Abstract: Dramatic accumulation of proline under a variety of stress conditions such as drought, salt and cold has been documented in many plants. Understanding the molecular mechanism of the accumulation of proline in plants may be helpful for improving abiotic stress tolerance of crop plants. Δ¹-pyrroline-5-carboxylate synthetase (P5CS) catalyzed the first two steps of proline biosynthesis in plants, is encoded by P5CS gene in higher plants. In this paper, the responses of PvP5CS2 gene in common bean (Phaseolus vulgaris) seedlings to drought, salt and cold stresses were investigated by Real-time quantitative PCR (RT-qPCR) approach. The accumulation of proline in common bean seedling was measured using aqueous sulfosalicylic acid method. In addition, subcellular localization of PvP5CS2 in onion epidermal cells was studied. The results showed that abiotic stresses significantly triggered the expression of PvP5CS2 gene in leaves and roots of common bean seedling, and caused the accumulation of proine. PvP5CS2 mRNA transcript level in common bean seedling leaves steadily increased to the maximum that was 1.51 times of control under the 4 d drought stress and then rapidly decreased to below control level in later period of drought stress. The proline content in leaves increased from the second day and up to the maximum (300.14 μg g^-1 FW) at 8 d of drought stress. Treatment with 200 mmol L^-1 NaCl caused a dramatic up-regulation of PvP5CS2 expression to the peak value, 7.67 fold of control at 2 h in leaves and 6.14 fold of control at 6 h in roots. The peak of proline accumulation appeared at 6–9 h (2.5 fold) in leaves and 9 h (14.2 fold) in roots when salt stress. A significant expression of PvP5CS2 was detected at 2 h under cold (4℃) stress, which was 2.38 fold of control in leaves and 7.55 fold of control in roots. The accumulation of proline steadily increased to the maximum at 24 h of treatment in leaves (43.29 μg g^-1 FW) and at 12 h of treatment (28.90 μg g^-1 FW) in roots. In addition, the accumulation of PvP5CS2 mRNA preceded the accumulation of proline in three stress treatments. These results indicated that PvP5CS2 gene is an abiotic stress-inducible gene with different transcript levels in response to abiotic stresses, which regulates the accumulation of proline in common bean seedlings under drought, salt and cold stresses. Transient expression of PvP5CS2- GFP fusion gene in onion epidermal cells showed that PvP5CS2 protein was distributed in nucleus and plasmalemma.

Key words: Common bean, PvP5CS2, Proline, Drought, Salt, and cold stress, Transient expression