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. 2002 Jun 15;89(7):813–823. doi: 10.1093/aob/mcf082

Proline Metabolism and Transport in Maize Seedlings at Low Water Potential

MARJORIE J RAYMOND 0, NICHOLAS SMIRNOFF *
PMCID: PMC4233804  PMID: 12102507

Abstract

The growing zone of maize seedling primary roots accumulates proline at low water potential. Endosperm removal and excision of root tips rapidly decreased the proline pool and greatly reduced proline accumulation in root tips at low water potential. Proline accumulation was not restored by exogenous amino acids. Labelling root tips with [14C]glutamate and [14C]proline showed that the rate of proline utilization (oxidation and protein synthesis) exceeded the rate of biosynthesis by five‐fold at high and low water potentials. This explains the reduction in the proline pool following root and endosperm excision and the inability to accumulate proline at low water potential. The endosperm is therefore the source of the proline that accumulates in the root tips of intact seedlings. Proline constituted 10 % of the amino acids released from the endosperm. [14C]Proline was transported from the scutellum to other parts of the seedling and reached the highest concentration in the root tip. Less [14C]proline was transported at low water potential but because of the lower rate of protein synthesis and oxidation, more accumulated as proline in the root tip. Despite the low biosynthesis capacity of the roots, the extent of proline accumulation in relation to water potential is precisely controlled by transport and utilization rate.

Key words: Endosperm, maize, metabolism, proline, roots, scutellum, water stress, Zea mays

INTRODUCTION

Proline accumulation is a widespread response of higher plants, algae, animals and bacteria to low water potential (Delauney and Verma, 1993; Samaras et al., 1995). Accumulation in leaves at low water potential is caused by a combination of increased biosynthesis and slower oxidation in mitochondria (Boggess et al., 1976; Stewart et al., 1977; Rhodes et al., 1986; Samaras et al., 1995). Proline synthesis from glutamate via Δ1‐pyrroline‐5‐carboxylate (P5C) is catalysed by P5C synthetase (P5CS) and P5C reductase (Samaras et al., 1995). P5CS transcripts increase on exposure to drought. P5C reductase activity and transcript levels show little or no increase, depending on the species (Delauney and Verma, 1990; Williamson and Slocum, 1992; Yoshiba et al., 1995). Proline oxidation to glutamate in mitochondria is catalysed by P5C dehydrogenase and proline dehydrogenase (PDH). Both PDH activity (Sells and Koeppe, 1981; Rayapati and Stewart, 1991) and its transcript levels are rapidly and strongly decreased in response to water stress (Kiyosue et al., 1996; Peng et al., 1996; Verbruggen et al., 1996; Nakashima et al., 1998). These observations show that the changes in biosynthesis and oxidation rate which lead to proline accumulation are at least partly controlled by changes in gene expression and enzyme amount. Proline transporters have been identified (Rentsch et al., 1996; Schwacke et al., 1999). Expression of one of these (ProT2) in arabidopsis is increased by drought and high salinity suggesting increased capacity for proline transport in droughted plants (Rentsch et al., 1996).

Proline is a compatible solute but there is presently no clear agreement about the function of drought‐induced accumulation, although a role in osmoregulation seems likely (Samaras et al., 1995). Other roles for proline have been proposed, including stabilization of macromolecules, a sink for excess reductant, and a store of carbon and nitrogen for use after relief of water deficit (Smirnoff and Stewart, 1985; Smirnoff and Cumbes, 1989; Samaras et al., 1995). Overexpression of P5CS in transgenic tobacco (Kavi‐Kishor et al., 1995; Verma and Hong, 1996) or expression of a feedback inhibition‐insensitive form of P5CS produced by site‐directed mutagenesis (Hong et al., 2000) results in proline overaccumulation with apparent improvement of growth under high salinity. These observations, along with the co‐ordinated regulation of proline synthesis, oxidation and transport at low water potential, argue strongly that proline accumulation has a role in acclimation to low water potential.

The response of the maize seedling primary root to water deficit has been studied in some detail and it has proved to be a useful model for investigating the mechanisms of acclimation to low water potential (Sharp et al., 1988, 1990; Saab et al., 1990; Spollen and Sharp, 1991; Voetberg and Sharp, 1991; Frensch and Hsiao, 1995). Early metabolic responses of maize seedling roots to osmotic shock have been investigated by in vivo 31P‐NMR (Spickett et al., 1992). Within the first 2 h after transfer to low water potential there is a transient increase in cytoplasmic Pi, an accumulation of vacuolar Pi and phosphocholine. Cytoplasmic pH becomes slightly more alkaline. These changes can be interpreted as reflecting membrane turnover and activation of the plasma membrane H+‐ATPase. The pH changes could be of significance in controlling the intracellular distribution of abscisic acid (ABA) (Hartung and Slovik, 1991). Activity of a protein kinase in the elongation zone is stimulated within 30 min of transfer to low water potential (Conley et al., 1997). Maize roots can maintain growth at water potentials down to –1·6 MPa (Sharp et al., 1988). This acclimation to low water potential is dependent on ABA which is rapidly accumulated in response to the low water potential (Saab et al., 1990). Growth appears to be maintained in the elongating cells near the meristem by a combination of osmotic adjustment and altered cell wall properties (Sharp et al., 1988, 1990; Spollen and Sharp, 1991; Frensch and Hsiao, 1995). Osmotic adjustment is effected by accumulation of free amino acids, proline and sugars in the growing regions of the root. The highest proline concentration occurs within 4 mm of the apex, where the meristem and newly elongating cells are located. Proline makes a significant contribution to the osmotic potential in this region (Voetberg and Sharp, 1991) reaching 0·1 mol. Measurements of local rates of cell expansion coupled with proline concentration in the maize root tip enable local deposition rates to be calculated and show that the rate of deposition increases ten‐fold in the apical 4 mm at low water potential (Voetberg and Sharp, 1991). Hexose accumulation accounts for a large proportion of the osmotic potential in the zone of cell elongation 4–10 mm behind the tip. However, its accumulation at low water potential is largely accounted for by reduced cell expansion (Sharp et al., 1990).

A series of experiments by Oaks and co‐workers has provided background information on the metabolism and transport of proline and other amino acids in maize seedlings (Oaks, 1966; Barnard and Oaks, 1970; Oaks et al., 1970). The biosynthetic capacity of the maize seedling root tips for proline and also threonine, valine, isoleucine, leucine, tyrosine, phenylalanine and lysine is limited (Oaks and Beevers, 1964; Oaks, 1966). When the endosperm is removed from germinating maize seedlings their growth is not greatly affected as long as they are supplied with a carbon source (glucose or sucrose) and inorganic N. However, despite maintenance of dry weight increase, the content of soluble nitrogen and protein decreases after endosperm removal (Oaks and Beevers, 1964). The protein content of the embryos is restored by incubation in an amino acid mixture that mimics the composition of the amino acids released by hydrolysis of the endosperm storage proteins (Oaks and Beevers, 1964). It is therefore apparent that the embryo and root tip are dependent on the endosperm for the supply of a range of amino acids and that biosynthesis enzymes are repressed by the amino acids supplied by the endosperm (Oaks, 1966). Excised 5‐mm root tips do have some capacity to synthesize proline from labelled acetate, but a large proportion of this is incorporated into protein and very little remains in the soluble pool (Oaks et al., 1970). Given the large accumulation of proline in maize roots at low water potential (Voetberg and Sharp, 1991), a re‐examination of proline synthesis capacity showed that neither excised maize seedling primary roots nor roots attached to seedlings without endosperms were able to accumulate proline at low water potential (Smirnoff and Raymond, 1994). Verslues and Sharp (1999) confirmed this observation and carried out labelling experiments that showed a very low rate of proline synthesis from glutamate and ornithine in primary maize roots. They suggested that accumulated proline must be derived from seed storage. In this paper we examine the source and metabolism of the proline accumulated in the root tips at low water potential. The results show that proline is transported from the endosperm and that its pool size in relation to water potential is controlled by the rate of its utilization by oxidation and protein synthesis.

MATERIALS AND METHODS

Growth conditions

Maize (Zea mays ‘LG11’) caryopses were surface sterilized with sodium hypochlorite solution and germinated in the dark at 25 °C on paper towelling moistened with one‐fifth strength Long Ashton nutrient medium containing nitrate as the N source (Hewitt and Smith, 1975). Three‐day‐old seedlings were used for the experiments. Five seedlings were transferred to Petri dishes containing two 9 cm diameter filter paper discs and 7 cm3 of embryo medium. The embryo medium contained one‐fifth strength Long Ashton solution plus 50 mol m–3 glucose and antibiotic/antimycotic mix (10 mm3 cm–3, Sigma‐Aldrich Co., Poole, UK) to minimize microbial contamination. Additions for individual experiments were proline, asparagine, glutamate, glutamine (all 25 mol m–3) or an endosperm amino acid mixture. The amino acid mixture (total concentration 25 mol m–3) was based on the maize endosperm storage protein amino acid composition (Oaks and Beevers, 1964) and contained the following (mol m–3): alanine (2·7), arginine (0·5), aspartate (1·5), cysteine (0·3), glutamate (4·8), glycine (1·1), histidine (0·6), isoleucine (0·9), leucine (3·9), lysine (0·25), methionine (0·4), phenylalanine (1·1), proline (2·8), serine (1·7), threonine (0·85), tyrosine (0·8) and valine (1·4). These were dissolved in the embryo medium and buffered at pH 6 with 10 mol m–3 MES. Water deficit was applied by adding polyethylene glycol 3350 (PEG 3350) to the medium to obtain the desired water potential (Money, 1989). For convenience, the high water potential treatment, in which PEG was not added, is referred to as 0 MPa. Root growth was measured as the increase in length over the duration of the experiments (20 h). Exposure of the seedlings to water deficit by placing them on filter papers moistened with PEG solutions minimized hypoxia which could be caused by submerging roots in viscous PEG solutions. As long as aeration is sufficient, there is no evidence that PEG is harmful to maize seedling roots (Verslues et al., 1998). Aeration was sufficient in our experiments as evidenced by the fact that PEG treatment did not cause enhanced accumulation of alanine or 4‐amino butyric acid (GABA) (Table 1), as occurs under hypoxia (Roberts et al., 1992; Good and Muench, 1993). The use of a liquid medium such as PEG solution, rather than a solid rooting medium, is essential to allow administration of substrates and radiolabelled compounds (Verslues and Sharp, 1999). Endosperms were dissected from the seedlings leaving the scutellum intact. For experiments with isolated root tips the apical 5 mm was excised and incubated in flasks containing aerated embryo medium.

Table 1.

The composition of the soluble amino acid pool (nmol per root tip) in 5‐mm tips of primary roots from maize seedlings which had previously been treated for 20 h with and without their endosperms at water potentials of 0 and –1·35 MPa

Intact seedlings Endosperm removed
No amino acids + Amino acids No amino acids + Amino acids
Amino acid 0 MPa –1·35 MPa 0 MPa –1·35 MPa 0 MPa –1·35 MPa 0 MPa –1·35 MPa
Ala 28·9 25·7 67·0 187·4 17·0 33·4 39·0 21·5
Arg 5·9 6·2 9·8 14·9 7·8 10·5 11·4 7·7
Asp 33·6 33·8 37·1 34·6 30·3 37·7 28·5 31·3
Asn 53·7 124·6 62·4 156·4 135·3 196·5 86·7 162·1
GABA 3·9 3·9 8·3 5·3 0 2·8 1·8 2·5
Glu 50·3 60·9 53·5 87·7 28·4 53·7 28·9 41·1
Gln 83·9 67·6 128·0 75·5 40·7 62·6 81·9 24·7
Gly 12·6 25·7 17·1 25·7 13·4 15·6 11·3 10·7
His 17·6 16·2 20·1 28·7 14·2 17·5 15·3 16·8
Ile 4·4 7·8 10·5 12·4 9·2 12·9 7·6 10·0
Leu 5·2 6·3 29·8 52·3 8·3 27·1 40·9 43·9
Lys 5·7 1·9 3·5 6·0 1·8 3·9 2·9 2·2
Phe 6·4 7·3 12·2 19·5 5·7 10·1 10·6 9·5
Ser 27·8 26·6 38·2 60·16 21·2 39·7 31·4 35·2
Tyr 4·9 3·0 7·8 3·0 0·5 4·3 4·9 3·7
Val 20·5 25·5 24·61 46·2 19·7 32·8 25·5 30
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Some of the seedlings were also incubated in an amino acid mixture mimicking the composition of the endosperm storage proteins. Values are means of three replicates.

Soluble proline, amino acid and carbohydrate pools

The apical 5 mm of the primary roots (five per sample) was excised and extracted in one of two ways. In the first method samples were placed in 1 cm3 80 % ethanol and steeped for 24 h at 4 °C to extract solutes. In the second method they were homogenized in 1 cm3 3 % sulfosalicylic acid and then centrifuged for 5 min at high speed in a microcentrifuge. The two methods gave comparable results. The supernatants were assayed for proline using acid ninhydrin (Bates et al., 1973). Individual amino acids were identified and quantified by high performance liquid chromatography (HPLC) of the 80 % ethanol extracts. HPLC of the o‐phthalaldehyde‐thiol (OPT) derivatives (Joseph and Marsden, 1986) was carried out with a Spherisorb ODS 2 (5 µm) column at 0·75 cm3 min–1. Mobile phase A was 200 mol m–3 sodium acetate/acetic acid buffer, pH 5·9, containing 1·5 % (v/v) tetrahydrofuran. Mobile phase B was methanol. The OPT derivatives were eluted with the following gradient: 0 min (85 % A), 15 min (75 % A), 41–50 min (10 % A). The derivatives were detected by absorbance at 340 nm. The internal standard was taurine. This method does not derivatize proline. Soluble carbohydrates were measured by the Dubois method (Dubois et al., 1956) with glucose as the standard.

Metabolism of 14C‐labelled proline and glutamate

Transport and metabolism of proline in 3‐d‐old maize seedlings was investigated by applying [U‐14C]proline to the scutellum. Endosperms were removed from seedlings and 4 mm3 of [U‐14C]proline (specific activity 9·5 GBq mmol–1) containing 14·8 kBq was placed on the exposed surface of the scutellum. A ring of petroleum jelly was placed between the scutellum and the remainder of the embryonic axis to prevent surface diffusion of label. The seedlings were then laid horizontally on dishes containing filter paper soaked with nutrient medium without or with PEG 3350 (–1·35 MPa). The dishes were placed in Plexiglass boxes. The seedlings were labelled for 4 h (pulse period). After this time some were extracted. A further set of seedlings was removed from the boxes, the unabsorbed label was washed from the scutellum surface and then the seedlings were incubated for another 2 h (chase). At the end of the labelling periods the seedlings were separated into coleoptile, scutellum, 5‐mm primary root tip, and remainder of the root and extracted in 80 % ethanol as described above. Glutamate and proline metabolism by 5‐mm root tips was followed by placing ten tips in small glass vials with 30 mm3 embryo medium with and without PEG 3350 (–1·35 MPa). [U‐14C]Proline (7·4 kBq) and [U‐14C]glutamate (18·5 kBq, specific activity 9·5 GBq mmol–1) were added and the roots incubated for 2 h in plexiglass boxes. Carbon dioxide was trapped on filter paper wicks soaked with 28 % (w/v) KOH. The roots were extracted with 80 % ethanol at the end of the labelling period.

The supernatants from the 80 % ethanol homogenates were separated into neutral + acidic, and basic fractions by cation exchange chromatography on a column (2 cm × 1 cm) of Dowex 50WX8‐400. Neutral and acidic compounds were eluted with 5 cm3 water, and basic compounds (mainly amino acids) were eluted with 5 cm3 of 6 m ammonium hydroxide. The basic fraction was freeze dried or dried under an air stream and redissolved in a small volume of water. The pellet was washed twice with 80 % ethanol and then resuspended in water. The 14C content of aliquots of the soluble fraction, the insoluble fraction (resuspended pellet), trapped CO2 and the neutral + acidic and basic fractions was determined by liquid scintillation counting (Packard Tri‐Carb 2500 TR liquid scintillation analyser) using Packard Emulsifier‐Safe scintillation cocktail (Packard, Meriden, CT, USA). Radioactivity in individual amino acids was determined after TLC separation of the basic fraction. TLC was carried out on silica gel plates (Whatman, 60 Å, 250 µm layer thickness, 20 × 20 cm) with a mobile phase of phenol (75 g) : water (25 cm3) : acetic acid (5 cm3). Radioactivity on the dried plates was detected with a Berthold LB2832 Linear TLC Analyser (Berthold Analytical, Gaithersburg, MD, USA). Amino acids and proline were visualized by spraying with ninhydrin (0·5 % in acetone) followed by gentle heating.

Amino acid and sugar release from isolated endosperms

Endosperms were detached from the scutellum and sliced longitudinally into six segments. The endosperm segments (from four endosperms per replicate) were rinsed thoroughly and incubated in 5 cm3 water at 30 °C. Samples of the water were taken at intervals, centrifuged for 3 min in a microcentrifuge to remove particulate material and analysed for proline and soluble sugars as described above. Total amino acids (excluding proline) were measured with ninhydrin. The extract (total volume 1 cm3) was added to 0·5 cm3 citrate buffer (16·8 g citric acid and 6·4 g NaOH in 100 cm3 water) and 1·2 cm3 ninhydrin reagent (1 % ninhydrin and 0·03 % sodium ascorbate in 2‐methoxyethanol). The mixture was boiled for 26 min, cooled on ice and diluted with 3 cm3 60 % ethanol. Absorbance was read at 570 nm. The standard was glutamate.

Data analysis

Each experiment was repeated two to three times with similar results. Error bars, where shown, are mean ± s.d. (n = 3).

RESULTS

Endosperm removal and root excision prevent proline accumulation

Proline accumulation in maize root tips started 4 h after transfer to low water potential. Accumulation then continued in a linear manner for a further 20 h (Fig. 1). Root tip proline content was linearly related to water potential after 20 h (Fig. 2). Proline in root tips from seedlings without endosperms was 30 % lower than in intact seedlings and proline hardly accumulated at low water potential (Fig. 2). Excised 5‐mm root tips also had a reduced free proline pool after 20 h incubation and did not accumulate proline at low water potential (Fig. 2). Proline accumulation in the coleoptiles at low water potential was also prevented by endosperm removal (data not shown). The endosperm is therefore required for proline accumulation by the roots and coleoptiles. Endosperm removal also decreased proline accumulation when the seedlings were allowed to recover for a further 24 h (data not shown). The endosperm is the source of carbohydrate and organic nitrogen for embryo growth so it is possible that its removal limits the substrate supply for proline synthesis even though glucose and nitrate were supplied. The effect of amino acid additions on proline accumulation was therefore investigated.

graphic file with name mcf082f1.jpg

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Fig. 1. The time course of proline accumulation in maize seedling root tips after transfer to low water potential (–0·9 MPa). Seedlings were 3‐d‐old at the start of treatment. Water potential was adjusted by adding PEG 3350 to the nutrient medium. Proline was measured in the 5‐mm root tips.

graphic file with name mcf082f2.jpg

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Fig. 2. Proline content of maize seedling primary roots in response to low water potential, root excision and endosperm removal. Three‐day‐old seedlings were exposed to the treatments for 20 h. Water potential was adjusted by adding PEG 3350 to the nutrient medium. Proline was measured in the 5‐mm root tips.

The effect of exogenous proline and amino acids on proline accumulation

Endosperm removal decreased the proline pool by 80 % at both the high and low water potential (Fig. 3). Neither glutamine nor glutamate and asparagine (data not shown) restored the proline pools in seedlings without endosperms. The amino acid mixture did allow increased proline accumulation but this did not reach the level found in intact seedlings (Fig. 3). In roots with and without endosperms, exogenous proline increased the size of the proline pool to the same level. Accumulation of exogenous proline at –1·35 MPa was 2·5 times (seedlings with endosperm) and 3·7 times (seedlings without endosperm) the level in roots at high water potential (Fig. 3). Low water potential had little effect on the total free amino acid pool and this was much less affected by endosperm removal (23 % decrease) than by proline (data not shown). The effect of low water potential and endosperm removal on individual amino acids is shown in Table 1. The major components of the amino acid pool were aspartate, asparagine, glutamate and glutamine. Low water potential caused a two‐fold increase in asparagine but had no consistent effect on any other amino acids. Endosperm removal decreased the glutamate and glutamine pools but increased the amount of asparagine. However, at –1·35 MPa, glutamate and glutamine levels were not affected by endosperm removal. The other amino acids were little affected by endosperm removal. The amino acid mixture had relatively little effect on the composition of the amino acid pool. The principal effects were an increase in glutamine and a very marked increase in leucine. Overall these results suggest that the lack of proline accumulation in embyros without endosperms is not caused by lack of suitable precursors but is possibly the result of a deficiency in biosynthetic capacity.

graphic file with name mcf082f3.jpg

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Fig. 3. The effect of endosperm removal and amino acid additions on the proline content of maize seedling primary roots grown at high (0 MPa) and low (–1·35 MPa) water potential. Amino acids were added to the nutrient medium at 25 mol m–3. Water potential was adjusted by adding PEG 3350 to the nutrient medium. Proline was measured in extracts of the 5‐mm root tips after 20 h. Additions: 1, none; 2, glutamine; 3, proline; 4, amino acid mixture.

Metabolism of [U‐14C]glutamate and [U‐14C]proline by root tips

The capacity for proline synthesis and turnover in isolated root tips was determined by following [U‐14C]glutamate and [U‐14C]proline metabolism, respectively. Seedlings with and without endosperms were exposed to low water potential for 4 and 24 h. They were then labelled for 2 h. After 24 h pre‐treatment, glutamate uptake was increased at low water potential (Fig. 4A). Proline uptake was unaffected (Fig. 4B). Endosperm removal increased both glutamate and proline uptake at high water potential after 24 h (Fig. 4B). At low water potential endosperm removal had no effect (Fig. 4B). The proportion of glutamate incorporated into the ethanol insoluble fraction and carbon dioxide was decreased at low water potential. Endosperm removal had no effect (Fig. 5A). Low water potential caused a very marked decrease in incorporation of proline into the insoluble fraction. Endosperm removal increased incorporation into the insoluble fraction at high water potential. Labelling of the insoluble fraction was decreased at low water potential but was still higher than in intact seedlings at high water potential (Fig. 5B). In all cases, 15 % or less of the soluble fraction comprised neutral and acidic compounds (data not shown). The remainder consisted of amino acids. Low water potential increased the proportion of [U‐14C]glutamate incorporated into proline in seedlings with and without endosperms (Fig. 6C). The other compounds labelled included aspartate and glutamine. The labelling pattern in these compounds was unaffected by low water potential or endosperm removal (Fig. 6C). Very much less [U‐14C]proline was converted to other amino acids at low water potential. Endosperm removal at high water potential increased conversion of proline to other amino acids, mainly aspartate, glutamate and glutamine (Fig. 6D).

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Fig. 4. Uptake of [U‐14C]glutamate and [U‐14C]proline by 5‐mm root tips from maize seedlings previously grown at 0 and –1·35 MPa for 4 (A) and 24 h (B) with or without endosperm removal. Label was supplied to excised root tips for 2 h after the pre‐treatments. Water potential was adjusted by adding PEG 3350 to the nutrient medium.

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Fig. 5. Distribution of 14C between 80 % ethanol soluble material, insoluble material and carbon dioxide in 5‐mm maize root tips after metabolism of [U‐14C]glutamate (A) and [U‐14C]proline (B). The root tips were excised from seedlings previously grown at 0 and –1·35 MPa for 4 h with or without endosperm removal. Label was supplied to excised root tips for 2 h after the pre‐treatments. Water potential was adjusted by adding PEG 3350 to the nutrient medium.

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Fig. 6. Distribution of 14C in soluble amino acids after metabolism of [U‐14C]glutamate (A and C) and [U‐14C]proline (B and D). Root tips were excised from seedlings previously grown at 0 and –1·35 MPa for 4 h (A and B) and 24 h (C and D) with or without endosperm removal. Label was supplied to excised root tips for 2 h after the pre‐treatments. Water potential was adjusted by adding PEG 3350 to the nutrient medium.

After a 4 h pre‐treatment at –1·35 MPa, proline had not begun to accumulate (Fig. 1). Neither low water potential nor endosperm removal affected uptake of [U‐14C]glutamate or [U‐14C]proline (Fig. 4A). The amount of glutamate taken up was higher than in the older seedlings (Fig. 4A and B). A smaller proportion of glutamate was incorporated into insoluble material and CO2 at low water potential, and endosperm removal had no effect (Fig. 5A). 14C incorporation from proline into insoluble material and CO2 was decreased at low water potential. Similarly, but less marked than the 24 h pre‐treatment, endosperm removal increased proline incorporation into the insoluble fraction (Fig. 5B). The distribution of label in the amino acid fraction after glutamate feeding was similar to 24 h pre‐treatment, with a small but consistent increase in the proportion of label in proline (Fig. 6A). The proportion of label remaining in proline after [U‐14C]proline feeding was greater at low potential. Endosperm removal, as previously, increased conversion of proline to other amino acids (Fig. 6B).

Overall, these results show that low water potential, even after 4 h, increased the proportion of labelled glutamate incorporated into proline. At the same time the proportion of proline respired, incorporated into insoluble material and converted to other amino acids decreased. To interpret these data as relative synthesis and turnover rates of proline it is necessary to take account of the pool sizes of the glutamate and proline precursors. In the short‐term experiment, pool sizes of proline and glutamate were unaffected which allows the conclusion that low water potential increases proline synthesis and decreases proline utilization (turnover and incorporation into protein). Estimates of the relative rates of synthesis and utilization can be made by estimating the mean specific activity of each precursor from total label uptake and pool size. A further assumption is that the labelling period is short enough to avoid extensive recycling of label. The estimated synthesis and utilization rates 4 h after transfer to low water potential are shown in Table 2. Proline synthesis increased after 4 h at –1·35 MPa. After 24 h, the rate of proline synthesis increased further at –1·35 MPa. Endosperm removal did not affect synthesis rate. Utilization at low water potential decreased by 67 % after 4 h. The decrease in seedlings with their endosperms removed was less (29 %). This reflects the increased metabolism and incorporation into protein caused by endosperm removal. The capacity for proline utilization by the root tips is five to ten times higher than the biosynthetic capacity.

Table 2.

Proline synthesis and utilization rate in 5‐mm tips of primary roots from maize seedlings which had been previously treated for 4 and 24 h with and without their endosperms at 0 and –1·35 MPa. Excised roots were then labelled for 2 h with [14C]‐glutamate or proline

Intact seedlings Endosperm removed
Treatment (h) 0 MPa –1·35 MPa 0 MPa –1·35 MPa
[14C]proline synthesis from [14C]glutamate 4 0·01–0·03 0·08–0·10 0 0·07–0·08
24 0–0·23 0·24–0·28 0–0·03 0·16–0·29
[14C]proline utilization 4 4·0–4·3 0·5–2·2 4·2–4·4 2·6–3·5
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Rates were estimated from the label from glutamate appearing in proline (synthesis) or from the label from proline appearing in compounds other than free proline (utilization) and the mean specific activity of the precursor pool through the labelling period of 2 h. The values are µmol proline 2 h–1 g–1 f. wt (range of values from two experiments).

Transport of [U‐14C]proline in maize seedlings

The above results suggest that root tips cannot accumulate proline in the absence of the endosperm because their proline utilization rate exceeds their biosynthetic capacity. The data also suggest that the endosperm is the source of the proline which accumulates in the root tips at low water potential. This was investigated further by measuring the release of proline from isolated endosperms and by following transport of [U‐14C]proline from the scutellum to other parts of the seedling.

Proline accumulates at low water potential in all parts of the seedling but accumulation is most marked in the 5‐mm root tip (Fig. 7A). [U‐14C]Proline was applied to the surface of scutella after removal of the endosperm. The seedlings were then left for 4 h (pulse period) and for a further 2 h (chase period) after washing unabsorbed label from the scutellum surface. After labelling, the seedlings were separated into coleoptile, scutellum, 5‐mm root tip and the remainder of the root. Identical results were obtained after the pulse and chase periods and the results presented are the mean of four experiments. Seedlings pre‐treated at 0 MPa respired 3·2 % of the absorbed label as CO2 while seedlings pre‐treated and labelled at –1·35 MPa respired 0·6 % of absorbed proline. The root tips were stronger sinks for labelled proline transported from the scutellum than the root and coleoptile (Fig. 7B). Low water potential decreased the amount and proportion of proline incorporated into the insoluble fraction. In the remaining soluble fraction, the amount remaining as free proline was much larger at low water potential. This was particularly marked in the root tip (Fig. 7B). The relative rate of transport of proline from the scutellum to root tip was calculated by estimating the mean specific activity of the source proline in the scutellum during the labelling period and using this to convert the amount of radioactivity in the tip to the amount of proline equivalents transported (Table 3). The transport of label to the root tip was 43 % lower at –1·35 MPa. However, at –1·35 MPa, 86 % of the label appearing in the root tips accumulated as free proline compared with 15 % at low water potential, so the overall estimated rate of proline transport and accumulation per 5‐mm tip was 3·3‐fold greater at low water potential. Proline accumulation in the root tip is therefore the result of decreased oxidation and incorporation into protein at low water potential.

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Fig. 7. A, Proline accumulation in the coleoptile, scutellum, 5‐mm root tip and remainder of the primary root after exposure to low water potential (–1·35 MPa) for 24 h. B, The effect of low water potential (–1·35 MPa) on distribution of 14C between the coleoptile, scutellum, 5‐mm root tip and remainder of the primary root after application of [U‐14C]proline to the scutellum. Three‐day‐old seedlings were exposed to low water potential by adding PEG 3350 to the nutrient medium 24 h before addition of label.

Table 3.

Transport of proline from the scutellum to the root tip of maize seedlings at high (0 MPa) and low (‐1·35 MPa) water potential

0 MPa –1·35 MPa
Total 14C transported to root tip 126 (100–152) 72 (34–100)
14C in root tip accumulating in soluble fraction 92 (70–113) 70 (31–100)
14C in root tip accumulating as free amino acids 68 (56–80) 69 (31–98)
14C accumulating as free proline 19 (17–21) 62 (31–86)
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[14C]proline was applied to scutella and the transport of label to the root tip measured after 4–6 h. The rate of transport was estimated from the 14C appearing in the root tips over the labelling period and the mean specific activity of the proline pool in the scutellum through the labelling period. The rates (nmol h–1 per root tip) are means, with ranges in parentheses.

Amino acid, proline and sugar release from isolated endosperms

Endosperms were removed from 3‐d‐old seedlings which had been exposed to –1·35 MPa for 24 h to determine if the capacity of the endosperm to hydrolyse storage products was affected by water deficit. Sliced endosperms were incubated in water and the release of sugars, amino acids and proline was monitored (Fig. 8). Release was the same at high and low water potential. Proline constituted 10 % of the amino acids released from the endosperm.

graphic file with name mcf082f8.jpg

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Fig. 8. Release of amino acids (A), proline (B) and sugars (C) from endosperm slices. Three‐day‐old maize seedlings were exposed to low water potential by adding PEG 3350 to the nutrient medium for 24 h before removing the endosperms. They were then incubated in water and aliquots taken at intervals for solute analysis.

DISCUSSION

Excision of maize root tips causes a rapid decrease (62 % in 3 h) of the soluble proline pool (Barnard and Oaks, 1970). We have found that endosperm removal has the same effect, supporting the suggestion that this is the major source of proline for the root tips in germinating seedlings (Oaks, 1966). Our results show that root excision or endosperm removal also decrease proline accumulation at low water potential. We have therefore addressed the question of how root tips, with apparently restricted proline biosynthesis capacity, can accumulate high proline concentrations at low water potential.

Proline accumulation is not limited by precursor supply

Addition of a range of amino acids to seedlings without endosperms did not restore proline accumulation to levels found in intact seedlings. An amino acid mixture previously shown to support the growth of maize embryos (Oaks, 1967) was most effective, while additions of glutamate, glutamine or asparagine were not effective. Glutamate is the predominant precursor of proline (Samaras et al., 1995). Additionally, glutamate pools are not greatly affected by endosperm removal. Other possible proline precursors are arginine and ornithine (Samaras et al., 1995). Competition experiments suggest that these are not proline precursors in maize roots while glutamate is incorporated (Oaks et al., 1970), and labelled ornithine is not a major source of proline in maize roots (Verslues and Sharp, 1999). Since the roots were supplied with glucose, carbon limitation is not the cause of lack of proline accumulation. The results suggest that substrate supply is not the major limitation on proline accumulation at low water potential.

Proline synthesis rate is lower than proline utilization rate at high and low water potential

The relative rates of proline synthesis and utilization (oxidation, conversion to other amino acids and incorporation into protein) were estimated by the incorporation of [14C]glutamate into proline and metabolism of [14C]proline over short time courses. The rates were estimated from the mean specific activity of the respective precursor pools (calculated from total uptake of label and pool concentration). The other major assumption is that there is no intracellular compartmentation of pools and that exogenous label is therefore metabolized in a similar way to the relevant endogenous pool. In the case of proline this condition is not strictly fulfilled because exogenous [14C]proline is oxidized more rapidly than would be expected from the rate of decrease in the proline pool after root excision (Barnard and Oaks, 1970). Nevertheless, the calculated rates provide a comparative estimate of proline synthesis and turnover. The rate of proline utilization was five to ten times greater than the rate of synthesis. This explains the rapid loss of the proline pool after root excision or endosperm removal. The low rate of proline biosynthesis is more likely to be caused by the low capacity of biosynthesis enzymes rather than by feedback inhibition of P5CS (Hong et al., 2000) since biosynthesis does not recover within 24 h of removing the endosperm. Low water potential increased proline synthesis from [14C]glutamate and decreased the rate of proline utilization. Both these effects were observed 4 h after transfer to low water potential which is just before proline accumulation can be measured in the roots. The response was more marked after 24 h. The labelling pattern suggests that low water potential decreases proline incorporation into protein and also its oxidation in mitochondria. The rate of protein synthesis decreases at low water potential in germinating seedlings (Bewley and Larsen, 1980). Proline is metabolized to glutamate and TCA cycle acids in mitochondria and the first enzyme on this pathway is PDH. Mitochondrial PDH activity decreases in maize seedlings exposed to low water potential (Rayapati and Stewart, 1991). This is consistent with the labelling data reported here. PDH has recently been cloned and its mRNA levels decrease rapidly in droughted plants suggesting that proline oxidation is controlled at least partly at the transcriptional level (Kiyosue et al., 1996; Peng et al., 1996; Verbruggen et al., 1996). Even though proline synthesis increased and oxidation decreased at low water potential, showing that maize root tips behave in a similar manner to other plant tissues, proline accumulation in the absence of the endosperm does not occur because the rate of utilization still exceeds the rate of synthesis. It must therefore be concluded that the proline that accumulates in the root tips at low water potential is derived as such from the endosperm.

Endosperm removal increases proline uptake and metabolism

When seedlings grown at high water potential were pre‐treated for 24 h by removing endosperms the subsequent uptake of [14C]proline and glutamate was increased. There was no response 4 h after removing the endosperm. Glutamate metabolism was not affected but, in the case of proline, there was a large increase in the proportion that was oxidized (i.e. appearing in other amino acids, organic acids and sugars) and incorporated into protein. This effect was detected 4 h after endosperm removal but was more marked after 24 h, suggesting that the amino acid starvation induced by endosperm removal increases uptake capacity. In the case of proline, incorporation into protein and oxidation via mitochondrial PDH is enhanced. At 4 h, the effect of low water potential on proline utilization was reduced in seedlings without endosperms suggesting that the proline starvation response counteracts the reduction in PDH activity at low water potential. The expression of PDH mRNA in arabidopsis at high water potential is increased by feeding exogenous proline. However, proline feeding does not reverse the down‐regulation of PDH mRNA at low water potential (Peng et al., 1996; Verbruggen et al., 1996). These results suggest another possible level of control to proline oxidation: the decrease in PDH activity at low water potential is partly reversed by proline starvation. Further work is required to determine whether PDH transcript levels are affected by endosperm removal. Uptake into the root tips could be mediated by the proline transport proteins (ProT1 and ProT2), which have been cloned from arabidopsis by functional complementation of yeast proline transport mutants (Rentsch et al., 1996). Transcript levels of ProT2 were increased by water stress in arabidopsis suggesting that proline transport capacity could be increased at low water potential. However, in maize root tips from intact seedlings, low water potential, unlike endosperm removal, did not induce increased uptake of [14C]proline, although uptake of glutamate was increased. Unlabelled exogenous proline was accumulated by maize root tips over 24 h to higher levels than at high water potential. Based on the above results, it would seem that this is largely caused by the inhibition of proline oxidation and incorporation into protein at low water potential rather than by faster uptake.

Proline transport from the endosperm

The capacity for proline biosynthesis in the roots is not sufficient to maintain the normal concentration and the proline that accumulates in the root tips at low water potential in intact seedlings must be transported from the endosperm. Proline constitutes about 10 % of the amino acid residues in maize endosperm storage proteins, due to the high proline content of zein (Casey et al., 1997). Isolated endosperms released amino acids, proline and soluble sugars. As expected, proline constituted 10 % of the amino acids. Endosperms removed from seedlings previously grown at low water potential had only a marginal, and statistically insignificant, increase in the rate of release of sugars and amino acids suggesting that the capacity of the hydrolytic enzymes in the endosperm was not altered by water status. It has been suggested that ABA, which accumulates in maize seedlings at low water potential (Saab et al., 1990), can decrease amylase expression and also induce an amylase inhibitor protein (Robertson et al., 1989) thereby matching the release of endosperm reserves to growth rate. This effect was not evident but since the embryos grow more slowly at low water potential they will potentially be supplied with relatively higher concentrations of proline, amino acids and sugars which could contribute to osmotic adjustment. Indeed, accumulation of hexose, the main osmoregulatory compound in the root elongation zone, is attributed to slower growth rather than increased deposition rate (Sharp et al., 1990), which implies that the endosperm supply must be maintained. The interface between the endosperm and embryo is the scutellum. The surface of the scutellum has a high uptake capacity for amino acids from the endosperm (Sopanen et al., 1980). Proline is taken up by barley scutella using a combination of a broad specificity amino acid transporter and a proline‐specific transporter (Väisänen and Sopanen, 1986) which has similar properties to the ProT tranporter identified in arabidopsis (Rentsch et al., 1996). The uptake capacity of the scutella for proline was not increased at low water potential, although the increased ProT2 transcript levels in arabidopsis seedlings at low water potential have been suggested to reflect increased proline transport capacity (Rentsch et al., 1996). The labelled proline applied to the scutellum was translocated to all parts of the seedling but the root tip was the strongest sink. Proline, asparagine and other neutral and basic amino acids synthesized from [14C]acetate applied to the maize scutellum are also preferentially translocated to the root tip (Oaks, 1966). When the relative rates of transport of 14C from the scutellum to root tip are calculated it is evident that the transport rate is lower at low water potential. However, because the oxidation and incorporation into protein is less at low water potential, a large proportion of the proline (86 %) arriving in the root tip accumulates as free proline. The results show that the increased deposition rate of free proline in the root tip is the result of transport from the scutellum combined with lower utilization in the root tip.

CONCLUSIONS

Proline accumulates in maize root tips where it provides a major part of the osmotic adjustment in the meristem at low water potential (Voetberg and Sharp, 1991; Verslues and Sharp, 1999). The mechanism behind this accumulation has been investigated. It is concluded that the rate of proline utilization in maize roots exceeds the biosynthesis capacity even at low water potential. This agrees with the results of Verslues and Sharp (1999). Our results show that the proline that accumulates at low water potential is translocated from the endosperm of germinating seedlings, supporting the speculation of Verslues and Sharp (1999). The accumulation is a consequence of sustained release from the endosperm (despite a reduced seedling growth rate at low water potential) and a reduced rate of both proline oxidation by PDH in mitochondria and utilization for protein synthesis at low water potential. These processes are controlled in a precise manner which results in proline accumulation being proportional to water potential. In contrast to this conclusion, Verslues and Sharp (1999) suggested that proline utilization is not reduced at low water potential and that the transport rate of proline to the tip is increased at low water potential. The capacity for proline biosynthesis in root tips from older plants after exhaustion of the endosperm reserves has not yet been investigated. Not all species (e.g. pea) lack autonomy in proline accumulation by their seedling root tips and we can speculate that this is related to the smaller proportion of proline in their storage proteins. Maize seedlings also have high PDH activity in comparison with pea (Overy and Smirnoff, unpubl. res.) which may allow them to utilize their abundant endosperm‐derived proline rapidly as a carbon and nitrogen source.

Supplementary Material

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Received: 30 October 2001; Returned for revision: 15 November 2001; Accepted: 11 January 2002.

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