A CDPK isoform participates in the regulation of nodule number in Medicago truncatula

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.     

Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti

Flavonoids play critical roles in legume–rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti , flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non- nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula .     

Suppression of NB-LRR genes by miRNAs promotes nitrogen-fixing nodule development in Medicago truncatula

Plant genomes contain two major classes of innate immune receptors to recognize different pathogens. The pattern recognition receptors perceive conserved pathogen-associated molecular patterns and the resistance genes with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains recognize specific pathogen effectors. The precise regulation of resistance genes is important since the unregulated expression of NB-LRR genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection. It was shown that a subset of miRNAs could target NB-LRR genes and act as an important regulator of plant immunity in the absence of pathogens. Plants not only interact with pathogens, but they can also establish symbiotic interactions with microbes. Nitrogen-fixing symbiotic interaction and nodule formation of legumes may also require the suppression of host defence to prevent immune responses. We found that upon symbiotic interactions, miRNAs repressing NB-LRR expression are upregulated in the developing nodules of Medicago truncatula. Furthermore, we show that the suppression of the activity of the NB-LRR genes targeted by these miRNAs is important during nodule development. Our results suggest that the downregulation of NB-LRR resistance genes in the developing nodule produces a suitable niche that facilitates bacterial colonization and the development of an N-fixing nodule.     

蒺藜苜蓿MtbHLH25基因克隆、亚细胞定位及表达分析

【目的】bHLH转录因子数量众多,能够广泛参与植物的生长发育和逆境胁迫等过程。本试验以蒺藜苜蓿R108为材料,初步探讨MtbHLH25基因的功能。【方法】通过PCR扩增技术从蒺藜苜蓿中克隆MtbHLH25基因和启动子,构建酵母表达载体并用LiAc转化法转移到Y2H Gold酵母菌株中进行酵母自激活检测,构建亚细胞定位载体并通过冻融法转入农杆菌EHA105,菌液注射到烟草下表皮细胞后利用SP8激光共聚焦显微镜观察,通过实时荧光定量PCR技术研究MtbHLH25基因的时空表达水平。【结果】（1）从蒺藜苜蓿中成功克隆出MtbHLH25基因和启动子,该基因总长882 bp,共编码293个氨基酸。启动子序列分析发现其包含了ABA、MeJA、GA和SA等响应元件。（2）进化树结果表明MtbHLH25蛋白与蚕豆和长柔毛野豌豆中bHLH蛋白高度同源。（3）亚细胞定位结果显示MtbHLH25蛋白定位于细胞核。（4）酵母自激活检测结果显示MtbHLH25蛋白具有自激活活性。（5）表达分析结果显示,MtbHLH25在蒺藜苜蓿根、茎、叶、花和果实中均有表达,其中在根中表达水平最高;外源SA、MeJA、ABA、GA以及盐胁迫使MtbHLH25基因表达量都呈下降趋势,推测SA、MeJA、ABA、GA以及盐胁迫对MtbHLH25基因的表达起到负调控作用。干旱胁迫能够显著诱导MtbHLH25基因表达量的上升,说明该转录因子可能在干旱胁迫中起到正调控作用。【结论】MtbHLH25基因可能对盐胁迫敏感,在干旱胁迫中可能发挥正调控作用。此外,MtbHLH25蛋白具有自激活活性,对下游启动子调控的报告基因可能具有激活作用。     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

蒺藜苜蓿MtSAG113基因的转化及表达特征分析

Senescence Associated Gene 113(SAG113)基因属于PP2Cc超家族,该基因的研究主要集中在植物衰老领域.为分析蒺藜苜蓿(Medicago truncatula)MtSAG113基因的表达特征,探究MtSAG113基因的功能.该基因从蒺藜苜蓿中克隆得到,以烟草(Nicotiana tab...     

Repeated leaf wounding alters the colonization of Medicago truncatula roots by beneficial and pathogenic microorganisms

In nature, plants are subject to various stresses that are often accompanied by wounding of the aboveground tissues. As wounding affects plants locally and systemically, we investigated the impact of leaf wounding on interactions of Medicago truncatula with root-colonizing microorganisms, such as the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the pathogenic oomycete Aphanomyces euteiches and the nitrogen-fixing bacterium Sinorhizobium meliloti. To obtain a long-lasting wound response, repeated wounding was performed and resulted in locally and systemically increased jasmonic acid (JA) levels accompanied by the expression of jasmonate-induced genes, among them the genes encoding allene oxide cyclase 1 (MtAOC1) and a putative cell wall-bound invertase (cwINV). After repeated wounding, colonization with the AM fungus was increased, suggesting a role of jasmonates as positive regulators of mycorrhization, whereas the interaction with the rhizobacterium was not affected. In contrast, wounded plants appeared to be less susceptible to pathogens which might be caused by JA-induced defence mechanisms. The effects of wounding on mycorrhization and pathogen infection could be partially mimicked by foliar application of JA. In addition to JA itself, the positive effect on mycorrhization might be mediated by systemically induced cwINV, which was previously shown to exhibit a regulatory function on interaction with AM fungi.     

黄花苜蓿与蒺藜苜蓿对土壤低磷胁迫适应策略的比较研究

土壤有效磷(P)含量低是限制植物生长的主要因素之一。根形态变化和根系大量分泌以柠檬酸为主的有机酸是植物适应土壤P素缺乏的重要机制。以广泛分布于我国北方的重要豆科牧草黄花苜蓿(Medicago falcata)和豆科模式植物蒺藜苜蓿(M. truncatula)为材料, 采用砂培方法, 研究了低P胁迫对其植株生长、根系形态和柠檬酸分泌的影响, 对比了两种苜蓿适应低P胁迫的不同策略。结果表明: 1)低P处理显著抑制了蒺藜苜蓿与黄花苜蓿的地上部生长, 而对地下部生长影响较小, 从而导致根冠比增加。2)低P胁迫显著降低黄花苜蓿的总根长和侧根长, 而对蒺藜苜蓿的上述根系形态指标没有显著影响。3)低P胁迫促进两种苜蓿根系的柠檬酸分泌, 无论是在正常供P还是低P胁迫条件下, 黄花苜蓿根系分泌柠檬酸量显著高于蒺藜苜蓿根系。上述结果表明, 黄花苜蓿和蒺藜苜蓿对低P胁迫的适应策略不同, 低P胁迫下, 黄花苜蓿主要通过根系大量分泌柠檬酸, 活化根际难溶态P来提高对P的吸收, 而蒺藜苜蓿维持较大的根系是其适应低P胁迫的主要策略。     

The Arabidopsis EIN2 restricts organ growth by retarding cell expansion

The growth of plant organ to its characteristic size is a fundamental developmental process, but the mechanism is still poorly understood. Plant hormones play a great role in organ size control by modulating cell division and/or cell expansion. ETHYLENE INSENSITVE 2 (EIN2) was first identified by a genetic screen for ethylene insensitivity and is regarded as a central component of ethylene signaling, but its role in cell growth has not been reported. Here we demonstrate that changed expression of EIN2 led to abnormity of cell expansion by morphological and cytological analyses of EIN2 loss-of-function mutants and the overexpressing transgenic plant. Our findings suggest that EIN2 controls final organ size by restricting cell expansion.     

Cytochemistry of proteolytic activity and pH status of vacuoles in Medicago truncatula root nodules

The vacuole development in root nodules of Medicago truncatula was analyzed by light and electron microscopy. Histochemistry of protease activity in root nodules was studied using fluorogenic substrates for proteolytic enzymes, 7-amino-4-methylcoumarin, CBZ-L-phenylalanyl-L-arginine amide, hydrochloride (AMC), and rhodamine 110, bis-(CBZ-L-phenylalanyl-L-arginine amide) dihydrochloride (RPA). Furthermore, the topology of acidification of the central vacuoles in infected and noninfected cells in root nodules of Medicago truncatula was analyzed with the fluorescent pH-sensitive acidotropic dye Neutral Red. It was shown that vacuoles were acidic, lytic organelles in noninfected cells and young infected cells of the nodule where they displayed protease activity. Mature vacuoles of infected cells had high pH and did not show any substantial protease activity. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 31–38. The text was submitted by the authors in English.     

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.     

The model legume Medicago truncatula A17 is poorly matched for N2 fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021

Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N(2) was evaluated. N(2) fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. The Sm1021-M. truncatula symbiosis is poorly matched for N(2) fixation and the strain could possess broader N(2) fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N(2) fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.     

Growth and senescence of Medicago truncatula cultured cells are associated with characteristic mitochondrial morphology

Here mitochondrial morphology and dynamics were investigated in Medicago truncatula cell-suspension cultures during growth and senescence. Cell biology techniques were used to measure cell growth and death in culture. Mitochondrial morphology was investigated in vivo using a membrane potential sensor probe coupled with confocal microscopy. Expression of a senescence-associated gene (MtSAG) was evaluated in different cell-growth phases. Mitochondria appeared as numerous, punctuate organelles in cells at the beginning of the subculture cycle, while interconnected networks were observed in actively growing cells. In senescent cells, giant mitochondria were associated with dying cells. The release of cytochrome c from mitochondria was detected in different growth phases of cultured cells. Studies on plant cell cultures allowed us to identify physiological and molecular markers of senescence and cell death, and to associate distinct mitochondrial morphology with cells under different physiological conditions.     

Medicago truncatula ENOD40-1 and ENOD40-2 are both involved in nodule initiation and bacteroid development

The establishment of a nitrogen-fixing root nodule on legumes requires the induction of mitotic activity of cortical cells leading to the formation of the nodule primordium and the infection process by which the bacteria enter this primordium. Several genes are up-regulated during these processes, among them ENOD40. Here it is shown, by using gene-specific knock-down of the two Medicago truncatula ENOD40 genes, that both genes are involved in nodule initiation. Further, during nodule development, both genes are essential for bacteroid development.