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[Role of the surface and extracellular substances of the phytopathogenic bacterium Xanthomonas campestris in its interactions with cabbage plants].

Changes in some physiological and biochemical characteristics of cabbage (cv. Slava) seedling roots in response to inoculation with the phytopathogen Xanthomonas campestris and its surface and extracellular substances were evaluated. Seven days after the inoculation, the growth of the roots was slightly suppressed and they contained increased amounts of peroxidase. The effect of the lipopolysaccharides stripped from the cell surface or isolated from the culture liquid of X. campestris was similar to that of the whole cells of the phytopathogen. The bacterial lectin isolated from the cell surface material did not induce any defense response in cabbage plants but, presumably, could play a role in the contact interactions between bacteria and plants.

Brassica↗

[Cloning a gene related to resistance to TuMV in cabbage].

A cDNA library was constructed from cabbage 84075 which resists TuMV. The degenerate primers was designed with the disease resistance gene conservative domain (NBS-LRR). The fragments of 513 bp were amplified by RT-PCR and genomic DNA PCR from resistant material 84075, then cloned and sequenced. Two recombinants which are highly homologous with the resistance genes cloned in other plants were used as probes, (named as Bor1, Bor2), the cDNA library was screened. A positive clone was obtained, named as TuR2, whose length was 762 bp, which encodes 226 amino acids, contains a long 681 bp open reading frame (ORF), and has different homology score of amino acid compared with the cloned resistance disease genes of other plants. TuR2 was used as probe. Southern blotting hybridization with genomic DNA shows that TuR2 is probably present in single copy; No. rthern blotting hybridization with RNA shows that the gene expression is constitutive and not differential in every part of the resistance plant 84075, the result of separating detection in F2 population shows that TuR2 gene is probably related to resistance to TuMV in cabbage.

Amino Acid Sequence↗

Purification and properties of chitinase from cabbage.

Chitinase has been purified from the extract of cabbage through successive steps of ammonium sulfate fractionation, chromatofocusing and Sephadex G-75 gel filtration. By these steps, the purity of the enzyme increased by 93.3 fold and the recovery of the enzyme activity was 20%. The purified enzyme had an optimal pH of 5.0, an optimal temperature between 40 to 50 degrees C and a Km of 76 microM for hydrolysis of ethylene glycol chitin. The molecular weight of the enzyme determined from filtration through Sephadex G-75 was 30,000 daltons. Heavy metal ions, Hg2+ (0.5 mM) and Ag+(2.5 mM) significantly inhibited the activity of the enzyme. NBSI1 (1.0 mM), DNFB (0.5 mM) and PMSF (0.5 mM) completely inhibited the activity of the enzyme. The enzyme also showed muramidase activity for hydrolysis of Micrococcus lysodeikticus cell wall. The presence of chitinase in cabbage may function as a defense enzyme against potential pathogens.

Ammonium Sulfate↗

[Mapping and analysis QTL controlling some morphological traits in Chinese cabbage (Brassica campestris L. ssp. pekinensis)].

An AFLP and RAPD genetic map with 352 markers and a RIL (recombinant inbred lines) population from the cross of two cultivated Chinese cabbage lines were employed in mapping and analysis quantitative trait loci (QTL). The number, location, variation explained and additive effect of QTL underlying nine morphological traits were determined by using composite interval mapping method. Fifty putative QTL, including five for plant growth habit, six for plant height, five for plant diameter, seven for leaf length, four for leaf width, six for leaf length/leaf width ratio, seven for petiole length, four for petiole width and six for bolting character, were mapped on 14 linkage groups. There were unequal gene effects and unequal variation explained on the expression of many morphological traits. These results are fundamentals for molecular assisted selection of morphological traits in Chinese cabbage breeding.

Brassica↗

[Establishment of an efficient regeneration system for Chinese cabbage cultured in vitro and histological observation of shoot development].

An efficient plant regeneration system from explants of petiole with cotyledon was developed for Chinese cabbage (Brassica campestris ssp. chinensis Makino) in our study. High frequency of shoot regeneration was obtained in the medium containing BAP 2 mg/L, NAA 0.45 mg/L and 5-7.5 mg/L AgNO3 solution to the half of NH4+ concentration's MS basic medium, with 84.3 percent being the highest frequency of shoot regeneration in seven Chinese cabbage cultivars and one parentage, and the new formed shoots were observed six days after inoculation. Factors influencing in vitro explant regeneration were further studied. The results showed that there was positive correlation between frequency of shoot regeneration and number of shoots per explant. The regeneration plants produced flowers and fruits after about 50 days with low temperature treatment from 8 degrees C to 16 degrees C. Moreover, histological observation indicated that the mode of plant regeneration in B. campestris ssp. chinensis was similar to that of other Brassica crops, and adventitions buds were formed from globular meristematic cluster which derived from the cells of vascular parenchyma at the cut surface of petiole.

Brassica↗

[Transformation of Bt-CpTi fusion protein gene to cabbage (Brassica oleracea var. capitata) mediated by Agrobacterium tumefaciens and particle bombardment].

Bt-CpTI fusion protein gene was transferred to the explants of hypocotyl, cotyledon with petiole and shoot apex of cabbage (Brassica oleracea L. var. capitata) variety "Zhonggan No 8" via Agrobacterium tumefaciens and particle bombardment, and 13 kanamycin-resistant plants were obtained. PCR and Southern blotting hybridization verified that all these plants of the kanr plants of type I regenerated from hypocotyl and cotyledon with petiole mediated by A. tumefaciens were transgenic plants, 2 karr plants of type II stemed from shoot apex mediated by A. tumefaciens were "false-positive"plants and one of 2 kanr plants of type III regenerated from shoot apex via particle bombardment was non-transformed plants. It was showed that part of transgenic plants had high activity of trypsin inhibitor and strong resistance to resist common cabbage worm through the analysis of CpTI relative capacity and insect-resistant test.

Agrobacterium tumefaciens↗

[The cytochemical observation of anthers of Chinese cabbage's male-sterile].

A Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) produces 1/4 male sterile and 3/4 fertility in offspring. The sterile plant can be identified from the color of corolla that is some white when it grows out. The fertile and sterile anthers were researched using cytological and cytochemical methods. Thick sections of both anthers of different developmental stages were stained with Toluidine blue for general cytological observation and stained with the periodic-acid-Schiff's (PAS) technique to detect polysaccharides (red), with Sudan black B (SBB) to detect lipids (black). Before meiosis of microspore mother cells, connective tissue of both fertile and sterile anthers stored a lot of starch grains. Neither starches nor lipid drops were in tapetal and microspore mother cells. The only difference of both anthers was that the tapetal cells of sterile anthers contained more vacuoles than those of fertile anthers. After meiosis of microspore mother cells, the starch grains in connective tissue of fertile anthers disappeared, the tapetal cells synthesized abundant lipid drops, and the microspores also began to accumulate lipid drops. In sterile anthers, the starch grains in connective tissue also disappeared, but only a few lipid drops appeared in tapetal cells. The tapetal cells, however, became red, suggesting the cell contained some polysaccharide material. Pollen abortion in sterile anthers occurred in this stage. The aborting microspores accumulated very less lipid drops in its cytoplasm than those in fertile the starch This result suggested that in the cabbage, the starch grains in connective tissue were transformed into polysaccharide and transported to tapetal cells, then these cells transformed polysaccharide into lipid material that was absorbed by developing microspore. In sterile anthers, however, polysaccharide in the tapetal cells could not be transformed to lipid. The functional default of tapetal cells during lipid metabolism led to microspore abortion. This is new sample in which the functional default of tapetal cells will make pollen abort, and will enhance research field in male sterile in higher plants.

Brassica↗

[Isolation and characterization of BcMF3, a gene expressed only in maintainer line in Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino var. communis Tsen et lee)].

Expression profiling was performed using cDNA-AFLP technology on floral buds of a genic male sterile line (A line) and a maintainer line (B line) in Chinese cabbage pak-choi (Brassica campestris L. ssp. chinensis Makino var. communis Tsen et Lee cv. Aijiaohuang). A differentially expressed cDNA fragment, BA18-T16, was obtained via selective amplification with A18/T16 primer pair. BA18-T16 was specifically associated with the maintainer line, and was expressed only in floral buds revealed by half-quantitative RT-PCR. The 3' end and 5' end sequences of BA18-T16 were obtained by 3' and 5' RACE amplification and the full cDNA,designated as BcMF3, is 2082 bp long containing a 1755 bp open reading frame. BcMF3 shares high homology with Bp19, a pectin methylesterase from Brassica rapa L. with 99% identity at nucleotide level and 85% identity at amino acid level. Suggesting that BcMF3 most likely encodes a pectin methylesterase in Chinese cabbage pak-choi.

Amino Acid Sequence↗

[Calcium distribution in fertile and sterile anthers of a genic male sterile Chinese cabbage].

Potassium antimonite was used to locate calcium in the fertile and sterile anthers of a genic male sterile Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) to probe the relation between Ca(2+) and fertility and sterility of anthers of the cabbage. During fertile anther development, calcium granules increase in number in anther wall cells after meiosis, and then appeared also in locule, suggesting a calcium influx into locule from anther wall cells (Plate I-4). Then the number of calcium granules in microspore cytoplasm also increased at early stage (Plate II-1), accumulated mainly on the membrane of small vacuoles which were fusing to form big ones to make a polarity in the cell and to prepare asymmetric division of microspore (Plate II-3,4). After microspore division and the big vacuole decomposition, many calcium granules accumulated again on the membrane of the vacuoles (Plate III-1,2), displaying calcium regulates vacuole formation and decomposition during pollen development. In sterile anthers, abnormal distribution of calcium granules first appeared in callus wall of microspore mother cell (Plate IV-1). However, only a few calcium granules appeared in early microspores, which then could not form small vacuoles and finally a big vacuole (Plate IV-2,3). The aborting microspores degenerate by cytoplasm shrinking (Plate IV-5,6). The difference pattern of distribution of calcium granules between the fertile and sterile anthers indicates that anomalies in the distribution of calcium accumulation are correlated with the failure of pollen development and pollen abortion.

Brassica↗

[The ultrastructural observation of anthers of Chinese cabbage's mail-sterility].

The fertile and sterile anthers of a Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) were observed using electron microscope to find the ultrastructural feature of sterile anthers. The earliest abnormal phenomenon in sterile anther was nucleolus of sporogenous cells locating in the edge of nucleus. During microspore mother cell development, callus wall surrounding the cell displayed uneven in the thick ness and was discontinuous,and the some cytoplasm leaked out of the cell from some rifts in the wall. After meiosis of microspore mother cells, the cells of tetrad were irregular and some of them contained several nuclei. The exine of pollen began to be formed in tetrad in this cabbage. The evident disorder during exine formation in the sterile pollen occurred during its primexine formation and then the sporopollenin was irregularly deposited to form a layer of uneven and discontinuous pollen exine. Cytoplasm of aborting microspores contracted and finally degenerated after them released from tetrad. The tapetal cells of fertile anther began to synthesis abundant lipid material during microspore development. However, the tapetal cells of sterile anther did not synthesis lipid material during microspore aborting. The microspore abortion was first and tapetal degeneration second. Therefore, aborting microspore induced the functional default of tapetal cells synthesizing lipid material. The ultrastructural results on this study further complete and correct our previous results obtained by light microscope.

Brassica↗

Cell-type-specific defense priming and NHP-dependent systemic immunity against Pectobacterium in Chinese cabbage.

Defense priming is an effective strategy for mounting the defensive capacity of plants. Primed plants undergo minimal changes, enabling rapid, robust responses to subsequent pathogen attack. The cell-type-specific mechanisms underlying priming and systemic immunity remain unclear. Using single-nucleus RNA sequencing (snRNA-seq), we reveal how primed Chinese cabbage coordinates N-hydroxypipecolic acid (NHP)-dependent systemic immunity against Pectobacterium carotovorum. NHP accumulates as a phloem-mobile priming signal. snRNA-seq identified a priming-specific epidermal cell state transition and transcriptional cascade: BrWRKY18-1 activates BrWRKY33-1 expression during priming, initiating NHP-dependent systemic immunity. At post-challenge primed state, pathogenesis-related genes (PRs), such as BrPR3, BrPR4-1, and BrPR4-2, are predominantly expressed in systemic tissues to inhibit infection. Notably, the functionality of these PR genes in immunity necessitates their expression in all cells to effectively enhance defensive resistance. This NHP-transcription factor-PR axis couples mobile signaling to systemic immunity, elucidating cell-type-specific defense priming.

Plant Immunity↗

Investigation of sulfhydryl groups in cabbage phospholipase D by combination of derivatization methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

All eight cysteine residues in 92 kDa cabbage phospholipase D (PLD), deduced from the cDNA sequence, were shown to have free sulfhydryl groups by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of tryptic peptides of PLD derivatized with p-chloromercurybenzoate, iodoacetic acid, and N-ethylmaleimide, as well as of underivatized PLD. Assignment of sulfhydryl groups by any one method was not conclusive. However, complementary information derived from tryptic peptides derivatized with different reagents made full assignment of sulfhydryl groups possible.

Brassica↗

Comparative analysis of two biliproteins, BP1 and BP2, from haemolymph of cabbage white butterfly, Pieris rapae.

Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs.

Amino Acid Sequence↗

Glycine conjugates in a lepidopteran insect herbivore--the metabolism of benzylglucosinolate in the cabbage white butterfly, Pieris rapae.

Herbivores have developed a wide array of countermeasures to overcome plants' chemical defences. Larvae of the cabbage white butterfly, Pieris rapae, feed exclusively on plants of the Brassicales order, which are defended by the glucosinolate-myrosinase system. The defensive function of this system comes from toxic isothiocyanates that are formed when glucosinolates are hydrolysed by myrosinases upon tissue damage. Here we show that P. rapae larvae convert benzylglucosinolate to phenylacetylglycine, which is released in their faeces. Feeding experiments with isotopic tracers suggest that phenylacetonitrile and phenylacetic acid are intermediates in this conversion. We also identified additional glycine and isoserine (2-hydroxy-3-aminopropanoic acid) conjugates with benzoate and indole-3-carboxylate from P. rapae faeces extracts. This is the first description of such conjugates from lepidopteran insects.

Animals↗

Turnip yellow mosaic virus RNA is aminoacylated in vivo in Chinese cabbage leaves.

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure as an integral part of its genome. This structure is located at the extreme 3' end of the viral RNA and is the acceptor of valine after 3'-terminal adenylation. It is known that in vitro (with bacterial, yeast, or plant systems) and in vivo (upon microinjection into Xenopus laevis oocytes) a series of tRNA-specific enzymes can recognize this structure in the viral RNA. We report that TYMV RNA is valylated and consequently adenylated in vivo in its natural host, Chinese cabbage leaves. This suggests that the acylated form of the viral RNA could play an important role in the life-cycle of the virus.

Journal Article↗

Disruption of responses to pheromone by (Z)-11-hexadecenyl trifluoromethyl ketone, an analogue of the pheromone, in the cabbage armyworm Mamestra brassicae.

The effects of (Z)-11-hexadecenyl trifluoromethyl ketone (Z11-16:TFMK) a fluorinated pheromone analogue, on the responses to sex pheromone of the male cabbage armyworm, Mamestra brassicae, have been investigated in an actograph and by electroantennography (EAG). In spite of its structural proximity with the natural pheromone, Z11-16:TFMK was poorly active in EAG, and not active on male behaviour. When permeated in the air, Z11-16:TFMK reversibly inhibited the electroantennographic responses to (Z)-11-hexadecenyl acetate (Z11-16:Ac), the main component of the sex pheromone. In the actograph, the latency of the activation was increased and the intensity of the behavioural activity of males in response to Z11-16:Ac was significantly reduced in the presence of Z11-16:TFMK. These results, along with others previously reported by us, provide new pointers to the possible use of Z11-16:TFMK in pest-control strategies.

Animals↗

Effects of cyazofamid against Plasmodiophora brassicae Woronin on Chinese cabbage.

Cyazofamid (4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide) is a novel fungicide with high levels of activity against Oomycetes fungi and Plasmodiophora brassicae Woronin. The effects of cyazofamid were investigated against P. brassicae, the causal agent of clubroot disease in Chinese cabbage. Cyazofamid at 0.3 mg litre(-1) inhibited resting spore germination of this pathogen by about 80%. Cyazofamid at 3-10 mg litre(-1) exhibited fungicidal activity to resting spores of P. brassicae 1-10 days after treatment. When cyazofamid was applied to infested soil, both root-hair infections and club formation caused by P. brassicae were strongly inhibited at 1-3 mg kg(-1) dry soil. These results suggest that cyazofamid directly inhibits resting spore germination, thereby leading to the inhibition of root-hair infection and club formation. Cyazofamid at 3 mg kg(-1) dry soil also exhibited complete control of clubroot disease. The effect of broadcast soil application using a dust formulation at 2 kg AI ha(-1) (equivalent to 1.3 mg AI kg(-1) dry soil), and plug seedling tray application by a suspension concentrate formulation at 200 and 400 mg AI tray(-1) (30 x 60 x 4 cm3) against P. brassicae was also evaluated. Cyazofamid exhibited good efficacy against the pathogen. The sequential treatment including plug seedling tray application with cyazofamid and pre-plant broadcast soil application with the fungicide fluazinam also exhibited excellent levels of control. These results indicate that cyazofamid has a high potential to be an effective fungicide for the control of clubroot disease.

Brassicaceae↗

The trypsin-inhibitory, immunostimulatory and antiproliferative activities of a napin-like polypeptide from Chinese cabbage seeds.

A heterodimeric 13.8 kDa napin-like polypeptide has previously been isolated from Chinese cabbage (Brassica parachinensis) seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. In the present study the N-terminal sequence of the 8.8 kDa subunit of the polypeptide (PQGPQQRPPKLLQQQTNEEHE) was found to have pronounced homology to napins, albumins and trypsin inhibitors, but demonstrated little similarity to the 5 kDa subunit. The polypeptide stimulated nitrite production by mouse peritoneal macrophages and reduced the viability of leukaemia (L1210) cells. It inhibited trypsin with a higher potency than it inhibited chymotrypsin, but was devoid of ribonuclease and antifungal activities.

Amino Acid Sequence↗