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Proline accumulation and Δ1-pyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance
Plant Science 165 (2003) 1059 /1068 www.elsevier.com/locate/plantsci
Proline accumulation and D1-pyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance Do Thu Hien a, Michel Jacobs a, Geert Angenon a,*, Christian Hermans b, Tran Thanh Thu a, Le Van Son a,1, Nancy He´le`ne Roosens a,b b
a Laboratory of Plant Genetics, Vrije Universiteit Brussel (VUB), Campus Etterbeek, Gebouw E Pleinlaan 2, B-1050 Brussels, Belgium Lab de Physiologie et de Ge´ne´tique Mole´culaire des Plantes, Universite´ Libre de Bruxelles (ULB), CP242, Boulevard du Triomphe, 1050 Bruxelles, Belgium
Received 17 September 2002; received in revised form 25 June 2003; accepted 3 July 2003
Abstract Three indica rice cultivars (Oryza sativa ) differing in their tolerance to salt and drought stress in field conditions in Vietnam were analyzed at the molecular and biochemical levels with the goal to reveal the basis for their differential behavior and in particular their ability to accumulate proline. An in vitro growth test showed that after a 7-day period of stress, the fresh weight of plantlet roots appears to be a relevant parameter for differentiating drought and salt tolerance of the concerned cultivars. Sodium level was lower in the salt tolerant cultivar than in the other rice cultivars. Proline accumulation in roots of tolerant cultivars starts earlier after the initiation of the stress treatment than that of the osmotic stress sensitive cultivar and also reaches a higher level. Proline accumulation was not related to proteolysis and so could be the result from induction of proline biosynthesis by osmotic stress. However, neither the sequence of amino acids involved in the proline feedback inhibition of the key regulatory enzyme D1-pyrroline5-carboxylate synthetase (P5CS; EC not assigned), nor the expression of the p5cs genes were modified in the tolerant cultivars. These observations suggest that proline accumulation in roots is a possible indicator of the osmotic tolerance in these rice cultivars. However, other mechanisms than those related to a change in P5CS regulation are responsible for the increased proline content. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Proline accumulation; D1-Pyrroline-5-carboxylate synthetase; Osmotic stress
1. Introduction Osmotic stress is the most serious problem affecting crop productivity and limiting agricultural expansion in many areas of the world. A large number of plant species accumulate proline in response to osmotic stress [1,2]. Therefore, it was suggested that this amino acid be involved in stress tolerance by playing a role in counteracting the effects of osmotic stress [2,3]. Direct evidence supporting this hypothesis was provided by some
* Corresponding author. Tel.: /32-2-629-1935; fax: /32-2-6291867. E-mail address: [email protected] (G. Angenon). 1 Present address: Laboratory of Plant Biotechnology, Institute of Biotechnology, Hanoi, Vietnam.
mutant and transgenic proline overproducing plants, which show increased osmotolerance [4 /7]. However, in some species or ecotypes, the data do not always indicate a positive correlation between proline accumulation and adaptation to drought and salt stress [2,8 / 10]. In rice, data are particularly contradictory. Some authors suggest that proline accumulation may be related to the degree of salt tolerance in Oryza sativa [11]. Others suggested that proline is a symptom of salt stress injury rather than an indicator of the resistance [9]. All these controversies may reflect, in these species or ecotypes, the predominance of the role of tolerance mechanisms other than osmotic adjustment, such as specific morphological and physiological modifications. Since these studies were often performed after submitting the plants to long periods of stress, part of the
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00301-7
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discrepancy could be due to the fact that proline accumulation is altered as a result of injury. In higher plants, proline accumulation appears to be essentially due to de novo synthesis [12,13] and catabolism rate minimization [13 /16] but lower proline utilization in protein synthesis and increased proteolysis may also contribute to net proline accumulation [12,15,17,18]. P5CS, which catalyzes the first two steps in proline biosynthesis, is critical for the control of the proline biosynthesis and consequently the increase in osmotolerance [5,6,19]. This essential role is probably due to two levels of control exerted at those steps: proline feedback inhibition on the P5CS protein [20,21] and regulation of the P5CS transcript level in response to osmotic stress conditions [11,21/23]. In plants, numerous studies suggest that P5CS is feedback regulated by proline and the release of feedback inhibition of P5CS enzyme can lead to an increase in proline accumulation in response to stress conferring better osmotolerance [21,24]. It has been previously proposed that the origin of salt tolerance in a mutant of Nicotiana plumbaginifolia was due to a mutation leading to a substantial reduction in the feedback inhibition by proline at the level of the P5CS enzyme [25]. Transgenic tobacco overexpressing a feedback insensitive form of the enzyme, accumulates more proline and shows more resistance to osmotic stress than the plant expressing the wild type P5CS [6]. Besides the proline feedback control of the P5CS activity, expression of the p5cs gene involves a second key level of regulation. Indeed, the p5cs gene is transcriptionally induced by osmotic stress and its induction precedes the accumulation of proline [26]. Accumulation of proline and expression of the p5cs gene were strongly induced after 48 h in salt-tolerant rice under high-salt conditions (250 mM) when compared with a sensitive one [11]. This strongly supports that proline accumulation and the induction of P5CS shortly after the start of salt stress may be related to the degree of salt tolerance in O. sativa . In the present study, we made use of three cultivars Cuom, DR2 and CR203 which are currently grown in Vietnam and which differ in salt and drought tolerance under field conditions. The cultivar CR203 is an elite breeding line from the International Rice Research Institute (Los Ban˜os, Philippines) [27], sensitive to salt and drought stress. DR2 is a drought tolerant elite line [28] resulting from somaclonal variation of the CR203 cultivar after in vitro selection under drought conditions. Cuom is a local cultivar grown in saline areas near the coast. In in vitro callus induction assays, Cuom showed higher survival rate and regeneration frequencies after salt stress than CR203 [29]. We assessed whether proline can be correlated with the observed differences in osmotic stress tolerance by measuring the content of this amino acid in these cultivars. We also
evaluated if differences in proline accumulation could be due to changes in the feedback sensitivity of P5CS by analyzing the structure of the P5CS allosteric site or to variation in the relative expression of the p5cs gene.
2. Materials and methods 2.1. Plant growth and stress treatment Three O. sativa (indica) cultivars Cuom, DR2 and CR203 from Vietnam were used in this study. These three cultivars were provided by the Institute of Biotechnology (Hanoi, Vietnam) and are cultivated in the North of Vietnam. The seeds were dark-germinated in vitro on solid MS medium [30] and grown in a culture room (24 8C, 16 h illumination, 8 h dark cycle). Eight-day-old plantlets were used for the stress treatment. Salt stress and osmotic stress treatment consisted in adding to the MS medium, respectively, 200 mM NaCl and an iso-osmotic solution of mannitol (66.4 g/l) causing an osmotic pressure of 0.380 Os/kg. 2.2. Determination of proline content and total amino acids After stress treatment, around 0.3 g fresh weight of rice plantlets was extracted with 3% (w/v) sulfosalicylic acid on ice. The supernatant containing total amino acids was transferred to a new tube for determination of proline and total amino acids. The tissue precipitates were saved for protein extraction (see below). Proline content was quantified according the method of Bates et al. [31]. Total amino acids was stained with ninhydrin reagent from Beckman Coulter (18.1 g ninhydrin and 0.72 g hydrindatin in 900 ml mixture of 76.7% dimethyl sulfoxide, 22.5% water, 0.7% lithium acetate and 0.1% acetic acid) at 100 8C during 5 min and read in a colorimeter at 570 nm. Amino acid mix from Beckman Coulter (System 7300/6300) was used as standard. Each analysis is the mean of minimum three independent measurements. 2.3. Determination of soluble protein Protein in 3% sulfosalicylic acid precipitates was solubilized in 1 N KOH on ice and the potassium sulfosalicylic acid precipitate was removed by centrifugation according to the method described by Williamson and Slocum [32]. Protein content was determined according to the method of Bradford [33], using bovine serum albumin (BSA) as standard.
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2.4. Mineral content analysis Around 0.2 g dried crushed material powder was ashed in a muffle furnace at 450 8C for 10 h. Ashes were digested with 5 ml of 7N HNO3. After appropriate dilutions, the filtrate was assayed for Na and K by atomic flame emission spectrometry (Perkin Elmer AAS 3110) at a wavelength of 589 and 766.5 nm, respectively. 2.5. DNA probes About 25 /50 ng of template (DNA fragment corresponding to P5CS-1 and P5CS-2 allosteric sites) were labeled with (a 32P) dCTP using the Random Prime Labeling Kit (Amersham). 2.6. Genomic DNA extraction and Southern blot hybridization Genomic DNA was prepared from leaves of O. sativa plantlets as described by Fedoroff et al. [34]. Twenty mg of genomic DNA were digested with appropriate restriction enzymes and electrophoresed in a 0.8% agarose gel. The genomic DNA was transferred by gravity blotting onto positively charged nylon membranes (Roche Diagnostics). The R2935 EST clone corresponding to the O. sativa P5CS was used as radioactive probe. Hybridization was carried out at 60 8C in the following solution: 10% SDS pH 8, 0.37 g/l EDTA, 67 g/l Na2HPO4.2H2O, 4 ml/l of 85% ortho phosphoric acid. The membranes were washed first in 2 /SSC at room temperature for 40 min, and then in 2 /SSC at 42 8C for 20 min before exposure to an X-ray film (Dupont).
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Vigna aconitifolia [22] and E. coli [35]. The PCR products were ligated to pUC18 using the Sure Clone Ligation Kit (Amersham). 2.8. DNA sequencing Plasmids were extracted from a midi scale culture via the alkaline lysis procedure [36]. The sequences of several clones were determined using the dideoxynucleotide chain termination method with automatic laser fluorescence detection (ALF Pharmacia). The sequences were assembled and analyzed using the GeneCompar program (Vauterin, Applied Maths, Kortrijk, Belgium). 2.9. Total RNA extraction and northern blot hybridization Total RNA was isolated from rice plantlets according to the method described by Rerie et al. [37]. RNA samples (20 /25 mg) were submitted to electrophoresis in formaldehyde agarose gel (1% agarose). Total RNA was transferred by gravity blotting onto positively charged nylon membranes (Roche Diagnostics). The DNA fragment corresponding to the P5CS-1 and P5CS-2 allosteric sites were used as radioactive probes. The hybridization was performed as described for the Southern blot. The membranes were first washed in 2 X SSC (sodium chloride/sodium citrate: 0.3 M sodium chloride/ 0.03 M tri-sodium citrate) at room temperature for 40 min, and then in 2/SSC with 0.1% SDS at 42 8C for 40 min before exposure to an X-ray film (Dupont). The membranes were stained with methylene blue to verify an equal amount of RNA transferred. 2.10. Statistical analysis
2.7. Isolation of the O. sativa P5CS allosteric site The DNA fragments of the p5cs gene were obtained by PCR amplification of rice genomic DNA with two sets of primers. The first set contains two homologous primers (AS7, 5?-CAG-ATG-GAG-TTA-GAT-GGAAAG-GCT-3?; AS8, 5?-CTC-ATA-TGG-AGC-CTTTCT-AGT-GCT-3?) corresponding to the japonica rice p5cs gene [11] from the position 356 to 380 and 577/ 601, respectively. The choice of the second set was based on the protein sequence alignment between the plant P5CS and the Escherichia coli g-glutamyl kinase, in which an aspartate residue involved in the proline allosteric site localized in position 107 of the bacterial enzyme corresponds to the same residue in plant P5CS at position 128 [35]. Therefore two degenerate primers (AS1, 5?-AGC-AGC-TTN-GCN-GAN-CTT-CAG-AA3?; AS2, 5?- CCC-ANA-ANA-TAC-CAG-AAG-AATCCT-3?) were synthesized on the basis of the conserved region of the P5CS allosteric site (assumed to be around the residue 128) between Arabidopsis thaliana [26],
One-way ANOVA followed by the T ?-multiple comparison test (a modified Tukey test) allowing multiple comparisons between treatments with unequal sample sizes [38] were used to analyze the data about growth and proline measurements. The differences are mentioned as significant for P smaller or equal to 0.05.
3. Results 3.1. Effect of NaCl and mannitol stresses on the growth of rice plantlets In order to analyze the possible causes of the differences in tolerance of three rice cultivars, we used young plantlets grown in a mineral medium in the presence or absence of NaCl or mannitol. Based on initial experiments with CR203 plantlets grown for 1 week in MS medium containing different NaCl concentrations (from 0 up to 400 mM), 200 mM NaCl was
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chosen for salt treatment. This NaCl concentration significantly reduced but did not fully inhibit the growth of the plantlets (data not shown). Fig. 1(A, B) shows that 1 week treatment with 200 mM NaCl or an iso-osmotic concentration of mannitol (66.4 g/l, 0.380 Os/kg) significantly affected the growth of the salt- and drought-sensitive cultivar, CR203. For this cultivar, mannitol stress appears to be the most detrimental. Root fresh weight of plantlets submitted to NaCl and mannitol stress conditions was significantly higher for both the drought and the salt-resistant cultivars (DR2 and Cuom) than for the sensitive line (CR203) (Fig. 1A). Root weight of the drought tolerant cultivar (DR2) was more affected by salt stress than by mannitol. Statistical analyses show that the salt and mannitol treatments have the same effect on the root biomass of the salt tolerant Cuom cultivar. In addition, root growth of Cuom is significantly less affected by NaCl treatment than the root growth of DR2. The leaf biomass of the Cuom plantlets is more affected by both
Fig. 1. Root fresh weight (A), and leaf fresh weight (B), of three rice cultivars (CR203, DR2 and Cuom) grown for 1 week in MS (black bars), or in MS supplemented with 200 mM NaCl (white bars) or in an iso-osmotic mannitol concentration (shaded bars). Results are expressed in percentage of the fresh weight of corresponding cultivars growing in non-stress conditions (fresh weight per 20 plantlets is 1.34 g for CR203 leaves; 0.73 g for CR203 roots; 2 g for DR2 leaves; 0.69 g for DR2 roots; 2.2 g for Cuom leaves; 0.62 g for Cuom roots). Each value represents two independent experiments with 20 plantlets and vertical bars represent standard error (S.E.).
stresses than the leaf biomass of CR203 and DR2 (Fig. 1B). 3.2. Ion content in rice plantlets In order to evaluate if any specific mechanism avoiding sodium toxicity is present in the salt tolerant cultivars Cuom, the Na and K content was measured in the roots and the leaves of salt-stressed rice plantlets (Table 1). When exposed 3 days to 200 mM NaCl, the salt tolerant cultivar (Cuom) accumulates slightly but significantly less Na than both the drought tolerant cultivar (DR2) and the drought and salt sensitive cultivar (CR203). Roots of NaCl-stressed plantlets exhibited lower K content than the nonstressed plants for all the cultivars but Cuom presented the highest K /Na ratio. 3.3. Proline content in rice plantlets To assess whether the accumulation of proline induced in plantlets growing on 200 mM NaCl or an iso-osmotic solution of mannitol could reach different levels in the cultivars differing in their salinity and drought tolerance, their proline content was measured in the roots and the leaves during a 3-day period following the stress induction. Fig. 2 reveals that under non-stress conditions the levels of proline in both shoots and roots are low and similar in the three cultivars. Rice plantlets exposed to salt and mannitol stresses accumulate significantly more free proline in their roots than in their shoots. In roots of drought and salt tolerant cultivars (DR2 and Cuom) treated with salt and mannitol, the relative increase in proline is high (four to six times) 2 days after the initiation of the stress treatment. In contrast, this level is not reached in CR203 even after 3 days (Fig. 2A /C). In leaves, mannitol treatment leads to a relatively low proline accumulation (not more than 1 mmol/g fresh weight). Proline accumulation at a somewhat higher level was only observed in leaves of the salt-tolerant cultivar (Cuom) upon salt stress (Fig. 2D/F). We thus conclude that under stress conditions, proline accumulates more rapidly and to a higher level in roots of the tolerant cultivars than in roots of the sensitive variety. Changes in the leaves after this short time period were not so striking than in roots. Therefore in further studies related to proline biosynthesis, we made only use of roots. 3.4. Total protein and free amino acid content in rice plantlets To determine whether proline accumulation under osmotic stress may be due to protein degradation, the level of total protein and free amino acids were measured in the roots and the leaves 3 days after the
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Table 1 Na and K concentration in tissues of three rice cultivars (CR203, salt and drought-sensitive; DR2, drought-tolerant; Cuom, salt-tolerant) incubated 3 days in nutrient solution with or without 200 mM NaCl. Each value is the mean of minimum three independent samples (9/S.E.) NaCl mM
0
200
mg/g DW
Na K K /Na Na K K /Na
Roots
Leaves
CR203
DR2
Cuom
CR203
DR2
Cuom
1.19/0.01 39.79/0.99 36.1 24.09/0.81 19.49/0.38 0.8
1.19/0.09 37.59/0.15 34.1 21.79/0.15 19.99/0.63 0.9
1.09/0.01 39.19/0.48 39.1 20.09/0.16 19.59/0.96 0.97
0.69/0.01 37.79/0.42 62.8 9.69/0.73 39.09/0.69 4.1
0.59/0.02 29.49/1.11 58.8 11.99/0.38 34.29/0.68 2.9
0.69/0.02 36.69/2.11 61.0 8.89/0.11 42.09/0.43 4.8
stress induction (Table 2). The level of total protein remains relatively stable while the level of free amino acids showed significant increase during both mannitol and salt stress treatment in three cultivars. This indicated that the increase in free amino acids is not due to proteolysis. Moreover, whereas the total free amino acid level increased up to three times under stress conditions, the proline level increased up to 15 times.
3.5. Southern blot analysis To estimate the number of p5cs genes present in the O. sativa indica genome, Southern blot analysis was performed using the R2935 EST clone as probe. The restriction of the CR203 genomic DNA by each enzyme (Eco RI, Xho I, Bam HI and StyI), leads to one strongly labeled band corresponding to one p5cs copy, probably
Fig. 2. Proline concentration in root (A /C) and leaf (D /F) tissues of three rice cultivars (CR203, salt and drought-sensitive; DR2, drought-tolerant; Cuom, salt-tolerant) incubated in MS (m, plain line), or in MS supplemented with 200 mM NaCl (', long dashes) or an iso-osmotic mannitol concentration (66.4 g/l, pressure is 0.380 Os/kg) (j, short dashes). Results are expressed in micromoles of proline per gram fresh weight (mmol proline/g FW). Each value is the mean of minimum three independent samples and vertical bars represent S.E.
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Table 2 Soluble protein, proline and total free amino acid contents in tissues of three rice cultivars (CR203, salt and drought-sensitive; DR2, droughttolerant; Cuom, salt-tolerant) incubated 3 days in nutrient solution (MS) supplemented with 200 mM NaCl (Salt) or an iso-osmotic concentration in mannitol (Man) (66.4 g/l, pressure is 0.380 Os/kg) Roots
MS
Salt
Man
Protein (mg/g FW) Proline (mmol/g FW) Amino acids (mmol/g FW) Proline/amino acids (%) Protein (mg/g FW) Proline (mmol/g FW) Amino acids (mmol/g FW) Proline/amino acids (%) Protein (mg/g FW) Proline (mmol/g FW) Amino acids (mmol/g FW) Proline/amino acids (%)
Leaves
CR203
DR2
Cuom
CR203
DR2
Cuom
5.879/0.18 0.389/0.07 24.779/1.33 1.53 6.269/0.15 1.969/0.42 40.239/2.08 4.87 6.019/0.14 3.519/0.18 52.939/1.36 6.63
6.669/0.24 0.649/0.22 28.109/1.05 2.28 5.989/0.24 3.069/0.10 44.209/1.44 6.92 6.949/0.33 4.389/0.55 59.89/0.33 7.32
5.379/0.05 0.319/0.06 24.219/0.72 1.28 5.379/0.17 3.119/0.39 38.359/1.23 8.10 6.149/0.27 4.779/0.57 73.219/3.52 6.51
13.139/0.28 0.339/0.06 20.429/0.60 1.62 13.829/0.89 1.109/0.18 30.939/0.51 3.55 14.389/0.35 1.039/0.12 45.989/1.41 2.24
12.899/0.69 0.469/0.03 28.389/0.82 1.62 13.649/0.26 1.129/0.15 39.979/1.28 2.80 13.689/0.14 0.659/0.01 40.509/1.72 1.60
12.709/0.26 0.559/0.03 32.519/1.57 1.69 13.439/0.39 2.339/0.44 41.509/0.71 5.61 13.889/0.37 1.139/0.04 36.079/1.82 3.13
Each value is the mean of minimum three independent samples (9/S.E.).
Fig. 3. Estimation of the number of p5cs genes in the O. sativa (indica ) genome by Southern blot analysis. O. sativa genomic DNA was digested with different restriction enzymes (lane 1, Eco RI; lane 2, Xho I; lane 3, Bam HI; lane 4, Sty I). The blot was hybridized with the R2935 EST clone corresponding to the O. sativa P5CS.
homologous to the EST probe (Fig. 3). The weakly labeled bands probably represent a second p5cs copy. Two P5CS copies are mentioned to be present in the genomes of A. thaliana and Medicago sativa [39,40] and can be deduced in O. sativa Japonica from the rice genome research data bank. 3.6. Isolation of the O. sativa P5CS allosteric sites Modifications in the p5cs gene sequence could lead to variation in the P5CS-proline feedback inhibition properties. To identify possible changes the DNA region corresponding to the O. sativa P5CS allosteric site of the three cultivars was sequenced. Two different p5cs DNA fragments (p5cs-1 obtained by AS7 and AS8 specific
primers and p5cs-2 obtained by degenerated AS1 and AS2 primers (see Section 2)) were isolated from the genomic DNA of the three rice cultivars. Analysis of the nucleotide and the deduced amino acid sequences demonstrated that P5CS-1 and P5CS-2 allosteric sites are identical for the three cultivars (data not shown). Moreover, the comparison of the O. sativa indica P5CS sequences with the available P5CS sequences from plant species (O. sativa japonica, M. sativa, A. thaliana , V. aconitifolia, and Lycopersicon esculentum ) shows that the amino acid sequences corresponding to the P5CS-1 and P5CS-2 allosteric sites are highly conserved in all species (Fig. 4). The O. sativa indica P5CS-1 and P5CS2 deduced amino acid sequences show 100% identity to O. sativa japonica sequences corresponding, respectively, to the published P5CS sequence [11] and a second P5CS gene, which is recently identified (NCBI data, accession number: BAB64280). Comparison of the indica P5CS-2 amino acid sequence to the indica P5CS-1 and japonica P5CS sequence [11] revealed 85% similarity and 78% identity, respectively.
3.7. Transcription of O. sativa indica p5cs-1 and p5cs-2 genes during salt and mannitol stresses To investigate whether the expression of the p5cs genes is related to proline accumulation and stress tolerance in the three cultivars, the level of p5cs-1 and p5cs-2 mRNAs was measured in roots of rice plantlets exposed for 2 days to 200 mM NaCl or an iso-osmotic mannitol concentration. The specificity of the probes for p5cs-1 and p5cs-2 was tested (Fig. 5A). The result showed that no crosshybridization appeared between the two p5cs genes indicating that the probe was specific for its target.
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Fig. 4. Comparison of the amino acid sequences corresponding to the region of the P5CS allosteric site of O. sativa (indica ) copy 1 and 2 with the A. thaliana copy 1 and 2, L. esculentum , V. aconitifolia , M. sativa and O. sativa higher plants P5CS. Shaded blocks indicate the conserved amino acid residues. The amino acids involved in proline feedback inhibition in plants are indicated by a rectangle.
Northern blot hybridization signals with the p5cs-1 specific probe were easily detected. In contrast, p5cs-2 signals were more difficult to obtain under the same experimental conditions and needed a three times longer exposure to X-ray film. This observation was repeated in different independent experiments. Fig. 5B shows that under non-stress conditions, the p5cs-1 and p5cs-2 mRNA levels in the roots of the three cultivars are low except in the CR203 roots where p5cs-1 is well expressed. Expression of p5cs-1 and p5cs-2 is substantially increased in response to stress. Exposures to NaCl and mannitol led to a similar increase in the two p5cs transcripts in CR203, whereas NaCl induced a higher increase in these transcripts compared with mannitol in both tolerant cultivars.
4. Discussion In this paper, we attempt to determine whether molecular and biochemical traits related to proline biosynthesis and accumulation could help to explain the improved osmotic tolerance observed in two cultivars developed in Vietnam in comparison to the sensitive cultivar CR203. We evaluated how their growth was differentially affected by a short period of osmotic stress. In the in vitro test, root fresh weight data
clearly correlate with the tolerance observed for these cultivars in the field. In addition, the specific tolerance of Cuom to NaCl stress is underlined by the smaller decrease of root growth as well as by the lower amount of toxic Na in salt-treated Cuom plantlets. Under the conditions of the in vitro test, proline content was measured to assess if the differences observed in the growth of rice plantlets were related to the level of proline. Proline content in the roots increased more rapidly after exposure to NaCl and mannitol stress and reached higher levels in the tolerant cultivars (Cuom and DR2) than in the sensitive cultivar (CR203). In contrast, leaves of plantlets submitted to stress display a relatively small increase in proline. The positive correlation established in the roots between proline accumulation and tolerance to both NaCl and mannitol stresses suggests a role for proline in the osmotic tolerance of the analyzed cultivars. The increase in proline level during osmotic stress may be essential for tolerance because it does not only contribute to osmotic adjustment, but also reduces the effect of salt and drought stress damage by protecting enzyme structure, stabilizing membranes and scavenging hydroxyl radicals [2,41 /44]. Proline accumulation resulting from high salt treatment in ‘‘natural’’ O. sativa cultivars has been described [9,11] but its role as an adaptive process is still a matter of debate. There is
Fig. 5. Analysis of p5cs-1 and p5cs-2 transcript levels in indica rice. (A) test of the specificity of the p5cs-1 and p5cs-2 probes. Around 15 ng of DNA fragment corresponding to the region of the allosteric site of p5cs-1 and p5cs-2 was transferred by gravity blotting onto positively charged nylon membranes. The blot was made in double and hybridized with, respectively, P5CS-1 and P5CS-2 fragments at 62 8C. (B) Northern blot analysis of p5cs-1 and p5cs-2 from the three rice cultivars (CR203, DR2 and Cuom). Total RNA was extracted from roots of plantlets incubated 2 days in MS medium (lane 1) or in MS medium supplemented with 200 mM NaCl (lane 2) or in iso-osmotic mannitol solution (lane 3). Blots were hybridized at 62 8C with DNA probes as used in A. The membranes were stained with methylene blue to verify that an equal amount of RNA was transferred (data not shown).
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considerable genetic variability in rice in terms of tolerance to osmotic stress [45,46] and probably several mechanisms are involved in the tolerance of these cultivars. Anyhow, experiments with transgenic plants, including rice [5 /7,47], established that proline overproduction can enhance osmotic stress tolerance. Because the differential proline accumulation observed between the three cultivars appears to be due to proline biosynthesis and not to be a consequence of protein degradation, the properties of P5CS, the key enzyme of the proline biosynthesis were studied in the three rice cultivars. One factor that could lead to differential proline levels in the cultivars submitted to salt or mannitol stresses, concerns the regulatory properties of the P5CS enzyme. Under osmotic stress, the P5CS conformation may change around the proline interaction site leading to partial but not complete loss of the proline allosteric regulation [6,25]. Mutagenesis to substitute each of the six amino acid residues between position 126 and 131 of the P5CS enzyme, demonstrated that the degree of feedback insensitivity of the enzyme can be effectively changed and results in an increased proline accumulation and protection of plants from osmotic stress [6]. Therefore, cloning of the cDNA fragment around these amino acids was performed for both P5CS-1 and P5CS-2 of all cultivars. However, no indication for a difference in P5CS feedback regulation was found, as the predicted amino acid sequence responsible for a potential relief of proline feedback is completely conserved in the investigated cultivars. Apart from feedback inhibition, changes in the transcriptional level of the p5cs gene can also lead to differential proline accumulation in response to stress. An analysis of the expression of the two p5cs genes was therefore performed to determine whether the accumulation of the p5cs-1 and p5cs-2 mRNA could have contributed to the differential proline production of the three cultivars under stress conditions. Osmotic stresses such as NaCl and drought induce p5cs transcription in japonica rice plantlets [11]. This japonica p5cs corresponds to our p5cs-1 in indica rice. The response of the p5cs-2 was unknown. The levels of p5cs mRNA monitored by Northern blot with specific probes reveals that the transcription of both p5cs-1 and p5cs-2 is regulated in indica rice by osmotic stress with a similar pattern of accumulation. However, the p5cs-1 transcript is more abundant and is likely to contribute more to proline biosynthesis in response to osmotic stress than p5cs-2. The comparison of the RNA expression pattern permitted the detection of some differences between rice cultivars differing in their salt- and droughttolerance. However, in roots these patterns are apparently not related to the level of proline accumulation observed in the different cultivars. Indeed, under nonstress conditions p5cs-1 was expressed to a higher level
in the roots of the sensitive cultivar than in the tolerant cultivars, while no difference in the proline level was observed. Moreover, the higher proline accumulation in the roots of the tolerant cultivars compared with the sensitive CR203 under stress conditions is not linked to a stronger p5cs mRNA induction. Finally, the differential proline accumulation observed in function of the stress treatment is inversely correlated to the p5cs mRNA detected. These observations are in contrast with the results of Igarashi et al. [11] who observed that both p5cs mRNA level and proline content were higher in the root of salt-tolerant cultivars than in sensitive cultivars of japonica rice. In conclusion, this work suggests that the proline accumulation induced by osmotic stress treatment is related to the degree of salt- and drought- tolerance shown in the in vitro test by three indica rice cultivars and a measurement of its short-term accumulation may be a good indicator of rice osmotic stress tolerance. We did not observe changes in the amino acid sequences known to be involved in the proline feedback inhibition of the P5CS activity [20]. Therefore, this is not responsible for the differential proline accumulation observed in Cuom, DR2 and cultivars. The stress inducible expression of the p5cs genes resulted in an increased proline accumulation but not related to the observed different proline content in the roots of the cultivars. Other phenomena rather than transcriptional or post-transcriptional regulation of the p5cs gene leading to differential proline accumulation in the roots of the tolerant lines appear to play a role in the response to osmotic stress conditions. Therefore, other targets beyond P5CS transcript or sequence should be considered.
Acknowledgements The authors are grateful to MAFF DNA Bank for the gift of R2935 EST clone and to the Institute of Biotechnology in Hanoi, Vietnam for providing rice seeds. The authors gratefully thank K. Loenders for technical assistance in molecular biology and C. Bouton and M. De Kerpel for DNA sequencing. The authors gratefully acknowledge financial support from the DG XII of European Commission (Contract number: ERBIC18CT980314). Nancy Roosens is a Postdoctoral Researcher for the Belgian ‘‘Fonds National de la Recherche Scientifique’’.
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Proline accumulation and D1-pyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance Do Thu Hien a, Michel Jacobs a, Geert Angenon a,*, Christian Hermans b, Tran Thanh Thu a, Le Van Son a,1, Nancy He´le`ne Roosens a,b b
a Laboratory of Plant Genetics, Vrije Universiteit Brussel (VUB), Campus Etterbeek, Gebouw E Pleinlaan 2, B-1050 Brussels, Belgium Lab de Physiologie et de Ge´ne´tique Mole´culaire des Plantes, Universite´ Libre de Bruxelles (ULB), CP242, Boulevard du Triomphe, 1050 Bruxelles, Belgium
Received 17 September 2002; received in revised form 25 June 2003; accepted 3 July 2003
Abstract Three indica rice cultivars (Oryza sativa ) differing in their tolerance to salt and drought stress in field conditions in Vietnam were analyzed at the molecular and biochemical levels with the goal to reveal the basis for their differential behavior and in particular their ability to accumulate proline. An in vitro growth test showed that after a 7-day period of stress, the fresh weight of plantlet roots appears to be a relevant parameter for differentiating drought and salt tolerance of the concerned cultivars. Sodium level was lower in the salt tolerant cultivar than in the other rice cultivars. Proline accumulation in roots of tolerant cultivars starts earlier after the initiation of the stress treatment than that of the osmotic stress sensitive cultivar and also reaches a higher level. Proline accumulation was not related to proteolysis and so could be the result from induction of proline biosynthesis by osmotic stress. However, neither the sequence of amino acids involved in the proline feedback inhibition of the key regulatory enzyme D1-pyrroline5-carboxylate synthetase (P5CS; EC not assigned), nor the expression of the p5cs genes were modified in the tolerant cultivars. These observations suggest that proline accumulation in roots is a possible indicator of the osmotic tolerance in these rice cultivars. However, other mechanisms than those related to a change in P5CS regulation are responsible for the increased proline content. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Proline accumulation; D1-Pyrroline-5-carboxylate synthetase; Osmotic stress
1. Introduction Osmotic stress is the most serious problem affecting crop productivity and limiting agricultural expansion in many areas of the world. A large number of plant species accumulate proline in response to osmotic stress [1,2]. Therefore, it was suggested that this amino acid be involved in stress tolerance by playing a role in counteracting the effects of osmotic stress [2,3]. Direct evidence supporting this hypothesis was provided by some
* Corresponding author. Tel.: /32-2-629-1935; fax: /32-2-6291867. E-mail address: [email protected] (G. Angenon). 1 Present address: Laboratory of Plant Biotechnology, Institute of Biotechnology, Hanoi, Vietnam.
mutant and transgenic proline overproducing plants, which show increased osmotolerance [4 /7]. However, in some species or ecotypes, the data do not always indicate a positive correlation between proline accumulation and adaptation to drought and salt stress [2,8 / 10]. In rice, data are particularly contradictory. Some authors suggest that proline accumulation may be related to the degree of salt tolerance in Oryza sativa [11]. Others suggested that proline is a symptom of salt stress injury rather than an indicator of the resistance [9]. All these controversies may reflect, in these species or ecotypes, the predominance of the role of tolerance mechanisms other than osmotic adjustment, such as specific morphological and physiological modifications. Since these studies were often performed after submitting the plants to long periods of stress, part of the
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00301-7
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discrepancy could be due to the fact that proline accumulation is altered as a result of injury. In higher plants, proline accumulation appears to be essentially due to de novo synthesis [12,13] and catabolism rate minimization [13 /16] but lower proline utilization in protein synthesis and increased proteolysis may also contribute to net proline accumulation [12,15,17,18]. P5CS, which catalyzes the first two steps in proline biosynthesis, is critical for the control of the proline biosynthesis and consequently the increase in osmotolerance [5,6,19]. This essential role is probably due to two levels of control exerted at those steps: proline feedback inhibition on the P5CS protein [20,21] and regulation of the P5CS transcript level in response to osmotic stress conditions [11,21/23]. In plants, numerous studies suggest that P5CS is feedback regulated by proline and the release of feedback inhibition of P5CS enzyme can lead to an increase in proline accumulation in response to stress conferring better osmotolerance [21,24]. It has been previously proposed that the origin of salt tolerance in a mutant of Nicotiana plumbaginifolia was due to a mutation leading to a substantial reduction in the feedback inhibition by proline at the level of the P5CS enzyme [25]. Transgenic tobacco overexpressing a feedback insensitive form of the enzyme, accumulates more proline and shows more resistance to osmotic stress than the plant expressing the wild type P5CS [6]. Besides the proline feedback control of the P5CS activity, expression of the p5cs gene involves a second key level of regulation. Indeed, the p5cs gene is transcriptionally induced by osmotic stress and its induction precedes the accumulation of proline [26]. Accumulation of proline and expression of the p5cs gene were strongly induced after 48 h in salt-tolerant rice under high-salt conditions (250 mM) when compared with a sensitive one [11]. This strongly supports that proline accumulation and the induction of P5CS shortly after the start of salt stress may be related to the degree of salt tolerance in O. sativa . In the present study, we made use of three cultivars Cuom, DR2 and CR203 which are currently grown in Vietnam and which differ in salt and drought tolerance under field conditions. The cultivar CR203 is an elite breeding line from the International Rice Research Institute (Los Ban˜os, Philippines) [27], sensitive to salt and drought stress. DR2 is a drought tolerant elite line [28] resulting from somaclonal variation of the CR203 cultivar after in vitro selection under drought conditions. Cuom is a local cultivar grown in saline areas near the coast. In in vitro callus induction assays, Cuom showed higher survival rate and regeneration frequencies after salt stress than CR203 [29]. We assessed whether proline can be correlated with the observed differences in osmotic stress tolerance by measuring the content of this amino acid in these cultivars. We also
evaluated if differences in proline accumulation could be due to changes in the feedback sensitivity of P5CS by analyzing the structure of the P5CS allosteric site or to variation in the relative expression of the p5cs gene.
2. Materials and methods 2.1. Plant growth and stress treatment Three O. sativa (indica) cultivars Cuom, DR2 and CR203 from Vietnam were used in this study. These three cultivars were provided by the Institute of Biotechnology (Hanoi, Vietnam) and are cultivated in the North of Vietnam. The seeds were dark-germinated in vitro on solid MS medium [30] and grown in a culture room (24 8C, 16 h illumination, 8 h dark cycle). Eight-day-old plantlets were used for the stress treatment. Salt stress and osmotic stress treatment consisted in adding to the MS medium, respectively, 200 mM NaCl and an iso-osmotic solution of mannitol (66.4 g/l) causing an osmotic pressure of 0.380 Os/kg. 2.2. Determination of proline content and total amino acids After stress treatment, around 0.3 g fresh weight of rice plantlets was extracted with 3% (w/v) sulfosalicylic acid on ice. The supernatant containing total amino acids was transferred to a new tube for determination of proline and total amino acids. The tissue precipitates were saved for protein extraction (see below). Proline content was quantified according the method of Bates et al. [31]. Total amino acids was stained with ninhydrin reagent from Beckman Coulter (18.1 g ninhydrin and 0.72 g hydrindatin in 900 ml mixture of 76.7% dimethyl sulfoxide, 22.5% water, 0.7% lithium acetate and 0.1% acetic acid) at 100 8C during 5 min and read in a colorimeter at 570 nm. Amino acid mix from Beckman Coulter (System 7300/6300) was used as standard. Each analysis is the mean of minimum three independent measurements. 2.3. Determination of soluble protein Protein in 3% sulfosalicylic acid precipitates was solubilized in 1 N KOH on ice and the potassium sulfosalicylic acid precipitate was removed by centrifugation according to the method described by Williamson and Slocum [32]. Protein content was determined according to the method of Bradford [33], using bovine serum albumin (BSA) as standard.
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2.4. Mineral content analysis Around 0.2 g dried crushed material powder was ashed in a muffle furnace at 450 8C for 10 h. Ashes were digested with 5 ml of 7N HNO3. After appropriate dilutions, the filtrate was assayed for Na and K by atomic flame emission spectrometry (Perkin Elmer AAS 3110) at a wavelength of 589 and 766.5 nm, respectively. 2.5. DNA probes About 25 /50 ng of template (DNA fragment corresponding to P5CS-1 and P5CS-2 allosteric sites) were labeled with (a 32P) dCTP using the Random Prime Labeling Kit (Amersham). 2.6. Genomic DNA extraction and Southern blot hybridization Genomic DNA was prepared from leaves of O. sativa plantlets as described by Fedoroff et al. [34]. Twenty mg of genomic DNA were digested with appropriate restriction enzymes and electrophoresed in a 0.8% agarose gel. The genomic DNA was transferred by gravity blotting onto positively charged nylon membranes (Roche Diagnostics). The R2935 EST clone corresponding to the O. sativa P5CS was used as radioactive probe. Hybridization was carried out at 60 8C in the following solution: 10% SDS pH 8, 0.37 g/l EDTA, 67 g/l Na2HPO4.2H2O, 4 ml/l of 85% ortho phosphoric acid. The membranes were washed first in 2 /SSC at room temperature for 40 min, and then in 2 /SSC at 42 8C for 20 min before exposure to an X-ray film (Dupont).
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Vigna aconitifolia [22] and E. coli [35]. The PCR products were ligated to pUC18 using the Sure Clone Ligation Kit (Amersham). 2.8. DNA sequencing Plasmids were extracted from a midi scale culture via the alkaline lysis procedure [36]. The sequences of several clones were determined using the dideoxynucleotide chain termination method with automatic laser fluorescence detection (ALF Pharmacia). The sequences were assembled and analyzed using the GeneCompar program (Vauterin, Applied Maths, Kortrijk, Belgium). 2.9. Total RNA extraction and northern blot hybridization Total RNA was isolated from rice plantlets according to the method described by Rerie et al. [37]. RNA samples (20 /25 mg) were submitted to electrophoresis in formaldehyde agarose gel (1% agarose). Total RNA was transferred by gravity blotting onto positively charged nylon membranes (Roche Diagnostics). The DNA fragment corresponding to the P5CS-1 and P5CS-2 allosteric sites were used as radioactive probes. The hybridization was performed as described for the Southern blot. The membranes were first washed in 2 X SSC (sodium chloride/sodium citrate: 0.3 M sodium chloride/ 0.03 M tri-sodium citrate) at room temperature for 40 min, and then in 2/SSC with 0.1% SDS at 42 8C for 40 min before exposure to an X-ray film (Dupont). The membranes were stained with methylene blue to verify an equal amount of RNA transferred. 2.10. Statistical analysis
2.7. Isolation of the O. sativa P5CS allosteric site The DNA fragments of the p5cs gene were obtained by PCR amplification of rice genomic DNA with two sets of primers. The first set contains two homologous primers (AS7, 5?-CAG-ATG-GAG-TTA-GAT-GGAAAG-GCT-3?; AS8, 5?-CTC-ATA-TGG-AGC-CTTTCT-AGT-GCT-3?) corresponding to the japonica rice p5cs gene [11] from the position 356 to 380 and 577/ 601, respectively. The choice of the second set was based on the protein sequence alignment between the plant P5CS and the Escherichia coli g-glutamyl kinase, in which an aspartate residue involved in the proline allosteric site localized in position 107 of the bacterial enzyme corresponds to the same residue in plant P5CS at position 128 [35]. Therefore two degenerate primers (AS1, 5?-AGC-AGC-TTN-GCN-GAN-CTT-CAG-AA3?; AS2, 5?- CCC-ANA-ANA-TAC-CAG-AAG-AATCCT-3?) were synthesized on the basis of the conserved region of the P5CS allosteric site (assumed to be around the residue 128) between Arabidopsis thaliana [26],
One-way ANOVA followed by the T ?-multiple comparison test (a modified Tukey test) allowing multiple comparisons between treatments with unequal sample sizes [38] were used to analyze the data about growth and proline measurements. The differences are mentioned as significant for P smaller or equal to 0.05.
3. Results 3.1. Effect of NaCl and mannitol stresses on the growth of rice plantlets In order to analyze the possible causes of the differences in tolerance of three rice cultivars, we used young plantlets grown in a mineral medium in the presence or absence of NaCl or mannitol. Based on initial experiments with CR203 plantlets grown for 1 week in MS medium containing different NaCl concentrations (from 0 up to 400 mM), 200 mM NaCl was
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chosen for salt treatment. This NaCl concentration significantly reduced but did not fully inhibit the growth of the plantlets (data not shown). Fig. 1(A, B) shows that 1 week treatment with 200 mM NaCl or an iso-osmotic concentration of mannitol (66.4 g/l, 0.380 Os/kg) significantly affected the growth of the salt- and drought-sensitive cultivar, CR203. For this cultivar, mannitol stress appears to be the most detrimental. Root fresh weight of plantlets submitted to NaCl and mannitol stress conditions was significantly higher for both the drought and the salt-resistant cultivars (DR2 and Cuom) than for the sensitive line (CR203) (Fig. 1A). Root weight of the drought tolerant cultivar (DR2) was more affected by salt stress than by mannitol. Statistical analyses show that the salt and mannitol treatments have the same effect on the root biomass of the salt tolerant Cuom cultivar. In addition, root growth of Cuom is significantly less affected by NaCl treatment than the root growth of DR2. The leaf biomass of the Cuom plantlets is more affected by both
Fig. 1. Root fresh weight (A), and leaf fresh weight (B), of three rice cultivars (CR203, DR2 and Cuom) grown for 1 week in MS (black bars), or in MS supplemented with 200 mM NaCl (white bars) or in an iso-osmotic mannitol concentration (shaded bars). Results are expressed in percentage of the fresh weight of corresponding cultivars growing in non-stress conditions (fresh weight per 20 plantlets is 1.34 g for CR203 leaves; 0.73 g for CR203 roots; 2 g for DR2 leaves; 0.69 g for DR2 roots; 2.2 g for Cuom leaves; 0.62 g for Cuom roots). Each value represents two independent experiments with 20 plantlets and vertical bars represent standard error (S.E.).
stresses than the leaf biomass of CR203 and DR2 (Fig. 1B). 3.2. Ion content in rice plantlets In order to evaluate if any specific mechanism avoiding sodium toxicity is present in the salt tolerant cultivars Cuom, the Na and K content was measured in the roots and the leaves of salt-stressed rice plantlets (Table 1). When exposed 3 days to 200 mM NaCl, the salt tolerant cultivar (Cuom) accumulates slightly but significantly less Na than both the drought tolerant cultivar (DR2) and the drought and salt sensitive cultivar (CR203). Roots of NaCl-stressed plantlets exhibited lower K content than the nonstressed plants for all the cultivars but Cuom presented the highest K /Na ratio. 3.3. Proline content in rice plantlets To assess whether the accumulation of proline induced in plantlets growing on 200 mM NaCl or an iso-osmotic solution of mannitol could reach different levels in the cultivars differing in their salinity and drought tolerance, their proline content was measured in the roots and the leaves during a 3-day period following the stress induction. Fig. 2 reveals that under non-stress conditions the levels of proline in both shoots and roots are low and similar in the three cultivars. Rice plantlets exposed to salt and mannitol stresses accumulate significantly more free proline in their roots than in their shoots. In roots of drought and salt tolerant cultivars (DR2 and Cuom) treated with salt and mannitol, the relative increase in proline is high (four to six times) 2 days after the initiation of the stress treatment. In contrast, this level is not reached in CR203 even after 3 days (Fig. 2A /C). In leaves, mannitol treatment leads to a relatively low proline accumulation (not more than 1 mmol/g fresh weight). Proline accumulation at a somewhat higher level was only observed in leaves of the salt-tolerant cultivar (Cuom) upon salt stress (Fig. 2D/F). We thus conclude that under stress conditions, proline accumulates more rapidly and to a higher level in roots of the tolerant cultivars than in roots of the sensitive variety. Changes in the leaves after this short time period were not so striking than in roots. Therefore in further studies related to proline biosynthesis, we made only use of roots. 3.4. Total protein and free amino acid content in rice plantlets To determine whether proline accumulation under osmotic stress may be due to protein degradation, the level of total protein and free amino acids were measured in the roots and the leaves 3 days after the
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Table 1 Na and K concentration in tissues of three rice cultivars (CR203, salt and drought-sensitive; DR2, drought-tolerant; Cuom, salt-tolerant) incubated 3 days in nutrient solution with or without 200 mM NaCl. Each value is the mean of minimum three independent samples (9/S.E.) NaCl mM
0
200
mg/g DW
Na K K /Na Na K K /Na
Roots
Leaves
CR203
DR2
Cuom
CR203
DR2
Cuom
1.19/0.01 39.79/0.99 36.1 24.09/0.81 19.49/0.38 0.8
1.19/0.09 37.59/0.15 34.1 21.79/0.15 19.99/0.63 0.9
1.09/0.01 39.19/0.48 39.1 20.09/0.16 19.59/0.96 0.97
0.69/0.01 37.79/0.42 62.8 9.69/0.73 39.09/0.69 4.1
0.59/0.02 29.49/1.11 58.8 11.99/0.38 34.29/0.68 2.9
0.69/0.02 36.69/2.11 61.0 8.89/0.11 42.09/0.43 4.8
stress induction (Table 2). The level of total protein remains relatively stable while the level of free amino acids showed significant increase during both mannitol and salt stress treatment in three cultivars. This indicated that the increase in free amino acids is not due to proteolysis. Moreover, whereas the total free amino acid level increased up to three times under stress conditions, the proline level increased up to 15 times.
3.5. Southern blot analysis To estimate the number of p5cs genes present in the O. sativa indica genome, Southern blot analysis was performed using the R2935 EST clone as probe. The restriction of the CR203 genomic DNA by each enzyme (Eco RI, Xho I, Bam HI and StyI), leads to one strongly labeled band corresponding to one p5cs copy, probably
Fig. 2. Proline concentration in root (A /C) and leaf (D /F) tissues of three rice cultivars (CR203, salt and drought-sensitive; DR2, drought-tolerant; Cuom, salt-tolerant) incubated in MS (m, plain line), or in MS supplemented with 200 mM NaCl (', long dashes) or an iso-osmotic mannitol concentration (66.4 g/l, pressure is 0.380 Os/kg) (j, short dashes). Results are expressed in micromoles of proline per gram fresh weight (mmol proline/g FW). Each value is the mean of minimum three independent samples and vertical bars represent S.E.
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Table 2 Soluble protein, proline and total free amino acid contents in tissues of three rice cultivars (CR203, salt and drought-sensitive; DR2, droughttolerant; Cuom, salt-tolerant) incubated 3 days in nutrient solution (MS) supplemented with 200 mM NaCl (Salt) or an iso-osmotic concentration in mannitol (Man) (66.4 g/l, pressure is 0.380 Os/kg) Roots
MS
Salt
Man
Protein (mg/g FW) Proline (mmol/g FW) Amino acids (mmol/g FW) Proline/amino acids (%) Protein (mg/g FW) Proline (mmol/g FW) Amino acids (mmol/g FW) Proline/amino acids (%) Protein (mg/g FW) Proline (mmol/g FW) Amino acids (mmol/g FW) Proline/amino acids (%)
Leaves
CR203
DR2
Cuom
CR203
DR2
Cuom
5.879/0.18 0.389/0.07 24.779/1.33 1.53 6.269/0.15 1.969/0.42 40.239/2.08 4.87 6.019/0.14 3.519/0.18 52.939/1.36 6.63
6.669/0.24 0.649/0.22 28.109/1.05 2.28 5.989/0.24 3.069/0.10 44.209/1.44 6.92 6.949/0.33 4.389/0.55 59.89/0.33 7.32
5.379/0.05 0.319/0.06 24.219/0.72 1.28 5.379/0.17 3.119/0.39 38.359/1.23 8.10 6.149/0.27 4.779/0.57 73.219/3.52 6.51
13.139/0.28 0.339/0.06 20.429/0.60 1.62 13.829/0.89 1.109/0.18 30.939/0.51 3.55 14.389/0.35 1.039/0.12 45.989/1.41 2.24
12.899/0.69 0.469/0.03 28.389/0.82 1.62 13.649/0.26 1.129/0.15 39.979/1.28 2.80 13.689/0.14 0.659/0.01 40.509/1.72 1.60
12.709/0.26 0.559/0.03 32.519/1.57 1.69 13.439/0.39 2.339/0.44 41.509/0.71 5.61 13.889/0.37 1.139/0.04 36.079/1.82 3.13
Each value is the mean of minimum three independent samples (9/S.E.).
Fig. 3. Estimation of the number of p5cs genes in the O. sativa (indica ) genome by Southern blot analysis. O. sativa genomic DNA was digested with different restriction enzymes (lane 1, Eco RI; lane 2, Xho I; lane 3, Bam HI; lane 4, Sty I). The blot was hybridized with the R2935 EST clone corresponding to the O. sativa P5CS.
homologous to the EST probe (Fig. 3). The weakly labeled bands probably represent a second p5cs copy. Two P5CS copies are mentioned to be present in the genomes of A. thaliana and Medicago sativa [39,40] and can be deduced in O. sativa Japonica from the rice genome research data bank. 3.6. Isolation of the O. sativa P5CS allosteric sites Modifications in the p5cs gene sequence could lead to variation in the P5CS-proline feedback inhibition properties. To identify possible changes the DNA region corresponding to the O. sativa P5CS allosteric site of the three cultivars was sequenced. Two different p5cs DNA fragments (p5cs-1 obtained by AS7 and AS8 specific
primers and p5cs-2 obtained by degenerated AS1 and AS2 primers (see Section 2)) were isolated from the genomic DNA of the three rice cultivars. Analysis of the nucleotide and the deduced amino acid sequences demonstrated that P5CS-1 and P5CS-2 allosteric sites are identical for the three cultivars (data not shown). Moreover, the comparison of the O. sativa indica P5CS sequences with the available P5CS sequences from plant species (O. sativa japonica, M. sativa, A. thaliana , V. aconitifolia, and Lycopersicon esculentum ) shows that the amino acid sequences corresponding to the P5CS-1 and P5CS-2 allosteric sites are highly conserved in all species (Fig. 4). The O. sativa indica P5CS-1 and P5CS2 deduced amino acid sequences show 100% identity to O. sativa japonica sequences corresponding, respectively, to the published P5CS sequence [11] and a second P5CS gene, which is recently identified (NCBI data, accession number: BAB64280). Comparison of the indica P5CS-2 amino acid sequence to the indica P5CS-1 and japonica P5CS sequence [11] revealed 85% similarity and 78% identity, respectively.
3.7. Transcription of O. sativa indica p5cs-1 and p5cs-2 genes during salt and mannitol stresses To investigate whether the expression of the p5cs genes is related to proline accumulation and stress tolerance in the three cultivars, the level of p5cs-1 and p5cs-2 mRNAs was measured in roots of rice plantlets exposed for 2 days to 200 mM NaCl or an iso-osmotic mannitol concentration. The specificity of the probes for p5cs-1 and p5cs-2 was tested (Fig. 5A). The result showed that no crosshybridization appeared between the two p5cs genes indicating that the probe was specific for its target.
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Fig. 4. Comparison of the amino acid sequences corresponding to the region of the P5CS allosteric site of O. sativa (indica ) copy 1 and 2 with the A. thaliana copy 1 and 2, L. esculentum , V. aconitifolia , M. sativa and O. sativa higher plants P5CS. Shaded blocks indicate the conserved amino acid residues. The amino acids involved in proline feedback inhibition in plants are indicated by a rectangle.
Northern blot hybridization signals with the p5cs-1 specific probe were easily detected. In contrast, p5cs-2 signals were more difficult to obtain under the same experimental conditions and needed a three times longer exposure to X-ray film. This observation was repeated in different independent experiments. Fig. 5B shows that under non-stress conditions, the p5cs-1 and p5cs-2 mRNA levels in the roots of the three cultivars are low except in the CR203 roots where p5cs-1 is well expressed. Expression of p5cs-1 and p5cs-2 is substantially increased in response to stress. Exposures to NaCl and mannitol led to a similar increase in the two p5cs transcripts in CR203, whereas NaCl induced a higher increase in these transcripts compared with mannitol in both tolerant cultivars.
4. Discussion In this paper, we attempt to determine whether molecular and biochemical traits related to proline biosynthesis and accumulation could help to explain the improved osmotic tolerance observed in two cultivars developed in Vietnam in comparison to the sensitive cultivar CR203. We evaluated how their growth was differentially affected by a short period of osmotic stress. In the in vitro test, root fresh weight data
clearly correlate with the tolerance observed for these cultivars in the field. In addition, the specific tolerance of Cuom to NaCl stress is underlined by the smaller decrease of root growth as well as by the lower amount of toxic Na in salt-treated Cuom plantlets. Under the conditions of the in vitro test, proline content was measured to assess if the differences observed in the growth of rice plantlets were related to the level of proline. Proline content in the roots increased more rapidly after exposure to NaCl and mannitol stress and reached higher levels in the tolerant cultivars (Cuom and DR2) than in the sensitive cultivar (CR203). In contrast, leaves of plantlets submitted to stress display a relatively small increase in proline. The positive correlation established in the roots between proline accumulation and tolerance to both NaCl and mannitol stresses suggests a role for proline in the osmotic tolerance of the analyzed cultivars. The increase in proline level during osmotic stress may be essential for tolerance because it does not only contribute to osmotic adjustment, but also reduces the effect of salt and drought stress damage by protecting enzyme structure, stabilizing membranes and scavenging hydroxyl radicals [2,41 /44]. Proline accumulation resulting from high salt treatment in ‘‘natural’’ O. sativa cultivars has been described [9,11] but its role as an adaptive process is still a matter of debate. There is
Fig. 5. Analysis of p5cs-1 and p5cs-2 transcript levels in indica rice. (A) test of the specificity of the p5cs-1 and p5cs-2 probes. Around 15 ng of DNA fragment corresponding to the region of the allosteric site of p5cs-1 and p5cs-2 was transferred by gravity blotting onto positively charged nylon membranes. The blot was made in double and hybridized with, respectively, P5CS-1 and P5CS-2 fragments at 62 8C. (B) Northern blot analysis of p5cs-1 and p5cs-2 from the three rice cultivars (CR203, DR2 and Cuom). Total RNA was extracted from roots of plantlets incubated 2 days in MS medium (lane 1) or in MS medium supplemented with 200 mM NaCl (lane 2) or in iso-osmotic mannitol solution (lane 3). Blots were hybridized at 62 8C with DNA probes as used in A. The membranes were stained with methylene blue to verify that an equal amount of RNA was transferred (data not shown).
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considerable genetic variability in rice in terms of tolerance to osmotic stress [45,46] and probably several mechanisms are involved in the tolerance of these cultivars. Anyhow, experiments with transgenic plants, including rice [5 /7,47], established that proline overproduction can enhance osmotic stress tolerance. Because the differential proline accumulation observed between the three cultivars appears to be due to proline biosynthesis and not to be a consequence of protein degradation, the properties of P5CS, the key enzyme of the proline biosynthesis were studied in the three rice cultivars. One factor that could lead to differential proline levels in the cultivars submitted to salt or mannitol stresses, concerns the regulatory properties of the P5CS enzyme. Under osmotic stress, the P5CS conformation may change around the proline interaction site leading to partial but not complete loss of the proline allosteric regulation [6,25]. Mutagenesis to substitute each of the six amino acid residues between position 126 and 131 of the P5CS enzyme, demonstrated that the degree of feedback insensitivity of the enzyme can be effectively changed and results in an increased proline accumulation and protection of plants from osmotic stress [6]. Therefore, cloning of the cDNA fragment around these amino acids was performed for both P5CS-1 and P5CS-2 of all cultivars. However, no indication for a difference in P5CS feedback regulation was found, as the predicted amino acid sequence responsible for a potential relief of proline feedback is completely conserved in the investigated cultivars. Apart from feedback inhibition, changes in the transcriptional level of the p5cs gene can also lead to differential proline accumulation in response to stress. An analysis of the expression of the two p5cs genes was therefore performed to determine whether the accumulation of the p5cs-1 and p5cs-2 mRNA could have contributed to the differential proline production of the three cultivars under stress conditions. Osmotic stresses such as NaCl and drought induce p5cs transcription in japonica rice plantlets [11]. This japonica p5cs corresponds to our p5cs-1 in indica rice. The response of the p5cs-2 was unknown. The levels of p5cs mRNA monitored by Northern blot with specific probes reveals that the transcription of both p5cs-1 and p5cs-2 is regulated in indica rice by osmotic stress with a similar pattern of accumulation. However, the p5cs-1 transcript is more abundant and is likely to contribute more to proline biosynthesis in response to osmotic stress than p5cs-2. The comparison of the RNA expression pattern permitted the detection of some differences between rice cultivars differing in their salt- and droughttolerance. However, in roots these patterns are apparently not related to the level of proline accumulation observed in the different cultivars. Indeed, under nonstress conditions p5cs-1 was expressed to a higher level
in the roots of the sensitive cultivar than in the tolerant cultivars, while no difference in the proline level was observed. Moreover, the higher proline accumulation in the roots of the tolerant cultivars compared with the sensitive CR203 under stress conditions is not linked to a stronger p5cs mRNA induction. Finally, the differential proline accumulation observed in function of the stress treatment is inversely correlated to the p5cs mRNA detected. These observations are in contrast with the results of Igarashi et al. [11] who observed that both p5cs mRNA level and proline content were higher in the root of salt-tolerant cultivars than in sensitive cultivars of japonica rice. In conclusion, this work suggests that the proline accumulation induced by osmotic stress treatment is related to the degree of salt- and drought- tolerance shown in the in vitro test by three indica rice cultivars and a measurement of its short-term accumulation may be a good indicator of rice osmotic stress tolerance. We did not observe changes in the amino acid sequences known to be involved in the proline feedback inhibition of the P5CS activity [20]. Therefore, this is not responsible for the differential proline accumulation observed in Cuom, DR2 and cultivars. The stress inducible expression of the p5cs genes resulted in an increased proline accumulation but not related to the observed different proline content in the roots of the cultivars. Other phenomena rather than transcriptional or post-transcriptional regulation of the p5cs gene leading to differential proline accumulation in the roots of the tolerant lines appear to play a role in the response to osmotic stress conditions. Therefore, other targets beyond P5CS transcript or sequence should be considered.
Acknowledgements The authors are grateful to MAFF DNA Bank for the gift of R2935 EST clone and to the Institute of Biotechnology in Hanoi, Vietnam for providing rice seeds. The authors gratefully thank K. Loenders for technical assistance in molecular biology and C. Bouton and M. De Kerpel for DNA sequencing. The authors gratefully acknowledge financial support from the DG XII of European Commission (Contract number: ERBIC18CT980314). Nancy Roosens is a Postdoctoral Researcher for the Belgian ‘‘Fonds National de la Recherche Scientifique’’.
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