Removal of Feedback Inhibition of Δ¹-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

Pro is known to play an important role as an osmoprotectant in plants subjected to hyperosmotic stresses such as drought and soil salinity (Delauney and Verma, 1993). Recent studies on Pro synthesis and catabolism genes have provided results that are consistent with diverse functions of Pro as a source of energy, nitrogen and carbon, and as an osmolyte in response to dehydration (Kohl et al., 1988; Kavi Kishor et al., 1995; Peng et al., 1996; Hua et al., 1997; Zhang et al., 1997). Synthesis, accumulation, and catabolism of Pro in plants are highly regulated processes. Pro is synthesized via two routes from either Glu or Orn (Adams and Frank, 1980; Delauney and Verma, 1993). We have demonstrated that the Glu pathway is predominant under the conditions of osmotic stress (Delauney et al., 1993). In Vigna aconitifolia and Arabidopsis, the first two steps of the Pro biosynthesis from Glu are catalyzed by Δ^{-pyrroline-5-carboxylate synthetase (P5CS), a bifunctional enzyme with activities of γ-glutamyl kinase (γ-GK) and Glu-5-semialdehyde (GSA) dehydrogensae (or γ-glutamyl phosphate reductase;} Hu et al., 1992; Savoure et al., 1995; Yoshiba et al., 1995). In tomato, it has been reported that there are two Pro loci in the nuclear genome: one specifies a bifunctional P5CS (tomPro2) and the other one (tomPro1) apparently encodes a polycistronic mRNA that directs the synthesis of γ-GK and GSA dehydrogenase as two separate peptides (Garcia-Rios et al., 1997). Two P5CS genes have also been shown to be present in both Arabidopsis and alfalfa (Strizhov et al., 1997; Ginzberg et al., 1998; Yoshiba et al., 1999). The Arabidopsis P5CS1 gene is expressed in most organs and is induced rapidly by stress (Strizhov et al., 1997; Zhang et al., 1997; Yoshiba et al., 1999). P5CS2 is expressed in dividing cell cultures and its induction by stress is dependent on protein synthesis (Ginzberg et al., 1998).

Earlier experiments suggested that Pro accumulation in plants under stress may involve the loss of feedback regulation due to a conformational change in the P5CS protein (Boggess et al., 1976a, 1976b). In bacteria, Pro biosynthesis has been shown to be regulated by the end product inhibition of γ-GK activity (Smith et al., 1984). A Salmonella typhimurium mutant resistant to a toxic Pro analog (3,4-dehydro-d,l-Pro) accumulated Pro and showed enhanced tolerance to osmotic stress (Csonka, 1981). The mutation was due to a change of an Asp residue (at position 107) to Asn, rendering the γ-GK much less sensitive to inhibition by Pro (Csonka et al., 1988; Dandekar and Uratsu, 1988). We showed that the conserved Asp residue (at position 128) in the V. aconitifolia P5CS is not involved in the feedback inhibition (Zhang et al., 1995). Using site-directed mutagenesis, a replacement of Phe at position 129 by Ala was made in V. aconitifolia P5CS (P5CSF129A). This mutant enzyme was shown to retain similar kinetic characteristics as wild-type P5CS, but its feedback inhibition was virtually eliminated (Zhang et al., 1995). In this report, we demonstrate that tobacco (Nicotiana tabacum) plants carrying P5CSF129A accumulate more Pro, produce fewer free radicals, and are more tolerant to osmotic stress than plants expressing the wild-type V. aconitifolia P5CS transgene only. The P5CS transgenic seeds germinated well in a high salinity (200 mm NaCl) environment, while the wild type did not. These results demonstrated that feedback regulation of P5CS by Pro plays a role in the control of Pro biosynthesis in plants, and that Pro accumulation reduces osmotic stress, which may be mediated by free radicals produced as a result of oxidative stress.

ACKNOWLEDGMENT

Footnotes

LITERATURE CITED