Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense

We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in the tissues of seeds treated with M. fujisawaense strains than in control seeds treated with MgSO₄. Though the increased activities of ACC synthase in the tissue extracts of the treated seedlings might be due to bacterial indole-3-acetic acid, the amount of ACC was reduced due to bacterial ACC deaminase activity. The activities of ACC oxidase, the enzyme catalyzing conversion of ACC to ethylene remained lower in M. fujisawaense treated seedlings. This consequently lowered the ethylene in plants and prevented ethylene inhibition of root elongation. Our results collectively suggest that Methylobacterium commonly found in soils, as well as on the surfaces of leaves, seeds, and in the rhizosphere of a wide variety of plants could be better exploited to promote plant growth.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

DNA sequences of the two homoeologous SLR1 genes of Brassica napus cv Westar

The DNA sequence data reported have been lodged in the Genbank, EMBL and DDBJ databases under the accession numbers Z21609 and Z26914     

Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay. Five of them tested positive for ACC deaminase. Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv. viciae 128C53K. This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 μM. The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.     

Stable sodium sulfate tolerance inBrassica napus cv. Westar callus cultures

Summary Sodium-sulfate-tolerant callus ofBrassica napus cv. Westar, selected on medium containing 105 mM Na₂SO₄, was maintained on medium without the salt to test for stability of tolerance. Tolerance to Na₂SO₄ was retained even after 18 subcultures on no salt. This tissue also showed tolerance to K₂SO₄, NaCl, and KCl. However, with the exception of callus grown on KCl fresh weight yields were less than that of tolerant callus maintained continuously on Na₂SO₄. Tolerant callus maintained on no salt had a mixture of the compact morphology of unselected callus and the friable morphology of tolerant callus. Both callus types expressed salt tolerance. Sucrose, reducing sugars and proline concentrations were measured in unselected callus, tolerant callus maintained continuously on Na₂SO₄, and tolerant callus maintained on no salt. Sucrose levels were similar in all cases. Whether maintained on or off Na₂SO₄., tolerant callus had reducing sugars levels three to fou times greater than unselected callus. Tolerant callus maintained on no salt had twice the amount of proline found in the unselected callus. Tolerant callus maintained in the absence of salt had an ash content, sodium concentration, and potassium concentration significantly lower than that of unselected callus. Supported by the Natural Sciences and Engineering Research Council of Canada Strategic Grant nos. G 0949 and G 1642 to T. A. Thorpe, D. M. Reid, and E.C. Yeung.     

A method for quantifying the gene flow that results from a single bumblebee visit using transgenic oilseed rape,Brassica napus L. cv. Westar

Genetically modified plants containing selectable markers offer a unique opportunity for pollination biologists to investigate some of the major, but intractable questions about paternity distributions and their causes. Here, a method is reported that uses transgenic plants to enable the quantification of the outcrossed fertilizations that result from a single pollinator visit. Gene flow mediated by worker bumblebees (Bombus terrestris) was studied among plants of oilseed rape (Brassica napus L. cv. Westar) where transgenic paternity in seeds of a non-transgenic plant was manifested as herbicide resistance. Overall, 91% of the resistant seeds resulted from the first four flowers that were visited after the bumblebee left the transgenic plant, and none was found beyond the 14th successively visited flower. The possibilities for developing the method to address various questions in pollination biology are discussed.     

The use of haploidy to develop plants that express several recessive traits using light-seeded canola (Brassica napus) as an example

Summary The use of haploidy to introgress recessive traits into Brassica napus canola is illustrated by describing the properties of doubled haploids obtained by microspore culture from crosses between a yellow-seeded rapeseed line (low erucic acid, high glucosinolate) and black-seeded canola. Of the 99 doubled haploid lines that were produced, 3 were yellow-seeded canola lines. This result was not significantly different than the predicted frequency of 1 in 64 for the homozygous recessive phenotype in a doubled haploid population segregating for six recessive genes. Thus, the study supports previous models of inheritance determined for yellow seededness and glucosinolate content in Brassica napus. Also, since the chances of obtaining a plant with the same characteristics in a F₂ population are 1 in 4,096, the underscore results the advantages of using haploidy to introgress recessive traits into Brassica napus canola.     

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.     

Effects of sowing time and plant age on critical nitrogen concentrations in canola (Brassica napus L.)

Critical concentrations of NO₃-N in fresh petiole tissue and total N in the dried lamina were determined for the youngest mature leaf (YML) of field-grown canola. For dry matter yield of canola sown on 4 May, critical NO₃-N concentration in the YML petiole at the rosette stage (RS) was 1.46 mg/g fresh wt. At the flower-buds-visible stage (BV) it was 0.45 mg/g fresh wt. For seed yield the values were 1.72 and 0.53 mg/g fresh wt. Critical total N concentration in the YML lamina for dry matter yield were 69 mg/g dry wt. at RS and 57 at BV. For seed yield they were 71 and 59 mg/g dry wt. Critical NO₃-N concentrations in the YML petiole of canola sown on 30 May were reduced by 50%; critical total-N concentrations in the YML lamina were not reduced to the same extent. Despite the reductions in critical N concentrations in the YML, critical N fertilizer rates for vegetative growth and seed yield were unaffected by sowing date or plant growth stage.     

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing <Emphasis Type="Italic">Methylobacterium oryzae</Emphasis> and interactions with auxins and ACC regulation of ethylene in canola (<Emphasis Type="Italic">Brassica campestris</Emphasis>)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacterium oryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.     

旱地小麦根际细菌中产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶菌株的分离和鉴定

采用富集定向筛选法,从旱地小麦的根际土壤中分离到2株产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶的菌株AS和CS。经测定菌株AS和CS的ACC脱氨酶的比活力分别为0.018 6 U/mg和0.016 7 U/mg蛋白。根据培养特征观察和生理生化指标测定结果,结合16S rDNA碱基序列测定和系统发育同源性分析,确定菌株AS和菌株CS分别属于霍氏肠杆菌(Enterobacter hormaechei)和变形斑沙雷氏菌(Serratia proteamaculans)。     

Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.     

Frequency and distance of pollen dispersal from transgenic oilseed rape (Brassica napus)

The objective of this study was to evaluate pollen dispersal inBrassica napus (oilseed rape). The selectable marker, used to follow pollen movement, was a dominant transgene (bar) conferring resistance to the herbicide glufosinate-ammonium. Transgenic and non-transgenic plants of the cultivar Westar were planted in a 1.1 ha field trial, with the transgenic plants in a 9 m diameter circle at the centre, surrounded by non-transgenic plants to a distance of at least 47 m in all directions. A 1 m circle of non-transgenic plants was sown in the centre of the transgenic area to allow estimation of the level of pollen dispersal when plants were in close contact. Honeybee hives were placed at the trial site to optimize the opportunity for cross-pollination. During the flowering period, regular observations were made of the number of plants flowering and the number and type of insects present in 60 1 m² areas. These areas were located uniformly around the plot at distances of 1, 3, 6, 12, 24, 36 and 47 m from the edge of the 9 m circle of transgenic plants. Seed samples were harvested from each of the 7 distances so that approximately 20% of the circumference of the plot was sampled at each distance. The centre non-transgenic circle was also sampled. Plants were grown from the seed samples and sprayed with glufosinate to estimate the frequency of pollen dispersal at each distance. In order to screen enough samples to detect low frequency cross-pollination events, seed samples were tested in the greenhouse and on a larger scale in the field. Results were confirmed by testing progeny for glufosinate resistance and by Southern blot analysis. The estimated percentage of pollen dispersal in the non-transgenic centre circle was 4.8%. The frequency was estimated to be 1.5% at a distance of 1 m and 0.4% at 3 m. The frequency decreased sharply to 0.02% at 12 m and was only 0.00033% at 47 m. No obvious directional effects were detected that could be ascribed to wind or insect activity.     

Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product

TheAspergillus niger gene encoding phytase(phyA) was expressed in canola (Brassicanapus). Phytase expression is controlled by the seed-specificcruciferin (CruA) promoter. Secretion of the enzyme was aimed for byincorporating the cruciferin signal peptide in the expression construct.Transgenic canola lines were generated by Agrobacteriummediated transformation using nptII as the selectable marker. Ninety-fiveindependent transgenic events were generated. Phytase expression in the T1seedsranged from 0 to 600 U/g seed. Single-copy lines were selected(based on segregation for kanamycin resistance, phytase expression and Southernanalyses) from originally multi-copy transgenic lines. Phytase was expressed inthese sub-lines up to 103 U/g. Expression levels were monitoredthrough an additional 3–4 generations (in the greenhouse and in thefield)and the accumulation of phytase appeared to be fairly stable. In the expressionrange studied, phytase expression was gene-dosage dependent.     

A simple method to estimate the percentage of hybridity in canola (Brassica napus) F1 hybrids

We have developed an efficient PCR-based system that uses RAPD markers for the certification of F₁ hybrids of canola. These markers were selected by screening five parental lines used in three crosses X, Y and Z with 131, 131 and 322 primers respectively. Stable DNA fragments that were homozygous and specific to the male inbreds were used to certify F₁ hybrid populations. The hybrid production system was based on self-incompatibility (SI) alleles that prevent self-pollination of the female parent. The efficiency of two S-alleles was compared under both field and greenhouse conditions. The percentage of hybridity was estimated in different F₁ populations. We found a significant difference between the two alleles for their efficiency in controlling selfing; both alleles were stable under greenhouse conditions, one allele appeared less reliable under field conditions.     

ACC氧化酶基因AhACOs对花生耐盐性的影响

ACC氧化酶(ACC oxidase,ACO)是催化乙烯合成的关键酶之一,乙烯参与植物的盐胁迫反应过程,而盐胁迫严重影响花生产量。本研究通过对AhACOs基因的克隆及功能验证,探究AhACOs在花生盐胁迫响应中的生物学功能,为花生耐盐品种的选育提供基因资源。以花生耐盐突变体M29的cDNA为模板扩增得到基因AhACO1和AhACO2,与植物表达载体pCAMBIA super1300重组后,通过农杆菌介导的花粉管注射法将重组质粒转化到花育22号中。收获后切取籽仁远胚端部分子叶,利用PCR检测筛选阳性籽仁。利用qRT-PCR分析AhACOs基因表达量,通过毛细管柱气相色谱法检测植株的乙烯释放量。阳性籽仁和对照籽仁种植21 d后浇盐水,观察其表型变化。结果发现,盐胁迫后,转基因植株生长状况好于对照组花育22号,并且其叶绿素相对含量SPAD(soil and plant analyzer development)值和净光合速率(net photosynthesis rate,Pn)均高于对照组花生。另外,AhACO1和AhACO_(2)转基因植株的乙烯释放量分别为对照组花生的2.79倍和1.87倍。这些结果表明AhACO1和AhACO2可显著提高花生的耐盐能力。     

Characterization of 1-aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation

The presence of 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity among the phyllosphere methylobacteria of rice was detected and its role in regulating plant ethylene level was assessed. Eighteen methylobacterial isolates from four different cultivars of rice were isolated and screened for ACCD. The 16S rRNA homology of ACCD positive methylobacterial isolate closely related to the species Methylobacterium radiotolerans. The accD gene sequence homology of the isolate was 98% similar to Rhizobium leguminosarum. Foliar spray of ACCD positive methylobacterial isolates enhanced the root and shoot length of rice and tomato seedlings under gnotobiotic condition and lower the ethylene level (60–80%) in the plant species.     

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

Aims: 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat‐resistant polypropylene chimney‐top 96‐well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC‐utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC‐utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR‐plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR‐plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.