Proline biosynthese in relation to osmotic adaptation in higher plants : a molecular and physiological approach.

Project Details


Physiological characterization of mutant tobacco plants modified in proline accumulation.

Research project RC 370. Promotor: G. Angenon (PLAN; earlier M. Jacobs, PLAN). Co-promotor: L. Slooten (DNTK). Report covering the period 1st jan 1999-31st december 2001, submitted by L. Slooten.


Proline production is one of the defense mechanisms of plants against osmotic stress. There are two alternative pathways for proline biosynthesis, starting from glutamate or ornithine, respectively. Key enzymes in these pathways are -pyrroline-5-carboxylate synthetase (P5CS) en ornithine-d-aminotransferase (d-OAT), respectively. P5CS is subjected to feedback inhibition by proline. In the laboratory of Plant Genetics, mutants of Nicotiana plumbaginifolia were selected in which this feedback inhibition is strongly inhibited [2]. This appears to be due to a single-point mutation resulting in the modification of one aminoacid in the P5CS protein [3]. When one of these mutants (designated RNa), and the wildtype (P2) were exposed to salt stress, the proline content increased in both lines, but in RNa it increased more strongly and especially more rapidly than in P2 [3]. RNa was also considerably more tolerant to osmotic stress than wildtype plants [2]. The purpose of the current project was to generate different proline under- or overproducing Arabidopsis thaliana transformants, and to characterize these transformants physiologically, among others with regard to proline content and osmotolerance. However to start with, the RNa mutant of N. plumbaginifolia was to be compared with the wildtype (P2). The experiments yielding the most significant results would be selected for examination of the A. thaliana transformants.


Plants were grown in a phytotron or in a growth room with 80-85 % RH, 12/12 h day/night length, and adjustable temperature and light intensity (in PAR; 1 E = 1 mol quanta.m-2.s-1). Plants were sown on pot soil. Prior to salt treatment, 31-33 day old plantlets were rinsed to remove most of the soil, and were then transferred to pots containing vermiculite which had been rinsed twice with demineralized water. The vermiculite was rinsed every other day (starting three days after the transfer) with a nutrient solution (Mn medium [4]). Fresh solutions were prepared twice per week. For the test plants, NaCl and CaCl2 were added to the nutrient solution (see below). Proline was determined according to Bates [5]. The chlorophyll fluorescence parameter Fv/Fm was determined as described before [6]. Results are expressed as the mean and standard error (SE), usually for 4 to 6 plants unless indicated otherwise. The significance (p) of the difference between means was determined in a two-sided Student t-test.


1. The proline content is in stem leaves of N. plumbaginifolia approx 2-5 times higher than in rozette leaves.The proline content increases by a factor of 10 to 50 in response to drought- and salt stress, but not in response to heat stress. Conversely, proline biosynthesis (elicited by salt stress) did not provide protection against subsequent heat stress.
2. RNa accumulates more biomass and develops somewhat more rapidly than P2. The difference in biomass arises during the first six weeks after sowing; therefter there is no significant difference in relative growth rate (RGR).
3. Exposure of young rozette plants (approx 42 days old) to salt stress leads to inhibition of the formation of new leaves, and to inhibition of stem growth. RNa is slightly less sensitive in this respect than P2. However, the proline content after salt stress is in RNa not higher than in P2. Remarkably, the RGR (measured as the increase in total leaf area) was hardly influenced by salt stress. This is because the inhibition of formation of new leaves in salt-stressed plants was compensated by further growth of the pre-existing leaves.
4. Inhibition of leaf formation was largely absent if the onset of salt stress was postponed by one week. This means that the difference in sensitivity to salt stress between RNa and P2 could be explained by assuming:
a. that salt stress causes damage to the top meristem
b. that this damage is less severe if organ differentiation of the top meristem is in a more advanced stage at the onset of salt stress
c. that the organ differentiation at the onset of salt stress was in RNa somewhat more advanced than in P2. The latter assumption was validated in detailed growth measurements.
5. There was no difference between RNa and P2 in respect to the increase in proline content after exposure to drought stress. Visually there was no difference in sensitivity to drought stress between RNa and P2 (as judged by wilting and growth inhibition).
6. RNa shows a higher increase in proline content in response to cold stress than P2. However, judging by the effect of cold stress on the RGR, and on visual characteristics, RNa is no more tolerant to cold stress than P2.

Conclusions and Prospects.

1. We could not confirm the increased salt tolerance of RNa as compared to P2. In other stress situations there was no evidence either, of an enhanced osmotolerance of RNa in comparison with P2. It cannot be excluded yet that a difference in salt tolerance can be demonstrated during germination, or in very young seedlings.
2. On the other hand, in these experiments we have accumulated useful experience concerning the exposure of plants to various kinds of osmotic stress; experience which can be used in testing proline over- or underproducing transformants.
3. For future experiments we recommend to replace the vermiculite by a matrix which is less stressing by itself.
4. Even aside from the effect of cold or salt on the top meristem, young plants seem to be considerably more sensitive to cold or salt stress than older plants. It seems worth while to investigate how that comes about, by determining the effect of cold or salt on gen expression, enzyme activities and physiological parameters in young and older plants.


[1] Roosens NHCJ, Thu TT, Iskandar HM, Jacobs M (1998). Isolation of the ornithine- -transferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana. Plant Physiol 117: 263-271.
[2] Sumaryati S, Negrutiu I, Jacobs M (1992) Characterization and regeneration of salt- and water-stress mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani) Theor Appl Genet 83: 613-619.
[3] Roosens NH, Willem R, Li Y, Verbruggen I, Biesemans M, Jacobs M (1999) Proline metabolism in the wild-type and in a salt-tolerant mutant of Nicotiana plumbaginifolia studied by 13-C magnetic resonance imaging. Plant Physiol 121:1280-1290.
[4] Negrutiu I, Dirks R, Jacobs M (1983) Regeneration of fully nitrate reductase deficient mutant from protoplast culture of Nicotiana plumbaginifolia (Viviani). Theor Appl Genet 66: 341-347
[5] Bates LS (1973) Rapid determination of free proline for water stress studies. Plant Soil 39: 205-207.
[6] Van Camp W, Capiau K, Van Montagu M, Inzé D, Slooten L (1995) Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts. Plant Physiol 112: 1703-1714.

Effective start/end date1/01/9931/12/01


  • salt and drought resistance
  • regulation
  • proline metabolism

Flemish discipline codes

  • Biological sciences
  • Materials engineering