The Arabidopsis peptide kiss of death is an inducer of programmed cell death.

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.

The NRT2.5 and NRT2.6 genes are involved in growth promotion of Arabidopsis by the plant growth-promoting rhizobacterium (PGPR) strain Phyllobacterium brassicacearum STM196.

The Phyllobacterium brassicacearum STM196 strain stimulates Arabidopsis thaliana growth and antagonizes high nitrate inhibition of lateral root development. A previous study identified two STM196-responsive genes, NRT2.5 and NRT2.6 (Mantelin et al., 2006, Planta 223: 591-603). We investigated the role of NRT2.5 and NRT2.6 in the plant response to STM196 using single and double Arabidopsis mutants. The single mutants were also crossed with an nrt2.1 mutant, lacking the major nitrate root transporter, to distinguish the effects of NRT2.5 and NRT2.6 from potential indirect effects of nitrate pools. The nrt2.5 and nrt2.6 mutations abolished the plant growth and root system architecture responses to STM196. The determination of nitrate content revealed that NRT2.5 and NRT2.6 do not play an important role in nitrate distribution between plant organs. Conversely, NRT2.5 and NRT2.6 appeared to play a role in the plant response independent of nitrate uptake. Using a nitrate reductase mutant, it was confirmed that the NRT2.5/NRT2.6-dependent plant signalling pathway is independent of nitrate-dependent regulation of root development. Our findings demonstrate that NRT2.5 and NRT2.6, which are preferentially expressed in leaves, play an essential role in plant growth promotion by the rhizospheric bacterium STM196.

The PGPR strain Phyllobacterium brassicacearum STM196 induces a reproductive delay and physiological changes that result in improved drought tolerance in Arabidopsis.

Understanding how biotic interactions can improve plant tolerance to drought is a challenging prospect for agronomy and ecology. Plant growth-promoting rhizobacteria (PGPR) are promising candidates but the phenotypic changes induced by PGPR under drought remain to be elucidated. We investigated the effects of Phyllobacterium brassicacearum STM196 strain, a PGPR isolated from the rhizosphere of oilseed rape, on two accessions of Arabidopsis thaliana with contrasting flowering time. We measured multiple morphophysiological traits related to plant growth and development in order to quantify the added value of the bacteria to drought-response strategies of Arabidopsis in soil conditions. A delay in reproductive development induced by the bacteria resulted in a gain of biomass that was independent of the accession and the watering regime. Coordinated changes in transpiration, ABA content, photosynthesis and development resulted in higher water-use efficiency and a better tolerance to drought of inoculated plants. Our findings give new insights into the ecophysiological bases by which PGPR can confer stress tolerance to plants. Rhizobacteria-induced delay in flowering time could represent a valuable strategy for increasing biomass yield, whereas rhizobacteria-induced improvement of water use is of particular interest in multiple scenarios of water availability.

Arabidopsis Growth-Promotion and Root Architecture Responses to the Beneficial Rhizobacterium Phyllobacterium brassicacearum Strain STM196 Are Independent of the Nitrate Assimilatory Pathway.

Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacterium isolated from roots of oilseed rape, stimulates Arabidopsis growth. We have previously shown that the NRT2.5 and NRT2.6 genes are required for this growth promotion response. Since these genes are members of the NRT2 family of nitrate transporters, the nitrogen assimilatory pathway could be involved in growth promotion by STM196. We address this hypothesis using two nitrate reductase mutants, G5 deleted in the major nitrate reductase gene NIA2 and G'4-3 altered in both NIA1 and NIA2 genes. Both mutants had a reduced growth rate and STM196 failed to increase their biomass production on a medium containing NO3- as the sole nitrogen source. However, they both displayed similar growth promotion by STM196 when grown on an NH4+ medium. STM196 was able to stimulate lateral roots development of the mutants under both nutrition conditions. Altogether, our results indicate that the nitrate assimilatory metabolism is not a primary target of STM196 interaction and is not involved in the root developmental response. The NIA1 transcript level was reduced in the shoots of nrt2.5 and nrt2.6 mutants suggesting a role for this nitrate reductase isoform independently from its role in nitrate assimilation.

The auxin-signaling pathway is required for the lateral root response of Arabidopsis to the rhizobacterium Phyllobacterium brassicacearum.

Plant root development is highly responsive both to changes in nitrate availability and beneficial microorganisms in the rhizosphere. We previously showed that Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacteria strain isolated from rapeseed roots, alleviates the inhibition exerted by high nitrate supply on lateral root growth. Since soil-borne bacteria can produce IAA and since this plant hormone may be implicated in the high nitrate-dependent control of lateral root development, we investigated its role in the root development response of Arabidopsis thaliana to STM196. Inoculation with STM196 resulted in a 50% increase of lateral root growth in Arabidopsis wild-type seedlings. This effect was completely abolished in aux1 and axr1 mutants, altered in IAA transport and signaling, respectively, indicating that these pathways are required. The STM196 strain, however, appeared to be a very low IAA producer when compared with the high-IAA-producing Azospirillum brasilense sp245 strain and its low-IAA-producing ipdc mutant. Consistent with the hypothesis that STM196 does not release significant amounts of IAA to the host roots, inoculation with this strain failed to increase root IAA content. Inoculation with STM196 led to increased expression levels of several IAA biosynthesis genes in shoots, increased Trp concentration in shoots, and increased auxin-dependent GUS staining in the root apices of DR5::GUS transgenic plants. All together, our results suggest that STM196 inoculation triggers changes in IAA distribution and homeostasis independently from IAA release by the bacteria.

A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots.

BACKGROUND: In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. RESULTS: We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. CONCLUSION: The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Interact to survive: Phyllobacterium brassicacearum improves Arabidopsis tolerance to severe water deficit and growth recovery.

Mutualistic bacteria can alter plant phenotypes and confer new abilities to plants. Some plant growth-promoting rhizobacteria (PGPR) are known to improve both plant growth and tolerance to multiple stresses, including drought, but reports on their effects on plant survival under severe water deficits are scarce. We investigated the effect of Phyllobacterium brassicacearum STM196 strain, a PGPR isolated from the rhizosphere of oilseed rape, on survival, growth and physiological responses of Arabidopsis thaliana to severe water deficits combining destructive and non-destructive high-throughput phenotyping. Soil inoculation with STM196 greatly increased the survival rate of A. thaliana under several scenarios of severe water deficit. Photosystem II efficiency, assessed at the whole-plant level by high-throughput fluorescence imaging (Fv/Fm), was related to the probability of survival and revealed that STM196 delayed plant mortality. Inoculated surviving plants tolerated more damages to the photosynthetic tissues through a delayed dehydration and a better tolerance to low water status. Importantly, STM196 allowed a better recovery of plant growth after rewatering and stressed plants reached a similar biomass at flowering than non-stressed plants. Our results highlight the importance of plant-bacteria interactions in plant responses to severe drought and provide a new avenue of investigations to improve drought tolerance in agriculture.

The ethylene pathway contributes to root hair elongation induced by the beneficial bacteria Phyllobacterium brassicacearum STM196.

In Arabidopsis roots, some epidermal cells differentiate into root hair cells. Auxin regulates root hair positioning, while ethylene controls cell elongation. Phyllobacterium brassicacearum STM196, a beneficial strain of plant growth promoting rhizobacteria (PGPR) isolated from the roots of field-grown oilseed rape, stimulates root hair elongation in Arabidopsis thaliana seedlings. We investigated the role of ethylene in the response of root hair cells to STM196 inoculation. While we could not detect a significant increase in ethylene biosynthesis, we could detect a slight activation of the ethylene signalling pathway. Consistent with this, an exhaustive survey of the root hair elongation response of mutants and transgenic lines affected in the ethylene pathway showed contrasting root hair sensitivities to STM196. We propose that local ethylene emission contributes to STM196-induceed root hair elongation.