Structure and development of root gall induced by Meloidogyne javanica in Glycine max L / Estrutura e desenvolvimento da galha radicular induzida por Meloidogyne javanica em Glycine max L

Galls formed by root-knot nematodes have been studied in several cultivated species focusing on understanding the intimate relationship between parasite and the host plant. Species of Meloidogyne induce the development of a feeding site in the cortex or vascular cylinder of the host plant and are totally dependent on this site formation to complete their life cycle. Therefore, we focused on anatomical, cytological and histochemical changes during the establishment and development of galls and giant cells induced by Meloidogyne javanica in the roots of Glycine max. Seeds of soybean (susceptible cultivar M8372 IPRO) were sown in trays with coconut fibre substrate and the seedlings were removed ten days after the seeds emerged for nematode inoculation. The roots from inoculated and non-inoculated (control) were sampled at different stages of development until 55 days after inoculation. Histological, cytological, histochemical analysis were performed in light and electron microscopy in non-galled tissue and galls induced by M. javanica. The galls showed different shapes and abundance in the roots inoculated by M. javanica. The induction of galls occurs by hypertrophy of the root cortex shortly after the initial infection process. Giant cells were formed 18 days after nematode inoculation. These giant or nourishing cells are multinucleated, and have a dense cytoplasm, a thick wall with invaginations, many mitochondria and small vacuoles. The anatomical sections indicated a disorganisation of the cells of the cortex and vascular cylinder in relation to the control root.

Galhas radiculares induzidas por nematoides têm sido estudadas em diversas espécies cultivadas, fornecendo subsídios para a compreensão da íntima relação entre parasita e hospedeiro. O estabelecimento do nematoide das galhas passa, necessariamente, pelo processo de formação das células gigantes, que garantem a nutrição do nematoide. As espécies de Meloidogyne estabelecem um sitio de alimentação no córtex ou cilindro vascular da raiz, sendo esses organismos totalmente dependentes deste sítio de indução. Desta forma o objetivo deste trabalho foi avaliar as alterações anatômicas, citológicas e histoquímicas durante o desenvolvimento de galhas induzidas por Meloidogyne javanica em raízes de Glycine max. A variedade de soja utilizada foi a M8372 IPRO, susceptível ao nematoide M. javanica. As sementes foram semeadas em bandejas e depois colocadas em tubetes, onde foram realizadas coletas de amostras radiculares em diferentes estádios de desenvolvimento, tanto de plantas inoculadas com o nematoide quanto de plantas controle. As amostras de galhas e raízes não galhadas foram utilizadas para as análises histológicas, citológicas e histoquímicas, em microscopia óptica e eletrônica. Verificou-se a presença de galhas após a inoculação de M. javanica, com variação na quantidade e na forma. A indução das galhas ocorre pela hipertrofia do córtex da raiz, logo após o processo inicial de infecção. Aos18 dias após a inoculação do nematoide já é possível observar a formação de células gigantes. Estas células gigantes ou nutridoras são multinucleadas, possuem citoplasma denso, parede espessa com invaginações, muitas mitocôndrias e pequenos vacúolos. As secções anatômicas indicaram uma desorganização das células do córtex e cilindro vascular em relação à raiz não inoculada (controle).

Structure and development of root gall induced by Meloidogyne javanica in Glycine max L / Estrutura e desenvolvimento da galha radicular induzida por Meloidogyne javanica em Glycine max L

Galls formed by root-knot nematodes have been studied in several cultivated species focusing on understanding the intimate relationship between parasite and the host plant. Species of Meloidogyne induce the development of a feeding site in the cortex or vascular cylinder of the host plant and are totally dependent on this site formation to complete their life cycle. Therefore, we focused on anatomical, cytological and histochemical changes during the establishment and development of galls and giant cells induced by Meloidogyne javanica in the roots of Glycine max. Seeds of soybean (susceptible cultivar M8372 IPRO) were sown in trays with coconut fibre substrate and the seedlings were removed ten days after the seeds emerged for nematode inoculation. The roots from inoculated and non-inoculated (control) were sampled at different stages of development until 55 days after inoculation. Histological, cytological, histochemical analysis were performed in light and electron microscopy in non-galled tissue and galls induced by M. javanica. The galls showed different shapes and abundance in the roots inoculated by M. javanica. The induction of galls occurs by hypertrophy of the root cortex shortly after the initial infection process. Giant cells were formed 18 days after nematode inoculation. These giant or nourishing cells are multinucleated, and have a dense cytoplasm, a thick wall with invaginations, many mitochondria and small vacuoles. The anatomical sections indicated a disorganisation of the cells of the cortex and vascular cylinder in relation to the control root.(AU)

Galhas radiculares induzidas por nematoides têm sido estudadas em diversas espécies cultivadas, fornecendo subsídios para a compreensão da íntima relação entre parasita e hospedeiro. O estabelecimento do nematoide das galhas passa, necessariamente, pelo processo de formação das células gigantes, que garantem a nutrição do nematoide. As espécies de Meloidogyne estabelecem um sitio de alimentação no córtex ou cilindro vascular da raiz, sendo esses organismos totalmente dependentes deste sítio de indução. Desta forma o objetivo deste trabalho foi avaliar as alterações anatômicas, citológicas e histoquímicas durante o desenvolvimento de galhas induzidas por Meloidogyne javanica em raízes de Glycine max. A variedade de soja utilizada foi a M8372 IPRO, susceptível ao nematoide M. javanica. As sementes foram semeadas em bandejas e depois colocadas em tubetes, onde foram realizadas coletas de amostras radiculares em diferentes estádios de desenvolvimento, tanto de plantas inoculadas com o nematoide quanto de plantas controle. As amostras de galhas e raízes não galhadas foram utilizadas para as análises histológicas, citológicas e histoquímicas, em microscopia óptica e eletrônica. Verificou-se a presença de galhas após a inoculação de M. javanica, com variação na quantidade e na forma. A indução das galhas ocorre pela hipertrofia do córtex da raiz, logo após o processo inicial de infecção. Aos18 dias após a inoculação do nematoide já é possível observar a formação de células gigantes. Estas células gigantes ou nutridoras são multinucleadas, possuem citoplasma denso, parede espessa com invaginações, muitas mitocôndrias e pequenos vacúolos. As secções anatômicas indicaram uma desorganização das células do córtex e cilindro vascular em relação à raiz não inoculada (controle).(AU)

In silico characterization of 1,2-diacylglycerol cholinephosphotransferase and lysophospha-tidylcholine acyltransferase genes in Glycine max L. Merrill.

The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.

The acetyl bromide method is faster, simpler and presents best recovery of lignin in different herbaceous tissues than Klason and thioglycolic acid methods.

We compared the amount of lignin as determined by the three most traditional methods for lignin measurement in three tissues (sugarcane bagasse, soybean roots and soybean seed coat) contrasting for lignin amount and composition. Although all methods presented high reproducibility, major inconsistencies among them were found. The amount of lignin determined by thioglycolic acid method was severely lower than that provided by the other methods (up to 95%) in all tissues analyzed. Klason method was quite similar to acetyl bromide in tissues containing higher amounts of lignin, but presented lower recovery of lignin in the less lignified tissue. To investigate the causes of the inconsistencies observed, we determined the monomer composition of all plant materials, but found no correlation. We found that the low recovery of lignin presented by the thioglycolic acid method were due losses of lignin in the residues disposed throughout the procedures. The production of furfurals by acetyl bromide method does not explain the differences observed. The acetyl bromide method is the simplest and fastest among the methods evaluated presenting similar or best recovery of lignin in all the tissues assessed.

The endoplasmic reticulum binding protein BiP displays dual function in modulating cell death events.

The binding protein (BiP) has been demonstrated to participate in innate immunity and attenuate endoplasmic reticulum- and osmotic stress-induced cell death. Here, we employed transgenic plants with manipulated levels of BiP to assess whether BiP also controlled developmental and hypersensitive programmed cell death (PCD). Under normal conditions, the BiP-induced transcriptome revealed a robust down-regulation of developmental PCD genes and an up-regulation of the genes involved in hypersensitive PCD triggered by nonhost-pathogen interactions. Accordingly, the BiP-overexpressing line displayed delayed leaf senescence under normal conditions and accelerated hypersensitive response triggered by Pseudomonas syringae pv tomato in soybean (Glycine max) and tobacco (Nicotiana tabacum), as monitored by measuring hallmarks of PCD in plants. The BiP-mediated delay of leaf senescence correlated with the attenuation of N-rich protein (NRP)-mediated cell death signaling and the inhibition of the senescence-associated activation of the unfolded protein response (UPR). By contrast, under biological activation of salicylic acid (SA) signaling and hypersensitive PCD, BiP overexpression further induced NRP-mediated cell death signaling and antagonistically inhibited the UPR. Thus, the SA-mediated induction of NRP cell death signaling occurs via a pathway distinct from UPR. Our data indicate that during the hypersensitive PCD, BiP positively regulates the NRP cell death signaling through a yet undefined mechanism that is activated by SA signaling and related to ER functioning. By contrast, BiP's negative regulation of leaf senescence may be linked to its capacity to attenuate the UPR activation and NRP cell death signaling. Therefore, BiP can function either as a negative or positive modulator of PCD events.

Enhanced lignin monomer production caused by cinnamic Acid and its hydroxylated derivatives inhibits soybean root growth.

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.

The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress- and osmotic stress-induced NRP-mediated cell-death signaling pathway.

BACKGROUND: The endoplasmic reticulum (ER) is a major signaling organelle, which integrates a variety of responses against physiological stresses. In plants, one such stress-integrating response is the N-rich protein (NRP)-mediated cell death signaling pathway, which is synergistically activated by combined ER stress and osmotic stress signals. Despite the potential of this integrated signaling to protect plant cells against different stress conditions, mechanistic knowledge of the pathway is lacking, and downstream components have yet to be identified. RESULTS: In the present investigation, we discovered an NAC domain-containing protein from soybean, GmNAC6 (Glycine max NAC6), to be a downstream component of the integrated pathway. Similar to NRP-A and NRP-B, GmNAC6 is induced by ER stress and osmotic stress individually, but requires both signals for full activation. Transient expression of GmNAC6 promoted cell death and hypersensitive-like responses in planta. GmNAC6 and NRPs also share overlapping responses to biotic signals, but the induction of NRPs peaked before the increased accumulation of GmNAC6 transcripts. Consistent with the delayed kinetics of GmNAC6 induction, increased levels of NRP-A and NRP-B transcripts induced promoter activation and the expression of the GmNAC6 gene. CONCLUSIONS: Collectively, our results biochemically link GmNAC6 to the ER stress- and osmotic stress-integrating cell death response and show that GmNAC6 may act downstream of the NRPs.

A new branch of endoplasmic reticulum stress signaling and the osmotic signal converge on plant-specific asparagine-rich proteins to promote cell death.

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins.

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

An original mutation in soybean (Glycine max (L.) Merrill) involving degeneration of the generative cell and causing male sterility.

A spontaneous mutation causing male sterility has been detected in line BR97-17739 from the soybean breeding program conducted by Embrapa-National Soybean Research Center. Meiotic division and male gametophyte development were analyzed in 10 male-sterile, female-fertile plants. Meiotic process had few irregularities related to chromosome segregation and affected about 2% of tetrads. Despite the high frequency of normal microspores, pollen sterility was total. After callose dissolution, microspores were released into the anther loculle and interphase nucleus was displaced from the center to one side of the cell. Displacement continued throughout normal microspore mitosis (PMI). After telophase, the hemispherical phragmoplast marked the place of cytokinesis. A typical generative cell, adjacent to the plasma membrane, and the vegetative one, containing most of the cytoplasm, were formed. In spite of the well-formed generative cell, pollen mitosis (PMII) failed to occur. The generative cell degenerated and was completely destroyed. The 3:1 segregation for male sterility in this line and its progenies indicate that a single recessive gene controls mutation.