Enzyme-assisted in vivo polymerisation of conjugated oligomer based conductors.

Conjugated polymers conduct both electronic and ionic carriers and thus can stimulate and translate biological signals when used as active materials in bioelectronic devices. Self- and on-demand organization of the active material directly in the in vivo environment can result in the seamless integration of the bioelectronic interface. Along that line, we recently demonstrated spontaneous in vivo polymerization of the conjugated oligomer ETE-S in the vascular tissue of plants and the formation of conducting wires. In this work, we elucidate the mechanism of the in vivo polymerization of the ETE-S trimer and demonstrate that ETE-S polymerizes due to an enzymatic reaction where the enzyme peroxidase is the catalyst and hydrogen peroxide is the oxidant. ETE-S, therefore, represents the first example of a conducting polymer that is enzymatically polymerized in vivo. By reproducing the reaction in vitro, we gain further insight on the polymerization mechanism and show that hydrogen peroxide is the limiting factor. In plants the ETE-S triggers the catalytic cycle responsible for the lignification process, hacks this biochemical pathway and integrates within the plant cell wall, forming conductors along the plant structure.

Transcriptome analysis of the differential effect of the NADPH oxidase gene RbohB in Phaseolus vulgaris roots following Rhizobium tropici and Rhizophagus irregularis inoculation.

BACKGROUND: Reactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. Rboh is a multigene family comprising nine members (RbohA-I) in common bean (Phaseolus vulgaris). The RNA interference-mediated silencing of RbohB (PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, the PvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed that PvRbohB positively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization by Rhizophagus irregularis in P. vulgaris. RESULTS: To corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots. CONCLUSIONS: Our research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P. vulgaris, as well as the differential roles that RbohB plays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.

The effect of cell wall encapsulation on macronutrients digestion: A case study in kidney beans.

Cotyledon cells in kidney beans naturally encapsulate starch and proteins limiting the access of digestive enzymes to their substrates. In this study, we investigated the effect of cell wall on bean protein digestibility and its relationship with starch digestion. Results showed that proteins contained in the cytoplasmic matrix influence the rate at which starch is digested in-vitro. Confocal laser scanning microscopy revealed that storage proteins in the cytoplasm act as a second encapsulation system preventing starch digestion. This microstructural organization only affected starch since no changes in protein digestion rate or extent were observed due to the presence of starch granules. Fourier transform infrared spectroscopy revealed that cellular entrapment limited protein denaturation induced by thermal treatments. High concentrations of a fraction resistant to digestion were found in proteins that were heated when entrapped within intact cotyledon cells, compared to those thermally treated as bean flour.

Wall porosity in isolated cells from food plants: Implications for nutritional functionality.

Macronutrients in whole plant foods are enclosed inside cells. The metabolic response from these entrapped nutrients may depend on cell-wall porosity, by controlling the passage of digestive enzymes. As non-interacting size mimics of digestive enzymes, we investigated the diffusion of fluorescently-labelled probes across the walls of isolated plant cells from potato tuber, red kidney bean and banana. Diffusion properties of permeable probes, dextran (20-kDa and 70-kDa) and albumin, were quantified, using fluorescence recovery after photobleaching. The consistent reduction of diffusion rate in the presence of cell walls (around 40%) compared to free-diffusion rate was attributed to the limiting porosity of the wall matrix. A combination of the physical barrier effects demonstrated here and non-catalytic binding of enzymes to cell walls limits the hydrolysis of intracellular macronutrients. This and further understanding of the structural basis for the physical barrier properties would help to design foods from plant materials with enhanced nutrition.

Effects of extracellular ATP on local and systemic responses of bean (Phaseolus vulgaris L) leaves to wounding.

Wounding increased the extracellular Adenosine 5'-triphosphate (eATP) level of kidney bean leaves. Treatment with wounding or exogenous ATP increased the hydrogen peroxide (H2O2) content, activities of catalase and polyphenol oxidase, and malondialdehyde content in both the treated and systemic leaves. Pre-treatment with ATP-degrading enzyme, apyrase, to the wounded leaves reduced the wound-induced local and systemic increases in H2O2 content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Application of dimethylthiourea (DMTU) and diphenylene iodonium (DPI) to the wounded and ATP-treated leaves, respectively, reduced the wound- and ATP-induced local and systemic increases in H2O2 content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Moreover, the wound- and ATP-induced systemic increases of these physiological parameters were suppressed when DMTU or DPI applied to leaf petiole of the wounded and ATP-treated leaves. These results suggest that eATP at wounded sites could mediate the wound-induced local and systemic responses by H2O2-dependent signal transduction.

Biochemical responses of common bean to white mold potentiated by phosphites.

Considering that the mechanisms for phosphite-afforded disease control remain elusive, this study investigated whether zinc (Zn) and copper (Cu) phosphites could possible potentiate common bean resistance to white mold, caused by Sclerotinia sclerotiorum, through the stimulation of biochemical defence responses. Lesion area and disease severity were decreased by phosphites spray, but Zn phosphite outcompeted Cu phosphite. Histopathological observations revealed fewer fungal hyphae and less collapse of the mesophyll cells in the Zn and Cu phosphite-sprayed plants compared to water-sprayed ones. The S. sclerotiorum-triggered accumulation of reactive oxygen species, oxalic acid (a fungal secreted toxin) and malondialdehyde (an indicator of cellular damage) were constrained as a result of Zn and Cu phosphites spray. Activities of antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase and glutathione-S-transferase at 12 h after inoculation (hai) and catalase at 60 and 84 hai) were higher for Zn and Cu phosphites-sprayed plants than for water-sprayed ones. Activities of defence-related enzymes chitinase (CHI) at 12 hai, ß-1,3-glucanase (GLU) and polyphenoloxidase (PPO) were higher at 12-84 hai for Zn, and Cu phosphites sprayed plants, phenylalanine ammonia-lyase at 36-84 hai for the Zn phosphite sprayed ones, CHI at 12-36 hai, GLU at 12-60 hai, PPO at 36 hai and PAL and lipoxygenase at 12 hai for the Cu phosphite sprayed ones upon inoculation with S. sclerotiorum relative to their water-sprayed counterparts. Concentrations of total soluble phenols and lignin-thioglycolic acid derivatives were not affected by Cu phosphite spray on infected plants but were higher and lower, respectively, for Zn phosphite sprayed plants at 60 hai compared to water-sprayed ones. Taken together, the findings from the present study shed light on the biochemical defence mechanisms involved in the Zn and Cu phosphites-mediated suppression of white mold in common bean.

Measurement of Gross Photosynthesis, Respiration in the Light, and Mesophyll Conductance Using H218O Labeling.

A fundamental challenge in plant physiology is independently determining the rates of gross O2 production by photosynthesis and O2 consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O2 production (NOP and GOP, respectively) by labeling ambient O2 with 18O while leaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas-exchange measurement. The petiole is immersed in water enriched to a δ18O of ∼9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers 18O from H2O to O2 GOP is calculated from the increase in δ18O of O2 as air passes through the cuvette. NOP is determined from the increase in O2/N2 Both terms are measured by isotope ratio mass spectrometry. CO2 assimilation and other standard gas-exchange parameters also were measured. Reproducible measurements are made on a single leaf for more than 15 h. We used this method to measure the light response curve of NOP and GOP in French bean (Phaseolus vulgaris) at 21% and 2% O2 We then used these data to examine the O2/CO2 ratio of net photosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study and understand leaf physiological traits.

Antifungal activity of Latarcin 1 derived cell-penetrating peptides against Fusarium solani.

Cell-penetrating peptides and antimicrobial peptides share physicochemical characteristics and mechanisms of interaction with biological membranes, hence, termed as membrane active peptides. The present study aims at evaluating AMP activity of CPPs. LDP-NLS and LDP are Latarcin 1 derived cell-penetrating peptides and in the current study we have evaluated antifungal and cell-penetrating properties of these CPPs in Fusarium solani. We observed that LDP-NLS and LDP exhibited excellent antifungal activity against the fungus. Cellular uptake experiments with LDP-NLS and LDP showed that LDP-NLS acted as a CPP but LDP uptake into fungal spores and hyphae was negligible. CPP and AMP activity of mutated version of LDP-NLS was also evaluated and it was observed that both the activities of the peptide were compromised, signifying the importance of arginines and lysines present in LDP-NLS for initial interaction of membrane active peptides with biological membranes. Dextrans and Propidium Iodide uptake studies revealed that the mode of entry of LDP-NLS into fungal hyphae is through pore formation. Also, both LDP-NLS and LDP showed no cytotoxicity when infiltered into leaf tissues. Overall, our results suggest that LDP-NLS and LDP are selectively cytotoxic to F. solani and can be a potent peptide based antifungal agents.

Agricultural practices to improve nitrogen use efficiency through the use of arbuscular mycorrhizae: Basic and agronomic aspects.

Nitrogen cycling in agroecosystems is heavily dependent upon arbuscular mycorrhizal fungi (AMF) present in the soil microbiome. These fungi develop obligate symbioses with various host plant species, thus increasing their ability to acquire nutrients. However, AMF are particularly sensitive to physical, chemical and biological disturbances caused by human actions that limit their establishment. For a more sustainable agriculture, it will be necessary to further investigate which agricultural practices could be favorable to maximize the benefits of AMF to improve crop nitrogen use efficiency (NUE), thus reducing nitrogen (N) fertilizer usage. Direct seeding, mulch-based cropping systems prevent soil mycelium disruption and increase AMF propagule abundance. Such cropping systems lead to more efficient root colonization by AMF and thus a better establishment of the plant/fungal symbiosis. In addition, the use of continuous cover cropping systems can also enhance the formation of more efficient interconnected hyphal networks between mycorrhizae colonized plants. Taking into account both fundamental and agronomic aspects of mineral nutrition by plant/AMF symbioses, we have critically described, how improving fungal colonization through the reduction of soil perturbation and maintenance of an ecological balance could be helpful for increasing crop NUE.

Expression of a phosphate-starvation inducible fructose-1,6-bisphosphatase gene in common bean nodules correlates with phosphorus use efficiency.

While increased P-hydrolysing acid phosphatases (APase) activity in bean nodules is well documented under phosphorus (P) limitation, gene expression and subcellular localization patterns within the N2-fixing nodule tissues are poorly understood. The aim of this research was to track the enzyme activity along with the intra-nodular localization of fructose-1,6-bisphosphatase (FBPase), and its contribution to P use efficiency (PUE) under symbiotic nitrogen fixation (SNF) in Phaseolus vulgaris. The FBPase transcript were localized in situ using RT-PCR and the protein activity was measured in nodules of two contrasting recombinant inbred lines (RILs) of P. vulgaris, namely RILs 115 (P-efficient) and 147 (P-inefficient), that were grown under sufficient versus deficient P supply. Under P-deficiency, higher FBPase transcript fluorescence was found in the inner cortex as compared to the infected zone of RIL115. In addition, both the specific FBPase and total APase enzyme activities significantly increased in both RILs, but to a more significant extent in RIL115 as compared to RIL147. Furthermore, the increased FBPase activity in nodules of RIL115 positively correlated with higher use efficiency of both the rhizobial symbiosis (23%) and P for SNF (14% calculated as the ratio of N2 fixed per nodule total P content). It is concluded that the abundant tissue-specific localized FBPase transcript along with induced enzymatic activity provides evidence of a specific tolerance mechanism where N2-fixing nodules overexpress under P-deficiency conditions. Such a mechanism would maximise the intra-nodular inorganic P fraction necessary to compensate for large amount of P needed during the SNF process.

The role of nitric oxide in basal and induced resistance in relation with hydrogen peroxide and antioxidant enzymes.

Nitric oxide (NO) is one of the main signal molecules, which is involved in plant growth and development and can change regular physiological activity in biotic and abiotic stresses. In this study, the role of NO in induced resistance with Pseudomonas fluorescent (CHA0) and basal resistance against Rhizoctonia solani in bean plant was investigated. Our results revealed that P. fluorescent and R. solani can increase NO production at 6h post inoculation (hpi). Also, using the NO donor S-nitroso-N-acetyl D-penicillamine (SNAP) led to increase NO and bean plant resistance against R. solani. Utilizing the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide (cPTIO), not only decreased basal resistance but also reduced induced resistance. In continue, the activity of antioxidant enzymes was studied in the former treatments. SNAP, CHA0 and R. solani increased the activity of peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) at 6, 12 and 24h post inoculation (hpi). In contrast, using cPTIO and R. solani simultaneously (cPTIO+R) showed reduction in activity of POX and APX at 6 hpi. The cPTIO+R treatment increased POX, APX and CAT activity at 12 and 24 hpi. Hydrogen peroxide (H2O2) monitoring in the leaf discs clarified that SNAP can increase H2O2 production like CHA0 and R. solani. On the other hand, SNAP increased the resistance level of leaf discs against R. solani. Treating the leaf discs with cPTIO led to decrease resistance against the pathogen. These leaf discs showed reduction in H2O2 production at 6 hpi and suddenly enhanced H2O2 generation was observed at 24hpi. This study showed that CHA0 can increase NO level in bean plants. NO induced H2O2 generation and regulated redox state of the host plant. This interaction resulted in significant defense against the pathogen.

Quinclorac-habituation of bean (Phaseolus vulgaris) cultured cells is related to an increase in their antioxidant capacity.

The habituation of bean cells to quinclorac did not rely on cell wall modifications, contrary to what it was previously observed for the well-known cellulose biosynthesis inhibitors dichlobenil or isoxaben. The aim of the present study was to investigate whether or not the bean cells habituation to quinclorac is related to an enhancement of antioxidant activities involved in the scavenging capacity of reactive oxygen species. Treating non-habituated bean calluses with 10 µM quinclorac reduced the relative growth rate and induced a two-fold increase in lipid peroxidation. However, the exposition of quinclorac-habituated cells to a concentration of quinclorac up to 30 µM neither affected their growth rate nor increased their lipid peroxidation levels. Quinclorac-habituated calluses had significantly higher constitutive levels of three antioxidant activities (class-III peroxidase, glutathione reductase, and superoxide dismutase) than those observed in non-habituated calluses, and the treatment of habituated calluses with 30 µM quinclorac significantly increased the level of class III-peroxidase and superoxide dismutase. The results reported here indicate that the process of habituation to quinclorac in bean callus-cultured cells is related, at least partially, to the development of a stable antioxidant capacity that enables them to cope with the oxidative stress caused by quinclorac. Class-III peroxidase and superoxide dismutase activities could play a major role in the quinclorac-habituation. Changes in the antioxidant status of bean cells were stable, since the increase in the antioxidant activities were maintained in quinclorac-dehabituated cells.

INDUCED CYTOMICTIC VARIATIONS AND SYNCYTE FORMATION DURING MICROSPOROGENESIS IN PHASEOLUS VULGARIS L.

The intercellular translocation of chromatin material along with other cytoplasmic contents among the proximate meiocytes lying in close contact with each other commonly referred as cytomixis was reported during microsporogenesis in Phaseolus vulgaris L., a member of the family Fabaceae. The phenomenon of cytomixis was observed at three administered doses of gamma rays viz. 100, 200, 300 Gy respectively in the diploid plants of Phaseolus vulgaris L. The gamma rays irradiated plants showed the characteristic feature of inter-meiocyte chromatin/chromosomes transmigration through various means.such as channel formation, beak formation or by direct adhesion between the PMC's (Pollen mother cells). The present study also reports the first instance of syncyte formation induced via cytomictic transmigration in Phaseolus vulgaris L. Though the frequency of syncyteformation was rather low yet these could play a significant role in plant evolution. It is speculated that syncyte enhances the ploidy level of plants by forming 2n gametes and may lead to the production ofpolyploid plants. The phenomenon of cytomixis shows a gradual inclination along with the increasing treatment doses of gamma rays. The preponderance of cytomixis was more frequent during meiosis I as compared to meiosis II. An interesting feature noticed during the present study was the channel formation among the microspores and fusion among the tetrads due to cell wall dissolution. The impact of this phenomenon is also visible on the development of post-meiotic products. The formation of heterosized pollen grains; a deviation from the normal pollen grains has also been reported. The production of gametes with unbalanced chromosomes is of utmost importance and should be given more attention in future studies as they possess the capability of inducing variations at the genomic level and can be further utilized in the improvement of germplasm.

Stress induced acquisition of somatic embryogenesis in common bean Phaseolus vulgaris L.

Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. We used an alternative strategy to induce SE in common bean based upon the use of a cytokinin (BAP) coupled with osmotic stress adaptation instead of SE response that is induced by auxins. Explants derived from zygotic embryos of common bean were subjected to osmotic stress (sucrose 12 % w/v, 0.5 M) in the presence of BAP 10 mg/L and adenine free base 40 mg/L to induce somatic embryos from specific competent cells of the apical meristem and cotyledonary node. Somatic embryos were obtained from the competent cells in a direct response (direct SE). In a secondary response (secondary SE), those somatic embryos formed proembryogenic masses (PEM) that originated/developed into secondary somatic embryos and showed the SE ontogeny. Maturation of somatic embryos was achieved by using different osmolality media and converted to plants. Full-visible light spectrum was necessary to achieve efficient plant regeneration. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and bioballistics as well as for basic biochemical and molecular biology experiments.

Spatial and temporal dynamics of primary and secondary metabolism in Phaseolus vulgaris challenged by Pseudomonas syringae.

Many defense mechanisms contribute to the plant immune system against pathogens, involving the regulation of different processes of the primary and secondary metabolism. At the same time, pathogens have evolved mechanisms to hijack the plant defense in order to establish the infection and proliferate. Localization and timing of the host response are essential to understand defense mechanisms and resistance to pathogens (Rico et al. 2011). Imaging techniques, such as fluorescence imaging and thermography, are a very valuable tool providing spatial and temporal information about a series of plant processes. In this study, bean plants challenged with two pathovars of Pseudomonas syringae have been investigated. Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tomato DC3000 elicit a compatible and incompatible interaction in bean, respectively. Both types of host-pathogen interaction triggered different changes in the activity of photosynthesis and the secondary metabolism. We conclude that the combined analysis of leaf temperature, chlorophyll fluorescence and green fluorescence emitted by phenolics allows to discriminate compatible from incompatible P. syringae-Phaseolus vulgaris interactions in very early times of the infection, prior to the development of symptoms. These can constitute disease signatures that would allow an early identification of emerging plagues in crops.

Apoplastic peroxidases are required for salicylic acid-mediated defense against Pseudomonas syringae.

Reactive oxygen species (ROS) generated by NADPH oxidases or apoplastic peroxidases play an important role in the plant defense response. Diminished expression of at least two Arabidopsis thaliana peroxidase encoding genes, PRX33 (At3g49110) and PRX34 (At3g49120), as a consequence of anti-sense expression of a heterologous French bean peroxidase gene (asFBP1.1), were previously shown to result in reduced levels of ROS following pathogen attack, enhanced susceptibility to a variety of bacterial and fungal pathogens, and reduced levels of callose production and defense-related gene expression in response to the microbe associated molecular pattern (MAMP) molecules flg22 and elf26. These data demonstrated that the peroxidase-dependent oxidative burst plays an important role in the elicitation of pattern-triggered immunity (PTI). Further work reported in this paper, however, shows that asFBP1.1 antisense plants are not impaired in all PTI-associated responses. For example, some but not all flg22-elicited genes are induced to lower levels by flg22 in asFPB1.1, and callose deposition in asFPB1.1 is similar to wild-type following infiltration with a Pseudomonas syringae hrcC mutant or with non-host P. syringae pathovars. Moreover, asFPB1.1 plants did not exhibit any apparent defect in their ability to mount a hypersensitive response (HR). On the other hand, salicylic acid (SA)-mediated activation of PR1 was dramatically impaired in asFPB1.1 plants. In addition, P. syringae-elicited expression of many genes known to be SA-dependent was significantly reduced in asFBP1.1 plants. Consistent with this latter result, in asFBP1.1 plants the key regulator of SA-mediated responses, NPR1, showed both dramatically decreased total protein abundance and a failure to monomerize, which is required for its translocation into the nucleus.

BAC-end microsatellites from intra and inter-genic regions of the common bean genome and their correlation with cytogenetic features.

Highly polymorphic markers such as simple sequence repeats (SSRs) or microsatellites are very useful for genetic mapping. In this study novel SSRs were identified in BAC-end sequences (BES) from non-contigged, non-overlapping bacterial artificial clones (BACs) in common bean (Phaseolus vulgaris L.). These so called "singleton" BACs were from the G19833 Andean gene pool physical map and the new BES-SSR markers were used for the saturation of the inter-gene pool, DOR364×G19833 genetic map. A total of 899 SSR loci were found among the singleton BES, but only 346 loci corresponded to the single di- or tri-nucleotide motifs that were likely to be polymorphic (ATT or AG motifs, principally) and useful for primer design and individual marker mapping. When these novel SSR markers were evaluated in the DOR364×G19833 population parents, 136 markers revealed polymorphism and 106 were mapped. Genetic mapping resulted in a map length of 2291 cM with an average distance between markers of 5.2 cM. The new genetic map was compared to the most recent cytogenetic analysis of common bean chromosomes. We found that the new singleton BES-SSR were helpful in filling peri-centromeric spaces on the cytogenetic map. Short genetic distances between some new singleton-derived BES-SSR markers was common showing suppressed recombination in these regions compared to other parts of the genome. The correlation of singleton-derived SSR marker distribution with other cytogenetic features of the bean genome is discussed.

SPX1 is an important component in the phosphorus signalling network of common bean regulating root growth and phosphorus homeostasis.

Proteins containing the SPX domain are believed to play vital roles in the phosphorus (P) signalling network in plants. However, the functions of SPX proteins in legumes remain largely unknown. In this study, three SPX members, PvSPX1-PvSPX3 were cloned from common bean (Phaseolus vulgaris L.). It was found that the transcripts of all three PvSPX members were significantly enhanced in both bean leaves and roots by phosphate (Pi) starvation. Among them, the expression of nuclear localized PvSPX1 showed more sensitive and rapid responses to Pi starvation. Consistently, only overexpression of PvSPX1 resulted in increased root P concentration and modified morphology of transgenic bean hairy roots, such as inhibited root growth and an enlarged root hair zone. It was further demonstrated that PvSPX1 transcripts were up-regulated by overexpressing PvPHR1, and overexpressing PvSPX1 led to increased transcripts of 10 Pi starvation-responsive genes in transgenic bean hairy roots. Taken together, it is suggested that PvSPX1 is a positive regulator in the P signalling network of common bean, and is downstream of PvPHR1.

Localization of the Bacillus subtilis beta-propeller phytase transcripts in nodulated roots of Phaseolus vulgaris supplied with phytate.

Soil organic phosphorus (Po) such as phytate, which comprises up to 80 % of total Po, must be hydrolyzed by specific enzymes called phytases to be used by plants. In contrast to plants, bacteria, such as Bacillus subtilis, have the ability to use phytate as the sole source of P due to the excretion of a beta-propeller phytase (BPP). In order to assess whether the B. subtilis BPP could make P available from phytate for the benefit of a nodulated legume, the P-sensitive recombinant inbred line RIL147 of Phaseolus vulgaris was grown under hydroaeroponic conditions with either 12.5 µM phytate (C6H18O24P6) or 75 µmol Pi (K2HPO4), and inoculated with Rhizobium tropici CIAT899 alone, or co-inoculated with both B. subtilis DSM 10 and CIAT899. The in situ RT-PCR of BPP genes displayed the most intense fluorescent BPP signal on root tips. Some BPP signal was found inside the root cortex and the endorhizosphere of the root tip, suggesting endophytic bacteria expressing BPP. However, the co-inoculation with B. subtilis was associated with a decrease in plant P content, nodulation and the subsequent plant growth. Such a competitive effect of B. subtilis on P acquisition from phytate in symbiotic nitrogen fixation might be circumvented if the rate of inoculation were reasoned in order to avoid the inhibition of nodulation by excess B. subtilis proliferation. It is concluded that B. subtilis BPP gene is expressed in P. vulgaris rhizosphere.

Nodulin 22, a novel small heat-shock protein of the endoplasmic reticulum, is linked to the unfolded protein response in common bean.

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing-based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions.