Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds

Desiccation tolerance (DT) in orthodox seeds is acquired during seed development and lost upon imbibition/germination, purportedly upon the resumption of DNA synthesis in the radicle cells. In the present study, flow cytometric analyses and visualization of microtubules (MTs) in radicle cells of seedlings of Medicago truncatula showed that up to a radicle length of 2 mm, there is neither DNA synthesis nor cell division, which were first detected in radicles with a length of 3 mm. However, DT started to be lost well before the resumption of DNA synthesis, when germinating seeds were dried back. By applying an osmotic treatment with polyethylene glycol (PEG) before dehydration, it was possible to re-establish DT in seedlings with a radicle up to 2 mm long. Dehydration of seedlings with a 2 mm radicle, with or without PEG treatment, caused disassembly of MTs and appearance of tubulin granules. Subsequent pre-humidification led to an almost complete disappearance of both MTs and tubulin granules. Upon rehydration, neither MTs nor tubulin granules were detected in radicle cells of untreated seedlings, while PEG-treated seedlings were able to reconstitute the microtubular cytoskeleton and continue their normal development. Dehydration of untreated seedlings also led to an apoptotic-like DNA fragmentation in radicle cells, while in PEG-treated seedlings DNA integrity was maintained. The results showed that for different cellular components, desiccation-tolerant seedlings may apply distinct strategies to survive dehydration, either by avoidance or further repair of the damages.     

NMR metabolite profiling analysis reveals changes in phospholipid metabolism associated with the re-establishment of desiccation tolerance upon osmotic stress in germinated radicles of cucumber

The adaptation of metabolism is thought to play a role in the acquisition of desiccation tolerance (DT). However, the importance of such a role and whether specific regulatory pathways exist remain to be assessed. Using in vitro 31P and 13C nuclear magnetic resonance (NMR) spectroscopy and biochemical assays, we analysed metabolite profiles of perchloric extracts from germinating radicles of cucumber to identify changes in carbon and phosphate metabolism associated with DT. Emerged radicles measuring 2 mm long can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution with a water potential of 1.5 MPa. However, in 4-mm-long emerged radicles, this treatment was ineffective. This manipulable system enabled the discrimination of changes in metabolites associated with DT from those associated with the response to osmotic stress. Independent of radicle length, the PEG treatment resulted in an increase in sucrose (Suc) content, whereas glucose (Glc), fructose (Fru) and the hexose phosphate pool, as well as phosphoenolpyruvate decreased three- to fourfold. In addition, three derivatives arising early during phospholipid catabolism (glycerylphosphorylcholine, glycerylphosphorylethanolamine and glycerylphosphorylinositol) appeared in the PEG-treated radicles. Interestingly, phospholipid degradation was much more pronounced in osmotically challenged radicles that remain sensitive to drying. This was proved by the appearance of catabolites, such as phosphocholine and phosphoethanolamine, solely in 4 mm PEG-treated radicles. Furthermore, glycerol-3-phosphate and its derivative 3-phosphoglycerate increased significantly. Our data suggest that the metabolic response leading to the re-establishment of DT is not entirely identical to that of an osmotic response. It is inferred that membrane remodelling and/or increased phospholipid catabolism is an adaptive response common to osmotic adjustment and DT but is controlled differently in tolerant and sensitive radicles.     

Respiratory pathways in germinating maize radicles correlated with desiccation tolerance and soluble sugars

We have assessed the activities of the cytochrome and alternative pathways in total respiration and their role in each stage of germination of Zea mays L. radicles. Throughout imbibition, the salicylhydroxamic acid (SHAM) concentration needed to inhibit the cyanide-resistant pathway, without any side effects, decreased from 15 m M in quiescent embryos to 5 m M at 72 h after imbibition. Electrons predominantly flowed through the cytochrome pathway although the alternative pathway was already present at early imbibition. The capacity of the alternative path was about 70% of the control rate of respiration. Its engagement progressively increased from 18% after 10 min of imbibition to 70% at the radicle emergence and then decreased to 50% at 96 h after imbibition, concomitant with the onset of radicle growth. The alternative pathway was, however, not essential for germination. The observed activity of the alternative path correlated with the monosaccharide (glucose + fructose) content, suggesting that the alternative pathway could be acting according to the 'energy overflow model'. On the other hand, up to 24 h after imbibition at 16°C, maize radicles tolerate a severe desiccation, becoming intolerant at 72 h. On reimbibition of tolerant radicles, respiration increased immediately and the alternative pathway was rapidly engaged. At 72 h, no respiration was measured, indicating a total loss of the respiratory systems. The possible correlation between carbohydrate content, loss of desiccation tolerance and activity of the two respiratory pathways is discussed.     

Comparative analysis of the heat stable proteome of radicles of Medicago truncatula seeds during germination identifies late embryogenesis abundant proteins associated with desiccation tolerance

A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting alpha helices and beta sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.     

Artificial seeds of alfalfa (Medicago sativa L.). Induction of desiccation tolerance in somatic embryos

Summary The use of somatic embryos from cell culture systems in the clonal propagation of plants would be greatly facilitated if the somatic embryos could be dried and stored in a dormant state similar to true seeds. A cell culture system was developed for alfalfa (Medicago sativa L.) line RL34 which gave high yields of somatic embryos in an approximately synchronized pattern. These somatic embryos were treated with abscisic acid (ABA) at the cotyledonary stage of development to induce desiccation tolerance. With no visual preselection, approximately 60% of the dried embryos converted into plants upon reimbibition. When high quality embryos were selected prior to drying, 90 to 100% conversion rates were observed. The timing of the application of ABA in terms of embryo development was critical with an optimum being at cotyledonary stage spanning approximately 4 days; thus, synchronized embryo development is required for optimal expression in bulk samples. The vigor of the seedlings from dried somatic embryos was greater than those from embryos which had not been dried, but remained substantially lower than those from true seeds.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Root system of Medicago sativa and Medicago truncatula: drought effects on carbon metabolism

Plant and Soil - Changes in snow cover can influence root growth and distribution of herbaceous species in water limiting desert ecosystems. However, how the growth of root systems of herbaceous...     

Maturation proteins and sugars in desiccation tolerance of developing soybean seeds

The desiccation-tolerant state in seeds is associated with high levels of certain sugars and maturation proteins. The aim of this work was to evaluate the contributions of these components to desiccation tolerance in soybean (Glycine max [L.] Merrill cv Chippewa 64). When axes of immature seeds (34 d after flowering) were excised and gradually dried (6 d), desiccation tolerance was induced. By contrast, seeds held at high relative humidity for the same period were destroyed by desiccation. Maturation proteins rapidly accumulated in the axes whether the seeds were slowly dried or maintained at high relative humidity. During slow drying, sucrose content increased to five times the level present in the axes of seeds held at high relative humidity (128 versus 25 μg/axis, respectively). Stachyose content increased dramatically from barely detectable levels upon excision to 483 μg/axis during slow drying but did not increase significantly when seeds were incubated at high relative humidity. Galactinol was the only saccharide that accumulated to higher levels in axes from seeds incubated at high relative humidity relative to axes from seeds that were slowly dried. This suggests that slow drying serves to induce the accumulation of the raffinose series sugars at a point after galactinol biosynthesis. We conclude that stachyose plays an important role in conferring desiccation tolerance.     

Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3–4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants. Received: 18 March 1997 / Revision received: 25 June 1997 / Accepted: 15 July 1997     

Purification and properties of alpha-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa).

alpha-Mannosidase of Medicago sativa (alfalfa) was purified 1340-fold. The purification method included dialysis of the crude extract against a citrate/phosphate buffer, pH 3.9, (NH4)SO4 precipitation, hydroxyapatite chromatography, chromatography on Sephadex G-200 and finally a preparatory electrophoresis on polyacrylamide-gel gradient by Doly & Petek's [(1977) J. Chromatogr. 137. 69--81] method. Each step of purification was checked by polyacrylamide-gel disc electrophoresis. The purified enzyme showed a single band, corresponding to alpha-mannosidase activity. alpha-Mannosidase has a mol.wt. 230 000 as estimated by Hedrick & Smith's [(1968) Arch. Biochem. Biophys. 126, 155--164] method and also by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by Weber & Osborn [(1969) J. Biol. Chem. 244, 4406--4412]. The enzyme comprises four subunits of different molecular weight. Optimum pH and Km values were determined with p-nitrophenyl alpha-D-mannoside as substrate. When incubated at a temperature between 20 and 62 degrees C before assay, alpha-mannosidase initially shows an increase in activity. alpha-Mannosidase is stable when the pH is about neutrality. It can be inactivated by several metal ions, including Zn2+. At a pH below 5 the enzyme undergoes irreversible inactivation. The presence of EDTA at acid pH considerably enhances the inactivation of the enzyme. This inactivation due to EDTA can be specifically reversed by incubation with Zn2+.     

Genome size and base composition in Medicago sativa and M. truncatula species.

The genome size (1C value) and base composition of 14 ecotypes of two species of tetraploid and diploid Medicago have been assessed by flow cytometry. These parameters vary both between and within species. The diploid annual Medicago truncatula Gaertn. had the smallest genome of the group studied (which also covered M. sativa L. subsp. sativa, M. sativa L. subsp. caerulea (Less. ex Ledeb.) Schmalh., M. sativa L. subsp. quasifalcata Sinsk., M. sativa L. subsp. x varia (Martyn) Arcangeli; however, its ecotypes revealed substantial intraspecific variation. The smallest M. truncatula genome observed was ecotype 108-1 with 1C = 0.49 pg and 38.1% GC and the largest was Jemalong with 1C = 0.57 pg and 38.6% GC. The degree of polysomaty in these Medicago was low, although in some tissues the frequency of cells with 4C nuclei reached 50%.     

Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer

Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.     

Manipulating the desiccation tolerance and vigor of dry somatic embryos of Medicago sativa L. with sucrose,heat shock and abscisic acid

Summary The effects of sucrose concentration in the maturation medium in combination with a heat shock treatment at 36°C were investigated in an attempt to improve the vigor of seedlings grown from dry somatic embryos of alfalfa (Medicago sativa L.). Callus was formed from petiole expiants and dispersed in liquid suspension medium in the presence of 5 M 2,4-D. The cell suspension was sieved to synchronize embryo development. The 200 – 500 m fraction was plated on embryo development medium without 2,4-D, grown for 14 days, and transferred to maturation medium. With 3% sucrose in the maturation medium, the somatic embryos germinated precociously and were unable to survive desiccation. At higher sucrose concentrations, germination was delayed and the embryos continued to accumulate dry matter. After 13 days on 6% sucrose medium (27 days after sieving), the somatic embryos were tolerant of drying to 12% moisture without exposure to exogenous ABA. Heat shock, which presumably stimulates endogenous ABA synthesis, improved the desiccation tolerance of somatic embryos if applied prior to day 27 after sieving, but its effects were minimal after day 27. High sucrose concentrations up to 9% in the maturation medium were optimal during the first 8 days on maturation medium (days 14 to 22 after sieving), but a lower concentration (6%) was optimal during the later stages of embryo maturation (days 22 to 30 after sieving). The inclusion of 10^–5 M ABA in the maturation medium with 6% sucrose further improved embryo quality if applied approximately 20 days after sieving.     

印度梨形孢促进蒺藜苜蓿生长及其提高耐盐性研究

【目的】研究盐胁迫下印度梨形孢定殖对豆科模式植物蒺藜苜蓿生长发育的影响。【方法】通过分析不同生境下植物的根长、根鲜重和茎鲜重,以及体内抗氧化物酶活性、脯氨酸含量、甜菜碱醛脱氢酶基因(BADH)的表达,确定印度梨形孢对蒺藜苜蓿生长的促进作用,并初步阐释印度梨形孢诱导植物耐盐性的机制。【结果】印度梨形孢能在蒺藜苜蓿根部定殖并能促进植物的生长发育,有效缓解盐胁迫造成的生长抑制。印度梨形孢能提高植物体内抗氧化物酶活性,增加游离脯氨酸含量并诱导BADH基因的表达。【结论】印度梨形孢作为植物生长促进因子可以用来提高植物耐盐性,实现盐碱土壤的间接改良。     

Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10(2) CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.     

Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10² CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.