JXB Advance Access originally published online on May 31, 2005
Journal of Experimental Botany 2005 56(417):1975-1981; doi:10.1093/jxb/eri195
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RESEARCH PAPER |
Effects of free proline accumulation in petunias under drought stress
1Central Research Laboratory, c/o Advanced Research Laboratory, Hitachi Ltd., 2520 Akanuma, Hatoyama-cho, Hiki-gun, Saitama 350-0395, Japan
2Research Resources Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
3Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
4Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ministry of Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
* To whom correspondence should be addressed. Fax: +81 49 296 6006. E-mail: yoshiba{at}harl.hitachi.co.jp
Received 25 March 2005; Accepted 8 April 2005
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Abstract |
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Petunias (Petunia hybrida cv. ‘Mitchell’) accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by
1-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57–1.01% of the total amino acids in the transgenic plants, or 1.5–2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal. Key words: Drought tolerance, genetic engineering, osmoprotectant, stress response, transgenic petunia
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Introduction |
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When plants experience environmental stresses, such as drought, high salinity, and low temperatures, they activate various metabolic and defence systems to survive. A number of genes corresponding to these stresses and their products were analysed in Arabidopsis (Seki et al., 2002
; Oono et al., 2003; Maruyama et al., 2004) and rice (Rabbani et al., 2003) plants using cDNA microarray analysis. Many genes and products commonly appear in response to drought, salinity, and low-temperature stresses. For example, osmoprotectants, such as proline (Pro), glycine betaine, mannitol, and sugars confer stress tolerance. Transgenic plants have enhanced tolerance to drought and salinity and to drought and cold (Kavi Kishor et al., 1995; Huang et al., 2000; Taji et al., 2002; Abebe et al., 2003). The focus here is on Pro.
Pro is synthesized from either Glu or Orn (Delauney and Verma, 1993), and as has been demonstrated, the Glu pathway dominates under osmotic stress conditions (Delauney et al., 1993). In Vigna aconitifolia and Arabidopsis, the first two steps of Pro biosynthesis from Glu were catalysed using 1-pyrroline-5-carboxylate synthetase (P5CS), a bifunctional enzyme with 
-glutamyl kinase (-GK) activities, and Glu-5-semialdehyde (GSA) dehydrogensae (Hu et al., 1992; Yoshiba et al., 1995). Under osmotic stress, P5CS plays a key role in the biosynthesis of Pro and catalyses the major regulated step (Kiyosue et al., 1996). Pro dehydrogenase (ProDH/PDH) catalyses the conversion of Pro back to P5C in Arabidopsis (Nanjo et al., 1999a). In tobacco (Nicotiana tabacum L.) plants, when P5CS was overexpressed the plants synthesized 10–18 times more Pro than the control plants and were more tolerant to water stress (Kavi Kishor et al., 1995). Transformed rice (Oryza sativa) plants that contained overexpressed P5CS were tolerant to salt and water stress (Zhu et al., 1998). Moreover, AtP5CS-antisense transgenic Arabidopsis plants, sensitive to low-humidity stress, survived by adding L-Pro (Nanjo et al., 1999b). Transgenic Arabidopsis plants with antisense-proline dehydrogenase (antisense-AtProDH) were more tolerant to freezing and high salinity conditions than wild-type plants (Nanjo et al., 1999a).
Because Pro accumulation in these transgenic plants improved stress tolerance, it was expected that the AtP5CS (from Arabidopsis) and OsP5CS (from rice) would display similar functions in other plants. While major success in the production of abiotic stress-tolerant transgenics has been achieved in model plants, ornamental plants have not yet been studied. Therefore, the environmental stress response mechanism and free Pro accumulation was investigated in petunia (Petunia hybrida) plants and the stress tolerance of transgenic plants was examined using a stress-induced AtP5CS or OsP5CS gene. Nanjo et al. (2003) reported that pdh, an Arabidopsis T-DNA-tagged mutant with an AtProDH defect, is hypersensitive to exogenous L-Pro, which suppresses root elongation and plant growth. Therefore, the influence of exogenous L-Pro on wild-type petunia and Arabidopsis plant growth was also investigated.
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Materials and methods |
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Pro measurement in stressed wild-type petunia plants
Sterile wild-type petunia (Petuniaxhybrida cv. Mitchell) seeds were germinated and grown on a solid MS medium (Murashige and Skoog (1962)
basal, 3% sucrose, 0.8% agar) for 1 week. The seedlings were then transplanted into soil (Super-soil:vermiculite 1:1 v:v, 100 g per pot), 10 per pot. After 3 d, the pots were placed on two paper towels for 30 min to absorb excess water and to equalize the soil moisture. For the drought stress conditions, the plants were grown without water in the culture room under 16/8 light/darkness at 25 °C and 60% humidity. For the salt-stress treatment, pots were placed in a salt solution (250 mM NaCl) and incubated in the same room. Cold-stress treatment was conducted under continuous illumination at 5 °C. These treatments were performed over 0, 3, 7, and 14 d. Plant tissue extracts were prepared as described by Flores and Galston (1982). Amino acids were extracted from the second or third leaves of 10 plants (Cohen and Strydom, 1988) and the amount/concentrations were measured using HPLC (HITACHI L-8500A).
Conditions of petunia plant transformation
pBIG35 plasmids containing P5CS coding from Arabidopsis thaliana ecotype Columbia (AtP5CS; Yoshiba et al., 1995), or Oryza sativa cv. ‘Akibare’ (OsP5CS; Igarashi et al., 1997) were used. The cauliflower mosaic virus 35S promoter was used for Agrobacterium-mediated transformation of the petunias (Petuniaxhybrida cv. Mitchell) as the control conditions. The mature seeds were harvested (T1) and those that exhibited kanamycin resistance were used for subsequent experiments.
RT-PCR analysis
The total RNA of the transgenic plants were subjected to RT-PCR using a OneStep RT-PCR kit (Qiagen). The PCR primer pairs used for amplification were 5'-TCAGAGGACTACGTGTTGGA-3' and 5'-ATGAGTACTAAGCAGAGAGG-3' for AtP5CS cDNA, and 5'-TTCATGGGAAAAAATTGGT-3' and 5'-CCGCAATAAGATGCTTGTAC-3' for the OsP5CS cDNA. A PCR reaction occurred for the reverse transcription (50 °C for 30 min) and the initial activation step (95 °C for 15 min), and then after 40 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension step at 72 °C for 10 min in a thermal cycler (GeneAmp PCR System 2400, Perkin Elmer).
Drought-stress treatment for young petunias
Wild-type petunia seeds were germinated and maintained in an MS medium. T1 transgenic plant seeds were germinated on an MS medium that contained kanamycin (70 mg l–1). After 7 d, the young plants were transferred to soil (Super-soil:vermiculite:soil 2:1:1 by vol., 100 g per pot), 10 per pot, and grown in the culture room under a 16/8 light/dark cycle at 25 °C. For the drought-stress treatment, they were grown without water in the culture room under the same conditions and 60% humidity. After 2 weeks, they were watered and allowed to recover.
Exogenous L-Pro treatment of wild-type petunias and Arabidopsis plants
The sterilized seeds were germinated and grown on a solid MS medium for 1 week. The 20 seedlings were then transferred to solid L-Pro medium (MS medium with 0, 5, 10, or 50 mM L-Pro, Wako). Each treatment was performed for 7 d. The amino acids were extracted from the leaves of 10 plants (Cohen and Strydom, 1988) and the amount/concentrations were measured using HPLC (Hitachi L-8500A).
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Results |
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Growth response and Pro accumulation in petunias during drought, salinity, and cold stress
Figure 1A shows the dry weights of the leaves of wild-type petunia plants according to the drought, salinity, and cold-stress conditions. The plants showed little or no growth under all stress conditions. The leaves from the plants subjected to cold did not grow. The number of new leaves increased in the plants subjected to salinity, but the old leaves became yellow and thick. Plants grown under these conditions recovered afterwards (data not shown). Under drought conditions, the leaves grew slightly but the rewatered plants did not survive. Free Pro did not accumulate in the control leaves (Fig. 1B). A lot of free Pro accumulated in the leaves stressed by drought for 14 d. On the other hand, it was verified that free Pro accumulated in the salinity-stressed leaves (about 20 pmol mg–1 DW) and that little accumulated under cold conditions. Table 1 shows the content of each amino acid as a percentage of the total amino acids for each 14 d period. Under drought-stress conditions, the free Pro amounted to 60% of all the amino acids. The Glu decreased under drought and salinity conditions, but increased under cold.
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Free Pro content in AtP5CS and OsP5CS transgenic petunia plants
RT-PCR gene expression was detected in the three AtP5CS lines and the two OsP5CS lines (Fig. 2B). However, it could not be detected in the wild-type plants. Figure 3A shows the detection of free Pro in each line (30-d-old). The free Pro content in the lines were 1.5–3.5 times greater than the content in the wild-type plants. All transgenic plants showed normal growth (Fig. 3B). Table 2 shows the content of each amino acid as a percentage of the total amino acids in wild-type and transgenic plants grown under normal conditions. The total amino acids of the transgenic plants increased more than for wild-type plants (data not shown). Under normal conditions, the Pro content of transgenic plants increased. The Glu content remained constant in four of the five transgenic plants.
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Drought tolerance in young transgenic petunia plants
The drought-stressed plants started with only two cotyledons and finished with about three leaves. After 10 d, the influence of drought conditions on growth became visible. A few of the wild-type plants that were watered after 14 d of drought treatment barely survived (Fig. 4). On the other hand, 20–53.3% of the transgenic plants were revived and resumed normal growth after watering.
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Influence of exogenous L-Pro on growth of wild-type Arabidopsis and petunias
The young wild-type plants (7-d-old) grown on the medium containing L-Pro, showed retarded root growth and yellowed leaves (Fig. 5A). The severity of phenotype was directly correlated to the concentrations of exogenous L-Pro. However, the mannitol treatment showed no effect on growth (data not shown). Low L-Pro concentrations suppressed the root growth and leaf production of the petunias more than Arabidopsis (Fig. 5B). At 5 mM L-Pro, the free Pro content in petunia leaves was about 100 times the content in the untreated leaves (Fig. 6). Additionally, the Pro content in petunia plants was much higher than that in Arabidopsis plants. The amounts of other amino acid concentrations also increased by L-Pro treatment in both plants (data not shown). The Glu content did not change, but the Gln content increased in the petunia leaves (Table 3).
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Discussion |
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The stress-induced Arabidopsis and rice genes are thought to be involved in the plants' response and tolerance to environmental stresses (Seki et al., 2002
; Rabbani et al., 2003). Many plants accumulate compatible osmolytes, such as Pro, Gly betaine, or sugars, under osmotic stress. Pro biosynthesis from Glu appears to be the predominant pathway under stress conditions, because the repressed salt-stress ornithine omega-aminotransferase expression (the enzyme responsible for synthesis of Pro from Orn), induced the synthesis of Pro from Glu (Delauney and Verma, 1993; Delauney et al., 1993). In adult Arabidopsis plants, the free Pro increase was mainly due to the enzyme activity in the Glu pathway. However, in young Arabidopsis plants, the Orn and Glu pathways together play an important role in accumulating Pro during osmotic stress (Roosens et al., 1998). In young petunia plants, most of the Pro accumulated under drought stress conditions (Fig. 1B), and the Glu content decreased under drought and salinity stresses (Table 1). It is suggested that P5CS is an important enzyme for Pro biosynthesis and accumulations in young petunia plants. It also increases their ability to withstand drought and salinity stresses.
It was examined whether AtP5CS and OsP5CS would improve stress tolerance in young transgenic petunia plants. In transgenic tobacco plants, overexpressing the mothbean (Vigna aconitifolia [Jacq.] Marechal) P5CS gene produced 10–18 times more Pro than in the control plants, and Pro accumulation occurred at the expense of Glu, a precursor for P5C under normal conditions (Kavi Kishor et al., 1995). The transgenic petunia plants used here accumulated more free Pro than the wild type (Fig. 3A), because the Glu content in proportion to the total amino acids did not decrease in transgenic plants (Table 2), and Pro production using AtP5CS and OsP5CS did not influence the amino acid metabolism of petunia plants. Further, the accumulated free Pro did not influence the growth of petunia plants (Fig. 3B). It is concluded that AtP5CS and OsP5CS function fully to produce free Pro in petunias and would be useful for genetically engineering Pro production.
The elevated Pro levels caused by overexpression of P5CS in transgenic tobacco and rice plants improved their tolerance to water and salt stresses (Kavi Kishor et al., 1995; Zhu et al., 1998). To clarify the role of Pro in drought tolerance, the drought tolerance of young transgenic plants was assessed. Although most wild-type plants died, the transgenic plants showed tolerance to drought stress (Fig. 4). This reveals a clear correlation between the survival rate and accumulated Pro in transgenic plants. Consequently, AtP5CS and OsP5CS have sufficient functionality in petunia plants.
To investigate the potential roles of Pro in the growth of petunia plants, the relationship between Pro accumulation and plant growth was analysed using exogenous L-Pro treatment. Up to 10 mM L-Pro on wild-type Arabidopsis seedlings had only a slight influence on growth (Fig. 5) (Nanjo et al., 2003). However, 5 mM L-Pro suppressed the growth of young wild-type petunia plants, and the internal Pro content increased from 0.001% to 0.018% of the total amino acids (Table 3). This increase was higher than the increase in Arabidopsis plants, suggesting that young petunias are hypersensitive to L-Pro. On the other hand, the internal Pro content of adult transgenic petunia plants ranged from 0.57% to 1.01% of the total amino acids (Table 2), and their phenotype was normal under normal conditions (Fig. 3). The internal Pro content of adult transgenic tobacco plants (normal phenotype) that overaccumulated Pro was 48.18% of the total amino acids, and the plants had an improved tolerance to drought and salinity stress (Kavi Kishor et al., 1995). It is suggested that the proportion of Pro to the total amino acids, rather than the concentration, influences the growth of petunias and that the capacity for free Pro accumulation might depend on the plant species or the growth stages.
The Pro accumulations in transgenic Arabidopsis plants with antisense-AtProDH cDNA showed an improved tolerance to freezing and high salinity stresses (Nanjo et al., 1999a). However, antisense-AtP5CS transgenic plants grown on media without L-Pro wilted and died, showing a hypersensitivity to stress (Nanjo et al., 1999b). Both P5CS and ProDH catalyse the first and the rate-limiting steps, respectively, and their activities could influence Pro accumulation and plant growth. The only ProDH responsible for Pro degradation in Arabidopsis is AtProDH (Mani et al., 2002; Nanjo et al., 2003). The Arabidopsis T-DNA-tagged pdh mutant with the AtProDH defect grew normally in the absence of exogenous L-Pro, but its growth was halved by 2 mM L-Pro. These results reveal a capacity for free Pro accumulation in Arabidopsis and show that ProDH activity is important for plant growth when free Pro accumulates (Nanjo et al., 2003). These results show that very low concentrations of exogenous L-Pro similarly influenced petunia growth (Figs 5, 6). The addition of exogeneous Pro and rehydration induced a high level of ProDH transcripts, which were repressed by dehydration (Nakashima et al., 1998; Satoh et al., 2002; Oono et al., 2003). Previous reports revealed the P5CS expression in Arabidopsis and rice plants during dehydration and the production of ProDH during rehydration (Yoshiba et al., 1995; Kiyosue et al., 1996; Igarashi et al., 1997; Nanjo et al., 2003). The expression pattern of AtP5CS in Arabidopsis plants was up-regulated by 2 h dehydration and repressed by rehydration, but the expression of AtProDH was up-regulated by 2 h rehydration (Oono et al., 2003). Previous reports revealed P5CS expression in Arabidopsis and rice plants during dehydration and the production of ProDH during rehydration (Yoshiba et al., 1995; Kiyosue et al., 1996; Igarashi et al., 1997; Nanjo et al., 2003). The AtP5CS expression pattern in Arabidopsis plants was up-regulated after 2 h dehydration and repressed by rehydration, but the AtProDH expression was up-regulated by 2 h rehydration (Oono et al., 2003). However, the ProDH promoter sequence and activity in petunias have not yet been analysed. Because the internal free Pro content in petunias was higher than that in Arabidopsis plants under 5 mM L-Pro conditions (Fig. 6), the ProDH activity of petunias might be lower than that of Arabidopsis plants. On the other hand, the free Pro content in Arabidopsis plants of different ages has not yet been reported (Nanjo et al., 2003). It was confirmed that the free Pro content in adult wild-type petunia plants (Fig. 3) was higher than young plants (Figs 1, 6), and the other amino acid contents changed too. The free Pro content of adult transgenic plants accumulated up to 70 pmol mg–1 FW. As shown, the Pro content percentage in transgenic plants was 1.5–2.6 times higher than in wild-type plants (Table 2), and transgenic plants also showed normal growth. However, the free Pro content of young wild-type petunia plants increased up to 14 pmol mg–1 FW under exogenous L-Pro conditions. It is also shown that the Pro content percentage in L-Pro-treated plants was 18 times higher than in non-treated plants (Table 3) and that their growth was suppressed. Therefore, it is thought that Pro content is likely to be a factor in the amino acid content imbalance, and it is proposed that the influence of Pro content varies depending on the age of the plant. These data show that the stress conditions changed nearly all the amino acids concentrations in young petunia plants (Table 1), and that environmental factors also affected the conversion and recycling of the key amino acids by regulating the gene expressions of the key enzymes and their activities. If so, the metabolic systems of petunias might change under drought-stress conditions and exogenous L-Pro treatment. In conclusion, while Pro toxicity is still questionable in petunias, it is shown that Pro accumulation is important for drought tolerance in transgenic petunia and Arabidopsis plants (Nanjo et al., 1999b; Mani et al., 2002).
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Acknowledgements |
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We are grateful to Dr H Takatsuji and Dr K Kubo of the National Institute of Agrobiological Resources (NIAR) for their helpful suggestions. This work was supported by a project grant from the Bio-oriented Technology Research Advancement Institution (BRAIN).
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Footnotes |
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Abbreviations: P5C,
1-pyrroline-5-carboxylate; P5CS, P5C synthetase; ProDH, proline dehydrogenase. |
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