Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes.

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host's roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial 59Fe2+ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for 52Mn2+ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.

Examining effects of rhizobacteria in relieving abiotic crop stresses using carbon-11 radiotracing.

In agriculture, plant growth promoting bacteria (PGPB) are increasingly used for reducing environmental stress-related crop losses through mutualistic actions of these microorganisms, activating physiological and biochemical responses, building tolerances within their hosts. Here we report the use of radioactive carbon-11 (t½ 20.4 min) to examine the metabolic and physiological responses of Zea mays to Azospirillum brasilense (HM053) inoculation while plants were subjected to salinity and low nitrogen stresses. Host metabolism of "new" carbon resources (as 11 C) and physiology including [11 C]-photosynthate translocation were measured in response to imposed growth conditions. Salinity stress caused shortened, dense root growth with a 6-fold increase in foliar [11 C]-raffinose, a potent osmolyte. ICP-MS analyses revealed increased foliar Na+ levels at the expense of K+ . HM053 inoculation relieved these effects, reinstating normal root growth, lowering [11 C]-raffinose levels while increasing [11 C]-sucrose and its translocation to the roots. Na+ levels remained elevated with inoculation, but K+ levels were boosted slightly. Low nitrogen stress yielded longer roots possessing high levels of anthocyanins. Metabolic analysis revealed significant shifts in "new" carbon partitioning into the amino acid pool under low nitrogen stress, with significant increases in foliar [11 C]-glutamate, [11 C]-aspartate, and [11 C]-asparagine, a noted osmoprotectant. 11 CO2 fixation and [11 C]-photosynthate translocation also decreased, limiting carbon supply to roots. However, starch levels in roots were reduced under nitrogen limitation, suggesting that carbon repartitioning could be a compensatory action to support root growth. Finally, inoculation with HM053 re-instated normal root growth, reduced anthocyanin, boosted root starch, and returned 11 C-allocation levels back to those of unstressed plants.

Examining the Effects of the Nitrogen Environment on Growth and N2-Fixation of Endophytic Herbaspirillum seropedicae in Maize Seedlings by Applying 11C Radiotracing.

Herbaspirillum seropedicae, as an endophyte and prolific root colonizer of numerous cereal crops, occupies an important ecological niche in agriculture because of its ability to promote plant growth and potentially improve crop yield. More importantly, there exists the untapped potential to harness its ability, as a diazotroph, to fix atmospheric N2 as an alternative nitrogen resource to synthetic fertilizers. While mechanisms for plant growth promotion remain controversial, especially in cereal crops, one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their own growth and biological functions. Biological nitrogen fixation (BNF), a microbial function that is reliant on nitrogenase enzyme activity, is extremely sensitive to the localized nitrogen environment of the microorganism. However, whether internal root colonization can serve to shield the microorganisms and de-sensitize nitrogenase activity to changes in the soil nitrogen status remains unanswered. We used RAM10, a GFP-reporting strain of H. seropedicae, and administered radioactive 11CO2 tracer to intact 3-week-old maize leaves and followed 11C-photosynthates to sites within intact roots where actively fluorescing microbial colonies assimilated the tracer. We examined the influence of administering either 1 mM or 10 mM nitrate during plant growth on microbial demands for plant-borne 11C. Nitrogenase activity was also examined under the same growth conditions using the acetylene reduction assay. We found that plant growth under low nitrate resulted in higher nitrogenase activity as well as higher microbial demands for plant-borne carbon than plant growth under high nitrate. However, carbon availability was significantly diminished under low nitrate growth due to reduced host CO2 fixation and reduced allocation of carbon resources to the roots. This response of the host caused significant inhibition of microbial growth. In summary, internal root colonization did little to shield these endophytic microorganisms from the nitrogen environment.

Plant-Growth-Promoting Bacteria Can Impact Zinc Uptake in Zea mays: An Examination of the Mechanisms of Action Using Functional Mutants of Azospirillum brasilense.

Among the PGPB, the genus Azospirillum-with an emphasis on A. brasilense-is likely the most studied microorganism for mitigation of plant stress. Here, we report the investigation of functional mutants HM053, ipdC and FP10 of A. brasilense to understand how the biological functions of these microorganisms can affect host Zn uptake. HM053 is a Nif+ constitutively expressed strain that hyper-fixes N2 and produces high levels of the plant's relevant hormone auxin. FP10 is a Nif- strain deficient in N2-fixation. ipdC is a strain that is deficient in auxin production but fixes N2. Zn uptake was measured in laboratory-based studies of 3-week-old plants using radioactive 65Zn2+ (t½ 244 days). Principal Component Analysis was applied to draw out correlations between microbial functions and host 65Zn2+ accumulation. Additionally, statistical correlations were made to our prior data on plant uptake of radioactive 59Fe3+ and 59Fe2+. These correlations showed that low microbial auxin-producing capacity resulted in the greatest accumulation of 65Zn. Just the opposite effect was noted for 59Fe where high microbial auxin-producing capacity resulted in the greatest accumulation of that tracer.