The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

Summary The presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH ₄ ⁺ ) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

Functional Activity of Exoglycans from Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Resistant Mutant M-71 during the Formation of Legume–Rhizobia Symbiosis against a High-Nitrogen Background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71, capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO₃ ^–), was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of the same or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5- to 2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix⁺ phenotype of strain 250a, capable of forming symbiosis with pea plants only against a low-nitrogen background. In study of the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72–75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25–28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

Pleiotropic effects of the<Emphasis Type="Italic"> male sterile33</Emphasis> (<Emphasis Type="Italic">ms33</Emphasis>) mutation in<Emphasis Type="Italic"> Arabidopsis</Emphasis> are associated with modifications in endogenous gibberellins,indole-3-acetic acid and abscisic acid

Earlier, we reported that mutation in the Male Sterile33 (MS33) locus in Arabidopsis thaliana causes inhibition of stamen filament growth and a defect in the maturation of pollen grains [Fei and Sawhney (1999) Physiol Plant 105:165–170; Fei and Sawhney (2001) Can J Bot 79:118–129]. Here we report that the ms33 mutant has other pleiotropic effects, including aberrant growth of all floral organs and a delay in seed germination and in flowering time. These defects could be partially or completely restored by low temperature or by exogenous gibberellin A₄ (GA₄), which in all cases was more effective than GA₃ Analysis of endogenous GAs showed that in wild type (WT) mature flowers GA₄ was the major GA, and that relative to WT the ms33 flowers had low levels of the growth active GAs, GA₁ and GA₄, and very reduced levels of GA₉, GA₂₄ and GA₁₅, precursors of GA₄. This suggests that mutation in the MS33 gene may suppress the GA biosynthetic pathway that leads to GA₄ via GA₉ and the early 13-H C₂₀ GAs. WT flowers also possessed a much higher level of indole-3-acetic acid (IAA), and a lower level of abscisic acid (ABA), relative to ms33 flowers. Low temperature induced partial restoration of male fertility in the ms33 flowers and this was associated with partial increase in GA₄. In contrast, in WT flowers GA₁ and GA₄ were very much reduced by low temperature. Low temperature also had little effect on IAA or ABA levels of ms33 flowers, but did reduce (>2-fold) IAA levels in WT flowers. The double mutants, ms33 aba1-1 (an ABA-deficient mutant), and ms33 spy-3 (a GA signal transduction mutant) had flower phenotypes similar to ms33. Together, the data suggest that the developmental defects in the ms33 mutant are unrelated to ABA levels, but may be causally associated with reduced levels of IAA, GA₁ and GA₄, compared to WT flowers.Abbreviations ABA Abscisic acid - GA Gibberellin - GC-MS-SIM Gas chromatography-mass spectrometry-selected ion monitoring - IAA Indole-3-acetic acid - ms33 Male sterile33 mutant - PP333 Paclobutrazol - WT Wild type     

Selection of Competitive Strains of Rhizobium Nodulating Phaseolus Vulgaris and Adapted to Environmental Conditions in Egypt,Using the Gus-Reporter Gene Technique

Two Rhizobium etli strains, EBRI 2 and EBRI 26, isolated from Egypt were tested for nodulation competitiveness on beans using Rhizobium tropici CIAT 899G as the competing strain. The insertion of the gus-reporter transposon mTn5ssgusA30 did not alter the nodulation or nitrogen fixation capacity of mutant strain CIAT 899G compared to the wild type. At neutral pH, R. etli strains EBRI 2 and EBRI 26 were more competitive than CIAT 899G with the bean cultivar Saxa. These two strains gave nodule occupancies of 52.1 and 61.1% competing with equal cell numbers of CIAT 899G. Nodule occupancies from these two native strains increased with the bean cultivar Giza 6 from Egypt to 66 and 67.5%. Based on these results, cultivar Giza 6 was used to select the most competitive strains under stress of salinity or alkalinity as a major problem for a large part of Egyptian soils. Under stress of salinity (0.2% NaCl or 34.2 mM NaCl), the salt-sensitive strain EBRI 2 was more competitive than the salt-resistant strain EBRI 26. Strain EBRI 2 gave 87.4% but strain EBRI 26 gave 63.7% nodule occupancy against CIAT 899G. The same trend of results was observed under stress of alkalinity (pH 8). Strain EBRI 2 occupied 83% while Strain EBRI 26 occupied 53.2%.     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Biological characteristics of plasmids of <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis> HN3015 from <Emphasis Type="Italic">Astragalus sinicus</Emphasis>

A Mesorhizobium huakuii strain HN3015 was isolated from Astragalus sinicus in a rice-growing field of Southern China. Strain HN3015 contained three large plasmids. The three indigenous plasmids, named as pMhHN3015a, pMhHN3015b and pMhHN3015c of M. huakuii HN3015, were, respectively, cured by Tn5-sacB insertion. The mutant strain HN3015-1 cured with its largest plasmid pMhHN3015c formed only white null nodules. Mutant HN3015-3 cured with its smallest plasmid pMhHN3015a could form pink effective nodules. However, mutant HN3015-2 cured of the second largest plasmid pMhHN3015b lost nodulation ability. Furthermore, curing of pMhHN3015a had enhanced competitive nodulation ability and symbiotic efficiency of HN3015-3. The results from acidity tolerance assays indicated that the three plasmids in M. huakuii HN3015 had a positive control effect on acidity tolerance of HN3015, and all indigenous plasmids of M. huakuii HN3015 had a negative control effect on the alkali tolerance capacity of HN3015. Surprisingly, all plasmids in M. huakuii HN3015 had also a negative control effect on its growth rate. The results showed an interactive and functional complexity of plasmids in strain HN3015.     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum

Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (<1.0 mM) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium‐induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH₄⁺ or NO₃^–) or for 3 weeks exposed to exogenous indole‐3‐acetic acid (IAA) or 6‐benzylaminopurine (BAP) at a range of concentrations (10^‐9?10^‐5 M). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant^?1) and specific nodulation (nodules g^?1 root DW), especially in 0.5 mM treatment in which nodulation was approximately 4‐fold of the mineral‐N‐free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t‐zeatin (Z) and lower t‐zeatin riboside (ZR) than that the control or nitrate‐treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10^‐7?10^‐5 M strongly inhibited nodulation. However, 10^?9 M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.     

Effects of temperature and sink activity on the transport of 14C-labelled indol-3yl-acetic acid in the intact pea plant (Pisum sativum L.)

The velocity and intensity of basipetal transport of ¹⁴C-labelled indol-3yl-acetic acid (IAA) applied to the apical bud of the intact pea plant were influenced by the temperature to which the stem was exposed and were not influenced by changes in the temperature of the root system when this was controlled independently between 5°C and 35°C. The velocity of transport increased steadily with temperature to a maximum in excess of 35°C and then fell sharply with further increase in temperature. The Q₁₀ for velocity, determined from Arrhenius plots, was low (ca. 1.3). Transport intensity increased to a maximum at about 25°C (Q₁₀=2.2) and then declined gradually with further increase in temperature. It is suggested that transport velocity and transport intensity are controlled independently.The characteristics of auxin transport through the stem were not affected by removal of the root system, or by the withdrawl of root aeration. Labelled IAA did not pass a region of the stem cooled to about 1.0°C, or through a narrow zone of stem tissue killed by heat treatment. In the latter case the heat treatment was shown not to interfere with the upward transport of water in the xylem. Labelled IAA continued to move into, and to accumulate in, the tissues immediately above a cooled or heat-killed region of the stem. It was concluded that the long-distance basipetal transport of auxin through the stem of the intact plant is driven by the transporting cells themselves and is independent of the activity of sinks for the transported auxin.The fronts of the observed tracer profiles in the stem were closely fitted by error function diffusion analogue curves. However, diffusion of IAA alone could not account for the observed characteristics of the transport and it is suggested that the curvilinear fronts of the profiles resulted from a diffusive mixing of exogenous IAA (or IAA-carrier complexes) with endogenous IAA already in the transport pathway.Abbreviations IAA indol-3yl-acetic acid - IAAsp indol-3yl-acetyl aspartic acid - CFM methyl 2-chloro-9-hydroxyfluorene-9-carboxylate (morphactin) - TIBA 2,3,5-triiodobenzoic acid - ABA abscisic acid     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Bradyrhizobium japonicum subspecies (USDA 110 and 26) characterized by fixed-nitrogen uptake and symbiotic indoleacetic acid production

Two dissimilar subspecies ofBradyrhizobium japonicum (USDA 110 and 26) differ in ammonia (NH₃) assimilation and symbiotic indoleacetic acid (IAA) production. Free-living cultures of type-strain USDA 26 grow on NH₃ as a sole N source and take up an NH₃ analog, methylamine, whereas USDA strain 110 does neither. Although both strains nodulate soybean effectively, root nodules infected with symbiont 26 contain 0.3–1.1 g IAA per gram fresh weight. Nodules infected by tryptophan catabolic variants 4b and 20d, derived from strain 26, also elicit an increased IAA content, two- to fourfold (2.0–3.9 g · g^–1). In contrast, nodules infected with the dissimilar subspecies (strains 110 and 123) contain significantly less IAA.     

Auxin gradient along the root of the maize seedling

[5-³H]Indol-3yl-acetic acid (IAA) applied to the shoot apices of intact 6-day-old maize (Zea mays L.) plants moved into the primary root and accumulated at the root apex. IAA from the shoot could partially satisfy the requirement of the primary root for IAA for growth.Abbreviation IAA indol-3yl-acetic acid     

Effect of morphactin on certain plant growth substances in bean roots

The effect of morphactin (methyl-2-chloro-9-hydroxyfluorene-9-carboxylate) on the content of several plant growth substances in bean roots was determined. Beans (Phaseolus vulgaris L. cv. Spartan) were soaked in aqueous solutions of morphactin, 1 x 10^-4, 1 x 10^-5, and 1 x 10^-6M and grown in moist vermiculite. As controls were used beans grown in water-moistened vermiculite either intact or having the root tips removed (decapped). The roots, morphactin-treated, controls, and the decapped ones were analyzed for indol-3-yl acetic acid (IAA), indol-3-yl acrylic acid (IAcA), indol-3-yl pyruvic acid (IPyA), indol-3-yl lactic acid (1LA), abscisic acid (ABA), gibberellins GA₁, GA₃, GA₄, and GA₉ using gas-liquid chromatographic methods. Morphactin, while affecting the geotropical responses, changed also the growth substance content of roots. IAA, ABA, GA₁, and GA₉ contents decreased, IPyA, IAeA, GA₃, and GA₄ contents were not affected and ILA content increased slightly with increasing dosages of morphactin. Growth substance pattern of decapped roots resembled that of the roots treated with the highest dose, 1 x 10^-4M, of morphactin.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Utilization of the Plant Hormone Indole-3-Acetic Acid for Growth by Pseudomonas putida Strain 1290

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.     

Exogenous Glutamate Inhibits the Root Growth and Increases the Glutamine Content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Previously, our work with ginseng hairy root shows that the tissue of low-branching and slow-growing phenotype contains high level of glutamine. In order to check if the high glutamine concentration inhibits the root growth, we applied exogenous glutamine or glutamate into growth medium and check the root growth of Arabidopsis. While glutamine did not affect root growth, over 0.1 mM glutamate inhibited severe root growth. However, when the amino acid solution was adjusted to pH 5.7 and added into medium, Arabidopsis seedlings show normal growth pattern on medium containing glutamate or aspartate. These results demonstrated that inhibition of the root growth by high concentration of exogenous glutamate was a result of the low pH toxicity caused by acidic amino acid, although low concentration (0.05 mM) of glutamate has an inhibitory effect on the primary root growth. The application of exogenous glutamine or glutamate increases glutamine concentration within root tissue about 3- to 4-fold. However, concentration of glutamate is not significantly increased. The KO mutant on each of the Gln1_1, Gln1_2, or Glu2 gene was little effective on the root growth. These results indicate that high concentration of endogenous glutamine observed in root tissue does not affect root growth.     

Boron and blue light reduce responsiveness of <Emphasis Type="Italic">Arabidopsis</Emphasis> hypocotyls to exogenous auxins

We reported earlier that boron stimulates hypocotyl growth in several Arabidopsis ecotypes but not in the boron-deficient mutant bor1-1. Others have shown that boron influences the metabolism and transport of the plant hormone auxin. We investigated how boron, in interaction with light, influences Arabidopsis hypocotyl growth responses to the exogenous auxins 1-NAA, 2,4-D and IAA. In either light condition, 1-NAA similarly inhibited hypocotyl growth in bor1-1 and the corresponding WT (Col-0), while in both genotypes, boron did not essentially affect the extent of the inhibition. Whatever the light conditions and in the absence of boron, 2,4-D inhibited hypocotyl elongation in WT, while in BL seedlings, high responsiveness to 2,4-D vanished when boron was added to the culture medium. Hypocotyl of bor1-1 seedlings in all boron concentrations tested and grown in the dark or RL responded to the auxin similar to WT plants. In BL, the mutant hypocotyls retained full sensitivity to 2,4-D at 0.1 mM H₃BO₃ but lost that sensitivity by 2 mM. In both genotypes tested, in the dark or RL, IAA inhibited hypocotyl growth. Conversely, IAA stimulated hypocotyl elongation in both genotypes developed in BL at 0.1 mM H₃BO₃. That stimulation disappeared when the boron supply increased to 2 mM. Our results suggest that specifically in BL, boron reduces hypocotyl responsiveness to auxins 2,4-D or IAA via the functional transporter BOR1. Our results lead to a discussion of how BL and BOR1 influence the mechanisms of auxin transport into and out of the cell.     

Cloning and characterization of indolepyruvate decarboxylase from <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k _cat value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k _cat/K _m). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.