Light-quality and irradiance effects on pigments, light-harvesting proteins and Rubisco activity in a chlorophyll- and light- harvesting-deficient soybean mutant

Soybean ( Glycine max [L.] Merrill) plants, normal green (Clark L1) and mutant yellow (Clark y₉y₉), were grown in (1) full-spectrum solar irradiation; (2) either red plus far-red or blue plus far-red; (3) either red or blue light with no far-red light. Young leaves harvested from first (1TF) trifoliolate or fifth (5TF) trifoliolate stages of development showed that the mutant plants express pigment and protein deficiencies as a direct function of irradiance. Response of the mutant to light quality indicates that blue light slightly enhances expression of the mutation at higher irradiances. Direct response of light-harvesting proteins of photosystem 2 (LHCP2) and light-harvesting protein of photosystem 1 (LHCP1) to light quality increases the ratio of LHCP1/LHCP2 in blue light compared to that in red or red/far-red light. Rubisco proteins and Rubisco activity (leaf area basis) are directly related to irradiance level but are enhanced in blue light over equal irradiance red. This enhancement is not shown in the presence of far-red light.     

Nuclear and cytoplasmic stay-green mutations of soybean alter the loss of leaf soluble proteins during senescence

In soybean ( Glycine max [L.] Merr.) the homozygous combination of the recessive alleles dI and d2 (i.e., dldld2d2 ) at two different nuclear loci or the cytoplasmic gene cytG inhibit chlorophyll degradation during senescence; i.e. their leaves are green when they are shed. The main objectives of the present work were: (J) to determine whether these stay-green genes also interfere with the loss of the bulk of leaf soluble proteins and ribulose bisphospnate carboxylase/oxygensase (Rubisco; EC 4.1.1.39) during senescence and (2) to relate this to alterations in leaf proteolytic activity. Leaves of the normal. Yellowing cvs Clark and Harosoy lost about 90% of their soluble proteins before abscission. The abscising leaves of these cultivars contained no detectable Rubisco. By contrast, protein degradation was significantly less in leaves of near-isogenic lines of Clark and Harosoy carrying dIdId2d2 , with or without G (a dominant nuclear gene in a third locus causing green seed coats). These leaves still retained 50% of the soluble protein and large amounts of both subunits of Rubisco at the time of abscission. Alone, neither dl nor d2 had any effect. The cytoplasmic gene cytG slowed the loss of Rubisco. although eventually when leaves were shed they contained as little Rubisco as Clark. Despite inhibition (i.e. dIdId2d2 and GGdIdId2d2 ) or retardation (i.e. cytG ) of protein loss, these mutant genotypes did not differ from Clark in the breakdown of endogenous Rubisco by leaf extracts (autodigestion). The wild-type alleles in the dI and d2 loci may control a central regulatory process of the senescence syndrome.     

Inhibition of the degradation of chloroplast membranes during senescence in nuclear 'stay green' mutants of soybean

Near-isogenic lines of soybean ( Glycine max [L.] Merr.) cv. Clark carrying nuclear 'stay green' genes were examined to determine the effects of these genes on the breakdown of thylakoid membranes during senescence. In order to accelerate their senescence, mature leaves were excised and incubated in darkness for 7 days. The homozygous combination of the recessive alleles d1 and d2 (at two different nuclear loci), with or without G (a dominant allele in another locus that causes green seed coat) inhibited the loss of chlorophyll and thylakoid proteins during senescence. Electron micrographs of leaves of cv. Clark during the yellowing process showed chloroplasts in various stages of disintegration; their thylakoid network was disrupted and abundant osmiophilic globuli formed. These senescent leaves also showed evident signs of deterioration of the plasma membrane, including discontinuities, invaginations and membrane 'whorls'. In contrast, leaves carrying d1d1d2d2 and GGd1d1d2d2 did not show signs of plasma membrane degradation, and their chloroplasts appeared intact, with a continuous, unbroken thylakoid network and tightly stacked grana.
Exogenous applications of abscisic acid (1 and 10 μ M ), methyl jasmonate (10 μ M ) or ethylene (1 and 10 μl]⁻¹) accelerated chlorophyll degradation in cv. Clark, but had no appreciable effect in d1d1d2d2 and GGd1d1d2d2 , which indicates that their pheno-types are not due to a deficiency in any of these hormones. The nuclear 'stay green' genotypes d1d1d2d2 and GGd1d1d2d2 exhibit a general incompetence for the degradation of chloroplast membranes and, thus, they may constitute useful tools in the study of the biochemistry and regulation of leaf senescence.     

Differential sensitivity of nodulation to ethylene in soybean cv. Bragg and a supernodulating mutant

We previously found that the ethylene inhibitor Ag⁺ could overcome the inhibitory effect of nitrate on nodulation of soybean ( Glycine max ) cv. Bragg. The same treatment increased nodulation quantitatively under non-inhibitory conditions, strongly suggesting involvement of ethylene in the control of nodulation in this species. Supernodulation mutants that lack internal autoregulation of nodulation, however, had biosynthesis capacity similar to the wild type. In the present work, the effects of ethylene on nodulation of 'Bragg' and two separate, but allelic, supernodulating mutants ( nts382 and nts1007 ) were compared. The nodulation process appeared much more sensitive than plant growth and development to ethylene, which reduced the number of nodules per plant, but nearly twofold more in the wild type than in the supernodulation mutants. The cause–effect relationship is established by the counteracting effect of Ag⁺ and the fact that the stronger the inhibition by ethylene, the higher the recovery of nodulation ability with the ethylene antagonist. This higher tolerance of or lower sensitivity to ethylene in nts382 persists even under low inoculum dose, where nodule number and mass could be decreased to wild-type levels. Differences between the mutant and the wild type in the triple response test do not appear to support differences in ethylene perception on a whole-plant basis. The results suggest that sensitivity of nodulation to ethylene might have been affected in supernodulation mutants.     

Relationship between autoregulation and nitrate inhibition of nodulation in soybeans

Ten of 11 supernodulating mutants of soybean [ Glycine max (L.) Merr.] cv. Bragg, in which nodulation was far in excess of that in the wild type, showed pronounced tolerance of nodulation to applied nitrate. Mutant nts (nitrate-tolerant symbiosis) 1116 had an intermediate nodulation response and also showed some inhibition by nitrate. Mutant 1029, a revertant of nts382 (an extreme supernodulator), showed a wild-type nodulation pattern and was equally sensitive to nitrate as cv. Bragg. Grafting experiments with cv. Bragg and nts382 indicated that both supernodulation and tolerance of nodulation to nitrate were dependent on shoot factors. Total leaf nitrate reductase (EC 1.6.6.1 and EC 1.6.6.2) activity of the supernodulating mutants was similar to that in cv. Bragg. We conclude from these results that the inhibitory effect of nitrate on nodule initiation and development in soybean depends on an interaction between nitrate and the autoregulation singal. In the supernodulating mutants, the autoregulation signal is either altered or absent and cosequently nodulation in these mutants is not sensitive to nitrate.     

Vascular transport and soybean nodule function. III: Implications of a continual phloem supply of carbon and water

Abstract. In soybean, stores of carbon within the leaf have been demonstrated to support nodule metabolism under both photosynthetic and non-photosynthetic conditions. Indeed, a net depletion of nodule starch is observed only under conditions of suboptimal rates of nodule metabolism. Therefore, maximal rates of nodule metabolism are associated with a continual supply of phloem sap to the nodule, delivering water, carbon and other solutes. A restriction of phloem supply to the nodule may result in changes in turgor between the apoplast of the export pathway and the symplast of the nodule. This change may cause the observed decrease in the permeability to gases and to the rate of product export from nodules deprived of a phloem supply. It is suggested that nodule metabolism is homeostatically regulated in terms of internal O₂ levels by the delivery of phloem water and solutes.     

Symbiotic dinitrogen fixation and host-plant growth during development of and recovery from phosphorus deficiency

Characterization of nodule growth and function, phosphorus and nitrogen status of plant tissues and host-plant growth of nodulated soybean ( Glycine max L. Merr.) plants developing and recovering from phosphorus deficiency was used to evaluate the role of phosphorus in symbiotic dinitrogen fixation. The sequence of physiological responses during recovery from phosphorus deficiency was; (1) rapid uptake of phosphorus, (2) rapid increases in the phosphorus concentration of leaves and nodules, (3) enhanced growth and function of nodules, (4) increased nitrogen concentrations in all plant organs and (5) enhanced plant growth. The sequence of physiological responses to onset of phosphorus deficiency was; (1) decreased phosphorus uptake, (2) decreased phosphorus concentrations in leaves and nodules, (3) decreased nodule function, (4) decreased nitrogen concentration in plant organs and (5) decreased plant growth. These results, in conjunction with previously published data (Sa and Israel, Plant Physiol. 97: 928–935, 1991), support an interpretation that the total response of symbiotic dinitrogen fixation in soybean plants to altered phosphorus supply is a function of both indirect effects on host-plant growth and more direct effects on the metabolic function of nodules.     

Development of protein bodies and accumulation of carbohydrates in a soybean (Leguminosae) shriveled seed mutant

The soybean seed mutant T311, when grown under specific environmental conditions, produces shriveled seed. This research investigated changes in development of protein bodies and accumulation of carbohydrates during seed development by comparing the mutant with P2180 seeds. The shriveled seeds contained larger protein bodies but fewer protein bodies per cell than round seeds. Protein bodies in T311 seeds included more dispersed crystals and less globoid regions than P2180 seeds. The elemental compositions of the crystals and of whole seeds in T311 were different from that in P2180 seeds. Starch breakdown was reduced with concomitant lower soluble sugar content in T311 seeds after the D11 stage (10.0-11.9 mm long seeds). The reduced starch breakdown and lowered soluble sugar content were consistent with lower a-amylase activity and earlier and greater water loss in T311 seeds. Changes in development of protein bodies and accumulation of carbohydrates were associated with the development of the shriveled seeds.     

The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean

The soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent is described. Soybean cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens for 3 days. Explants were cultured on Gamborg's B5 medium supplemented with 1.67 mg l^-1 BAP and glufosinate at levels of 3.3 mg l^-1 or 5.0 mg l^-1 for 4 weeks. After 4 weeks explants were subcultured to medium containing MS major and minor salts and B₅ vitamins (MS/B5) supplemented with 1.0 mg l^-1 zeatin-riboside, 0.5 mg l^-1 GA₃ and 0.1 mg l^-1 IAA amended with 1.7 mg l^-1 or 2.0 mg l^-1 glufosinate. Elongated shoots were rooted on a MS/B5 rooting medium supplemented with 0.5 mg l^-1 NAA without further glufosinate selection. Plantlets were transplanted to soil and grown to maturity and set seed in the greenhouse. Primary transformants and their progeny were characterized by Southern blot analysis and a leaf paint assay.     

Soybean (Glycine max) modulation and N2-fixation as affected by exposure to a low root-zone temperature

Low root-zone temperatures (RZTs) are known to reduce soybean N₂-fixation. However, the relative sensitivity of the various stages of symbiosis establishment and function (N₂-fixation) to suboptimal RZTs is unresolved. We conducted experiments to examine the effect of exposure to a RZT of 15°C on nodulation. The control RZT was 25°C. Root temperatures were controlled by circulating cooled water around pots on a growth bench. Soybean seedlings [ Glycine max (L.) Merr. cv. Maple Arrow] were inoculated with 1 ml of a log-phase culture (approximately 10⁻⁸ cells) of Bradyhizobium japonicum strain 532C. They were then (1) maintained continuously at RZTs of 15 or 25°C, transferred to 15 or 25°C from the alternate temperature 7 days after inoculation (DAI), or transferred to 15 or 25°C at 14 DAI, and (2) maintained at 15 or 25°C, or transferred at either 1, 4 or 7 DAI. When seedlings were maintained at a RZT of 25°C nodule primordia (<1 mm) were visible at 7 DAI and N₂-fixation commenced at 14 DAI. Nodule function (N₂-fixation) appeared to be relatively insensitive to low RZTs since exposure of plants to 15°C following the onset of N₂-fixation (14 DAI) resulted in 68% of the N fixed and 78% of the dry weight of the 25°C RZT, although N partitioning to shoot tissues was reduced. In contrast, exposure to the low RZT shortly after inoculation declayed the onset of N₂-fixation for 4 to 6 weeks, primarily by inhibiting the early stages of nodulation. This resulted in fixed N and dry weight levels of 9% and 22% of controls, respectively.     

Determination of the distribution of three nitrate reductase isoforms in soybean seedlings by chromatography and a simple method based on assay conditions

Blue Sepharose affinity chromatography was used to study the distribution of the constitutive NAD(P)H-nitrate reductase (EC 1.6.6.2: Cl-NR) and of the constitutive and inducible NADH-nitrate reductases (EC 1.6.6.1; C2-NR and i-NR, respectively), in the unifoliolate leaf (F0), the first and the second trifoliolate leaves (F1 and F2) and the roots of urea- and nitrate-grown soybean ( Glycine max [L.] Merr.) plants. The C1-NR eluted by NADPH is present in the F0 and F1 leaves and nearly absent in the F2 leaf. The activity pattern of this isoform is not modified by nitrate nutrition. The C2-NR eluted by NADH is high in the F0 leaf, low in the F1 leaf and nearly absent in the F2 leaf of urea-grown plants. The NADH elution from leaves of nitrate-grown plants is a mixture of C2-NR and i-NR, requiring careful interpretation of results. However, i-NR appears the principal isoform in the leaves especially in the F2 leaf. This i-NR is the only NR present in the roots.
The pH effect on the assay of the 3 partially purified isoforms was studied using LNR2 and LNR5 soybean mutants to remove the cross contamination. It appears that C1-NR and C2-NR activities are negligible at pH 8.5, which allows the assay of only the i-NR in a crude extract at this pH, even when C1-NR and C2-NR are present. It appears also that the assay of C1-NR activity at pH 6.5 with NADPH is free of interference by the i-NR. To estimate the C2-NR activity with NADH at pH 6.5 in a crude extract in the presence of C1-NR and i-NR, we propose a simple calculation using the coefficient from the pH responses. These calculations are used to compare the development of C1-NR, C2-NR and i-NR activities in the F0 and F1 leaves of plants previously grown on urea and transferred to nitrate. Only the activity of the inducible isoform is modified by the nitrogen treatment. Activity of the constitutive isofroms appear stable during the 48 h treatment, with only a slight decrease in C1-NR activity being observed with time.     

Immunological cross-reactivity between the light-harvesting chlorophyll a/b-proteins of a marine green alga and spinach

Immunoblotting was used to probe the reactivity of rabbit polyclonal antibodies against PS1I and PSI light-harvesting chlorophyll a/b-proteins of spinach ( Spinacea oleracea L.) with the light-harvesting complexes of a siphonaceous marine alga, Codium , that have more chlorophyll b, siphonaxanthin and siphonein instead of the lutein. The spinach LHCII antibodies cross-reacted only with the apoproteins of Cod-ium LHCII. Antisera against the spinach LHCI apoproteins showed strong affinity for the apoproteins of Codium LHCI, and also reacted with the polypeptides of spinach LHCII and Codium LHCII. Our results indicate some similarities in the amino acid sequences between the Codium siphonaxanthin-Chl a/fe-proteins of LHCII and LHCI and the corresponding spinach lutein-chlorophyll a/b-proteins.     

DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase-α. When DNA replication was partially inhibited by 10 mg l^-1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l^-1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l^-1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.     

Development and seed number in indeterminate soybean as affected by timing and duration of exposure to long photoperiods after flowering

BACKGROUND AND AIMS: Long photoperiods from flowering to maturity have been found to delay reproductive development in soybean (Glycine max) and to increase the number of seeds per unit land area. This study was aimed to evaluate whether sensitivity to photoperiod after flowering (a) is quantitatively related to the length of exposure to long days and (b) persists throughout the whole pod-setting period. It was also evaluated whether seed number was related to changes in the duration of post-flowering phenophases. METHODS: Two field experiments were conducted with an indeterminate cultivar of soybean of maturity group V. In expt 1, photoperiods 2 h longer than natural daylength were applied during different numbers of days from the beginning pod stage (R3) onwards, while in expt 2 these photoperiod extensions were imposed during 9 consecutive days starting at different times between R3 and R6 (full seed) stages. KEY RESULTS: There was a quantitative response of development to the number of cycles with a long photoperiod. The exposure to long photoperiods from R3 to R5 (beginning of seed growth) increased the duration of R3-R6 regardless of the timing of exposure. The stages of development comprised in the R3-R6 phase were delayed by current as well as by previous exposure to long days. A positive relationship was found between seed number and the duration of R3-R6, irrespective of the timing and length of exposure to the long photoperiod. CONCLUSIONS: Sensitivity to photoperiod remained high during the reproductive period and was highly and positively coupled with the processes of generation of yield.     

Spectral balance and UV-B sensitivity of soybean: a field experiment

Soybean [Glycine max (L.) Merr.] cultivar Essex was grown and tested for sensitivity to UV-B radiation (280–320 nm) under different combinations of UV-A (320–400 nm) and PFD (400–700 nm) radiation in four simultaneous field experiments. The radiation conditions were effected with combinations of filtered solar radiation and UV-B and UV-A lamps electronically modulated to track ambient radiation. Significant UV-B-caused decreases in total aboveground production and growth were seen only when PFD and UV-A were reduced to less than half their flux in sunlight. When PFD was low, UV-A appeared to be particularly effective in mitigating UV-B damage. However, when PFD was high, substantial UV-A did not appear to be required for UV-B damage mitigation. Leaf chlorophyll fluorescence did not indicate photosynthetic damage under any radiation combination. With UV-B, leaves in all experiments exhibited increased UV-absorbing pigments and decreased whole-leaf UV transmittance. Results of these field experiments indicate difficulties in extrapolating from UV-B experiments conducted in glasshouse or growth cabinet conditions to plant UV-B sensitivity in the field. Implications for UV radiation weighting functions in evaluating atmospheric ozone reduction are also raised.     

Ergosterol accumulation and oil quality changes in stored soybean invaded by Aspergillus ruber (A. glaucus group)

Soybean seed samples inoculated with spores of Aspergillus ruber were stored for 20 to 140 days at 25 ± 1 °C with moisture content varying from 11.3% to 17.7%. Seeds were colonised by the fungus within 20 days at all moisture levels. Ergosterol concentration in seeds increased with time of storage, being slow in samples with moisture content of 11.3% to 13.1% and more rapidly in those with higher moisture content. Free fatty acid (FFA) content also increased following the pattern of ergosterol. Equations were used to predict minimum safe storage period of the seeds at 25 °C. The rise in FFA is due to increase of A. ruber mass within the seed. Fungal growth did not affect fatty acids profiles or iodine index of the extracted oil. The results suggest revision of the present recommendations regarding seed moisture during storage. The FFA concentration of a seed lot can be used as a sensitive indicator of seed deterioration due to storage fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Xylem development and cell wall changes of soybean seedlings grown in space

BACKGROUND AND AIMS: Plants growing in altered gravity conditions encounter changes in vascular development and cell wall deposition. The aim of this study was to investigate xylem anatomy and arrangement of cellulose microfibrils in vessel walls of different organs of soybean seedlings grown in Space. METHODS: Seeds germinated and seedlings grew for 5 d in Space during the Foton-M2 mission. The environmental conditions, other than gravity, of the ground control repeated those experienced in orbit. The seedlings developed in space were compared with those of the control test on the basis of numerous anatomical and ultrastructural parameters such as number of veins, size and shape of vessel lumens, thickness of cell walls and deposition of cellulose microfibrils. KEY RESULTS: Observations made with light, fluorescence and transmission electron microscopy, together with the quantification of the structural features through digital image analysis, showed that the alterations due to microgravity do not occur at the same level in the various organs of soybean seedlings. The modifications induced by microgravity or by the indirect effect of space-flight conditions, became conspicuous only in developing vessels at the ultrastructural level. The results suggested that the orientation of microfibrils and their assembly in developing vessels are perturbed by microgravity at the beginning of wall deposition, while they are still able to orient and arrange in thicker and ordered structures at later stages of secondary wall deposition. CONCLUSIONS: The process of proper cell-wall building, although not prevented, is perturbed in Space at the early stage of development. This would explain the almost unaltered anatomy of mature structures, accompanied by a slower growth observed in seedlings grown in Space than on Earth.     

Development of microbial consortia as a biocontrol agent for effective management of fungal diseases in Glycine max L.

Thirty isolates of bacteria and six isolates of Trichoderma were isolated from fertile agricultural soil and evaluated for their antagonistic activity against phytopathogens like Macrophomina phaseolina and Sclerotinia sclerotiorum, under in vitro conditions. Different isolates showed varying degrees of antagonism. The three most antagonistic bacteria Pseudomonas aeruginosa (MBAA1), Bacillus cereus (MBAA2) and Bacillus amyloliquefaciens (MBAA3) and one fungi Trichoderma citrinoviride (MBAAT) were selected as the most effective isolates as biocontrol agents. The present study was undertaken to develop a plant growth promoting microbial consortium to reduce the disease incidence in Glycine max both under in vitro and in vivo conditions. Biocontrol attributes such as ammonia, siderophore, enzymes like β-1,3 glucanase, chitinase and cellulase were more potential in consortia in comparison to single isolates. Plants treated with consortia?+?pathogen showed lower disease incidence in comparison to single antagonist?+?pathogen and pathogen infested control (p?≤?0.05). Maximum disease control was observed in potted plants treated with S. sclerotiorum?+?MBAA1?+?MBAAT showing only 15.8% disease incidence in comparison to Sclerotinia infested control, in which disease incidence was 97%. Seed bacterised with MBAA1?+?MBAAT exhibited enhanced seed germination of G. max up to 68% along with subsequent increase in other plant growth parameters. Considerable increase in seedling vigour index (1863.2) and chlorophyll content (13.518?mg/g) was observed in seeds treated with MBAA1?+?MBAAT in plants infected with M. phaseolina.