P. Pardha Saradhi

Proline Accumulation Under Heavy Metal Stress

Summary The effect of nitrate salts of the four heavy metals cadmium, cobalt and zinc in B 5 medium and lead in distilled water was tested on seed germination and seedling growth in Cajanus cajan (cv. BS-15), Vigna mungo (cv. T-9) and Triticum aestivum (cv. C-306) under controlled sterile conditions. All four heavy metals caused proportionate reduction in growth depending on the concentration. However, zinc stimulated the growth of seedlings at low concentrations. A considerable and proportionate increase in proline content was recorded with increase in concentration of heavy metals. Cadmium proved to be the strongest inducer for proline accumulation, zinc the weakest. Equimolar concentrations of potassium did not show any proportionate change in the level of proline when compared with controls. It is therefore inferred that proline accumulation can be used as a marker to test the level of heavy metal pollution. Accumulation of large quantities of proline in seedlings raised in media with lead nitrate having higher osmotic potential compared with those grown in B 5 medium (not supplemented with heavy metal) having lower osmotic potential suggests that proline accumulation is not related to osmotic adjustment.

EFFECT OF ZINC ON FREE RADICALS AND PROLINE IN BRASSICA AND CAJANUS

Abstract Investigations were carried out to study the effect of zinc on growth, free radical production and proline accumulation in shoots of Brassica juncea (Brassicaceae) and Cajanus cajan (Fabaceae). Seedlings were raised in modified B 5 medium supplemented with zinc sulphate under controlled aseptic conditions. In general, small concentrations of zinc (up to 0.1 mM) promoted growth of seedlings in both plant species. In contrast, production of free radicals (measured in terms of malondialdehyde) and the level of proline were low in the seedlings raised in the presence of these concentrations of zinc. However, zinc at higher concentrations significantly reduced growth, but promoted generation of free radicals as well as the accumulation of proline. Irrespective of the concentration of zinc sulphate, the shoots of C. cajan showed significantly higher levels of malondialdehyde as well as proline as compared with those of B. juncea . These results suggest the existence of a correlation between the generation of free radicals and the accumulation of proline. In this communication we propose that accumulation of proline is related to non enzymatic detoxification of free radicals that are generated excessively under stress.

INVOLVEMENT OF PROLINE IN PROTECTING THYLAKOID MEMBRANES AGAINST FREE RADICAL-INDUCED PHOTODAMAGE

Abstract Investigations were carried out to determine the involvement of proline in protecting thylakoid membranes against photodamage. The exposure of thylakoids isolated from the cotyledons of Brassica juncea to photoinhibitory light caused a linear increase in lipid peroxidation with time as measured by the assay of conjugated dienes and malondialdehyde (MDA). Proline produced a considerable reduction in the lipid peroxidation-linked formation of both conjugated dienes and MDA in the thylakoids during exposure to strong light. This reduction in lipid peroxidation reflects the probable involvement of proline in the reduction of the free radicals generated on strong light exposure. The thylakoids exposed to strong light intensity also generated singlet oxygen (1O2) and superoxide radical anion (O2−1). The level of these radicals increased with the time of exposure. The presence of proline significantly curtailed the production of 1O2 by the thylakoids during strong illumination. However, the presence of proline did not affect the O2− induction capacity of the thylakoid membranes during strong light exposure. Peroxidation of linolenic acid micelles occurred on exposure to UV radiation. The almost total suppression of UV-induced lipid peroxidation of linolenic acid micelles in the presence of sodium azide and L-histidine (singlet oxygen quenchers) suggested the involvement of 1O2 in UV-induced lipid peroxidation. The presence of proline during the exposure of linolenic acid micelles to UV radiation caused a considerable reduction in the formation of conjugated dienes as well as MDA. The proline-induced reduction in the peroxidation of these micelles further confirms the involvement of proline in reducing the level of 1O2. Our results therefore demonstrate that proline is involved in reducing the photodamage in the thylakoid membranes by scavenging and/or reducing the production of 1O2.

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.

Proline enhances primary photochemical activities in isolated thylakoid membranes of Brassica juncea by arresting photoinhibitory damage

The presence of L-proline in the reaction mixture enhances the photosystem II (H2O----DCPIP) and whole chain (H2O----MV) catalysed electron transport activities of thylakoids isolated from the cotyledonary leaves of Brassica juncea seedlings raised in the absence and the presence of NaCl. The extent of stimulation in activities was higher in the thylakoids of NaCl raised plants than the controls. The extent of proline mediated stimulation was seen even in the presence of uncoupler NH4Cl suggesting that this stimulation is not due to uncoupling. However, photosystem I (DCPIPH2----MV) catalysed photoreaction remained almost insensitive to proline. The presence of proline in the incubation medium brought about a significant reduction in the time dependent loss in photochemical activity of thylakoids exposed to strong light suggesting that proline prevents photoinhibitory loss in chloroplast activity. Also, proline brought about a considerable reduction in the production of lipid peroxidation linked maiondialdehyde during strong illumination. We suggest that proline protects the components involved in water oxidation capacity by reducing the production of free radicals and/or scavenging the free radicals and thereby reducing thylakoid lipid peroxidation.

Proline in relation to free radical production in seedlings of Brassica Juncea raised under sodium chloride stress

The production of malondialdehyde (MDA) was higher in cotyledons from NaCl-raised Brassica juncea seedlings than in control seedlings. Light accelerated the MDA-producing capacity of thylakoids isolated from both control and treated seedlings. When exposed to strong white light (920 μmol photons m−2 s−1) the thylakoids from NaCl seedlings produced nearly 5 times more MDA than control thylakoids. In the cotyledons of NaCl seedlings, the proline level was 24-fold higher than in controls. The presence of proline during exposure of thylakoids to white light decreased MDA levels. The reduction in MDA production was higher in the thylakoids of NaCl seedlings than of controls. It is proposed that proline accumulation has an adaptive significance as it lowers the generation of free radicals and thus reduces the lipid peroxidation linked membrane deterioration under stress.

Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes

Summary Exposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (∼65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide 14C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSI. Immunoblotting analysis of several PSII polypeptides such as D1/D2 reaction dimer and Cyt b559 showed no major changes due to mild heat exposure except for the PSII core antenna polypeptide (CP43), which could reflect the reduction in PSII activity observed in light saturation studies. Similarly, haeme staining did not indicate any change in other cytochrome related polypeptides. Our results therefore clearly suggest thatin vivo exposure of rice seedlings to elevated (40 °C) temperature caused thylakoid structural disorganization, and this disorganization of some of the thylakoid complexes resulted in a loss in thylakoid photochemical function.

Vesicular arbuscular mycorrhizal fungi improves establishment of micropropagated Leucaena leucocephala plantlets

Investigations were carried out to achieve cent per cent transplantation success of micropropagated Leucaena leucocephala (a fast growing multipurpose leguminous tree species) plantlets using two vesicular arbuscular mycorrhizal fungi, Glomus fasciculatum and Glomus macrocarpum. Plantlets were obtained by rooting the shoots [obtained through; hypocotyl callus in presence of 10-5M BAP + 10-6M NAA; and axillary bud sprouting from cotyledonary and other nodes in presence of 10-5M BAP, on Gamborgs B5 medium], on half strength B5 medium supplemented with 5×10-6M IBA. Subsequent to the nodulation of their roots with Rhizobium (strain PRGL 001)in soilrite, these plantlets were tranferred to sterilized garden soil by laying inoculum of either Glomus fasciculatum or Glomus macrocarpum around their roots. Only 20% of the plantlets survived in soils lacking VAM fungus. In contrast, cent per cent of the plantlets of Leucaena leucocephala established very well and showed good growth in VAM inoculated soil. Roots of the later plantlets showed presence of both external and internal hyphae with well formed arbuscules and vesicles confirming the establishment of good mycorrhizal association. These studies convincingly demonstrate that the mycorrhizal association help in successful establishment of tissue culture raised plantlets of Leucaena leucocephala in the field conditions by alleviating the transplantation shock.

Glomus fasciculatum alleviates transplantation shock of micropropagated Sesbania sesban.

Abstract Investigations were carried out using the vesicular arbuscular mycorrhizal fungus, Glomus fasciculatum, to improve the success in transplanting micropropagated plantlets of Sesbania sesban. Plantlets were developed from somatic embryos and/or adventitious buds (induced from various explants on Gamborgs medium supplemented with 6-benzylaminopurine), in the presence of 10–7mα-naphthaleneacetic acid and 5×10–6m gibberellic acid. Subsequent to nodulating the roots with Rhizobium, plantlets were transplanted into sterile garden soil and inoculated with or without G. fasciculatum. Only 30% of plantlets transferred to soil without G. fasciculatum survived. In contrast, all the plantlets inoculated with G. fasciculatum survived. Histochemical study revealed the presence of intracellular hyphae with well-developed arbuscules and intercellular hyphae with vesicles, suggesting that G. fasciculatum formed a good mycorrhizal association with S. sesban roots. These observations showed that mycorrhizal association helped to increase the potential of micropropagated plantlets to successfully withstand transplantation shock.

Effect of Sodium Chloride on Primary Photochemical Activities in Cotyledonary Leaves of Brassica juncea

Summary The effect of varying concentrations of sodium chloride (NaCl) (0, 100, 150 and 200 mM) on seedling growth, pigment composition and the primary photochemical activities in cotyledonary leaves of Brassica juncea cv. DIRA 367 was investigated. NaCl-stress resulted in a considerable decrease in growth of the seedlings without causing any significant change in dry weight. While, chlorophyll a content was higher in cotyledonary leaves of seedlings raised in high salt containing media, chlorophyll b content did not show any significant variation till 11th day of growth. Thus the high Chl a/Chl b ratio in NaCl treatments reflects changes in the photosynthetic antenna size. The carotenoid content in 11 day old cotyledonary leaves, on the other hand, was much reduced at high concentrations of NaCl. NaCl treatments caused an increase in water oxidation capacity, as measured through H2O → MV (whole chain) or H2O → PDOX (PS II) photoreactions, by 1.2 to 2.0 fold. But, PS I mediated photochemical activities showed no or only a minor increase by NaCl treatment. The enhancement in photochemical activities noted even in presence of uncouplers (NH4Cl and methyl amine) suggest that the NaCl induced enhancement of primary photochemical activity is not linked to the uncoupling of electron transport from photophosphorylation. Also, the studies on the interactive effect of in vivo ageing and salinity on the pigment composition and primary photochemical activities suggest that ageing and NaCI-stress result in partly independent and partly interactive changes in the pigment composition and photochemical activities.