro accumulation is also necessary for other functions such as osmotic adjustment.