Proline suppresses Rubisco activity by dissociating small subunits from holoenzyme.

Proline caused irreversible inhibition (involving reduction in V(max) without altering K(m) for RuBP) in Rubisco activity. Proline-induced suppression in Rubisco activity did not exceed beyond approximately 65% of the original activity even upon exposure to higher levels of proline for prolonged duration. However, NaCl-induced reduction in Rubisco activity was reversible. Native PAGE analysis of Rubisco-incubated with proline showed the presence of two distinct bands corresponding to approximately 430 and approximately 28 kDa, but that incubated with NaCl showed a single band. SDS-PAGE analysis revealed that the approximately 430- and approximately 28-kDa bands represent octamers of large subunits and dimers of small subunits, respectively. These results demonstrated for the first time that proline suppresses Rubisco activity by bringing about dissociation of the small subunits from the octamer core of large subunits, probably by weakening hydrophobic interactions between them.

Characterization of high temperature induced stress impairments in thylakoids of rice seedlings.

Exposure of isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40 degrees C) for 24 hr in dark on rice seedlings to characterize the extent of damage by in vivo heat stress on photofunctions of photosystem II (PSII). Chl a fluorescence transient analysis in the intact rice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited >50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thylakoid membranes from heat exposed seedlings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by approximately 40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40 degrees C heat exposure for 24 hr, the PSII reaction centers were impaired by in vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII.

Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase.

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis. This gene encodes choline oxidase, an enzyme that converts choline to glycinebetaine. The photosynthetic activity, monitored in terms of chlorophyll fluorescence, of transformed plants was more tolerant to light stress than that of wild-type plants. This enhanced tolerance to light stress was caused by acceleration of the recovery of the photosystem II (PS II) complex from the photo-inactivated state. The transformed plants synthesized glycinebetaine, but no changes were detected in the relative levels of membrane lipids or in the relative levels of fatty acids in the various membrane lipids. Transformation with the codA gene increased levels of H2O2, a by-product of the reaction catalyzed by choline oxidase, by only 50% to 100% under stress or non-stress conditions. The activity of ascorbate peroxidase and, to a lesser extent, that of catalase in transformed plants were significantly higher than in the wild-type plants. These observations suggest that H2O2 produced by choline oxidase in the transformed plants might have stimulated the expression of H2O2 scavenging enzymes, with resultant maintenance of the level of H2O2 within a certain limited range. It appears that glycinebetaine produced in vivo, but not changes in membrane lipids or in the level of H2O2, protected the PS II complex in transformed plants from damage due to light stress.

Proline suppresses Rubisco activity in higher plants.

Seedlings of Brassica juncea, Sesbania sesban, and Oryza sativa exposed to salt stress accumulated proline to levels as high as 4- to 20-fold over those of controls. Because chloroplasts are the major site for synthesis of stress induced proline accumulation, in vitro studies were conducted to see how the over-accumulation of this solute influences the activity of the major chloroplastic enzyme Ribulose 1,5-bis-phosphate carboxylase (Rubisco) purified from the above plant species. Surprisingly, proline (believed to be a compatible solute) suppressed the activity of Rubisco significantly even when present at a concentration as low as 100 mM. The extent of this inhibition in Rubisco activity increased with an increase in the concentration of proline. Irrespective of the plant species from which Rubisco was purified, its activity declined by about 50% in the presence of 1 M proline. Rubisco from all three plant species was sensitive to NaCl and proline accelerated salt induced suppression in its activity. To the best of our knowledge this is the first report wherein a negative effect of proline (which is believed to protect enzymes under stress) has been clearly demonstrated. This perturbing effect of proline on Rubisco from higher plant species belonging to three distinct families cautions targeting of gene(s)/gene products for over-production of proline into chloroplasts.

Proline accumulates in plants exposed to UV radiation and protects them against UV induced peroxidation.

Proline accumulated in the shoots of seedlings of rice (Oryza sativa), mustard (Brassica juncea) and mung bean (Vigna radiata) exposed to UV radiations. The level of proline in the seedlings increased significantly with increase in UV exposure time. The production of malondialdehyde (an indice of lipid peroxidation) was also higher in the shoots of seedlings exposed to UV radiation as compared to controls, suggesting that UV radiations promote lipid peroxidation. The extent of UV radiation promoted enhancement in the levels of proline as well as that of malondialdehyde was higher in the seedlings of rice than those of mung bean or mustard. This lead us to believe that UV radiation induced proline accumulation protects plants against UV radiation promoted peroxidative processes. UV radiations also promoted peroxidation in linolenic acid micelles. The presence of proline along with linolenic acid micelles during UV exposure caused a considerable reduction in the production of malondialdehyde. This study, for the first time shows that plants exposed to UV radiations accumulate proline and proline can protect plant cells against UV radiation induced peroxidative processes.

Suppression in mitochondrial electron transport is the prime cause behind stress induced proline accumulation.

Exposure of six day old rice (Oryza sativa) seedlings to salt or cadmium stress lead to an increase in the level of proline with a simultaneous decline in the mitochondrial electron transport activity. Mitochondrial electron transport inhibitors - rotenone, antimycin A or potassium cyanide also stimulated proline accumulation in rice seedlings with a concurrent decline in the mitochondrial electron transport activity. Four to five fold enhancement in proline level was noted in seedlings after 48 h exposure to electron transport inhibitors. A significant rise in the level of NADH was also noted in seedlings exposed to salt stress, cadmium stress or any of the electron transport inhibitors. These results show for the first time that the suppression in the mitochondrial electron transport activity lead to proline accumulation. Our results also suggest that the increase in the ratio of NADH to NAD+ due to the suppression in mitochondrial electron transport might be the prime reason behind proline accumulation in plants exposed to environmental stresses.

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B5 medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10(-7),10(-6) and 10(-5) M). Best callus induction was noted on medium with 10(-7) M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10(-7)M or 10(-6)MNAA. However, 10(-5)M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10(-6)M BAP+10(-6)MNAA. Both IBA and NAA at 10(-6) M proved to be equally effective in induction of roots from the cut ends of 15-20 mm long shoots (excised from callus) in half-strength B5 liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.

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.