Correlation of xylem sap cytokinin levels with monocarpic senescence in soybean

Cytokinins (CKs) coming from the roots via the xylem are known to delay leaf senescence, and their decline may be important in the senescence of soybean (Glycine max) plants during pod development (monocarpic senescence). Therefore, using radioimmunoassay of highly purified CKs, we quantified the zeatin (Z), zeatin riboside (ZR), the dihydro derivatives (DZ, DZR), the O-glucosides, and DZ nucleotide in xylem sap collected from root stocks under pressure at various stages of pod development. Z, ZR, DZ, and DZR dropped sharply during early pod development to levels below those expected to retard senescence. Pod removal at full extension, which delayed leaf senescence, caused an increase in xylem sap CKs (particularly ZR and DZR), while depodding at late podfill, which did not delay senescence, likewise did not increase the CK levels greatly. The levels of the O-glucosides and the DZ nucleotide were relatively low, and they showed less change with senescence or depodding. The differences in the responses of individual CKs to senescence and depodding suggest differences in their metabolism. Judging from their activity, concentrations and response to depodding, DZR and ZR may be the most important senescence retardants in soybean xylem sap. These data also suggest that the pods can depress CK production by the roots at an early stage and this decrease in CK production is required for monocarpic senescence in soybean.     

Waterlogging effect on xylem sap glutamine of nodulated soybean

Waterlogging of soybean plants (Glycine max L.) led to impaired symbiotic N₂ fixation and a marked decline in glutamine (Gln) concentration in xylem bleeding sap. Xylem Gln concentration increased during the growth cycle of the plant and was correlated with nodule formation. Treatments known to impair N₂ fixation, such as exposing the root system to pure N₂ gas or a mixture of Ar and O₂ (80:20; v/v), led to specific declines in xylem sap Gln. The decrease in Gln observed during waterlogging was also seen on transfer of nodulated plants to aerated hydroponics, where the decline was highly correlated with ureide content in the xylem sap. Upon flooding the nodulated root system, the specific decline in xylem sap Gln could be detected within 10 min and reached a minimum within 60 min, indicating that waterlogging has an immediate effect on N₂ fixation. It is concluded that xylem Gln arises directly from N₂-fixation and is a useful indicator of N₂ fixation activity of symbiotic soybean plants.     

Characterization and kinetics of soybean maturation and monocarpic senescence

Daring monocarpic senescence in potted soybeans ( Glycine maxi (L.) Merrill cv. Anoka) grown in controlled-environment chambers, foliar chlorophyll, soluble protein nitrogen, total nitrogen, and starch decline (roughly in that order). All of these precede visible yellowing and, of course, abscission. The pattern of yellowing within a leaf is not uniform and is closely paralleled by starch loss. Unexpectedly, acid-soluble nitrogen rises slightly before the total foliar nitrogen declines. Foliar fresh weight and total dry matter/cm2 of leaf surface decline little if at all before shedding. Preceding and even during the foliar yellowing, the seeds rapidly accumulate dry matter and nitrogen. Yellowing appears first in the radicle tip, then in the rest of the axis and the leaves and finally in the carpels. Ability to germinate is acquired at about the time the radicle + hypocotyl turns yellow. The relationship between these changes and their role in senescence is discussed.     

Evidence supporting a non-phloem source of water for export of solutes in the xylem of soybean root nodules

The vascular anatomy of soybean nodules [Glycine max (L.) Merr.] suggests that export of solutes in the xylem should be dependent on influx of water in the phloem. However, after severing of stem xylem and phloem by shoot decapitation, export of ureides from nodules continued at an approximately linear rate for 5h. This result was obtained with decapitated roots remaining in the sand medium, but when roots were disturbed by removal from the rooting medium prior to shoot decapitation, export of ureides from nodules was greatly reduced. Stem exudate could not be collected from disturbed roots, indicating that flow in the root xylem had ceased. Thus, ureide export from nodules appeared to be dependent on a continuation of flow in the root xylem. When seedlings were fed a mixture of ³H₂O and ¹⁴C-inulin for periods of 14–21 min, nodules had higher ³H/¹⁴C ratios than roots from which they were detached. The combined results are not consistent with the proposal that export of nitrogenous compounds from nodules is dependent on import of water via the phloem. The results do support the view that a portion of the water required for xylem export from soybean nodules is supplied via a symplastic route from root cortex to nodule cortex to the nodule vascular apoplast.     

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.     

Stress-induced changes in the free amino acid composition in transgenic soybean plants having increased proline content

Following drought stress at supraoptimal temperature the increase in proline (Pro) content in transgenic (T) soybean [Glycine max (L.) Merr. cv. Ibis] plants overexpressing the gene coding for the last enzyme of Pro biosynthesis, L-Δ¹-pyrroline-5-carboxylate reductase, was much greater than in wild type (W) plants (105-fold versus 19-fold after 7 d). Under control conditions arginine accounted for nearly 60 % of the total free amino acid content. After stress treatment the content of Pro was more than 50 % in both T and W genotypes, and at the end of recovery the γ-aminobutyrate content reached 27 and 53 % in the W and T plants, respectively. Without stress treatment there was only a 2-fold difference between T and W in the tyrosine content. However, during the stress period and the subsequent recovery a similar difference was found for many amino acids. The present results indicate that manipulating of the content of a single amino acid influences the whole free amino acid composition in soybean.     

Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

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.     

Growth,nodulation and nitrogen fixation in soybean as affected by air humidity and root temperature

Growth, nodulation and N₂ fixation inGlycine max L. Merr., cv. Biison as affected by the relative humidity of air (RH) during the dark period (95 or 50 – 65 %) and day/night root temperature (T_r) (28/28, 25/25, 18/18, 22/28, 22/18 °C) were studied. The growth parameters (plant fresh and dry mass, yield), nodulation (nodule number and fresh mass) and N₂ fixation abilities (total nitrogen content, nitrogenase activity) increased significantly with the increasing T_r. In addition, at the same T_r during the day all studied parameters were increased at the higher T_r during the dark period. Growth, nodulation and N₂ fixation were significantly enhanced at low RH. The findings indicate that all studied parameters could be regulated by environmental factors during the dark period.     

Polymerase chain reaction detect polymorphisms and trait association in soybean

Five sets of synthetic oligonucleotide (20-to 24-mers containing no intenal repeats) primers of known gene sequences [yellow lupin nodule specific (hydroxyl) proline-rich protein, pearl millet alcohol dehydrogenase, Pisum sativum heat shock proteins, Drosophila homeobox, and tRNA] were used to differentiate 73 soybean accessions, including 56 Glycine max (L.) Merr. and 17 G. soja Zucc. & Sieb. The amplified genetic markers revealed polymorphic bands for most genotypes studied. The χ²-analyses of the results showed that several fragments produced with these gene primers were associated non-randomly with resistance to Phytophothora, maturity, seed size, flower colour, seed coat colour, seed hilum colour, growth type, and leaf shape. These markers appear to be valuable for differentiation of G. max and G. soja species and genotypes within these species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Grafting experiments on the nature of the decline in N2 fixation during fruit development in soybean

A decline in nitrogen fixation at the time of pod-filling is persistently seen in soybeans. This phenomenon was studied by grafting experiments. Young scions with 3 nodes were grafted near the base of fruiting stocks which had passed their peak of nitrogen fixation. These grafts produced a second peak of nitrogen fixation on the same root system, indicating that the decline is reversible. If the scions were grafted near the apex of the fruiting stocks then the second peak of nitrogen fixation was very small. Thus, the translocation system could have an important role in regulating the decline in nitrogen fixation. Grafting of a second shoot of the same age as the rootstock, after the decline in nitrogen fixation, did not reduce the rate of decline even when the fruits from the scion were removed. It appears that physiological changes in different components of the shoot jointly regulate the decline in the rate of nitrogen fixation in soybeans.     

Activities of chlorophyllase, phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase in the primary leaves of soybean during senescence and drought

The effects of senescence and drought on the levels and activities of chlorophyllase (EC 3.1.1.14), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) in the intact primary leaves of soybean ( Glycine max L. cv. Jackson) were monitored. Plants were grown either (1) for 2 to 8 weeks and the primary leaves harvested every week or (2) for 2 weeks and the plants subjected to drought stress and compared to control plants that were watered daily. In the senescence experiment, chlorophyllase activity changed in parallel with water content, leaf chlorophyll and total protein per unit dry weight of leaf tissue, with all factors increasing in concert during expansion of the primary leaves in the first 4 to 5 weeks of seedling development. Thereafter, all factors, including chlorophyllase activity, declined reaching markedly reduced values at weeks 7 and 8 when the primary leaves were yellow and ready to abscise. PEPC and Rubisco activities peaked in the third week, i.e. well before full leaf expansion, and then declined. In contrast to its response during senescence, chlorophyllase activity per unit leaf dry weight did not change during drought stress, but the specific activity of the enzyme rose and showed an inverse relationship to total leaf chlorophyll and protein content. Rubisco activity was highly sensitive to drought, with decrements observed in the activity and in levels of the large subunit within 2 days of withholding water and before significant changes in leaf water content were detected.     

Sodium partitioning within the shoot of soybean

Uptake and partitioning of Na⁺ and Cl⁻ in plants of soybean ( Glycine max L. Merr. cv. Hodgson) exposed to moderate NaCl concentrations were studied over an 8-day period. Plants showed marked retention of Na⁺ in the stems and low transport to laminae of young leaves. The xylem sap ascending the main axis was progressively depleted in Na⁺. The oldest leaf greatly contributed to Na⁺ depletion of the sap flowing to younger leaves. These results in combination with estimates of phloem recirculation indicated that Na⁺ accumulation in the young leaf was prevented both by depletion of Na⁺ from the xylem stream, and by a high recirculation of Na⁺ via the phloem. However, this protection of young leaves was effective only for very mild salinity treatment.     

Vascular transport and soybean nodule function: nodule xylem is a blind alley, not a throughway

Abstract. The only published consideration of product removal from the soybean root nodule hypothesizes that the peripheral xylem circuit of this determinate nodule structure is flushed by the transpiration stream. However, dyes fed to the transpiration stream through a cut root distal to the nodule do not enter the nodule, and the observed movement of radio-tracers from the root into the nodule can be explained by simple diffusion, Also, there are few xylem elements in the nodule, and these elements are of a small diameter, such that this path can not act as a functional loop of the root system. Further, in this study, nodule vascular strands were never observed to be continuous about the nodule, but were observed to end at the nodule tip in a loop within an intact, closed endodermal sac. Nodule vascular tissue was invested in a pericycle of at least three cell layers. These cells are suggested to be active in the loading of the xylem apoplast with ureides, such that the xylem of the nodule always operates in an export role. Nodule water requirements may be supplied via the phloem or the root cortex apoplasm.     

大豆microRNA基因GmMIR160A负调控植物叶片衰老进程

叶片衰老是受内外多种因子影响的遗传发育进程。生长素、细胞分裂素和乙烯等多种植物激素是调控叶片衰老的重要内部因子,它们通过长或短距离运输形成叶片组织内特定的区域分布和浓度梯度,从而直接或间接参与植物叶片衰老过程。分子遗传学表明,细胞分裂素和乙烯分别是叶片衰老的抑制子和正调节子,而生长素如何参与叶片衰老的分子机制目前还不清晰。植物体内成熟小分子RNA由小RNA基因转录并通过特定酶加工形成的21~23bp的双链RNA分子。这些小分子通过不完全配对方式抑制其靶基因转录和/或表达,参与植物生长发育多个过程,然而这类小RNA分子如何调控植物叶片衰老发育过程目前则还鲜有报告。大豆是重要的油料作物,具有典型的单次结实性衰老特征。研究大豆叶片衰老具有重要的科学意义和深远的应用价值。该文采用实时荧光定量PCR(qPCR)技术分析大豆(Glycine max)micro RNA基因GmMIR160A的表达模式,发现大豆第一复叶中GmMIR160A表达受外源生长素和黑暗处理的诱导,暗示该基因是生长素快速响应的叶片衰老相关基因。为进一步探究GmMIR160A在大豆叶片发育中的功能,构建了肾上腺皮质激素(Glucocorticoid,GR)类似物地塞米松(Dexamethasone,DEX)诱导表达GmMIR160A双元表达载体并通过农杆菌介导的子叶节方法转化野生型大豆。通过抗性筛选和基因组PCR鉴定并结合表型分析,共获得了4株诱导表达的稳定遗传转基因植株(株系OX-3、OX-5、OX-7和OX-8)。GmMIR160A过表达植株根、茎、叶、花和果实在形态学上与野生型相比无显著差异,但叶片的叶绿素含量增加、最大光量子效率(Fv/Fm)增强。进一步分子分析发现,转基因大豆叶片中GmARFs和衰老标记基因(GmCYSP1)表达明显下降,表明大豆Gma-miR160通过抑制靶基因GmARFs的表达来负调控植物叶片的衰老进程。该文揭示了生长素通过小分子RNA调控叶片发育一条新途径,为研究植物激素调控植物叶片衰老提供了新的思路。     

Hydrogen sulfide and rhizobia synergistically regulate nitrogen (N) assimilation and remobilization during N deficiency-induced senescence in soybean

Hydrogen sulfide (H₂S) is emerging as an important signalling molecule that regulates plant growth and abiotic stress responses. However, the roles of H₂S in symbiotic nitrogen (N) assimilation and remobilization have not been characterized. Therefore, we examined how H₂S influences the soybean (Glycine max)/rhizobia interaction in terms of symbiotic N fixation and mobilization during N deficiency-induced senescence. H₂S enhanced biomass accumulation and delayed leaf senescence through effects on nodule numbers, leaf chlorophyll contents, leaf N resorption efficiency, and the N contents in different tissues. Moreover, grain numbers and yield were regulated by H₂S and rhizobia, together with N accumulation in the organs, and N use efficiency. The synergistic effects of H₂S and rhizobia were also demonstrated by effects on the enzyme activities, protein abundances, and gene expressions associated with N metabolism, and senescence-associated genes (SAGs) expression in soybeans grown under conditions of N deficiency. Taken together, these results show that H₂S and rhizobia accelerate N assimilation and remobilization by regulation of the expression of SAGs during N deficiency-induced senescence. Thus, H₂S enhances the vegetative and reproductive growth of soybean, presumably through interactions with rhizobia under conditions of N deficiency.     

Changes in the composition of xylem sap during development of the spadix of skunk cabbage (Symplocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 degrees C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2+/-24.7 microl h(-1) g(-1)) and post-thermogenic (50.5+/-30.4 microl h(-1) g(-1)) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.     

Terpenoids are transported in the xylem sap of Norway spruce

Norway spruce is a conifer storing large amounts of terpenoids in resin ducts of various tissues. Parts of the terpenoids stored in needles can be emitted together with de novo synthesized terpenoids. Since previous studies provided hints on xylem transported terpenoids as a third emission source, we tested if terpenoids are transported in xylem sap of Norway spruce. We further aimed at understanding if they might contribute to terpenoid emission from needles. We determined terpenoid content and composition in xylem sap, needles, bark, wood and roots of field grown trees, as well as terpenoid emissions from needles. We found considerable amounts of terpenoids—mainly oxygenated compounds—in xylem sap. The terpenoid concentration in xylem sap was relatively low compared with the content in other tissues, where terpenoids are stored in resin ducts. Importantly, the terpenoid composition in the xylem sap greatly differed from the composition in wood, bark or roots, suggesting that an internal transport of terpenoids takes place at the sites of xylem loading. Four terpenoids were identified in xylem sap and emissions, but not within needle tissue, suggesting that these compounds are likely derived from xylem sap. Our work gives hints that plant internal transport of terpenoids exists within conifers; studies on their functions should be a focus of future research.