Ultrastructural and Photosynthetic Responses of Pod Walls in Alfalfa to Drought Stress.

Increasing photosynthetic ability as a whole is essential for acquiring higher crop yields. Nonleaf green organs (NLGOs) make important contributions to photosynthate formation, especially under stress conditions. However, there is little information on the pod wall in legume forage related to seed development and yield. This experiment is designed for alfalfa (Medicago sativa) under drought stress to explore the photosynthetic responses of pod walls after 5, 10, 15, and 20 days of pollination (DAP5, DAP10, DAP15, and DAP20) based on ultrastructural, physiological and proteomic analyses. Stomata were evidently observed on the outer epidermis of the pod wall. Chloroplasts had intact structures arranged alongside the cell wall, which on DAP5 were already capable of producing photosynthate. The pod wall at the late stage (DAP20) still had photosynthetic ability under well-watered (WW) treatments, while under water-stress (WS), the structure of the chloroplast membrane was damaged and the grana lamella of thylakoids were blurry. The chlorophyll a and chlorophyll b concentrations both decreased with the development of pod walls, and drought stress impeded the synthesis of photosynthetic pigments. Although the activity of ribulose-1,5-bisphosphate carboxylase (RuBisCo) decreased in the pod wall under drought stress, the activity of phosphoenolpyruvate carboxylase (PEPC) increased higher than that of RuBisCo. The proteomic analysis showed that the absorption of light is limited due to the suppression of the synthesis of chlorophyll a/b binding proteins by drought stress. Moreover, proteins involved in photosystem I and photosystem II were downregulated under WW compared with WS. Although the expression of some proteins participating in the regeneration period of RuBisCo was suppressed in the pod wall subjected to drought stress, the synthesis of PEPC was induced. In addition, some proteins, which were involved in the reduction period of RuBisCo, carbohydrate metabolism, and energy metabolism, and related to resistance, including chitinase, heat shock protein 81-2 (Hsp81-2), and lipoxygenases (LOXs), were highly expressed for the protective response to drought stress. It could be suggested that the pod wall in alfalfa is capable of operating photosynthesis and reducing the photosynthetic loss from drought stress through the promotion of the C4 pathway, ATP synthesis, and resistance ability.

A distinct class of vesicles derived from the trans-Golgi mediates secretion of xylogalacturonan in the root border cell.

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three-dimensional structures and macromolecular compositions of these Golgi stacks, we examined high-pressure frozen/freeze-substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans-Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi-associated vesicles. Margins of trans-Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan-I (PGA/RG-I) are detected in the trans-most cisternae and TGN compartments. LVs produced from TGN compartments (TGN-LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN-LVs containing the XG and PGA/RG-I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans-Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans-cisternae accompanying polysaccharide synthesis with a mathematical model.

REORGANIZATION OF INTERPHASE MICROTUBULES IN ROOT CELLS OF MEDICAGO SATIVA L. DURING ACCLIMATION TO OSMOTIC AND SALT STRESS CONDITION.

We have investigated the organization of microtubule system in interphase cells of Medicago sativa L. roots during acclimation to salt and osmotic stress at different concentrations of NaCl, Na2SO4 and mannitol. We have identified several morphological changes in tubulin cytoskeleton that appear during the acclimation to salt and osmotic stress in the cells of different root tissues: 1) decreased density of cortical microtubule network, 2) random orientation of cortical microtubule bundles, 3) non-uniform density of the bundles, 4) thickening of the bundles, 5) fragmentation of the bundles, 6) formation of centers of converging microtubule. Reduced density of the microtubule network and thickening of the bundles was detected during osmotic and salt stress, yet random orientation of cortical microtubules was observed under osmotic stress and not found during salt stress. Fragmentation of microtubule bundles was apparent during salt stress and less evident at high concentration of mannitol. Formation of centers of converging microtubule was common under prolonged action of sodium sulfate, less common under sodium chloride and not found after mannitol treatment. Our data show that cortical microtubules in alfalfa root cells rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered not only by sodium cations but also by sulfate and chloride anion at the concentrations that do not cause irreversible cell damage. Our study show that the osmotic and salt stress differently affect the cortical microtubules, and their reorganization in response to stress depends on the salt cations as well as anions might also show additional effect under salt stress.

Mercury localization and speciation in plants grown hydroponically or in a natural environment.

Better understanding of mercury (Hg) accumulation, distribution, and speciation in plants is required to evaluate potential risks for the environment and to optimize phytostabilization strategies for Hg-contaminated soils. The behavior of Hg in alfalfa (Medicago sativa) plants grown under controlled conditions in a hydroponic system (30 µM HgCl2) was compared with that of naturally occurring Horehound (Marrubium vulgare) plants collected from a mining soil polluted with Hg (Almadenejos, Spain) to characterize common mechanisms of tolerance. Synchrotron X-ray Fluorescence microprobe (µ-SXRF) showed that Hg accumulated at the root apex of alfalfa and was distributed through the vascular system to the leaves. Transmission electron microscopy (TEM) implied association of Hg with cell walls, accompanied by their structural changes, in alfalfa roots. Extended X-ray absorption fine structure (EXAFS) determined that Hg was principally bound to biothiols and/or proteins in M. sativa roots, stems, and leaves. However, the major fraction of Hg detected in M. vulgare plants consisted of mineral species, possibly associated with soil components. Interestingly, the fraction of Hg bound to biothiols/proteins (i.e., metabolically processed Hg) in leaves of both plants (alfalfa and M. vulgare) was similar, in spite of the big difference in Hg accumulation in roots, suggesting that some tolerance mechanisms might be shared.

[Quality standard of uygur medicine Medicago sativa seeds].

In this paper, microscopic identification method was adopted to observe the microscopic characters of ten batches of Medicago sativa seeds. And M. sativa seeds were identificated by TLC method in contrast to trigonelline and stachydrine hydrochloride. The impurities, moisture, ash, sour insoluble ash were detected based on Chinese Pharmacopoeia 2010 version (Vol I ). An HPLC method was also established for determination of trigonelline in the M. sativa seeds. The contents of impurities, moisture, ash, sour insoluble ash should not exceed 5%, 10%, 6%, and 2%, respectively. The content of trigonelline should be not less than 0.795 6 mg x g(-1). The experimental methods were accurate and reliable, and can be used as the quality control of the seeds of M. sativa.

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress.

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate-glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.

NaCl effect on the distribution of wall ingrowth polymers and arabinogalactan proteins in type A transfer cells of Medicago sativa Gabès leaves.

We studied the distribution of wall ingrowth (WI) polymers by probing thin sections of companion cells specialized as transfer cells in minor veins of Medicago sativa cv Gabès blade with affinity probes and antibodies specific to polysaccharides and glycoproteins. The wall polymers in the controls were similar in WIs and in the primary wall but differently distributed. The extent of labeling in these papillate WIs differed for JIM5 and JIM7 homogalacturonans but was in the same range for LM5 and LM6 rhamnogalacturonans and xyloglucans. These data show that WI enhancement probably requires arabinogalactan proteins (JIM8) mainly localized on the outer part of the primary wall and WIs. By comparison, NaCl-treated plants exhibited cell wall polysaccharide modifications indicating (1) an increase in unesterified homogalacturonans (JIM5), probably implicated in Na(+) binding and/or polysaccharide network interaction for limiting turgor variations in mesophyll cells; (2) enhancement of the xyloglucan network with an accumulation of fucosylated xyloglucans (CCRC-M1) known to increase the capacity of cellulose binding; and (3) specific recognition of JIM8 arabinogalactan proteins that could participate in both wall enlargement and cohesion by increasing the number of molecular interactions with the other polymers. In conclusion, the cell wall polysaccharide distribution in enlarged WIs might (1) participate in wall resistance to sequestration of Na(+), allowing a better control of hydric homeostasis in mesophyll cells to maintain metabolic activity in source leaves, and (2) maintain tolerance of M. sativa to NaCl.

A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro.

In vitro experiments were conducted to examine the characteristics and mode of action of a protease that increased the ruminal fiber digestibility of alfalfa hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY), already characterized for its main activities, was further analyzed to determine protease activity in response to pH, molecular size by SDS-PAGE, specificity to degrade model or feed substrates, response to autoclaving, and action of specific protease inhibitors in the absence or presence of ruminal fluid. In addition, batch culture in vitro incubations in buffered ruminal fluid were conducted to compare the enzyme product with purified protease sources, and dose-response studies (0 to 10 microL/g of forage DM) were carried out using alfalfa hay as a substrate. The enzyme product was shown to be an alkaline protease (optimum pH >8.5) of approximately 30 kDa. Specificity in the absence of ruminal fluid showed that the enzyme was active against gelatin and casein to the same extent, whereas it had limited (21% of the total) activity on BSA. In the presence of ruminal fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs. 68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively). Inhibitor studies in the absence of ruminal fluid indicated that the enzyme was inhibited most by a serine protease inhibitor but not by cysteine- or metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to that of the control treatment. Comparisons among different proteases revealed that only pure subtilisin achieved increases in IVDMD that were similar to those with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62). Dose-response studies using alfalfa hay as substrate showed quadratic responses in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is postulated that this enzyme acts by removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.

The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis.

Sinorhizobium meliloti is a beneficial legume symbiont, closely related to Brucella species, which are chronic mammalian pathogens. We discovered that the S. meliloti MsbA2 protein is essential to ensure the symbiotic interaction with the host plant, alfalfa. S. meliloti invades plant cells via plant-derived structures known as infection threads. However, in the absence of MsbA2, S. meliloti remains trapped within abnormally thickened infection threads and induces a heightened plant defence response, characterized by a substantial thickening of the nodule endodermis layer and the accumulation of polyphenolic compounds. The S. meliloti MsbA2 protein is homologous to the Escherichia coli lipopolysaccharide/phospholipid trafficking protein MsbA. However, MsbA2 was not essential for the membrane transport of either lipopolysaccharide or phospholipids in S. meliloti. We determined that the msbA2 gene is transcribed in free-living S. meliloti and that in the absence of MsbA2 the polysaccharide content of S. meliloti is altered. Consequently, we propose a model whereby the altered polysaccharide content of the S. meliloti msbA2 mutant could be responsible for its symbiotic defect by inducing an inappropriate host response.

[TolC mutant of Sinorhizobium meliloti strain SKHM1-188 fails to establish effective symbiosis with alfalfa].

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain SKhM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix- nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.

Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12.

An insight into a previously unknown step in B(12) biosynthesis was unexpectedly obtained through our analysis of a mutant of the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti. This mutant was identified based on its unusually bright fluorescence on plates containing the succinoglycan binding dye calcofluor. The mutant contains a Tn5 insertion in a gene that has not been characterized previously in S. meliloti. The closest known homolog is the bluB gene of Rhodobacter capsulatus, which is implicated in the biosynthesis of B(12) (cobalamin). The S. meliloti bluB mutant is unable to grow in minimal media and fails to establish a symbiosis with alfalfa, and these defects can be rescued by the addition of vitamin B(12) (cyanocobalamin) or the lower ligand of cobalamin, 5,6-dimethylbenzimidazole (DMB). Biochemical analysis demonstrated that the bluB mutant does not produce cobalamin unless DMB is supplied. Sequence comparison suggests that BluB is a member of the NADH/flavin mononucleotide (FMN)-dependent nitroreductase family, and we propose that it is involved in the conversion of FMN to DMB.

Localization of superoxide dismutases and hydrogen peroxide in legume root nodules.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to O2 and H2O2 and thus represent a primary line of antioxidant defense in all aerobic organisms. H2O2 is a signal molecule involved in the plant's response to pathogen attack and other stress conditions as well as in nodulation. In this work, we have tested the hypothesis that SODs are a source of H2O2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized by in situ RNA hybridization and immunogold electron microscopy, respectively, whereas H2O2 was localized cytochemically by electron microscopy of cerium-perfused nodule tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the meristem (I) and invasion (II) zones, interzone II-III, and distal part of the N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected cells. At the subcellular level, CuZnSOD was found in the infection threads, cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the bacteroids, bacteria within infection threads, and mitochondria. The distinct expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in nodules. Large amounts of H2O2 were found at the same three nodule sites as CuZnSOD but not in association with MnSOD. This colocalization led us to postulate that cytosolic CuZnSOD is a source of H2O2 in nodules. Furthermore, the absence or large reduction of H2O2 in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that at least some of the H2O2 originates by the sequential operation of an NADPH oxidase-like enzyme and CuZnSOD. Results also show that there is abundant H2O2 associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.

Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose.

Hydrolysis of cellulose requires two different types of cellulases: exo- and endocellulase. Here, we investigated for the hydrolysis of cellulose by two types of cellulases, an endoglucanase (Cel5) from Ruminococcus albus fused with the xylanase A cellulose binding domain II (CBM6) of Clostridium stercorarium and Thermobifidus fusca E3, an exoglucanase (Cel6B). Cel5-CBM6 or Cel6B showed a linear relationship between the production of soluble sugars and the incubation time when native alfalfa cellulose was used as a substrate. Cel5-CBM6 produces more soluble sugars than Cel6B and the hydrolysis of cellulose by a mixture of the two enzymes produces substantially more (22%) soluble sugars than the total amount produced by these enzymes individually. Although Cel5-CBM6 solubilized high quantities of sugars from alfalfa cellulose, it did not significantly decrease its crystallinity, while Cel6B decreased the crystallinity of cellulose by 34%. When the two cellulases were combined, a decrease of more than 50% in the content of crystalline cellulose was observed. The enzyme-gold labeling experiments revealed that both enzymes showed a high affinity for all substrates. Furthermore, simultaneous visualization of the enzyme-binding sites revealed the preferred substrates in native lignocellulosic material. When plant cellulose was pre-incubated with Cel5-CBM6, density of the gold labeling greatly increased suggesting that preliminary exposure of lignocellulosic material to Cel5-CBM6 may have enhanced the accessibility of the substrate to Cel5-CBM6 and Cel6B. This result provides a plausible explanation for the observed endo/exo cellulase synergism during hydrolysis.

Structural changes induced by NaCl in companion and transfer cells of Medicago sativa blades.

Medicago sativa var. Gabes is a perennial glycophyte that develops new shoots even in high salinity (150 mM NaCl). In the upper exporting leaves, K(+) is high and Na(+) is low by comparison with the lower leaves, where Na(+) accumulation induces chlorosis after 4 weeks of NaCl treatment. By secondary ion mass spectroscopy, a low Na(+)/K(+) ratio was detected in the phloem complex of blade veins in these lower leaves. By transmission electron microscopy, the ultrastructural features were observed in the phloem complex. In the upper leaves of both control and NaCl-treated plants, companion cells in minor veins were found to be transfer cells. These cells may well be involved in the intravenous recycling of ions and in Na(+) flowing out of exporting leaves. Under the effect of NaCl, companion cells in the main veins develop transfer cell features, which may favor the rate of assimilate transport from exporting leaves toward meristems, allowing the positive balance necessary for the survival in salt conditions. These features no longer assist the lower leaves when transfer cells are necrotized in both minor and main veins of NaCl-treated plants. As transfer cells are the only degenerating phloem constituent, our observations emphasize their role in controlling nutrient (in particular, Na(+)) fluxes associated with the stress response.

Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and their crucial role during the infection process.

Sinorhizobium meliloti possesses three distinct catalases to cope with oxidative stress: two monofunctional catalases (KatA and KatC) and one bifunctional catalase-peroxydase (KatB). The katB gene is constitutively expressed during growth in batch culture and is not induced under oxidative stress conditions. In contrast, the expression of katA and katC genes is mainly regulated at the transcription level in these conditions. A differential expression of kat genes was observed during the development of the nodule. A high expression of katA gene was detected in bacteroids, suggesting that the nitrogen-fixation process induces a strong oxidative stress. In contrast, bacteria express katB and katC genes and not the H2O2-inducible katA gene in infection threads despite the detection of H2O2 around the bacteria. A katB katC double mutant nodulated poorly and displayed abnormal infection. After nonefficient release into plant cells, bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Our results indicate that these two catalases are essential for the establishment of the symbiosis. They also suggest that the bacteria are in a nonexponential growth phase in infection threads and corroborate previous studies on the growth rate of bacteria inside the plant.

Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts.

A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were also determined. The treatment (10 or 20 min) of seeds in water (4 degrees C) containing an initial concentration of 21.8 +/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05) reduction in populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for 20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/- 0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05) reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water. Treatments (2 min) with up to 23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and natural microbiota compared to soaking in water (control) but did not enhance the lethality compared to the sprouts not treated with continuous sparging. The treatment of sprouts with ozonated water (20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the sensory quality during subsequent storage at 4 degrees C for 7 to 11 days. Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth, and biofilm formation that provide evidence of factors contributing to the difficulty of killing microorganisms by treatment with ozone and other sanitizers.

Ultrastructural studies on nodules induced by pyrimidine auxotrophs of Sinorhizobium meliloti.

Twenty three pyrimidine auxotrophs of Sinorhizobium meliloti Rmd201 were generated by random mutagenesis with transposon Tn5. On the basis of biochemical characters these auxotrophic mutants were classified into car, pyrC and pyrE/pyrF categories. All auxotrophs induced white nodules which were ineffective in nitrogen fixation. Light and electron microscopic studies revealed that the nodules induced by pyrC mutants were more developed than the nodules of car mutants. Similarly the nodules induced by pyrE/pyrF mutants had more advanced structural features than the nodules of pyrC mutants. The nodule development in case of pyrE/pyrF mutants was not to the extent observed in the parental strain. These results indicated that some of the intermediates and/or enzymes of pyrimidine biosynthetic pathway of S. meliloti play a key role in bacteroidal transformation and nodule development.

Demonstration of prominent actin filaments in the root columella.

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.

Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts.

Laser scanning confocal microscopy (LSCM) was used to observe the interaction of Salmonella Stanley with alfalfa sprouts. The green fluorescent protein (gfp) gene was integrated into the chromosome of Salmonella Stanley for constitutive expression, thereby eliminating problems of plasmid stability and loss of signal. Alfalfa seeds were inoculated by immersion in a suspension of Salmonella Stanley (ca. 10(7) CFU/ml) for 5 min at 22 degrees C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12 microm within intact sprout tissue. An initial population of ca. 10(4) CFU/g seed increased to 7.0 log CFU/g during a 24-h germination period and then decreased to 4.9 log CFU/g during a 144-h sprouting period. Populations of Salmonella Stanley on alfalfa seeds decreased from 5.2 to 4.1 log CFU/g and from 5.2 to 2.8 log CFU/g for seeds stored 60 days at 5 and 22 degrees C, respectively. The efficacy of 100, 200, 500, or 2,000 ppm chlorine in killing Salmonella Stanley associated with sprouts was determined. Treatment of sprouts in 2,000 ppm chlorine for 2 or 5 min caused a significant reduction in populations of Salmonella Stanley. Influence of storage on Salmonella Stanley populations was investigated by storing sprouts 4 days at 4 degrees C. The initial population (7.76 log CFU/g) of Salmonella Stanley on mature sprouts decreased (7.67 log CFU/g) only slightly. Cross-contamination during harvest was investigated by harvesting contaminated sprouts, then directly harvesting noncontaminated sprouts. This process resulted in the transfer of ca. 10(5) CFU/g Salmonella Stanley to the noncontaminated sprouts.