Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non-canonical natures. The presence of non-canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.     

蒺藜苜蓿糖基转移酶基因SMALL AND EMERALD1的克隆和功能研究

类黄酮代谢对于植物生长发育和植物-环境互作至关重要,其中糖基转移酶介导的糖基化修饰在类黄酮代谢中发挥着重要作用。为了研究蒺藜苜蓿中糖基转移酶的生物学功能,通过定向筛选蒺藜苜蓿Tnt1逆转座子插入突变体库,获得了一类植株矮小、叶片深绿的突变体small and emerald1 (se1)。通过基因表型连锁性分析成功克隆了SE1基因,该基因编码1个糖基转移酶,与拟南芥中调控类黄酮生物合成的AtUGT84A1氨基酸同源性为52.8%。对野生型和se1突变体叶片的类黄酮含量进行测定发现类黄酮总量在se1突变体中显著降低(P<0.01)。进一步研究发现在se1突变体中类黄酮合成途径关键基因CHS、F3H和F3’H表达水平下降。亚细胞定位显示SE1可能在细胞质和细胞核中发挥生物学功能。研究表明糖基转移酶基因SE1可能参与蒺藜苜蓿类黄酮合成代谢调控,进而影响其生长发育。此外,研究还发现SE1基因对于叶绿素合成可能具有负向调控作用。     

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...     

Transformation of floral organs with GFP in Medicago truncatula

 A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108–1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants. Received: 25 May 1999 / Revision received: 2 August 1999 / Accepted: 2 August 1999     

蒺藜苜蓿花器官特异基因的表达分析

蒺藜苜蓿(Medicago truncatula G)花器官特异表达基因是参与其花器官形成与发育的重要基因。筛选蒺藜苜蓿的花器官特异表达基因,寻找这类基因在其他模式植物中的直系同源基因,并将其表达模式在不同植物间进行比较,有利于深入的理解这类基因在蒺藜苜蓿花器官发育中的功能。根据蒺藜苜蓿表达谱,并以其PISTILLAZA(PI)基因为模板,文章筛选了97个蒺藜苜蓿花器官特异表达基因(Ratio≥10,且Z≥7.9).通过同源比对,确定了这类基因在拟南芥(Arabidopsis thaliana L.)、大豆(Glycinemax L.)、百脉根(Lotusjaponicus L.)和水稻(Oryzasativa L.)中的直系同源基因。对这类基因在5种植物中的表达量、表达部位和功能进行比较,发现进化关系较近的植物,直系同源基因的表达变异较小,而进化关系较远的植物,直系同源基因的表达变异较大。进一步对表达分化较大的直系同源基因进行启动子分析,发现不同植物中直系同源基因表达模式的变化与启动子中调控元件的特性有关。     

蒺藜苜蓿花器官特异基因的表达分析

蒺藜苜蓿(Medicago truncatula G.)花器官特异表达基因是参与其花器官形成与发育的重要基因。筛选蒺藜苜蓿的花器官特异表达基因, 寻找这类基因在其他模式植物中的直系同源基因, 并将其表达模式在不同植物间进行比较, 有利于深入的理解这类基因在蒺藜苜蓿花器官发育中的功能。根据蒺藜苜蓿表达谱, 并以其PISTILLATA(PI)基因为模板, 文章筛选了97个蒺藜苜蓿花器官特异表达基因(Ratio≥10, 且Z≥7.9)。通过同源比对, 确定了这类基因在拟南芥(Arabidopsis thaliana L.)、大豆(Glycine max L.)、百脉根(Lotus japonicus L.)和水稻(Oryza sativa L.)中的直系同源基因。对这类基因在5种植物中的表达量、表达部位和功能进行比较, 发现进化关系较近的植物, 直系同源基因的表达变异较小, 而进化关系较远的植物, 直系同源基因的表达变异较大。进一步对表达分化较大的直系同源基因进行启动子分析, 发现不同植物中直系同源基因表达模式的变化与启动子中调控元件的特性有关。     

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.     

Nucleocytoplasmic-localized acyltransferases catalyze the malonylation of 7-O-glycosidic (iso)flavones in Medicago truncatula

(Iso)flavonoids are commonly accumulated as malonylated or acetylated glycoconjugates in legumes. Sequence analysis on EST database of the model legume Medicago truncatula enabled us to identify nine cDNA sequences encoding BAHD super-family enzymes that are distinct from the most of the characterized anthocyanin/flavonol acyltransferase genes in other species. Functional characterization revealed that three of these corresponding enzymes, MtMaT1, 2 and 3, specifically recognize malonyl CoA as an acyl donor and catalyze the malonylation of a range of isoflavone 7- O- glucosides in vitro . These malonyltransferase genes displayed distinct tissue-specific expression patterns and responded differentially to biotic and abiotic stresses. Consistent with gene expression, the level of the accumulated malonyl isoflavone glucoside was altered in the roots of M. truncatula grown under normal and drought-stressed conditions. Overexpression of the MtMaT1 gene in a previously engineered Arabidopsis line that accumulates genistein glycosides ( Proc. Natl Acad. Sci. USA , 99 , 2002:14578) led to a malonylated product. Confocal microscopy of the transiently expressed MtMaT1–GFP fusion revealed strong fluorescence in both the cytoplasm and nucleus of M. truncatula and tobacco leaf cells. A truncated MtMaT1 lacking the C-terminal polypeptide of 110 amino acid residues that include the DFGWG motif, the single conserved sequence signature of BAHD super-family members, retained considerable catalytic efficiency, but showed an altered optimum pH preference for maximum activity. Such C-terminal polypeptide deletion or deletion of the DFGWG motif alone led to improper folding of the transiently expressed GFP fusion protein in living cells, and impaired nuclear localization of the enzyme.     

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。     

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.     

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。     

High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures

The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.     

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.     

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis , with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.