Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of Δ1-Pyrroline-5-Carboxylate Synthetase mRNA or Protein1

Abstract

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 mol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera ⌬ 1 -pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steadystate levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.The high levels of free Pro observed in some plant tissues and organs suggest that this amino acid may have an important function in normal plant growth and development. Very high levels of free Pro have been reported in the flowers and seeds of Arabidopsis (Chiang and Dandekar, 1995;Savouré et al., 1995), in inflorescences and siliques of Brassica napus (Flasinski and Rogozinska, 1985), in ovules of broad bean (Venekamp and Koot, 1984), in pollen grains of petunia and tomato (Zhang et al., 1982;Fujita et al., 1998), and in the mature fruits of citrus species (Clements and Leland, 1962), pear species (Ulrich and Thaler, 1955), and grapevine (Vitis vinifera; Lafon-Lafourcade and Guimberteau, 1962;Kliewer, 1968;Ough and Stashak, 1974). The role of free Pro in the development or function of these organs remains unknown. Similarly, the regulation and temporal patterns of Pro biosynthesis and accumulation during normal plant development, in the absence of abiotic stress, remain essentially uncharacterized.Most of the research concerning Pro metabolism in plants has been focused on its accumulation in vegetative tissues in response to abiotic stresses such as drought and salinity. Stress-induced accumulation occurs predominantly through the enhanced biosynthesis of Pro from Glu via the pathway catalyzed by P5CS and P5CR rather than from Orn via the pathway catalyzed by OAT and P5CR (Boggess et al., 1976;Rhodes and Bressan, 1986;Delauney and Verma, 1993). The onset of stress-induced Pro accumulation is correlated with transcriptional activation of the gene encoding P5CS, which is the key regulatory and ratelimiting enzyme in this biosynthetic pathway (Hu et al., 1992;Delauney and Verma, 1993;Kishor et al., 1995;Savouré et al., 1995;Yoshiba et al., 1995;Zhang et al., 1995;Peng et al., 1996;Strizhov et al., 1997). Transcriptional regulation of the gene encoding PDH, the firs...

Proline Metabolism and Its Implications for Plant-Environment Interaction

“…The interconversion of proline and glutamate is sometimes referred to as the "proline cycle". The transcriptional upregulation of proline synthesis from glutamate and downregulation of proline catabolism during stress are thought to control proline levels, although exceptions to this pattern have been observed (Stines et al, 1999). Much data are consistent with this straightforward model of proline metabolism being mainly regulated by transcription of genes encoding the key enzymes.…”

Effects of UV radiation on five marine microphytobenthic Wadden sea diatoms, isolated from the Solthörn tidal flat (Lower Saxony, southern North Sea) – Part II: changes in carbohydrate, amino acid and fatty acid composition

“…The reaction velocity was measured as the rate of consumption of NADPH, monitored as decrease in absorption at 340 nm as a function of time, as described by Stines et al (1999). Δ 1 -pyrroline-5-carboxylate reductase (P5CR) activity was determined spectrophotometrically at 20°C according to the method of Treichel (1986), using an extinction coefficient of 6.317 l −1 mM −1 cm −1 of NADPH at 340 nm, in a reaction mixture containing 75 mM DDT, 5 mM Δ 1 -pyrroline-5-carboxylate (P5C), 15 mM NADPH, 50 µl of enzyme solution, and 500 mM K 3 PO 4 buffer (pH 7.0), in a total volume of 900 µl.…”

Compatible solutes in three marine intertidal microphytobenthic Wadden Sea diatoms exposed to different salinities

“…6H 2 O, 5 mM ATP, 10 mM NADPH and 100 ml enzyme extract at 20 C. The reaction velocity was measured as the rate of consumption of NADPH, monitored as decrease in absorption at 340 nm as a function of time as described by Stines et al (1999). D 1 -pyrroline-5-carboxylate reductase (P5CR) activity was determined spectrophotometrically at 20 C according to the method of Treichel (1986), using an extinction coefficient of 6.317 l À1 mM À1 cm À1 of NADPH at 340 nm, in a reaction mixture containing 75 mM DDT, 5 mM D 1 -pyrroline-5-carboxylate (P5C), 15 mM NADPH, 50 ml of enzyme solution, and 500 mM K 3 PO 4 buffer (pH 7.0), in a total volume of 900 ml.…”