Exogenous Pro Research Articles

Osmotolerance and growth stimulation of transgenic tobacco cells accumulating free proline by silencing proline dehydrogenase expression with double‐stranded RNA interference technique

Many higher plants accumulate free proline (Pro) to counteract osmotic stress. To examine the role of free Pro in salt resistance, we suppressed the tobacco Pro dehydrogenase (NtProDH) gene using a double‐stranded RNA interference technique in tobacco Bright Yellow 2 cells. Northern blot analysis showed reduced levels of the NtProDH transcripts in the transgenic line. The free Pro level in transgenic cells was about 1.2‐ to 3.0‐fold, and the Pro dehydrogenase activity was about 4.9–32.2% of those in wild‐type (WT) cells. The transgenic cells had an appearance markedly different from that of WT cells. Microscopic analysis revealed that the transgenic tobacco cells were mostly barrel shaped as in a filament, cylindrical and small. In synchronous cultures, transgenic cells showed more active cell division than WT cells. Hypersensitivity to exogenous Pro increased in the transgenic tobacco cells. The transgenic cells showed an increased osmotolerance, perhaps by free Pro accumulation.

Altered Levels of Proline Dehydrogenase Cause Hypersensitivity to Proline and Its Analogs in Arabidopsis

Pro dehydrogenase (PDH) catalyzes the first and rate-limiting step in the Pro catabolic pathway. In Arabidopsis, this enzyme is encoded byAt-PDH. To investigate the role of Pro catabolism in plants, we generated transgenic Arabidopsis plants with altered levels of PDH by sense (PDH-S plants) and antisense (PDH-AS plants) strategies. Free Pro levels were reduced by up to 50% in PDH-S plants under stress and recovery conditions and enhanced by a maximum of 25% in PDH-AS plants, despite large modifications of theAt-PDH transcript and At-PDH protein levels. A similar trend in free Pro levels was observed in the PDH-S and PDH-AS seeds without visible effects on germination or growth. Under stress conditions, PDH transgenic plants showed no signs of change in osmotolerance. However, addition of exogenous Pro increased survival rates of salt-stressed PDH-S plants by 30%. Isotope-labeling studies showed that the conversion of [14C]Pro to Glu was reduced in PDH-AS plants and increased in PDH-S plants, especially under stress conditions. Furthermore, PDH-AS plants were hypersensitive to exogenous Pro, whereas PDH-S plants were sensitive to Pro analogs. These findings demonstrate that altered At-PDH levels lead to weakly modified free Pro accumulation with a limited impact on plant development and growth, suggesting a tight control of Pro homeostasis and/or gene redundancy.

Altered Levels of Proline Dehydrogenase Cause Hypersensitivity to Proline and Its Analogs in Arabidopsis

Pro dehydrogenase (PDH) catalyzes the first and rate-limiting step in the Pro catabolic pathway. In Arabidopsis, this enzyme is encoded by At-PDH. To investigate the role of Pro catabolism in plants, we generated transgenic Arabidopsis plants with altered levels of PDH by sense (PDH-S plants) and antisense (PDH-AS plants) strategies. Free Pro levels were reduced by up to 50% in PDH-S plants under stress and recovery conditions and enhanced by a maximum of 25% in PDH-AS plants, despite large modifications of the At-PDH transcript and At-PDH protein levels. A similar trend in free Pro levels was observed in the PDH-S and PDH-AS seeds without visible effects on germination or growth. Under stress conditions, PDH transgenic plants showed no signs of change in osmotolerance. However, addition of exogenous Pro increased survival rates of salt-stressed PDH-S plants by 30%. Isotope-labeling studies showed that the conversion of [14C]Pro to Glu was reduced in PDH-AS plants and increased in PDH-S plants, especially under stress conditions. Furthermore, PDH-AS plants were hypersensitive to exogenous Pro, whereas PDH-S plants were sensitive to Pro analogs. These findings demonstrate that altered At-PDH levels lead to weakly modified free Pro accumulation with a limited impact on plant development and growth, suggesting a tight control of Pro homeostasis and/or gene redundancy.

Proline Metabolism in the Wild-Type and in a Salt-Tolerant Mutant of Nicotiana plumbaginifolia Studied by13C-Nuclear Magnetic Resonance Imaging

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. (13)C-Nuclear magnetic resonance with [5-(13)C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mM NaCl stress in the presence of [5-(13)C]Glu, a significant enrichment in [5-(13)C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-(13)C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-(13)C]Glu into [5-(13)C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Delta-pyrroline-5-carboxylate synthetase enzyme.