Over-expression of PIP2;5 aquaporin in a Populus tremula×P.alba clone and its effects on plant responses to low root temperature and osmotic stress

Abstract

This dissertation describes the studies that examined: 1. Production of transgenic poplar over-expressing the poplar aquaporin PIP2;5 and 2. Comparison of the physiological properties of a transgenic poplar with the wild-type under different stress conditions including a) short-term low root temperature (LRT), b) prolonged LRT and c) polyethyleneglycol (PEG 6000) induced osmotic stress. Aquaporin PIP2;5 was selected due to its water-transporting capacity and increased expression under water limitation. The coding sequence of PtdPIP2;5 was cloned in the expression vector pCambia1305.2, under the control of maize ubiquitin promoter and the construct was introduced into in vitro grown Populus tremula × P. alba INRA Clone 717-1B4 by Agrobacterium-mediated transformation. Rooted plantlets of transformed poplar were transferred to the growth chamber and transformed lines were confirmed by PCR using primers against the hygromycin resistance gene and quantitative RT-PCR. Two lines exhibiting the highest expression of PIP2;5 were selected and ex vitro plants were generated for experimentation. When the root temperature (RT) was lowered from 20°C to 5°C in solution culture, for 30 minutes, PtdPIP2;5-over-expressing plants had significantly higher net gas exchange rates and root hydraulic conductivities (Lp) compared with the wild-type plants. Recovery was rapid in transgenic lines when the RT was raised back to 20°C. In the transgenic lines, the transcript abundance of PIP2;5 was significantly higher at 20°C and remained highly significant at 5°C for 30 minutes, when compared with the wild-type plants. When the plants were exposed to LRT for 3 weeks in solution culture, transgenic lines showed improved gas exchanging properties and Lp values than the wild-type plants. The transcript abundance of PIP2;5 significantly increased in both transgenic lines at 20°C and PIP2;1 levels increased in one transgenic line, after 3 weeks at 5°C. Transgenic lines had significantly higher growth rates, leaf size, gas exchange parameters and Lp than the wild-type poplars when subjected to PEG 6000 induced osmotic stress, at 50 g L-1. The results suggest that transgenic lines may be more resistant to LRT and osmotic stresses than the wild-type plants. This study confirms the contribution of PIP2;5 on plant water transport, gas exchange and growth.