Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.     

内源茉莉酸甲酯的积累改变了大豆叶片与根的发育

茉莉酸甲酯是一种调节植物形态发生、诱导防御相关基因的植物信号转导分子。为了解内源茉莉酸甲酯在植物发育中的作用,将编码茉莉酸甲基转移酶的NTR1基因与CaMV 35S启动子连接并导入大豆植株。PCR及Northern杂交结果表明,NTR1基因稳定整合在大豆基因组并得到过量表达。与野生型植株相比,转基因大豆叶片与根的形态发生了显著的变化。大部分转基因大豆叶片变得细长,初生根生长受到抑制而侧根的生长却受到促进。定量分析结果表明,转基因大豆植株叶片中茉莉酸甲酯的含量比对照高出 2～2.5 倍。这些结果表明,内源茉莉酸甲酯的积累参与了大豆形态发生的调控。     

Two Methyl Jasmonate-Insensitive Mutants Show Altered Expression of AtVsp in Response to Methyl Jasmonate and Wounding

Jasmonates are plant signal molecules that are derived from lipids through the action of lipoxygenase. Jasmonates regulate gene expression during plant development and in response to water deficit, wounding, and pathogen elicitors. The signal transduction chain that mediates jasmonate action was investigated by isolating and studying two methyl jasmonate (MeJA)-insensitive mutants of Arabidopsis thaliana. The recessive mutants, jin1 and jin4, are nonallelic and neither corresponds to coi1, a previously identified MeJA-insensitive mutant. Both mutants showed reduced sensitivity to MeJA-mediated root growth inhibition as well as reduced MeJA induction of AtVsp in leaves. Expression of AtVsp in flowers was not altered in the mutants. Furthermore, MeJA modulation of the jasmonate-responsive lipoxygenase and phenylalanine ammonia lyase genes was not altered in the mutants. jin4 plants exhibited increased sensitivity to abscisic acid in seed germination assays, whereas jin1 plants showed wild-type sensitivity. Neither mutant showed altered sensitivity to ethylene in hypocotyl growth inhibition assays. jin1 and jin4 identify genes that modulate the response of AtVsp to MeJA in leaves of A. thaliana.     

Shoot versus Root Signal Involvement in Nodulation and Vegetative Growth in Wild-Type and Hypernodulating Soybean Genotypes

Grafting studies involving Williams 82 (normally nodulating) and NOD1-3 (hypernodulating) soybean (Glycine max [L.] Merr.) lines and Lablab purpureus were used to evaluate the effect of shoot and root on nodulation control and plant growth. A single- or double-wedge graft technique, with superimposed partial defoliation, was used to separate signal control from a photosynthate supply effect. Grafting of hypernodulated soybean shoots to roots of Williams 82 or L. purpureus resulted in increased nodule numbers. Grafting of two shoots to one root enhanced root growth in both soybean genotypes, whereas the nodule number was a function of shoot genotype but not of the photosynthetic area. In double-shoot, single-root-grafted plants, removing trifoliolate leaves from either Williams 82 or NOD1-3 shoots decreased root and shoot dry matter, attributable to decreased photosynthetic source. Concurrently, Williams 82 shoot defoliation increased the nodule number, whereas NOD1-3 shoot defoliation decreased the nodule number on both soybean and L. purpureus roots. It was concluded that (a) soybean leaves are the dominant site of autoregulatory signal production, which controls the nodule number; (b) soybean and L. purpureus have a common, translocatable, autoregulatory control signal; (c) seedling vegetative growth and nodule number are independently controlled; and (d) two signals, inhibitor and promoter, may be involved in controlling legume nodule numbers.     

Jasmonate-dependent plant defenses mediate soybean thrips and soybean aphid performance on soybean

Insect herbivores from different feeding guilds induce different signaling pathways in plants. In this study, we examined the effects of salicylic acid (SA)- and jasmonic acid (JA)-mediated defenses on performance of insect herbivores from two different feeding guilds: cell-content feeders, soybean thrips and phloem feeders, soybean aphids. We used a combination of RT-qPCR analysis and elicitor-induced plant resistance to determine induction of SA and JA signaling pathways and the impact on herbivore performance. In the early interaction between the host plant and the two herbivores, SA and JA signaling seems to occur simultaneously. But overall, soybean thrips induced JA-related marker genes, whereas soybean aphids increased SA and ABA-related marker genes over a 24-h period. Populations of both soybean thrips and soybean aphids were reduced (47 and 25 %, respectively) in methyl jasmonate (MeJA)-pretreated soybean plants. SA treatment has no effect on either herbivore performance. A combination pretreatment of SA and MeJA did not impact soybean thrips population but reduced soybean aphid numbers which was comparable with MeJA treatment. Our data suggest that SA–JA antagonism could be responsible for the effect of hormone pretreatment on thrips performance, but not on aphid performance. By linking plant defense gene expression and elicitor-induced resistance, we were able to pinpoint the role for JA signaling pathway in resistance to two herbivores from different feeding guilds.     

Methyl jasmonate regulated diploid and tetraploid black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) tolerance to salt stress

Methyl jasmonate (MeJA) is an essential and promising plant growth regulation factor that can improve plant development and growth. Here, we explored the mechanism by which MeJA regulates the tolerance of black locust (Robinia pseudoacacia L.) to salt stress. In this study, diploid and tetraploid R. pseudoacacia were subjected to three treatments: 500 mM NaCl; 100 μM MeJA; and 500 mM NaCl and 100 μM MeJA, and the changes in plant growth, endogenous MeJA levels and the anti-oxidative metabolism of leaves were investigated. The results showed that salt stress significantly inhibited plant growth and induced the accumulation of Na⁺ and Cl^? ions, malondialdehyde (MDA) content and reactive oxygen species. However, these adverse effects could be alleviated by applying MeJA, which was followed by a marked increase in the activities of antioxidant enzymes. In addition, some genes encoding several antioxidant enzymes were also up-regulated. Simultaneously, the endogenous MeJA content in MeJA-treated plants was lower than in salt-treated plants. It is noteworthy that tetraploids always possessed higher salt tolerance and obtained greater positive effects from MeJA than diploids. These results suggested that MeJA might play a protective role in defense responses, enabling diploid and tetraploid black locust, especially tetraploid, to better tolerate the adverse effects of salt stress.     

Phosphorus runoff from a phosphorus deficient soil under common bean (Phaseolus vulgaris L.) and soybean (Glycine max L.) genotypes with contrasting root architecture

The selection and breeding of crop genotypes with root traits that improve soil resource extraction is a promising avenue to improved nutrient and water use efficiency in low-input farming systems. Such genotypes may accelerate nutrient extraction (“nutrient mining”), but may also reduce nutrient loss via soil erosion by producing greater shoot biomass and by direct effects of root traits on aggregate formation and water infiltration. Little is known about the effects of root architecture on phosphorus (P) runoff and soil erosion, and the relative importance of root and shoot traits on runoff P loss has not been determined. Four genotypes of common bean (Phaseolus vulgaris L.) and two genotypes of soybean (Glycine max) selected for contrasting root architecture were grown in a low P soil (Aquic Fragiudult, <20 mg kg^?1 Mehlich-3 P, 3% slope) and subjected to rainfall-runoff experiments with and without shoot removal. Plots with intact shoots had significantly lower runoff volumes (1.3–7.6 mm) and total P loads in runoff (0.005–0.32 kg ha^?1) than plots with shoots removed (7.0–16.8 mm; 0.025–1.95 kg ha^?1). Dissolved reactive P leached from plant material did not contribute significantly to P loss in runoff. Total root length acquired from soil cores differed significantly among genotypes. Root length densities in the upper 15 cm of soil mid-way between rows were less than 4.0 cm cm^?3 and variation in root length density was not correlated with runoff or P loss. Root length density also did not affect rainfall infiltration or surface runoff volume. We conclude that for annual dicotyledonous crops such as bean and soybean with relatively low root length densities, root traits have little direct effect on soil erosion.     

Reaction of selected soybean cultivars to Rhizoctonia root rot and other damping-off disease agents

Eight soybean cultivars; Giza 21. Giza 22, Giza 35, Giza 82, Giza 83, Crawford, Holladay and Toamo were evaluated to Rhizoctonia root rot using agar plate and potted plant techniques. Data cleared that, in agar plate assay all soybean cultivars were moderately susceptible (MS), although the differences between them were significant (P=0.05). Generally, in potted assay, the reactions were resistant (R) or moderately resistant (MR) to root rots. Also, the differences between cultivars were significant (P=0.05). These cultivars were inoculated under greenhouse conditions with Fusarium solani, Macrophomina phaseolina, Rhizoctonia solani and Sclerotium rolfsii Generally, G21 had the least pre-emergence damping-off followed by Giza 35, Crawford and Giza 83 with averages of 19.0, 20.0, 20.5 and 21.5%, respectively. In case of post-emergence, Giza 35 had the least values, followed by Giza 21, Crawford and Giza 82 with averages 3.95, 4.10, 4.10 and 4.25%, respectively. Under naturally infested soil in the field conditions the reactions of the same cultivars to damping-off were evaluated in two successive seasons. In 2002 season, G35 had the least pre-emergence damping-off % followed by Giza 21 and Giza 22 with averages of 22.61, 24.33 and 29.33%, respectively. Also, G35 had the least post-emergence damping-off % followed by Toamo and Giza 21 with averages of 9.40, 10.33 and 10.41%, respectively. In 2003 season, the same trend was appeared with light grade where Giza 35 had the least pre-emergence damping of % followed by Giza 22 and Giza 21 with averages of 30.67, 31.00 and 36.67%, respectively and Giza 35 was the most resistant cultivar against post-emergence damping-off, followed by Giza 21 and Giza 22 with averages of 10.91, 11.32 and 11.80%, respectively. Generally, Giza 21 significantly surpassed the other cultivars in plant height, number of pods per plant and 100-seed weight. Moreover, also it had second grade with the other traits.     

Pre-incubation of Bradyrhizobium japonicum with jasmonates accelerates nodulation and nitrogen fixation in soybean (Glycine max) at optimal and suboptimal root zone temperatures

Jasmonic acid (JA) and methyl jasmonate, collectively known as jasmonates, are naturally occurring in plants; they are important signal molecules involved in induced disease resistance and mediate many physiological activities in plants. We studied the effect of JA and its methyl ester, methyl jasmonate (MeJA), on the induction of nod genes in Bradyrhizobium japonicum GG4 (USDA3) carrying a plasmid with a translational fusion between B. japonicum nodY and lacZ of Escherichia coli, and the expression activity was measured by β-galactosidase activity. Both JA and MeJA strongly induced the expression of nod genes. They have little or no deleterious effects on the growth of B. japonicum cells, while genistein (Gen) showed inhibitory effects. We further studied the effect of JA- and MeJA-induced B. japonicum on soybean nodulation and nitrogen fixation under optimal (25°C) and suboptimal (17°C) root zone temperature (RZT) conditions. B. japonicum cells were grown in liquid yeast extract mannitol media and induced with a range of Gen, JA, and MeJA concentrations, including a treatment control with no inducer added. Soybean seedlings were grown at 25 or 17°C RZT with a constant air temperature (25°C) and inoculated, at the vegetative cotyledonary stage, with various B. japonicum induction treatments. Addition of Gen or jasmonates to B. japonicum, prior to inoculation, enhanced nodulation, nitrogen fixation, and plant growth at suboptimal RZT conditions. A higher concentration of Gen was inhibitory at 25°C, while this same concentration was stimulatory at 17°C. Interestingly, pre-incubation of B. japonicum with JA and MeJA enhanced soybean nodulation and nitrogen fixation under both optimal and suboptimal RZTs. We show that jasmonates are thus a new class of signaling molecules in the B. japonicum-soybean symbiosis and that pre-induction of B. japonicum with jasmonates can be used to enhance soybean nodulation, nitrogen fixation, and early plant growth.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.     

Vegetative micro-cloning to sustain biodiversity of threatened Moringa species

Efficient vegetative cloning in vitro requires definition of plant growth regulator regimes for each genotype, and therefore formulation of a uniform culture protocol for a genetically heterogeneous wild or uncultivated plant population is often impossible. The likelihood of cloning a wide array of plant genotypes by avoiding the use of plant growth regulator(s) was explored with Moringa oleifera Lamk., Moringa stenopetala (Baker f.) Cufod, and Moringa peregrina Forssk. ex Fiori tree seedlings. Propagation was achieved by multiple shoot regeneration from the cotyledonary node of decapitated seedlings, followed by axillary shoot growth from single node shoot segments and rooting of excised shoots. All steps were accomplished on basal Murashige and Skoog medium without plant growth regulator supplements. The results revealed competence for generation of multiple shoots from cotyledonary node tissue, stimulated by repeated shoot harvest, in seedlings of all three tree species. Tens of plants per seedling were regenerated in about 4 mo from culture initiation. In a given species clone size was seedling-dependent, which presumably stems from genotypic variability among seedlings in regeneration ability in vitro. By this means the laborious search for a plant growth regulator regime suitable for organogenesis induction and adapted per genotype became redundant, and biodiversity of the seed germplasm could be maintained. The approach ideally suits establishment of clones of wild plants of endangered species, like those of the Moringaceae, species with high ability for producing supplementary shoots, and without the need to add plant growth regulators, including the rooting stage.     

Root-shoot signalling in sunflower plants with confined root systems

Sunflower plants were grown hydroponically under controlled conditions with the root systems confined in small containers. Root confinement inhibited the growth of sunflower plants as indicated by reduction in both leaf and cotyledon area and root and shoot fresh weight. This effect was more pronounced in shoots. Root confinement favored the accumulation of potassium in the roots and shoots, and the exudation of potassium and water in excised roots. Xylem sap from root confined plants inhibited cotyledon expansion as revealed by bioassay with decapited sunflower seedlings. In addition decapited control plants incubated in ABA solution also showed cotyledon growth reduction. Xylem sap ABA analysis indicated a 7-times higher concentration in root confined than control plants. Our results suggest the synthesis of a chemical signal in the roots of plants subjected to mechanical stress which can be responsible for the inhibition of plant growth.     

Expression of Flowering Repressor Gene CsSVP,Carbohydrates, and Antioxidants Affected by Plant Growth Regulators in Saffron

Improving flower yield through lengthening flowering duration is a primary breeding objective in saffron (Crocus sativus L.). Asexual reproduction in saffron limits biodiversity and conventional breeding. Hence, eliciting flowering-related gene expression by plant growth regulators is one way to achieve this aim. The phytohormones methyl jasmonate (MeJA) and 6-benzyl amino purine (BAP) signals are received by the MADs-box gene family. In this study, to elucidate the role of phytohormones on flower development, plant were treated with BAP (0 and 5 mg L^?1), and methyl jasmonate (MeJA) (0, 20, and 100 mM) at three developmental stages of the saffron life cycle. Then, the expression of the SHORT VEGETATIVE PHASE (CsSVP) gene as a MADS-box gene family was assessed in the saffron corm. The activities of antioxidant enzymes, soluble sugar, starch content, and soluble protein content were also measured in corm, leaf, and root tissues. The application of MeJA and BAP treatments resulted in down-regulation of CsSVP expression in the corm during dormancy. At the dormancy stage, catalase, peroxidase activity decreased, and ascorbate peroxidase activity increased following MeJA treatment. In contrast, an increment in catalase and peroxidase activity and reduction of ascorbate peroxidase activity were observed after treatment with MeJA during the flowering stage. This change in enzyme activity is most likely due to flowering, which demands the re-allocation of resources. As flowering is a process heavily influenced by the environment, plants treated with MeJA, which may mimic environmental stress, showed changes in antioxidant enzyme activity. Overall, these results suggested that MeJA and BAP treatments play a significant role in the vegetative-to-reproductive phase change in saffron.

     

An efficient in vitro regeneration system of fieldpea (Pisum sativum L.) via. shoot organogenesis

In-vitro regeneration in fieldpea was achieved from immature embryonic axes and cotyledonary node explants of six genotypes on modified MS media supplemented with different concentration of plant growth regulators, 6-Benzylamino purine (BAP) and Naphthalene acetic acid (NAA). The best regeneration response, leading to multiple shoot formation efficiency (22.34 shoots/explant) was observed in the medium supplemented with 1.0 mg/L BAP and 0.2 mg/L NAA and best frequency (67.55?±?4.74) was achieved on medium containing 2.0 mg/L BAP and 0.4 mg/L NAA. The shoots were subcultured on a medium supplemented with a combination of 1.0 mg/L GA₃, 2.0 mg/L BAP and 0.4 mg/L NAA, which resulted in elongation of 85 % of shoots. Rooting attempted from the elongated shoots, on half strength MS medium and supplemented with three different auxins IBA, IAA and NAA separately, exhibited similar results. Alternatively, micro-grafting of in vitro regenerated shoots onto pre-germinated root stocks raised in green house facility was attempted with high success rate (75 %). The grafted plants could be successfully hardened, fertigated with Hoagland solution and distilled water in a ratio of (1:10) for acclimatization and further development. All the genotypes tested, produced multiple shoots that could be established to mature fertile plant, hence, the medium combinations used were found to be genotype neutral.     

Multiannual effects of induced plant defenses: Are defended plants good or bad neighbors?

Defenses induced by herbivore feeding or phytohormones such as methyl jasmonate (MeJA) can affect growth, reproduction, and herbivory, not only on the affected individual but also in its neighboring plants. Here, we report multiannual defense, growth, and reproductive responses of MeJA‐treated bilberry (Vaccinium myrtillus) and neighboring ramets. In a boreal forest in western Norway, we treated bilberry ramets with MeJA and water (control) and measured responses over three consecutive years. We observed the treatment effects on variables associated with herbivory, growth, and reproduction in the MeJA‐treated and untreated ramet and neighboring ramets distanced from 10 to 500 cm. MeJA‐treated ramets had fewer grazed leaves and browsed shoots compared to control, with higher effects in 2014 and 2015, respectively. In 2013, growth of control ramets was greater than MeJA‐treated ramets. However, MeJA‐treated ramets had more flowers and berries than control ramets 2 years after the treatment. The level of insect and mammalian herbivory was also lower in untreated neighboring ramets distanced 10–150 cm and, consistent with responses of MeJA‐treated ramets, the stronger effect was also one and 2 years delayed, respectively. The same neighboring ramets had fewer flowers and berries than untreated ramets, indicating a trade‐off between defense and reproduction. Although plant–plant effects were observed across all years, the strength varied by the distance between the MeJA‐treated ramets and its untreated neighbors. We document that induced defense in bilberry reduces both insect and mammalian herbivory, as well as growth, over multiple seasons. The defense responses occurred in a delayed manner with strongest effects one and 2 years after the induction. Additionally, our results indicate defense signaling between MeJA‐treated ramets and untreated neighbors. In summary, this study shows that induced defenses are important ecological strategies not only for the induced individual plant but also for neighboring plants across multiple years in boreal forests.     

Effects of varied water regimes on root length,dry matter partitioning and endogenous plant growth regulators in sunflower (Helianthus annuus L.)

Abstract

A field experiment was conducted to quantify the effect of varied water regimes on root length, partitioning of dry matter and plant growth regulators by using sunflower genotypes differing in maturity and drought tolerance. Significant depressing effect of drought stress was evident on traits (i.e., reproductive dry matter, leaf area index and cytokinin concentrations in leaves). However, root/shoot, reproductive/vegetative ratios and Abscisic acid (ABA) concentration were found to increase under drought stress. Drought stress also changed the dry matter accumulation pattern of genotypes. In most cases it reduced the days to reach the maximum peak showing early senescence.

ABA was identified as a multi-functional plant growth regulator under drought stress, causing early senescence of plants and translocation of assimilates to the roots and reproductive part while root growth under drought stress was explained by the indole-acetic acid (IAA) concentrations. Maintaining higher cytokinin contents were involved in accumulation of higher reproductive dry matter under drought stress. Although ABA and IAA were both involved in the development of defense responses during the adaptation and survival to drought stress but higher productivity under drought stress was only realized through maintaining higher cytokinin contents.