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Plant Physiol. Sep 2011; 157(1): 292–304.
Published online Jul 26, 2011. doi:  10.1104/pp.111.183210
PMCID: PMC3165878

Essential Role of Tissue-Specific Proline Synthesis and Catabolism in Growth and Redox Balance at Low Water Potential1,[W][OA]

Abstract

To better define the still unclear role of proline (Pro) metabolism in drought resistance, we analyzed Arabidopsis (Arabidopsis thaliana) Δ1-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 (ψw). 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 ψw. PDH1 maintained high expression in the root apex and shoot meristem at low ψw 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 ψw. 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 ψw. These results indicate a new model of Pro metabolism at low ψw 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.

Plant responses to low water potential (ψw) that occur during drought include changes in metabolite levels and the activity of specific metabolic pathways (Wilson et al., 2009). A continuing challenge is to identify the adaptive metabolic changes and determine how they contribute to drought resistance. In many plant species, including Arabidopsis (Arabidopsis thaliana), Pro accumulation is one of the main metabolic responses to abiotic stress. The highest levels of Pro accumulation are typically seen in response to low ψw with lower levels accumulating in response to salt or cold (Kaplan et al., 2007; Sharma and Verslues, 2010). Ecophysiological observations have also suggested a role of Pro in drought adaptation (Ben Hassine et al., 2008; Parida et al., 2008; Evers et al., 2010). However, even in Arabidopsis, the role of Pro in low-ψw stress is not clearly established and the specific effects of Pro on low-ψw resistance have been relatively less studied than salt or cold stress (Verslues and Sharma, 2010).

Early molecular studies of Pro in salt stress or under severe dehydration established a “standard model” whereby transcriptional up-regulation of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE1 (P5CS1), which catalyzes the first step of Pro synthesis (Hu et al., 1992; Yoshiba et al., 1995, 1999; Abrahám et al., 2003; Armengaud et al., 2004), and down-regulation of PROLINE DEHYDROGENASE (PDH1), which catalyzes the first step of Pro catabolism, were both required and sufficient for stress-induced Pro accumulation (Kiyosue et al., 1996; Peng et al., 1996; Yoshiba et al., 1997; Miller et al., 2005). Consistent with this standard model, p5cs1 mutants have greatly reduced levels of Pro under both salt stress and low ψw (Székely et al., 2008; Sharma and Verslues, 2010). Székely et al. (2008) showed that p5cs1 mutants had reduced growth and increased hydrogen peroxide (H2O2) content and reactive oxygen species (ROS) damage under salt stress, demonstrating the importance of Pro in salt resistance. Conversely, the standard model would suggest that suppression of PDH1 would increase Pro content and enhance stress resistance. Some reports have been consistent with this model: antisense suppression of PDH1 improved resistance to freezing and possibly salinity in one study (Nanjo et al., 1999a). However, another study of PDH1 overexpression and antisense lines found no difference in growth or stress damage in salt or high-osmolarity treatments (Mani et al., 2002). Many of these studies focused on salt or cold stress; there has been relatively little examination of p5cs1 or pdh1 mutants at low ψw indicative of drought.

Exceptions to the standard model of up-regulated Pro synthesis and down-regulated catabolism leading to Pro accumulation have been reported. For example, Kaplan et al. (2007) saw increased expression of PDH1 in longer term (24–96 h) cold treatments even while Pro contents were high and increasing. Stines et al. (1999) found high levels of Pro despite low P5CS1 expression in grape (Vitis vinifera) berries. Verslues and Sharp (1999) showed evidence that the high levels of Pro accumulated in the maize (Zea mays) root tip at low ψw were not due to de novo synthesis in the root tip but more likely came from Pro transport from other parts of the plant. Tissue-specific differences were also noted by Skirycz et al. (2010), who found that Pro accumulated in mature leaf tissue but not in expanding or meristematic tissue during long-term growth at moderate severity of osmotic stress, and Ueda et al. (2007), who found high utilization of Pro in the root apex during salt stress. Such observations suggest that stress-specific and tissue-specific differences in Pro metabolism may be more important than previously thought.

The function of drought-induced Pro accumulation in plants and how it enhances drought resistance is a long-standing question. Traditionally, Pro has been thought of mainly as a compatible solute that accumulates as part of osmotic adjustment; for example, there is strong evidence to support such a role in the root growth zone at low ψw (Voetberg and Sharp, 1991). In Arabidopsis seedlings at low ψw, osmotically significant levels of Pro (50–100 μmol g−1 fresh weight, roughly equivalent to 50–100 mm) are routinely observed in our laboratory (Sharma and Verslues, 2010). Such osmotic roles, as well as other potential roles in protecting cellular structure or ROS detoxification, depend on high bulk levels of Pro and have led to a “more-is-better” strategy of generating transgenic plants that constitutively overexpress P5CS1 or suppress PDH1 to increase Pro contents and, presumably, salt or drought resistance (Kishor et al., 1995; Zhu et al., 1998; Nanjo et al., 1999a, 1999b; Hong et al., 2000; Sawahel and Hassan, 2002; Parvanova et al., 2004; Vendruscolo et al., 2007).

Other proposed functions of Pro include redox buffering and storage or transfer of energy and reductant (Szabados and Savouré, 2010; Verslues and Sharma, 2010). These functions depend on spatial and temporal control of Pro synthesis and catabolism to either take up or release reductant and energy at the correct place and time to meet the plant’s needs and do not lend themselves as well to the more-is-better theory of Pro accumulation. Of particular interest is the fact that both P5CS1 and Δ1-PYRROLINE-5-CARBOXYLATE REDUCTASE (P5CR), which catalyzes the second step of Pro synthesis, prefer NADPH to NADH as an electron donor (Zhang et al., 1995; Murahama et al., 2001). It has been suggested that Pro synthesis could be a mechanism to regenerate NADP+ in the chloroplast and thus prevent ROS production and photoinhibition caused by a lack of appropriate electron acceptors (Hare and Cress, 1997; Hare et al., 1998; Szabados and Savouré, 2010; Verslues and Sharma, 2010). However, there is no experimental evidence to test the role of Pro metabolism in controlling NADP+ and NADPH levels during low-ψw stress.

These functions all require appropriate regulation of Pro metabolism and the coordination of Pro metabolism with many other stress-induced metabolic changes. Abscisic acid (ABA) is the main candidate for such a regulator and at the physiological level is well established to coordinate growth under low ψw by protecting root growth to maximize water uptake while restricting shoot growth to avoid water loss (Saab et al., 1990; Sharp et al., 1994). Pro accumulation is partially controlled by ABA, as ABA-deficient mutants such as aba2-1 in Arabidopsis and viviparous14 in maize had reduced Pro at low ψw (Ober and Sharp, 1994; Sharma and Verslues, 2010). However, whether Pro is an important factor in ABA regulation of growth at low ψw is unclear.

We used Arabidopsis Pro metabolism mutants subjected to constant, precisely controlled low-ψw stress to demonstrate the essential role of both Pro synthesis and Pro catabolism in promoting growth and maintaining a more oxidized NADP/NADPH ratio at low ψw. Analysis of p5cs1, p5cs1/aba2-1, and pdh1 mutants, tissue-specific expression of Pro metabolism genes, and the effects of root- and shoot-applied Pro all suggest that Pro supply from the shoot and its catabolism in the root are essential for continued growth at low ψw. The p5cs1/aba2-1 mutants demonstrated that these roles of Pro metabolism are a key part of ABA-mediated growth responses to low ψw. Both Pro synthesis and catabolism were needed to maintain a more oxidized NADP/NADPH ratio in the shoot. These results provide a new model that emphasizes dynamic transport and turnover of Pro with tissue-specific synthesis and catabolism, rather than static cell-autonomous Pro accumulation, as fundamental to the protective role of Pro during drought.

RESULTS

Pro Is Required for ABA Protection of Root Elongation and Stimulates Growth at Low ψw

We generated p5cs1-4/aba2-1 and p5cs1-1/aba2-1 double mutants in which Pro accumulation is only about 15% of the wild-type level (Sharma and Verslues, 2010) and ABA content is only a few percent of the wild-type level (Schwartz et al., 1997; Verslues and Bray, 2006). These p5cs alleles have been shown to be null (p5cs1-4) or to have very low expression (p5cs1-1; Székely et al., 2008). To test the low-ψw response of these mutants, 5-d-old seedlings were transferred from normal medium to polyethylene glycol (PEG)-infused agar plates (Verslues et al., 2006) to impose a precisely controlled low-ψw stress. As transpiration was low in this system, dehydration caused by impaired stomatal regulation in aba2-1 was minimized, allowing the growth response to low ψw to be accurately compared among genotypes. Root elongation, fresh weight, and dry weight were all measured as indicators of growth at low ψw. All media were prepared without added sugar to better mimic the environment of soil-grown plants.

At −0.25 MPa (unstressed control), all genotypes grew similarly to the ecotype Columbia (Col-0) wild type over 7 d after transfer except aba2-1 and the p5cs1/aba2-1 mutants, which had an approximately 25% reduction in root elongation (Fig. 1). When seedlings were exposed to a low-ψw stress (−1.2 MPa), root elongation of all genotypes was reduced; however, the root elongation of aba2-1, p5cs1-1, p5cs1-4, and the p5cs1/aba2-1 double mutant was further reduced to half that of the wild type, and overall seedling growth indicated by dry weight and fresh weight was also reduced (Fig. 1).

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 ψw (−1.2 MPa) or ...

Root elongation and dry weight of aba2-1 were restored to the wild-type level by the addition of 2 μm ABA to the media (Fig. 1). However, ABA treatment did not increase the root growth or dry weight of p5cs1-1, p5cs1-4, or the p5cs1/aba2-1 double mutant. This indicated that Pro accumulation mediated by P5CS1 was required for the protective effect of ABA on root elongation and growth at low ψw. In contrast, application of 10 mm Pro could restore the root elongation and dry weight of all genotypes (Fig. 1). Pro application also had a stimulatory effect on dry weight, fresh weight (Fig. 1A), and shoot growth (Fig. 1B) of ABA-deficient seedlings. Because ABA normally acts to restrict shoot growth under low ψw (Saab et al., 1990; Verslues et al., 2006), seedlings that did not accumulate ABA (aba2-1 and p5cs1/aba2-1 mutants) were able to respond more to the stimulation of growth by Pro. Adding Pro to seedlings under control conditions (−0.25 MPa) had no effect on root growth or fresh weight (Supplemental Fig. S1).

Measurement of root elongation at −1.2 MPa in the presence of a range of Pro concentrations from 0 to 10 mm indicated that as little as 0.2 mm exogenous Pro was sufficient to stimulate root elongation of both aba2-1 and p5cs1-4 as well as the double mutant (Fig. 2). We also tested whether other amino acids could duplicate the effects of Pro. The branched-chain amino acids (Leu and Ile) have also been observed to accumulate in response to dehydration (Nambara et al., 1998; Joshi and Jander, 2009), but neither Leu, Ile, Val, nor Ala could restore the root elongation of aba2-1, p5cs1-4, or p5cs1-4/aba2-1 to the wild-type level at low ψw (Supplemental Fig. S2). In fact, these amino acids had a general inhibitory effect on root elongation. γ-Aminobutyrate (GABA) also could not restore root elongation (see below), and the Pro-related amino acids Glu, Gln, Arg, and Orn were strongly inhibitory to root elongation. Inhibition of plant growth by exogenous amino acids has been observed in a number of studies (Bonner and Jensen, 1997; Chen et al., 2010), and this is likely related to feedback inhibition or disturbance of metabolic equilibrium caused by applied amino acids or disruption of metabolite transport. Such observations also illustrate the uniqueness of Pro in promoting growth at low ψw.

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 ...

Tissue-Specific Gene Expression Suggests Spatial Separation of Pro Synthesis and Catabolism as Well as Continued Pro Catabolism at Low ψw

To understand the basis of the unique growth-promoting effect of Pro, we quantified Pro metabolism gene expression and found dramatic differences between shoot and root. Shoot tissue had the well-established pattern of P5CS1 induction and PDH1 repression after 96 h at −1.2 MPa (Fig. 3A). In contrast, PDH1 expression was induced by low ψw rather than repressed in the 0- to 10-mm root section containing the root meristem and expanding cells (Fig. 3A). Thus, PDH1 went from being expressed at a relatively even level across root and shoot in unstressed seedlings to being expressed 12-fold higher in the 0- to 10-mm root section than in the bulk of the shoot at low ψw. Arabidopsis also contains a second Pro dehydrogenase (PDH2), which has only more recently been studied (Funck et al., 2010). PDH2 had a similar pattern as PDH1, although its level of expression was much lower.

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-ψw (−1.2 MPa) medium for ...

A contrasting pattern was observed for P5CS1, which was expressed at a much lower level in root than in shoot (Fig. 3A). P5CR, which encodes the other enzyme of Pro synthesis, was also significantly induced in shoot but not in root. Δ1-PYRROLINE-5-CARBOXYLATE DEHYDROGENASE (P5CDH) and ORNITHINE AMINOTRANSFERASE had increased root expression, especially in the 0- to 10-mm section. Overall, the gene expression pattern of the root apex during low ψw was similar to that observed in whole seedlings during stress release when rapid Pro catabolism was occurring (Sharma and Verslues, 2010). However, this pattern was different from the combination of high PDH1 expression but low P5CDH expression that was associated with ROS production during pathogen infection (Cecchini et al., 2011).

To further define the tissue-specific expression of PDH1, we generated PDH1pro:GUS plants. After 96 h at −1.2 MPa, there was high PDH1 promoter activity in the apical few millimeters of the root containing the root meristem and expanding cells, consistent with the quantitative PCR results, as well as high GUS activity in the shoot meristematic region and young expanding leaves (Fig. 3B). The combined gene expression results suggested that expanding and dividing cells in both the root and shoot meristem had substantial Pro catabolism at low ψw, while the bulk of the photosynthetic tissue in the shoot had high Pro synthesis but repressed Pro catabolism.

PDH Expression Is Required to Sustain Growth at Low ψw

To test the importance of continued Pro catabolism at low ψw, two homozygous T-DNA mutant lines were isolated for PDH1. The PDH1 mutants were null or had extremely low expression based both on a previous report (Funck et al., 2010) and our own data (Supplemental Fig. S3A). The pdh1 mutants grew similarly to the Col-0 wild type in the control (−0.25 MPa; Fig. 4A). At −1.2 MPa, however, root growth, fresh weight, and dry weight of pdh1 were reduced, indicating the importance of Pro catabolism for both root and shoot growth. Exogenous Pro could not restore the growth of pdh1 mutants (Fig. 4, A and B). Tests of a range of exogenous Pro concentrations from 0.2 to 10 mm found that none could restore the root elongation of pdh1-2 (Fig. 4C). Higher exogenous Pro levels (10 mm) inhibited the root growth of pdh1-2 at both low ψw (Fig. 4, A and C) and high ψw (Supplemental Fig. S3B). This inhibition could be caused by high levels of Pro inhibiting the transport of other metabolites to the root tip, which may be more critical to sustaining root growth when Pro catabolism is blocked. ABA application also could not restore the root elongation of pdh1-2 (Supplemental Fig. S3C).

Figure 4.
pdh1 growth response to low ψw and Pro. Five-day-old seedlings were transferred to control (−0.25MPa), low-ψw (−1.2 MPa), or low-ψw plus Pro (10 mm) medium. A, Root elongation, fresh weight, and dry weight data ...

As a further test of the importance of Pro metabolism for growth at low ψw, we quantified root elongation of both p5cs1 and pdh1 mutants at the moderate stress level of −0.7 MPa, where growth continues at a more rapid rate than at −1.2 MPa. Root elongation was reduced in p5cs1-4 and to an even greater extent in pdh1-2 (Supplemental Fig. S4A), demonstrating that both Pro synthesis and catabolism were required for maximal growth across a range of low-ψw severities. It was also seen that Pro application at −0.7 MPa inhibited the root growth of pdh1-2 seedlings even more than low ψw alone (Supplemental Fig. S4B), suggesting that when Pro catabolism was blocked, simply adding additional Pro was not sufficient to promote growth and may in fact interfere with the transport or metabolism of other substrates.

Lack of PDH1 Expression Decreases Root Respiration Despite Increased Pro Content

One possible explanation for the reduced growth of pdh or p5cs1 mutants is that inhibited Pro catabolism or Pro supply caused a decrease in respiration needed to sustain growth. Consistent with this hypothesis, we found reduced root tip oxygen consumption of pdh1-2 at low ψw while the Col-0 wild type and p5cs1-4 were unaffected (Fig. 5A). Adding 10 mm Pro to seedlings at −1.2 MPa could not restore oxygen consumption of pdh1-2 and in fact further decreased oxygen consumption, consistent with the effects on root elongation. The impaired oxygen consumption of pdh1-2 but not p5cs1-4 suggests that although both p5cs1 and pdh1 mutants have similar effects on root elongation, the main factor limiting growth may be different.

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-ψw (−1.2 MPa), or low-ψw plus ...

To further determine how root oxygen consumption and growth were related to Pro, the same shoot and root section sampling scheme used for gene expression measurements was used to measure Pro content of seedlings at −1.2 MPa. pdh1-2 had Pro content more than double that of the wild type in the root apex (0- to 10-mm section; Fig. 5B), while Pro contents of the shoot, 20- to 30-mm root, and 10- to 20-mm root sections were only slightly increased or unchanged. This again suggested an active Pro catabolism in the root apex at low ψw, such that in pdh1-2, where this catabolism was blocked, Pro accumulated to high levels while root respiration was inhibited.

In contrast, Pro content of p5cs1-4 was much lower than that of the wild type in all tissues measured (Fig. 5B). Interestingly, p5cs1-4 maintained a relatively higher Pro content in the 0- to 10-mm root section (22% of the wild type) than in the shoot (9% of the wild type), despite higher P5CS1 expression in the shoot (Fig. 3A). One possible explanation for this would be shoot-to-root movement of Pro. aba2-1 was also analyzed to see if its reduced growth at low ψw may be caused by altered Pro distribution. Pro content was reduced to half the wild-type level in both the shoot and 0- to 10-mm root sections, although Pro content was not different from the wild type in the other root sections (Fig. 5B). Thus, aba2-1 may be affected by Pro shortage in the root growing region at low ψw as well as by reduced Pro synthesis in the shoot tissue.

Stimulation of Root Growth by Shoot-Applied Pro Suggests Active Shoot-to-Root Transport

The gene expression pattern, oxygen consumption, and Pro contents all suggested that Pro synthesized in the shoot may be transported to the root to support growth at low ψw. Consistent with this idea, we found that Pro applied specifically to the shoot could stimulate root elongation of the wild type, p5cs1-1, and p5cs1-4 at low ψw. Split agar plates were constructed by removing the regular agar medium from half of a PEG-infused agar plate (−1.2 MPa) and replacing it with the same low-ψw medium supplemented with Pro (Supplemental Fig. S5). Seedlings were then transferred to these plates such that only the root or shoot was in contact with the Pro-containing medium. Pro diffusion across the boundary separating the two plate halves was not detected (Supplemental Fig. S5).

When split plates were constructed with both halves lacking Pro, both root elongation and Pro content of the 0- to 10-mm root section (Fig. 6A) were similar to those seen in other experiments (compare Fig. 6A with Figs. 1A and and5B).5B). Addition of Pro specifically to the root stimulated root elongation, and this was associated with a large increase in root tip Pro content (Fig. 6A). Interestingly, however, addition of Pro specifically to the shoot stimulated root elongation to a greater extent than either Pro applied to whole seedlings or specifically to the root (compare Fig. 6A with Fig. 1A). Shoot-applied Pro only increased root Pro content back to the wild-type level or even slightly below the wild-type level, showing that it was not a large buildup of Pro in the root that was most needed to enhance growth. The observation that shoot-applied Pro could stimulate root elongation suggests shoot-to-root movement of Pro; however, we cannot rule out the possibility that shoot-applied Pro stimulated the production of other metabolites that were transported to the root.

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 ψw (−1.2MPa) with the addition of 10 mm Pro to either root or shoot using split plates (the ...

GABA can stimulate root elongation in unstressed seedlings (Mirabella et al., 2008), is often thought to have similar stress-protective roles as Pro, and is also catabolized in the mitochondria. However, GABA application to either root or shoot could not duplicate the effect of Pro in restoring root elongation of p5cs1 mutants (Fig. 6B), again demonstrating a specific effect of Pro on growth at low ψw.

Pro Metabolism Is Required to Maintain an Oxidized NADP/NADPH Ratio

These data still left open the question of why Pro synthesis would be greater in the shoot than in the root. Our data, plus recent observations that P5CS1 may be in, or closely associated with, the chloroplast (Székely et al., 2008; Reiland et al., 2009) led us to test the possibility that Pro synthesis serves to regenerate NADP+ and maintain an appropriate NADP/NADPH ratio (Hare and Cress, 1997; Szabados and Savouré, 2010; Verslues and Sharma, 2010). In the Col-0 wild type, low ψw (−1.2 MPa for 96 h) increased NADP content by approximately 4-fold while NADPH content increased by nearly 6-fold (Fig. 7A). Thus, the NADP/NADPH ratio was decreased by half from 4.9 at high ψw to 2.4 at low ψw. This trend to a more reduced ratio at low ψw was exacerbated significantly in both p5cs1-4 and pdh1-2, where the NADP/NADPH ratio at −1.2 MPa was 1.5 and 1.6 for p5cs1-4 and pdh1-2, respectively. This change in ratio resulted from higher NADPH and lower NADP contents, such that the total pyridine nucleotide content was little changed in the mutants compared with the wild type. It was particularly interesting that we found similar NADP/NADPH in both p5cs1-4 and pdh1-2 at −1.2 MPa, as PDH1 expression in most of the shoot tissue was reduced to a low level in this treatment (Fig. 3). Also interesting was the observation that pdh1-2 had a significantly more reduced NADP/NADPH ratio at high ψw (2.9 versus 4.9 for the wild type). This demonstrated a role for Pro metabolism in maintaining an oxidized NADP/NADPH ratio even at high ψw, when Pro content was low. A specific effect of Pro metabolism on NADP/NADPH was further indicated by the lack of any significant difference between the wild type and p5cs1-4 or pdh1-2 in either NAD or NADH content or NAD/NADH ratio at high or low ψw (Fig. 7B).

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-ψw treatment. Seven-day-old seedlings were transferred to control (−0.25 MPa) or low-ψw (−1.2 ...

We also assayed H2O2 to see if any of the Pro metabolism mutants had increased overall ROS content. Consistent with previous reports (Verslues et al., 2007; Fujii et al., 2011), low ψw caused an increase in H2O2 content of the wild type (Supplemental Fig. S6). This increase also occurred in aba2-1, p5cs1, and prodh mutants; however, none of the mutants had H2O2 levels above that of the wild type at either high or low ψw. This lack of H2O2 accumulation suggested that general ROS accumulation or damage was unlikely to cause the altered growth or other phenotypes observed for p5cs1 or pdh1.

DISCUSSION

Several novel observations presented here shed new light on the role of Pro metabolism in low-ψw resistance. First, the p5cs1/aba2-1 double mutants demonstrated that Pro was essential for ABA protection of root elongation and overall growth at low ψw. Second, the similar phenotypes of p5cs1 and pdh1 mutants demonstrated that both Pro synthesis and catabolism were required to maintain growth at low ψw. Third, the localized expression of PDH1 and P5CS1, the ability of shoot-applied Pro to greatly enhance root elongation, and the large buildup of Pro in the root apex of pdh1-2 all suggest the transport of Pro and/or related metabolites from shoot to root and catabolism in the root apex as essential to continued growth at low ψw. Fourth, the NADP/NADPH ratio, but no change in NAD/NADH, in both p5cs1-4 and pdh1-2 suggest a specific role of Pro turnover in maintaining a more oxidized NADP/NADPH ratio.

Together, these data lead to a new model of Pro metabolism (Fig. 8). At low ψw, Pro synthesis is focused in “Pro source” tissue (primarily the photosynthetic tissue of the shoot), where it regenerates NADP to help maintain an appropriately oxidized NADP/NADPH ratio. Some of this Pro accumulates in the leaf as a compatible solute. However, a portion of the Pro is transported to “Pro sink” tissue in the root and shoot meristem, where it can be catabolized both to support continued growth at low ψw and to maintain a sink for continued Pro export from the source tissue. Thus, the effectiveness of Pro metabolism in buffering energy and redox imbalances within the plant, in addition to Pro function as a protective compatible solute for osmotic adjustment, is key to its role in low-ψw resistance.

Figure 8.
Model of tissue-specific Pro synthesis and catabolism at low ψw. At low ψw, 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 ...

Pro Metabolism Maintains a More Oxidized NADP/NADPH Ratio

Low ψw caused a shift in NADP/NADPH to a more reduced state, and this was exacerbated in both p5cs1-4 and pdh1-2. The NADP/NADPH ratio we observed at high ψw was consistent with previous reports (Wang and Pichersky, 2007). However, we could find no previous data on NADP/NADPH ratio at low ψw. Consistent with our hypotheses, De Ronde et al. (2004) found increased levels of NADP in drought-stressed soybean (Glycine max) plants overexpressing P5CR and decreased NADP in P5CR antisense plants; however, they did not measure NADPH levels or calculate NADP/NADPH ratio, the key measure of redox status. The specific effect of Pro metabolism on NADP/NADPH, but not NAD/NADH, further suggests a link of Pro metabolism to redox buffering in the chloroplast, which is prone to overreduction under changing environmental conditions (Dietz and Pfannschmidt, 2011). The NADP/NADPH ratio is critical not only for photosynthetic electron transport but also for ROS generation and signaling by NADPH oxidases, activity of many NADPH-dependent enzymes, and nitric oxide production (Zhang et al., 2009; Takahara et al., 2010; Dietz and Pfannschmidt, 2011). Therefore, control of NADP/NADPH ratio by Pro metabolism could impact other metabolic and signaling pathways. It will be of particular interest to investigate the possible coupling of Pro synthesis and the oxidative pentose phosphate pathway, which is also known to have a role in controlling the NADP/NADPH ratio (Verslues and Sharma, 2010; Dietz and Pfannschmidt, 2011).

The surprising observation that pdh1-2 had a substantial role in maintaining the NADP/NADPH ratio even in unstressed seedlings suggests that the cycle of Pro synthesis and catabolism was important at both high and low ψw. Thus, restricted Pro catabolism in pdh1-2 may have caused a reduction in NADPH consumption by Pro synthesis by mechanisms such as reduced regeneration of Glu for Pro synthesis. However, other more indirect effects of PDH1 on NADP/NADPH, such as a restricted supply of Glu generated by Pro catabolism causing altered glutathione production, cannot be ruled out.

Growth Maintenance at Low ψw Depends on Both Pro Accumulation and Pro Metabolism

The reduced dry weight and root elongation of p5cs1 and pdh1 mutants demonstrated the essential role of Pro metabolism in growth at low ψw. The role of Pro was particularly prominent in root elongation, and the ability of Pro, but not ABA, to rescue p5cs1 and p5cs1/aba1-2 root elongation demonstrated that Pro metabolism was part of the well-established function of ABA in promoting root elongation at low ψw. The observation that both p5cs1 and pdh1 mutants had decreased root elongation (as well as reduced dry weight) suggests that two mechanisms are likely to be involved in Pro promotion of growth. In the case of p5cs1-4, root oxygen consumption remained high but root Pro content was low (Fig. 5). Pro could be catabolized for respiration, but then insufficient Pro was left for other essential functions such as osmotic adjustment. For pdh1-2, oxygen consumption was reduced but there was nearly twice as much Pro in the root as in the wild type. In this case, the catabolism of Pro as a respiratory substrate was the limiting factor.

In both of these cases, the data suggest that delivery of Pro to the root growing region was critical for continued growth at low ψw. This is consistent with previous work describing a stress-induced, root-specific Pro transporter from barley (Hordeum vulgare; Ueda et al., 2001) and increased root growth (at high ψw) of plants with root tip-specific overexpression of this transporter (Ueda et al., 2008). Also consistent with our model of Pro movement are previous suggestions that the root growth zone has limited Pro synthesis but high Pro utilization (Verslues and Sharp, 1999; Ueda et al., 2007) and reports of high levels of Pro in the phloem of drought-stressed plants (Girousse et al., 1996; Lee et al., 2009). Identifying the transport mechanisms that move Pro from leaf to root at low ψw will be of interest for future experiments.

While we focused more on the maintenance of root growth, our data are also consistent with those of Skirycz et al. (2010), who performed a detailed analysis of gene expression and metabolite profiles in shoots of Arabidopsis plants grown under long-term mild osmotic stress. They found high expression of PDH1 in leaf primordial tissue, which was not down-regulated in plants adapted to osmotic stress, all consistent with our PDH1pro:GUS staining pattern. Also, Pro content was higher in mature leaf than in primordial or expanding tissue. In agreement with our model, they postulated a role for Pro in protecting photosynthesis in mature leaves and a role of mitochondrial metabolism in stress adaptation of the primordial leaf tissue.

Pro Metabolism Is a Means to Buffer Energy and Redox Imbalances at Low ψw

The combined data point to Pro metabolism and transport as part of a strategy to deal with a fundamental imbalance caused by low ψw: photosynthetic tissue has an overabundance of reductant and a decreased ability to use it effectively because of reduced CO2 uptake and restricted shoot growth. Conversely, the root is still growing and has an increased need for energy to support processes such as solute accumulation and cell wall metabolism. A need to correct energy imbalances within the plant at low ψw has also been indicated by the observation that alternative oxidase overexpression lines had less reduction in leaf growth during soil drying (Skirycz et al., 2010).

The movement of Pro from shoot to root can be one way for the plant to correct such energy and redox imbalances. An obvious question is why Pro transport, instead of other sugars or amino acids, would be beneficial under low ψw. Part of the answer seems to be the benefit of continued Pro synthesis in the shoot to generate NADP+. On the catabolism side, the unique catabolic pathway of Pro (PDH1 donates electrons directly to the mitochondrial electron transport chain) and the high energy content of Pro (30 ATP equivalents per Pro molecule; Mattioli et al., 2009), as well as the possibility that Suc supply from the shoot may be diminished (Boyer, 2010), make Pro a good substrate for continued respiration at low ψw. Function of Pro as a specialized respiratory substrate has been demonstrated in other biological systems (Verslues and Sharma, 2010). In addition, PDH1 is known to be up-regulated during darkness-induced energy starvation (Dietrich et al., 2011), where Pro may also substitute for other respiratory substrates. Our observations place Pro metabolism within the emerging view that energy and redox management, including important roles for chloroplast and mitochondrial metabolism, are key to drought adaptation (Atkin and Macherel, 2009; Skirycz et al., 2010; Dietz and Pfannschmidt, 2011). We believe that this role of Pro metabolism is complementary to the role of Pro as a compatible solute for osmotic adjustment.

A number of studies have used transgenic strategies to modify Pro metabolism and, presumably, enhance drought resistance. Such studies have generally been designed on the premise that Pro accumulation is a cell autonomous process and that any combination of increased synthesis and decreased catabolism that leads to higher bulk Pro levels would increase drought resistance. Our model suggests that tissue-specific modifications of Pro metabolism, such as enhanced Pro synthesis in the shoot coupled with increased transport to the root, could be a more effective strategy.

MATERIALS AND METHODS

Plant Material, Growth, and Stress Treatments

T-DNA insertion lines of Arabidopsis (Arabidopsis thaliana) were obtained from the Arabidopsis Biological Resource Center, and homozygous plants were identified by PCR screening as described (http://signal.salk.edu/tdnaprimers.2.html). Double mutants of p5cs1/aba2-1 were isolated as described previously (Sharma and Verslues, 2010). A summary of the T-DNA lines used is presented in Supplemental Table S1.

Seedling growth and stress treatments were performed in a manner similar to previous experiments (Verslues et al., 2006; Sharma and Verslues, 2010). Briefly, sterilized Arabidopsis seeds were plated on half-strength Murashige and Skoog (MS) medium (pH 5.7) without the addition of sugar. After 48 h of cold treatment at 4°C, the plates were placed in a growth chamber (25°C, continuous light at 80–100 μmol photons m−2 s−1) oriented vertically so that seedlings grew along the surface of the agar. Five- or 7-d-old seedlings were transferred to PEG-infused agar plates to impose low-ψw stress (Verslues et al., 2006). Where indicated, S(+)ABA or Pro was added to the medium after sterilization. For root elongation and fresh weight measurements, seedlings were transferred to the indicated medium at 5 d of growth and root elongation was monitored over the subsequent 10 d for stress treatments and 7 d for the control.

To apply Pro or other metabolites specifically to root or shoot, split agar plates were prepared by removing half of the agar from PEG-infused plates of −1.2 MPa and replacing that half of the agar with PEG-infused agar containing Pro or other amino acid. Five-day-old seedlings were transferred to these split plates such that only the root or shoot was in contact with the Pro-containing medium, and root growth was monitored for the subsequent 10 d. The split agar plate experiment is diagrammed and further explained in Supplemental Figure S5.

Quantitative Real-Time PCR

Gene expression analysis was carried out with shoot tissue and three different root sections each of 10 mm from the root tips. Three or four biological samples were collected, and total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) with DNase treatment. RNA was quantified using a Nanodrop spectrophotometer, and 0.5 to 1.0 μg of RNA was reverse transcribed using the SuperScript III (Invitrogen) cDNA synthesis kit. cDNAs were diluted appropriately to fall within the range of standard curves generated using known copy numbers of cloned cDNAs, and real-time PCR was performed using FAM fluorophore/BHQ quencher-labeled TaqMan probes as described previously (Sharma and Verslues, 2010).

Oxygen Consumption

Oxygen uptake rate in 10-mm root tips was measured by Clark-type electrode (Hansatech) in a stirred 1-mL reaction volume at 25°C. The incubation mixture contained half-strength MS (pH 5.7) for control samples or half-strength MS with mannitol added to match the ψw of the PEG-infused agar plates. Each sample consisted of 40 to 50 (control) or 30 to 35 (low ψw) root tips. Three or four samples were assayed in each independent experiment.

Assay of Nicotinamide Coenzyme

Oxidized and reduced nicotinamide coenzyme contents were measured using enzymatic assays as described previously by Hayashi et al. (2005) with some modifications. Samples of 100 to 200 mg (control) or 30 to 50 mg (low ψw) were ground in liquid N2 and homogenized with 0.1 m HCl (for NAD and NADP assay) or 0.1 m NaOH (for NADH and NADPH assay). Samples were heated at 95°C for 2 min, cooled in an ice bath, and pH adjusted using NaOH (for NAD and NADP assay) or HCl (for NADH and NADPH assay). Samples were centrifuged for 10 min at 4°C, and the supernatants were used for coenzyme assay. For NADP and NADPH measurement, sample aliquots were added to a reaction mixture containing 50 mm glyglycine buffer (pH 7.4), 5 mm Glc-6-P, 0.25 mm phenazine methosulfate, 0.5 mm thiazolyl blue, 20 mm nicotinamide, and Glc-6-P dehydrogenase at a final concentration of 0.5 μg mL−1. For NAD and NADH measurement, the reaction mixture consisted of 50 mm glyglycine buffer (pH 8.8), 8% ethanol, 0.25 mm phenazine methosulfate, 0.5 mm thiazolyl blue, 20 mm nicotinamide, and alcohol dehydrogenase at a final concentration of 2.5 μg mL−1. Assays were performed using a reaction volume of 200 μL on 96-well plates, and absorbance was measured at 565 nm from 0 to 30 min after the start of the reaction. The concentration of NAD(P)(H) was determined by comparing sample values with standard curves.

Construction of the PDH1pro:GUS Transgenic Plants and Histochemical GUS Assay

The PDH1 promoter and 5′ untranslated region from −1,500 to +115 was amplified (forward primer, 5′-AAAAAGCAGGCTTGAAGGAACTTCTCAAAA-3′; reverse primer, 5′-AGAAAGCTGGGTAAAATTCAAAGATTTTGT-3′; these primers include Gateway cloning sequences, and a second nested PCR was performed to add the remaining portion of the Gateway recognition sequences) from Col-0 genomic DNA, cloned into entry vector pDONR207 (Invitrogen) by BP reaction, and subsequently transferred by LR reaction into the pGWB433 binary vector (Nakagawa et al., 2007) to generate a promoter:GUS fusion. The construct was transferred into Agrobacterium tumefaciens strain GV3101 and used for transformation of the Arabidopsis Col-0 wild type. Transgenic plants were selected on half-strength MS medium containing 50 μg mL−1 kanamycin. Lines exhibiting segregation ratios consistent with a single locus insertion were selected, and three independent homozygous T3 lines were used for GUS staining. Similar staining patterns were obtained with all lines, and representative results are shown. GUS staining was performed following standard protocols (Weigel and Glazebrook, 2002).

Pro and H2O2 Measurement

Free Pro was assayed using the ninhydrin assay (Bates et al., 1973) adapted to 96-well format (Verslues, 2010). For H2O2 assay, 50 to 100 mg of plant material was collected and extracted as described previously (Shin and Schachtman, 2004), and H2O2 was quantified using an Amplex Red H2O2 assay kit (Invitrogen) following the manufacturer’s protocol.

Statistical Analysis

Data were analyzed by two-factor ANOVA with the Holm-Sidak posttest as implemented in Sigma Plot 11 (Systat Software). Other comparisons were performed by two-tailed t test as indicated in the text and figure legends.

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We thank Na Lin for assistance in the laboratory and Mei-Jane Fang for assistance with photography of the GUS-stained plants.

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