Connecting Proline and γ-Aminobutyric Acid in Stressed Plants through Non-Enzymatic Reactions

The accumulation of proline (Pro) in plants exposed to biotic/abiotic stress is a well-documented and conserved response in most vegetal species. Stress conditions induce the overproduction of reactive oxygen species which can lead to cellular damage. In vitro assays have shown that enzyme inactivation by hydroxyl radicals (^·OH) can be avoided in presence of Pro, suggesting that this amino acid could act as an ^·OH scavenger. We applied Density Functional Theory coupled with a polarizable continuum model to elucidate how Pro reacts with ^·OH. In this work we suggest that Pro reacts favourably with ^·OH by H–abstraction on the amine group. This reaction produces the spontaneous decarboxylation of Pro leading to the formation of pyrrolidin-1-yl. In turn, pyrrolidin-1-yl can easily be converted to Δ¹-pyrroline, the substrate of the enzyme Δ¹-pyrroline dehydrogenase, which produces γ-aminobutyric acid (GABA). GABA and Pro are frequently accumulated in stressed plants and several protective roles have been assigned to these molecules. Thereby we present an alternative non-enzymatic way to synthetize GABA under oxidative stress. Finally this work sheds light on a new beneficial role of Pro accumulation in the maintenance of photosynthetic activity.     

Denitrification ability of rhizobial strains isolated from Lotus sp.

Ten rhizobial strains isolated from Lotus sp. have been characterized by their ability to denitrify. Out of the 10 strains, the five slow-growing isolates grew well under oxygen-limiting conditions with nitrate as a sole nitrogen source, and accumulated nitrous oxide in the growth medium when acetylene was used to inhibit nitrous oxide reductase activity. All five strains contained DNA homologous to the Bradyrhizobium japonicum nirK, norBDQ and nosZ genes. In contrast, fast-growing lotus rhizobia were incapable of growing under nitrate-respiring conditions, and did not accumulate nitrous oxide in the growth medium. DNA from each of the five fast-growing strains showed a hybridization band with the B. japonicum nirK gene but not with norBDQ and nosZ genes. Partial 16S rDNA gene sequencing revealed that fast-growing strains could be identified as Mesorhizobium loti species and the slow-growers as Bradyrhizobium sp.     

Heat stress results in loss of chloroplast Cu/Zn superoxide dismutase and increased damage to Photosystem II in combined drought‐heat stressed Lotus japonicus

Drought and heat stress have been studied extensively in plants, but most reports involve analysis of response to only one of these stresses. Studies in which both stresses were studied in combination have less commonly been reported. We report the combined effect of drought and heat stress on Photosystem II (PSII) of Lotus japonicus cv. Gifu plants. Photochemistry of PSII was not affected by drought or heat stress alone, but the two stresses together decreased PSII activity as determined by fluorescence emission. Heat stress alone resulted in degradation of D1 and CP47 proteins, and D2 protein was also degraded by combined drought–heat stress. None of these proteins were degraded by drought stress alone. Drought alone induced accumulation of hydrogen peroxide but the drought–heat combination led to an increase in superoxide levels and a decrease in hydrogen peroxide levels. Furthermore, combined drought–heat stress was correlated with an increase in oxidative damage as determined by increased levels of thiobarbituric acid reactive substances. Heat also induced degradation of chloroplast Cu/Zn superoxide dismutase (SOD: EC 1.15.1.1) as shown by reduced protein levels and isozyme‐specific SOD activity. Loss of Cu/Zn SOD and induction of catalase (CAT: EC 1.11.1.6) activity would explain the altered balance between hydrogen peroxide and superoxide in response to drought vs combined drought–heat stress. Degradation of PSII could thus be caused by the loss of components of chloroplast antioxidant defence systems and subsequent decreased function of PSII. A possible explanation for energy dissipation by L. japonicus under stress conditions is discussed.     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Cyanobacteria in Uruguayan rice fields: diversity, nitrogen fixing ability and tolerance to herbicides and combined nitrogen

It has been established that cyanobacteria play a vital role in the maintenance of flooded rice field fertility. To evaluate the potential use of nitrogen-fixing cyanobacteria as a natural biofertilizer for rice in Uruguay, the diversity, abundance and nitrogen fixing ability of these microorganisms were studied in the field and in the laboratory. The effect of urea fertilization on population density and diversity of heterocystous cyanobacteria was determined on a 3-year assay. The highest number of cyanobacteria, 1.6x10(4) CFU x m(-2), was found at the control 8 weeks after flooding. About 90% of the heterocystous cyanobacteria found in both treatments belong to the genera Nostoc and Anabaena. Maximal nitrogenase activity was reached after 12 weeks of flooding in both treatments, with an average of about 20 micromol C2H4 x m(-2) x h(-1). To improve the understanding of the environmental factors that can limit nitrogenase activity in rice fields, two of the most abundant cyanobacteria isolates were tested for tolerance to combined nitrogen and two herbicides. In both isolates 0.2 mM ammonium inhibited nitrogenase activity after 24 h of culture. The addition of field-recommended doses of quinclorac and propanil affected oxygen photoevolution but nitrogenase activity was only inhibited by propanil.     

Cyanobacterial inoculation and nitrogen fertilization in rice

During three rice-growing seasons in Uruguay, field experiments were conducted to study the contribution of cyanobacterial inoculation and chemical N fertilization to rice production. Neither grain yield nor fertilizer recovery by the plant were affected by inoculation with native cyanobacterial isolates. A low fertilizer use efficiency (around 20%) was observed when labelled (NH₄)₂SO₄ was applied at sowing. Recovery of applied ¹⁵N by the soil–plant system was 50%. Inoculation did not modify ¹⁵N uptake by the plant when the fertilizer was three-split applied either. The total N-fertilizer recovery was higher when the fertilizer was split than when applied in a single dose. Plant N-fertilizer uptake was higher when the fertilizer was applied at tillering. Uptake of ¹⁵N from cyanobacteria by rice was studied in a greenhouse pots experiment without chemical nitrogen addition. Recovery of ¹⁵N from labelled cyanobacteria by rice in greenhouse growth conditions was similar to that of partial recovery of (NH₄)₂SO₄ applied at sowing in the field. Cyanobacterial N mineralization under controlled conditions was fast as cyanobacterial N was detected in plants after 25 days. Moreover 40 days after inoculation non-planted and inoculated soil had more inorganic N than the non-inoculated one.