Calcium oxalate crystal production and density at different phenological stages of soybean plants (Glycine max L.) from the southeast of the Pampean Plain, Argentina.

Glycine max L. (soybean) is one of the major crops of the world. Although the process of biomineralisation has been reported in some organs of soybean, we now report the description and quantification of calcium oxalate crystals in vegetative and reproductive organs of soybean during its life cycle, as they act as an important source of calcium to the soil, once the harvesting is finished. Through diaphanisation, cross-sectioning, optical and scanning electron microscopy analysis of the organs, morphology, size and location of the crystals were identified. In addition, crystal density (n° crystals·mm-2 ) and the input of crystals to soil (n° crystals·ha-1 ) were calculated. Soybean produced prismatic calcium oxalate crystals in vegetative and reproductive organs, generally associated with vascular bundles, resulting in a potencial transfer to the soil of 81.4 x 107 crystals·ha-1 throughout its life cycle. Pods were the organs with higher calcium oxalate crystal production (1112.7 ± 384.6 crystals·mm-2 ), but with the smaller size (12.3 ± 2.1 µm long). However, cotyledons were the organs that produce the larger crystals (21.3 ± 3.5 µm long), but in lesser amounts (150.9 ± 64.4 crystals·mm-2 ). In leaves, although crystal size did not differ from vegetative to reproductive stage (14.5 ± 4.2 and 14.5 ± 4 µm in length, respectively), the crystal density increased (293.2 and 409 crystals·mm-2 , respectively). These results will contribute to knowledge of the amount of calcium oxalate crystals involved in the process of Ca recycling through cultivated vegetation in fields from humid plains at medium latitudes, which therefore have biological, botanical, biogeochemical and pedological relevance.

Bioaccumulation and effects of lanthanum on growth and mitotic index in soybean plants.

Rare earth elements such as lanthanum (La) have been used as agricultural inputs in some countries in order to enhance yield and improve crop quality. However, little is known about the effect of La on the growth and structure of soybean, which is an important food and feed crop worldwide. In this study, bioaccumulation of La and its effects on the growth and mitotic index of soybean was evaluated. Soybean plants were exposed to increasing concentrations of La (0, 5, 10, 20, 40, 80, and 160 µM) in nutrient solution for 28 days. Plant response to La was evaluated in terms of plant growth, nutritional characteristics, photosynthetic rate, chlorophyll content, mitotic index, modifications in the ultrastructure of roots and leaves, and La mapping in root and shoot tissues. The results showed that the roots of soybean plants can accumulate sixty-fold more La than shoots. La deposition occurred mainly in cell walls and in crystals dispersed in the root cortex and in the mesophyll. When La was applied, it resulted in increased contents of some essential nutrients (i.e., Ca, P, K, and Mn), while Cu and Fe levels decreased. Moreover, low La concentrations stimulated the photosynthetic rate and total chlorophyll content and lead to a higher incidence of binucleate cells, resulting in a slight increase in roots and shoot biomass. At higher La levels, soybean growth was reduced. This was caused by ultrastructural modifications in the cell wall, thylakoids and chloroplasts, and the appearance of c-metaphases.

Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A:AtDREB1A for the improvement of drought tolerance.

We evaluated the molecular, anatomical and physiological properties of a soybean line transformed to improve drought tolerance with an rd29A:AtDREB1A construct. This construct expressed dehydration- responsive element binding protein DREB1A from the stress-inducible rd29A promoter. The greenhouse growth test included four randomized blocks of soybean plants, with each treatment performed in triplicate. Seeds from the non-transformed soybean cultivar BR16 and from the genetically modified soybean P58 line (T(2) generation) were grown at 15% gravimetric humidity for 31 days. To induce water deficit, the humidity was reduced to 5% gravimetric humidity (moderate stress) for 29 days and then to 2.5% gravimetric humidity (severe stress). AtDREB1A gene expression was higher in the genetically modified P58 plants during water deficit, demonstrating transgene stability in T(2) generations and induction of the rd29A promoter. Drought-response genes, including GmPI-PLC, GmSTP, GmGRP, and GmLEA14, were highly expressed in plants submitted to severe stress. Genetically modified plants had higher stomatal conductance and consequently higher photosynthetic and transpiration rates. In addition, they had more chlorophyll. Overexpression of AtDREB1A may contribute to a decrease in leaf thickness; however, a thicker abaxial epidermis was observed. Overexpression of AtDREB1A in soybean appears to enhance drought tolerance.

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway.

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

Irradiance regulates genotype-specific responses of Rhizobium-nodulated soybean to increasing iron and two manganese concentrations in solution culture.

The growth of soybean plants were examined when subjected to three contrasting irradiance levels and to various combinations of nutrient solution Fe and Mn concentrations. Two Rhizobium-nodulated soybean genotypes (PI 227557 and Biloxi), which had been previously found to differ in their growth response to various Fe and Mn solutions, were studied. Both genotypes displayed the poorest growth, nodulation and the lowest chlorophyll and nodule ureide concentration at high irradiance (HI), regardless of the solution Fe and Mn concentrations. However, the genotypes differed under HI in their accumulation of Fe. For solution concentrations greater than 13 microM, PI 227557 accumulated up to 1200 microg Feg(-1) leaf dry wt mainly in the form of ferritin crystals within chloroplasts. In contrast, leaf Fe concentrations in Biloxi only reached 300 microg Feg(-1) dry wt and there were no ferritin crystals. Also, in PI 227557 HI induced more severe distortions in leaf cells and nodule ultrastructure than in Biloxi. Based on its poor growth under HI, PI 227557 could be categorized as an Fe-inefficient genotype prone to undergo photoinhibition at HI, in spite of the ferritin crystals in the chloroplasts. Enhanced growth, nodulation, chlorophyll and ureide concentrations in nodules as well as leaf ureide catabolism occurred in both genotypes grown at moderate irradiance (MI) in Fe solutions from 13 to 60 microM supplied with 20 microM Mn. At low irradiance (LI), plant growth and nodulation were lower than at MI values, but higher than those of plants at HI. Irradiance and solution Fe concentration did not alter leaf Cu and Zn concentration in either genotype, with the higher concentrations of these two elements detected in Biloxi. Solutions with Fe concentrations greater than 100 microM were deleterious for both genotypes at all irradiances. Low Fe and high Mn concentrations in leaves was bound to result in the best growth at HI.

Soybean (Glycine max) root lignification induced by ferulic acid. The possible mode of action.

Ferulic acid, in the form of feruloyl CoA, occupies a central position as an intermediate in the phenylpropanoid pathway. Due to the allelopathic function, its effects were tested on root growth, H(2)O(2) and lignin contents, and activities of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) and peroxidase (POD, EC 1.11.1.7) from soybean (Glycine max (L.) Merr.) root seedlings. Three-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 1.0 mM ferulic acid in a growth chamber (25 degrees C, 12/12 hr light/dark photoperiod, irradiance of 280 micromol m(-2) s(-1)) for 24 or 48 hr. Exogenously supplied ferulic acid induced premature cessation of root growth, with disintegration of the root cap, compression of cells in the quiescent center, increase of the vascular cylinder diameter, and earlier lignification of the metaxylem. Moreover, the allelochemical decreased CAD activity and H(2)O(2) level and increased the anionic isoform PODa5 activity and lignin content. The lignin monomer composition of ferulic acid-exposed roots revealed a significant increase of guaiacyl (G) units. When applied jointly with piperonylic acid (an inhibitor of the cinnamate 4-hydroxylase, C4H), ferulic acid increased lignin content. By contrast, the application of 3,4-(methylenedioxy) cinnamic acid (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with ferulic acid did not. Taken together, these results suggest that ferulic acid may be channeled into the phenylpropanoid pathway (by the 4CL reaction) and, further, may increase the lignin monomer amount solidifying the cell wall and restricting the root growth.

Soybean genotypic difference in growth, nutrient accumulation and ultrastructure in response to manganese and iron supply in solution culture.

BACKGROUND AND AIMS: The objective of this research was to characterize the physiology and cell ultrastructure of two soybean genotypes subjected to nutrient solutions with increasing concentrations of manganese (Mn) at two contrasting iron (Fe) concentrations. Genotypes 'PI227557' and 'Biloxi' were selected based on their distinctly different capacities to accumulate Mn and Fe. * METHODS: Bradyrhizobium-inoculated plants were grown in hydroponic cultures in a greenhouse. Nutrient solutions were supplied with Mn concentrations ranging from 0.3 to 90 microm, at either 5 or 150 microm Fe as FeEDTA. * KEY RESULTS: For both genotypes and at both Fe concentrations, Mn concentrations from 6.6 to 50 microm did not affect shoot, root and nodule mass, or leaf and nodule ureide concentration. Mn concentrations of 70 and 90 microm did not result in visible toxicity symptoms, but hindered growth and nodulation of 'Biloxi'. An Mn concentration of 0.3 microm was, however, deleterious to growth and nodulation for both genotypes, and caused an accumulation of ureides in leaves and major alterations in the ultrastructure of chloroplasts, nuclei and mitochondria, regardless of the Fe concentration. In 'PI227557', there was also a proliferation of Golgi apparatus and endoplasmic reticulum in the cytoplasm of leaf cells, and nodules showed disrupted symbiosomes lacking poly-beta-hydroxybutirate grains concomitantly with a proliferation of endoplasmic reticulum as well as arrested bacterial division. At 15 microm Fe, ferritin-like crystals were formed in the lumen of chloroplasts of 'PI227557' plants. For both genotypes, there was an antagonism between the Fe and Mn concentrations in leaves, the higher values of both microelements being detected in 'PI227557'. The absence of any detectable relationship between Fe or Mn and zinc, phosphorus and copper concentrations in leaves ruled out those micronutrients as relevant for Mn and Fe nutrition in soybeans. * CONCLUSIONS: The results confirmed the greater capacity of 'PI227557' for Mn and Fe accumulation than 'Biloxi' for most nutrient treatments. Hence, 'PI227557' may be a very useful genetic resource both in developing soybean cultivars for growth on low nutrient soils and in physiological studies to understand differing approaches to nutrient accumulation in plants.

Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean.

Vacuolar compartments associated with leaf senescence and the subcellular localization of the senescence-specific cysteine-protease SAG12 (senescence-associated gene 12) were studied using specific fluorescent markers, the expression of reporter genes, and the analysis of high-pressure frozen/freeze-substituted samples. Senescence-associated vacuoles (SAVs) with intense proteolytic activity develop in the peripheral cytoplasm of mesophyll and guard cells in Arabidopsis and soybean. The vacuolar identity of these compartments was confirmed by immunolabeling with specific antibody markers. SAVs and the central vacuole differ in their acidity and tonoplast composition: SAVs are more acidic than the central vacuole and, whereas the tonoplast of central vacuoles is highly enriched in gamma-TIP (tonoplast intrinsic protein), the tonoplast of SAVs lacks this aquaporin. The expression of a SAG12-GFP fusion protein in transgenic Arabidopsis plants shows that SAG12 localizes to SAVs. The analysis of Pro(SAG12):GUS transgenic plants indicates that SAG12 expression in senescing leaves is restricted to SAV-containing cells, for example, mesophyll and guard cells. A homozygous sag12 Arabidopsis mutant develops SAVs and does not show any visually detectable phenotypical alteration during senescence, indicating that SAG12 is not required either for SAV formation or for progression of visual symptoms of senescence. The presence of two types of vacuoles in senescing leaves could provide different lytic compartments for the dismantling of specific cellular components. The possible origin and functions of SAVs during leaf senescence are discussed.

Characterization of the biosynthesis of beta(1-2) cyclic glucan in R. Fredii. Beta(1-2) glucan has no apparent role in nodule invasion of Mc Call and Peking soybean cultivars.

Three wild type strains of Rhizobium fredii, USDA 191, USDA 257 and HH 303, do not synthesize in vivo or in vitro beta(1-3), beta(1-6) cyclic glucans, all strains form in vitro and in vivo cyclic beta(1-2) glucans. Approximately 80% of the recovered R. fredii cellular cyclic beta(1-2) glucans were anionic and the substituent was identified as phosphoglycerol. Inner membranes prepared from these R. fredii strains have a beta(1-2) glucan-intermediate-protein with apparent molecular mass undistinguishable from Agrobacterium tumefaciens beta(1-2) glucan intermediate protein. Studies of the degree of polymerization of the oligosaccharides recovered from the protein-intermediate after short pulse incubations with UDP-14C-glucose suggested that the rate limiting step in the biosynthesis of cyclic glucan is cyclization. Kinetic studies revealed that the K(m) for UDP-glucose was 0.33 mM. No difference was detected between the K(m) for initiation/elongation and cyclization reactions. Nodulation studies of a ndvB R. fredii mutant with Mc Call and Peking soybean cultivars, revealed that beta(1-2) glucans do not seem to be required for normal nodule invasion of these soybean cultivars.