Effect of Water Stress Induced by Polyethylene Glycol on Growth,Proline Accumulation in <i>Agave americana</i> L.

The effect of water deficit was determined on both in vitro and soil seedling as well as in cells in suspension of Agave americana L. In order to do the establishment of cells, the formation of callus was induced; for it two auxins were evaluated: 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-mino-3,5,6-trichloropicolinic acid (picloram) at three concentrations (0.25, 0.5 and 0.75 mg L⁻¹) in three explants (leaf, root and meristems) cultured in MS semisolid medium. The callogenesis response was related to the type and section of the explant, as well as the regulator used, and a cell suspension was established using 0.5 mg L⁻¹ naphthaleneacetic acid (NAA) + 0.5 mg L⁻¹ Benzylaminopurine (BAP). Seedlings were exposed to polyethyleneglycol (15% and 30% w/v) with a water potential of −0.87 and −2.67 MPa, respectively, under soil conditions. Water stress was applied through restricted irrigation. Fresh weight, root system growth, and chlorophyll concentration were some of the parameters that were affected by the effect of water deficit on A. americana L. Chlorophyll concentration values were significantly decreased by 15 at 30% PEG (19.6 SPAD units) compared to the control treatment. In in vitro plants, the highest concentration of proline was found in the roots, being the treatment with 30% polyethylene glycol where the highest concentration of this osmoregulator was obtained (62.5 mg g⁻¹ DW). Under restricted irrigation conditions, an increase in proline concentration was observed both in the aerial part (2.2 µg 100 g⁻¹ DW) and in the root system (1.8 µg 100 g⁻¹ DW). However, the concentrations found were approximately ten times greater, less than those found under in vitro conditions. Therefore, the accumulation of proline can be considered an indicator of stress in Agave Americana L. growth in vitro.     

The accumulation of proline in Prasiola crispa during winter in Antarctica

Samples of Prasiola crispa were collected in Antarctica throughout a 13-month period and analysed for free amino acids by HPLC. There was a marked increase in the levels of proline with the onset of winter, concurrent with a decrease in the other predominant amino acids. In January, proline constituted 1.2 ± 0.1 μ mol (g dry weight)⁻¹. whereas by mid-April it was the major component at 28.4 ± 2.9 μ mol (g dry weight)⁻¹. When winter samples were thawed in a growth cabinet, their proline content declined to 4.3 ± 0.5 μ mol (g dry weight)⁻¹ after 7 days. Measurements of photosynthetic quantum yield indicated that winter samples of P. crispa also recovered photosynthetic activity upon thawing. Amino acids and other solutes are involved in the preservation of photosynthetic activities during freezing and it seems probable that proline is involved in cryoprotection in this species. In summer samples, there was no evidence that proline levels in P. crispa increased with the conductivity of the water in which they were found growing.     

Does altered nitrogen metabolism and H2O2 accumulation explain the vitrified status of the fully habituated callus of Beta vulgaris (L.)?

The habituated callus is a vitrified tissue which has two main biochemical characteristics both leading to production of toxic forms of oxygen: first the blockage of the porphyrin pathway and a lack of H₂O₂ detoxifying enzymes (catalase and peroxidases); secondly a deviation of the nitrogen metabolism induced by NH₃ accumulation. Ammonia detoxification is ensured by increased glutamate dehydrogenase activity and accumulation of both proline and polyamines. A putative linkage between proline synthesis and the HMP pathway, as proposed for animal proliferating tissues (Phang 1985), might explain a high purine biosynthesis and cytokinin autonomy.Abbreviations FFA free fatty acids - 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - GLU glutamate - GDH glutamate dehydrogenase - GR glutathion reductase - H habituated callus - HMP hexoses-monophosphate - IAA indolyl-acetic acid - LOX lipoxygenase - MDA malondialdehyde - N normal callus - OAT ornithine aminotransferase - ORN ornithine - PAs polyamines - P₅C pyrroline-5-carboxylate - P₅CR pyrroline-5-carboxylate reductase - PP-ribose-P phosphoribosyl pyrophosphate - SOD superoxide dismutase     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

Role of α-helical conformation of histatin-5 in candidacidal activity examined by proline variants

Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent. The aim of this study was to investigate the importance of α-helical structure of Hsn-5 for its candidacidal activity. The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent α-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes. Our results indicated that 3P variant displayed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P), while 1P and 2P variants showed lower cidal activity. The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P. These results suggested that the α-helical content of Hsn-5 proline variants does not correlate with the candidacidal activity. Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of β-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of α-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests α-helix may not be important for anticandidal activity of Hsn-5.