Reactive oxygen species from type-I photosensitized reactions contribute to the light-induced wilting of dark-grown pea (Pisum sativum) epicotyls.

Type-II, singlet oxygen-mediated photosensitized damage has already been shown to occur in epicotyls of dark-germinated pea (Pisum sativum L.) seedlings upon illumination, resulting in fast turgor loss and wilting. In this study we show evidence that the palette of reactive oxygen species (ROS) is more complex. Hydrogen peroxide, superoxide and hydroxyl radicals are also formed, suggesting the occurrence of type-I reactions as well. Moreover, hydrogen peroxide injection into the epicotyls in the dark was able to provoke wilting directly. Formation of hydroxyl radicals could also be triggered by the addition of hydrogen peroxide in the dark, preferentially in the mid-sections where wilting occurs, showing that potential mediators of a Fenton reaction are present in the epicotyls, but unevenly distributed. Localization of light-inducible ROS formation fully (hydrogen peroxide) or partially (superoxide radicals) overlaps with the distribution of monomer protochlorophyllide complexes, showing that these pigment forms are capable of provoking both type-I and type-II reactions.

RNAi knock-down of ENOD40s leads to significant suppression of nodule formation in Lotus japonicus.

ENOD40 is one of the most intriguing early nodulin genes that is known to be induced very early in response to interaction of legume plants with symbiotic Rhizobium bacteria, but its function in the nodulation process is still not known. Lotus japonicus has two ENOD40 genes: LjENOD40-1 is abundantly induced in very early stages of bacterial infection or Nod factor application, whereas LjENOD40-2 is abundantly expressed only in mature nodules. We generated transgenic lines of L. japonicus with an RNAi (RNA interference) construct that expresses hairpin double-stranded RNA for LjENOD40-1 to induce sequence-specific RNA silencing. In the transgenic plants, expression of both LjENOD40-1 and -2 was significantly reduced, and no accumulation of ENOD40 transcripts was detected upon Mesorhizobium loti inoculation. The transgenic plants exhibited very poor nodulation (only 0-2 nodules per plant) and could not grow well without additional nitrogen supply. Analysis of segregation in the T(2) progeny indicated that the suppression of nodulation is perfectly linked with the presence of the transgene. Microscopic observation of the infection process using lacZ-labeled M. loti, together with expression analysis of infection-related nodulin genes, demonstrated that ENOD40 knock-down did not inhibit the initiation of the bacterial infection process. In contrast, nodule primordium initiation and subsequent nodule development were significantly suppressed in the transgenic plants. These results clearly indicate that ENOD40 is required for nodule initiation and subsequent organogenesis, but is not involved in early infection events.

Microtubule dynamics in living root hairs: transient slowing by lipochitin oligosaccharide nodulation signals.

The incorporation of a fusion of green fluorescent protein and tubulin-alpha 6 from Arabidopsis thaliana in root hairs of Lotus japonicus has allowed us to visualize and quantify the dynamic parameters of the cortical microtubules in living root hairs. Analysis of individual microtubule turnover in real time showed that only plus polymer ends contributed to overall microtubule dynamicity, exhibiting dynamic instability as the main type of microtubule behavior in Lotus root hairs. Comparison of the four standard parameters of in vivo dynamic instability--the growth rate, the disassembly rate, and the frequency of transitions from disassembly to growth (rescue) and from growth to disassembly (catastrophe)--revealed that microtubules in young root hairs were more dynamic than those in mature root hairs. Either inoculation with Mesorhizobium loti or purified M. loti lipochitin oligosaccharide signal molecules (Nod factors) significantly affected the growth rate and transition frequencies in emerging and growing root hairs, making microtubules less dynamic at a specific window after symbiotic inoculation. This response of root hair cells to rhizobial Nod factors is discussed in terms of the possible biological significance of microtubule dynamics in the early signaling events leading to the establishment and progression of the globally important Rhizobium/legume symbiosis.