A water-soluble chlorophyll protein in cauliflower may be identical to BnD22, a drought-induced, 22-kilodalton protein in rapeseed.

A water-soluble chlorophyll protein (WSCP) in cauliflower (Brassica oleracea L.) was purified and its N-terminal sequence was determined. Forty-six of 48 residues of the sequence completely matched those of the drought-induced 22-kD protein (BnD22) in rapeseed (Brassica napus L.). All 40 sequenced residues of WSCP from rapeseed were perfectly matched to those of BnD22. Thus, WSCP may be identical to BnD22. The abundance of WSCP was increased in detached cauliflower leaves.     

油菜矮秆突变WRKY转录因子cDNA克隆及表达分析

以甘蓝型油菜为材料,利用已建立的抑制性消减文库(SSH),采用RACE技术克隆到1个植物WRKY转录因子相关基因,命名为BnD11,其cDNA全长1034 bp,含有810 bp的完整开放阅读框,编码269个氨基酸。该基因编码的氨基酸序列与拟南芥WRKY40氨基酸序列相似性为79%,与拟南芥中编码WRKY-DNA结合蛋白40基因的氨基酸序列相似性达78%,与其它多种植物的WRKY转录因子的氨基酸序列也有较高的相似性。半定量RT-PCR对BnD11进行组织特异性表达分析显示:在正常生长条件下,BnD11在野生型和矮秆油菜的各个组织中均有表达,但在矮秆突变的根、茎、茎尖的相对表达量明显高于野生型。研究表明,BnD11功能区段具有很高的保守性,可能参与了油菜的茎秆发育。     

A fern spore storage protein is genetically similar to the 1.7 S seed storage protein ofBrassica napus

The ostrich fern, Matteuccia struthiopteris L., contains two globulin spore storage proteins of 2.2 S and 11.3 S, with physical characteristics similar to those of seed storage proteins of Brassica napus (rapeseed) and Raphanus sativus (radish). By the use of a cloned cDNA that encodes the 1.7 S B. napus storage protein (napin), gene sequences that hybridized with napin were detected in fern nuclear DNA, and a 900-nucleotide homologous mRNA was detected in developing spores. In vitro translation of this fern mRNA produced a 22-kD polypeptide comparable in size to the 21-kD precursor polypeptide identified in Brassica. No hybridizations were observed between the Brassica 12 S clone and either fern DNA or developing spore mRNA.     

渗透胁迫下外源茉莉酸甲酯对油菜种子生理特性的影响

采用PEG模拟水分胁迫的方法,研究在5%PEG-6000胁迫下,不同浓度(0.025、0.05、0.1 mmol·L-1)茉莉酸甲酯(MeJA)对油菜种子萌发、幼苗生长、叶片氧化损伤和保护酶活性等生理生化特性的影响.结果显示:0.1mmol·L-1MeJA处理能显著缓解5%PEG-6000的胁迫伤害,使油菜种子发芽率、发芽势、发芽指数和活力指数分别显著提高62.3%、11.2%、55.2%、126.3%,主根长度降低45.0%,不定根数提高59.4%,但对株高无显著影响;同时叶片MDA含量降低20.6%,SOD、CAT、POD和APX活性分别提高100.0%、109.1%、452.6%和134.8%.研究表明,MeJA在一定程度上能够缓解渗透胁迫的抑制作用,提高SOD等保护酶的活性,缓解渗透胁迫造成的氧化损伤,有效促进渗透胁迫下油菜种子的萌发和幼苗生长.     

Importance of the Chiral Centers of Jasmonic Acid in the Responses of Plants (Activities and Antagonism between Natural and Synthetic Analogs)

The importance of the two chiral centers at C-3 and C-7 in the molecular structure of jasmonic acid in plant responses was investigated. We separated methyl jasmonate (MeJA) into (3R)- and (3S)-isomers with a fixed stereochemistry at C-3, but epimerization at C-7 is possible. The four isomers of the nonepimerizable analog 7-methyl MeJA were synthesized. These six esters and their corresponding acids were tested in three bioassays: (a) senescence in sunflower (Helianthus annuus) cotyledons; (b) proteinase inhibitor II gene expression in transgenic tobacco (Nicotiana tabacum) with [beta]-glucuronidase as a biochemical reporter; and (c) seed germination in Brassica napus and wheat (Triticum aestivum). The esters and acids had similar activities in the three assays, with the ester being more effective than its acid. The (3R)-stereochemistry was critical for jasmonate activity. Although activity was reduced after substituting the C-7 proton with a methyl group, the analogs with (3R,7R)- or (3R,7S)-stereochemistry were active in some of the assays. Although the four isomers of 7-methyl MeJA were inactive or only weakly active in the senescence assay, they could overcome the senescence-promoting effect of (3R)-MeJA. The strongest antagonistic effect was observed with the (3R,7S)-isomer.     

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: effects of methyl jasmonate on nitrate uptake, senescence, growth, and VSP accumulation

The role of methyl jasmonate (MeJa) in promoting senescence has been described previously in many species, but it has been questioned in monocarpic species whether induced senescence is a result of a potential death hormone like MeJa, or a consequence of an increased metabolic drain resulting from the growth of reproductive tissue. In oilseed rape (Brassica napus L.), a polypeptide of 23 kDa has been recently identified as a putative vegetative storage protein (VSP). This polypeptide could be used as a storage buffer between N losses from senescing leaves putatively promoted by methyl jasmonate that might be produced by flowers, and grain filling which occurs later on, while N uptake is strongly reduced. In order to describe causal relationships during Brassica napus L. plant responses to MeJa treatment, a kinetic experiment was performed to determine the order and the amplitude with which general processes such as growth, photosynthesis, chlorophyll content, N uptake, and N storage under the form of the 23 kDa VSP are affected. One of the most immediate consequences of MeJa treatment was the strong reduction of nitrate uptake within 6 h, relative to control plants. However, this was not a specific effect as K(+) uptake was similarly affected. Photosynthesis was reduced later (after 24 h), while chlorophyll content as well as leaf growth also decreased in a similar way. Moreover, this was concomitant with a remobilization of endogenous unlabelled N from senescing leaves to roots. Accumulation of the 23 kDa VSP was induced in the taproot after 24 h of MeJa treatment and was increased 10-fold within 8 d. On the other hand, the reversible effect of a MeJa pretreatment was tested in the long term (i.e. along the growth cycle) using plants previously grown in field conditions induced for flowering. Results show that a MeJa pulse induced a reversible effect on N uptake inhibition. In parallel, protein immunologically related to the 23 kDa VSP was detected in stems with a similar molecular weight (23 kDa), and in flowers and leaves with a molecular weight of 24 kDa. This accumulation was concomitant with the remobilization of both subunits of Rubisco. During stem and pod development, this protein induced by MeJa is fully hydrolysed. The external and intermittent supply of MeJa mimic some of the plant physiological processes previously reported under natural conditions. This suggests that in oilseed rape, methyl jasmonate could be considered as a possible monocarpic senescence factor while accumulation/mobilization of the 23 kDa VSP in taproot could be a marker for the cessation of N uptake and the initiation of a massive leaf senescence.     

油菜抗冷相关基因BnCOR14的克隆及序列分析

以油菜沪油15幼叶为材料,采用RACE技术克隆获得1条新的抗冷相关基因(BnCOR14,GenBank登录号AY456378).该基因全长为564bp,含有1个387bp的开放阅读框(ORF),编码129个氨基酸的多肽,其理论分子量约为14kD.序列分析表明BnCOR14与拟南芥及荠菜等COR蛋白具有较高的相似性,且BnCOR14具有典型的LEA蛋白序列特征,表明BnCOR14可能在油菜抵抗冷胁迫的过程中具有重要的作用.     

Extraction and Isolation of Antifreeze Proteins from Winter Rye (Secale cereale L.) Leaves

Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.     

Plants water soluble chlorophyll binding proteins act as enzyme-inhibitor pair

The hydrophilic water-soluble chlorophyll binding proteins (WSCP) which form complex with chlorophyll molecules have been numerously isolated from the chloroplasts of plants. Although, their molecular properties have been partly characterized, but their physio-biochemical roles are still unclear in the photosynthesizing organs. In this study, using bioinformatic tools WSCP pair were predicted to act as hydrolase and hydrolase inhibitor towards chlorophyll molecules. To enhance our information regarding the possible functions of WSCP, we cloned WSCP1 and WSCP2 cDNAs from Chenopodium album L. and Brassica oleracea L. leaves and expressed them as soluble maltose-binding fusion proteins in Escherichia coli. The purified fused products were subjected to chlorophyll hydrolyzing activity in vitro. The results showed that WSCP1 and WCSP2 are antagonistically involved in chlorophyll breakdown, while WSCP1 acts as chlorophyll hydrolyzing enzyme (with the hydrolysis rate of about 40% per 12 h), WSCP2 exerts inhibitory activity (with the inhibition rate of about 38% per 12 h) towards chlorophyll hydrolysis. This is the first ever time report speculates the hydrolase/inhibitory roles for WSCP and proposes that the relative activity of WSCP pair might balance and regulate the chlorophyll breakdown process in the photosynthetic apparatus of plants. It may open the new gate to investigate the potent roles of WSCP in plant system.     

Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique

Water-soluble chlorophyll protein (WSCP) has been found in many Brassicaceae, most often in leaves. In many cases, its expression is stress-induced, therefore, it is thought to be involved in some stress response. In this work, recombinant WSCP from Arabidopsis thaliana (AtWSCP) is found to form chlorophyll-protein complexes in vitro that share many properties with recombinant or native WSCP from Brassica oleracea, BoWSCP, including an unusual heat resistance up to 100°C in aqueous solution. A polyclonal antibody raised against the recombinant apoprotein is used to identify plant tissues expressing AtWSCP. The only plant organs containing significant amounts of AtWSCP are the gynoecium in open flowers and the septum of developing siliques, specifically the transmission tract. In fully grown but still green siliques, the protein has almost disappeared. Possible implications for AtWSCP functions are discussed.     

Compartmentation of ATP:citrate lyase in plants

Extracts prepared from young leaves of Pea (Pisum sativum), tobacco (Nicotiana tabacum), rape (Brassica napus), and spinach (Spinacia oleracea) all contained ATP:citrate lyase (ACL) activity, which was most active in rape leaflets (130 nmol min(-1) g fresh weight). In rape and spinach, ACL activity was predominantly localized in the plastids (between about 78% and 90% of the total activity), whereas in pea and tobacco, distribution was mainly cytosolic (about 85% and 78%, respectively, of the total). These distributions were calculated from the relative distributions of plastid and cytosol marker enzymes. Cross-reactivity between plant and rat ACL antibody was carried out by immunoblot analysis and, in rape and spinach, showed that a 120-kD protein, presumably indicating homomeric ACL proteins, was present in both cytosolic and plastidic fractions. In pea, two cross-reacting proteins were detected, the major material being in the cytosol fraction. Therefore, ACL occurs both in the cytosol and plastids of higher plants, but the distribution of activity changes according to the species. The plastidic ACL is proposed to function for the supply of acetyl-coenzyme A for lipid biosynthesis de novo, whereas the cytosolic ACL may provide acetyl-coenzyme A for the mevalonate pathway or fatty acid elongation.     

Characterization of phosphorus starvation-induced gene BnSPX3 in Brassica napus

Plant and Soil - The molecular studies of Pi starvation in Brassica napus have been limited. To explore the molecular responses and mechanisms involved in Pi starvation in Brassica napus, two SPX...     

Involvement of phospholipases C and D in early response to SAR and ISR inducers in Brassica napus plants.

Phospholipid signaling is an important component in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as in responses to environmental stresses, including pathogen attack. We investigated the involvement of both phospholipase C (PLC, EC 3.1.4.11) and D (PLD, EC 3.1.4.4) in early responses to the treatment of Brassica napus plants with the chemical inducers of systemic acquired resistance (SAR): salicylic acid (SA), benzothiadiazole (BTH), and with the inducer mediating the induced systemic resistance (ISR) pathway, methyl jasmonate (MeJA). Rapid activation (within 0.5-6 h treatment) of the in vitro activity level was found for phosphatidyl inositol 4,5 bisphosphate (PIP2)-specific PLC (PI-PLC) and three enzymatically different forms of PLD: conventional PLDalpha, PIP2-dependent PLD beta/gamma, and oleate-stimulated PLDdelta. The strongest response was found in case of cytosolic PIP2-dependent PLD beta/gamma after BTH treatment. PLDdelta was identified in B. napus leaves and was very rapidly activated after MeJA treatment with the highest degree of activation compared to the other PLD isoforms. Interestingly, an increase in the amount of protein was observed only for PLDgamma and/or delta after ISR induction, but later than the activation occurred. These results show that phospholipases are involved in very early processes leading to systemic responses in plants and that they are most probably initially first activated on post translational level.     

Influence of cytokinins on the methyl jasmonate-promoted senescence in Helianthus annuus cotyledons

Cotyledons of seven-day-old sunflower seedlings were pretreated withcytokinins prior to exposure to methyl jasmonate (MeJA). The rate of senescencewas then followed by measuring chlorophyll loss, electrolyte leakage, ethyleneproduction, and 1-aminocyclopropane-1-carboxylase (ACC) oxidase activity. MeJApromoted all these senescence parameters and the MeJA effects were partiallyblocked by cytokinin pretreatment. 8-azaguanine, which has been reported tohaveanticytokinin activity, blocked the ability of benzyl adenine (BA) to reversethe effect of MeJA on senescence. MeJA also increased SOD and catalaseactivity,decreased protein content. However, while the cytokinin BA more than overcamethe MeJA effect on protein content and SOD activity it did not antagonize theeffect of MeJA on catalase.     

The effects of drought stress on free amino acid accumulation and protein synthesis in Brassica napus

Changes in the concentrations of free amino acids and specific organic acids were analysed during the induction of drought stress in Brassica napus . Most of the amino acids showed a characteristic linear increase with the induction of drought stress in Brassica leaves, increasing an average of 5.9-fold over control levels, followed by a reduction in concentration upon rehydration of the plants. Pyruvate concentrations doubled after 4 days of drought stress whereas 2-oxoglutarate concentrations remained relatively constant. The activities of two of the enzymes involved in amino acid biosynthesis, alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1), were also measured. Neither enzyme showed any increase in activity, except when the plants were rehydrated. This suggests that the increase in both alanine and aspartate levels results from the increase in their precursors pyruvate and glutamate and may not require increased enzyme activity. The effect of drought stress upon changes in protein synthesis was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that there was an overall decrease in protein synthesis with the induction of drought stress, followed by a resumption of synthesis upon rehydration. In addition, the synthesis of a number of specific polypeptides was found to decrease upon water loss in the leaves.