Glutathione S-transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of toxic xenobiotics and oxidatively produced compounds to reduced glutathione, which facilitates their metabolism, sequestration, or removal. We report here that soybean (Glycine max) root nodules contain at least 14 forms of GST, with GST9 being most prevalent, as measured by both real-time reverse transcription-polymerase chain reaction and identification of peptides in glutathione-affinity purified extracts. GST8 was prevalent in stems and uninfected roots, whereas GST2/10 prevailed in leaves. Purified, recombinant GSTs were shown to have wide-ranging kinetic properties, suggesting that the suite of GSTs could provide physiological flexibility to deal with numerous stresses. Levels of GST9 increased with aging, suggesting a role related to senescence. RNA interference studies of nodules on composite plants showed that a down-regulation of GST9 led to a decrease in nitrogenase (acetylene reduction) activity and an increase in oxidatively damaged proteins. These findings indicate that GSTs are abundant in nodules and likely function to provide antioxidant defenses that are critical to support nitrogen fixation.

Several tropical grasses harbor symbiotic nitrogen-fixing bacteria within their stem and rhizome tissue that may contribute to the nitrogen nutrition of the host plant. We present evidence here that sand dune grasses (Ammophila arenaria and Elymus mollis) from Oregon also contain nitrogen-fixing bacteria. Surface-sterilized stem and rhizome tissue from these species possess acetylene reduction (nitrogen fixation) activity and large populations (10(5) to 10(6) cfu/g fresh weight) of bacteria. These bacteria were cultured on N-free media and identified by sequencing of 16S rRNA genes or by GC-FAME. Random sequencing of numerous colonies from the initial isolation plates of mixed isolates showed that pseudomonads (Stenotrophomonas and Pseudomonas) were by far the most common microorganism. One isolate -Burkholderia sp. strain Aa1 - reduced acetylene in culture with maximum activity at an O(2) concentration of 2% (v/v) in liquid media or 10% on solid media. PCR screening of all the isolates with nifH and nifD primers was positive only for this species. Immunolocalization studies with antibodies to nitrogenase resulted in labeling within plant cell walls of stems and rhizomes. Evidence for a similar nitrogen-fixing association was also detected in Uniola paniculata (sea oats) and Ammophila brevigulata (American beachgrass). We conclude that these grasses, and probably other dune grasses from temperate climates, contain endophytic, diazotrophic bacteria that may contribute to the phenomenal success of these grasses on nutrient-poor sand.

Ascorbate peroxidase (AP) is a key enzyme that scavenges potentially harmful H₂O₂and thus prevents oxidative damage in plants, especially in N₂-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago sativa) root nodules contains elevated levels of AP protein, as well as the corresponding mRNA transcript and substrate (ascorbate). Enhanced AP protein levels were also found in cells immediately peripheral to the infected region of soybean (Glycine max), pea (Pisum sativum), clover (Trifolium pratense), and common bean (Phaseolus vulgaris) nodules. Regeneration of ascorbate was achieved by (homo)glutathione and associated enzymes of the ascorbate-glutathione pathway, which were present at high levels. The presence of high levels of antioxidants suggests that respiratory consumption of O₂in the endodermis or nodule parenchyma may be an essential component of the O₂-diffusion barrier that regulates the entry of O₂ into the central region of nodules and ensures optimal functioning of nitrogenase.

Several tropical grasses harbor symbiotic nitrogen-fixing bacteria within their stem and rhizome tissue that may contribute to the nitrogen nutrition of the host plant. We present evidence here that sand dune grasses (Ammophila arenaria and Elymus mollis) from Oregon also contain nitrogen-fixing bacteria. Surface-sterilized stem and rhizome tissue from these species possess acetylene reduction (nitrogen fixation) activity and large populations (10⁵ to 10⁶ cfu/g fresh weight) of bacteria. These bacteria were cultured on N-free media and identified by sequencing of 16S rRNA genes or by GC-FAME. Random sequencing of numerous colonies from the initial isolation plates of mixed isolates showed that pseudomonads (Stenotrophomonas and Pseudomonas) were by far the most common microorganism. One isolate – Burkholderia sp. strain Aa1 – reduced acetylene in culture with maximum activity at an O₂ concentration of 2% (v/v) in liquid media or 10% on solid media. PCR screening of all the isolates with nifH and nifD primers was positive only for this species. Immunolocalization studies with antibodies to nitrogenase resulted in labeling within plant cell walls of stems and rhizomes. Evidence for a similar nitrogen-fixing association was also detected in Uniola paniculata (sea oats) and Ammophila brevigulata (American beachgrass). We conclude that these grasses, and probably other dune grasses from temperate climates, contain endophytic, diazotrophic bacteria that may contribute to the phenomenal success of these grasses on nutrient-poor sand.