Stable isotope carbon study: long-term partitioning during progressive drought stress in Brassica napus var. oleifera

Abstract. Long-term carbon partitioning was analysed by stable carbon isotope labelling of CO₂ during the adaptive response of Brassica napus to progressive drought stress. This method allowed us to distinguish between the pre-existing carbon, which had accumulated before the change in CO₂ isotope composition (on 20 d) and the recent photosynthetic input occurring during the following period. Three successive adaptive phases characterized the plant response to drought. In the first period of water shortage (20–30 d), growth was progressively slowed down: the recent C input (28.3 mg per plant) was mainly allocated to mature leaves (14 mg) and roots (8.9 mg); the pre-existing C chains were partly lost by respiration (12.1 mg) and partly translocated to the apex (2.3 mg). The increase in dry matter (13.0 mg) was mainly due to the recent C input (19.1 mg) in shoots but the roots appeared also as an important sink despite their small dry matter increase (3.2 mg). Root dry matter turnover corresponded to the elimination of pre-existing C chains (6.0 mg) and their renewal with the recent C input (9.2 mg). Root sink strength was related also to the development of drought-induced short tuberized roots (0.1 mg from pre-existing C and 0.3 mg from recent C). In the second period of water stress (from 30 to 40 d), the whole plant biomass remained constant in spite of efficient allocations from pre-existing and recent C sources to two main sinks: shoot apex (3.1 and 5.4 mg, respectively) and short tuberized roots (1.0 and 0.2 mg, respectively). The hypocotyl acted as a transient storage organ for the recent C input (2.0 mg). In the third period of recovery upon rehydration (from 46 to 51 d), the short tuberized roots gave rise to a new root system using C chains from pre-existing and recent sources (1.7 and 2.8 mg, respectively). Such concomitant sink-source behaviour of tuberized roots underlines the adaptive value of drought rhizogenesis.     

Brassica napus hsp90 can autophosphorylate and phosphorylate other protein substrates

A Brassica napus cDNA encoding the 90 kDa heat shock protein, hsp90, was modified to add 6 histidines at the C-terminus and expressed in insect cells to prepare a recombinant histidine-tagged hsp90. The recombinant protein was purified over Ni²⁺-NTA agarose columns and its identity was confirmed by Western blotting, using a plant hsp90-specific antiserum. Incubation of purified hsp90 with [-³²P] ATP in the presence of Mn²⁺ resulted in its autophosphorylation on serine residues. The purified hsp90 could also phosphorylate other protein substrates such as histones and casein in the presence of Mn²⁺. Analysis of phosphorylated casein revealed that serine residues are phosphorylated by hsp90. This is the first demonstration that a cytosolic hsp90 homolog can phosphorylate other protein substrates.     

Fatty acid desaturation in monogalactosyldiacylglycerol of Brassica napus leaves during low temperature acclimation

Brassica napus plants grown at 5°C have the potential to desaturate fatty acids in the major membrane diacylglycerols of leaves at rates much higher than those of plants grown at 30°C. This low temperature-induced desaturation (LTD) is rapidly deactivated if plants grown at 5°C are transferred to 30°C for several hours. By exposure to ¹⁴CO₂ and a computer simulation program, we estimated the rate of desaturation of monogalactosyldiacylglycerol by (ω9-, ω6- and ω3-desaturases of plants grown at 5, 10, 20 and 30°C. Data show that LTD can be induced in mature leaves of plants grown at 20 and 30°C after transfer to 5°C. Full activation of LTD takes several weeks and occurs more rapidly in plants grown at 20°C than 30°C. This activation is largely due to the increased activity of ω9- and ω6-desaturases on C16 fatty acids and ω6-desaturase on C18 fatty acids.     

Oxalic acid‐mediated stress responses in Brassica napus L.

Oxalic acid (OA) occurs extensively in nature and plays diverse roles, especially in pathogenic processes involving various plant pathogens. However, proteome changes and modifications of signaling and oxidative network of plants in response to OA are not well understood. In order to investigate the responses of Brassica napus toward OA, a proteome analysis was conducted employing 2‐DE with MS/MS. A total of 37 proteins were identified as responding to OA stress, of which 13 were up‐regulated and 24 were down‐regulated. These proteins were categorized into several functional groups including protein processing, RNA processing, photosynthesis, signal transduction, stress response, and redox homeostasis. Investigation of the effect of OA on phytohormone signaling and oxidative responses revealed that jasmonic acid‐, ethylene‐, and abscisic acid‐mediated signaling pathways appear to increase at later time points, whereas those pathways mediated by salicylic acid appear to be suppressed. Moreover, the activities of the antioxidant enzymes catalase, peroxidase, superoxide dismutase and oxalic acid oxidase, but not NADPH oxidase, were suppressed by OA stress. Our findings are discussed within the context of the proposed role(s) of OA during infection by Sclerotinia sclerotiorum and subsequent disease progression.     

模拟硫酸型、硝酸型及其混合型酸雨对油菜生理特性、生长和产量的影响

为了了解我国酸雨污染由硫酸型向硫-硝酸复合型转变所引起的环境效应,以油菜(Brassicanapus)为供试材料,在大田试验条件下,系统研究了模拟硫酸型(SAR)、硝酸型(NAR)及其混合型(MAR)酸雨对农作物生理特性、生长和产量的影响。结果表明,3种酸雨胁迫均能抑制油菜生理、生长和产量形成,但不同类型的酸雨间的抑制效应存在差异。当pH≤4.1时,SAR、NAR、MAR能破坏油菜叶质膜系统、降低光合色素含量及光合速率,从而抑制作物的光合作用;当pH≤3.1时,油菜叶面积减小,叶受害百分率明显增加。pH=4.1可作为酸雨对油菜产量的影响阈值。在pH=7.0–1.5的酸度范围内,油菜叶片膜透性、丙二醛含量、叶受害百分率表现为NARMARSAR,光合速率、光合色素含量、叶面积及产量则表现出SARMARNAR的变化特征。当pH4.1时,3种酸雨处理间差异均不明显,pH≤3.1时,3种酸雨间的胁迫效应差异显著增加(p0.05),且酸度越强,差异越大,其变化趋势为NARMARSAR。说明NAR和MAR胁迫对油菜生理、生长及产量的抑制较大。     

Identification of molecular markers associated with linoleic acid desaturation in Brassica napus

 Linolenic acid is a component of canola oil that is readily oxidized, which results in a reduced frying stability and shelf life of the oil. The reduction of linolenic acid in canola seed has therefore been an important breeding objective for many years. The inheritance of linolenic acid concentrations in seed oil is polygenic and is also strongly influenced by the environment. For these reasons, molecular markers are sought to assist in early and reliable selection of desired low linolenic acid genotypes in breeding programmes. Molecular markers associated with low linolenic acid loci were identified in a doubled-haploid population derived from a cross between the Brassica napus lines, ‘Apollo’ (low linolenic)×YN90-1016 (high linolenic) using RAPDs and bulked segregant analysis. A total of 16 markers were distributed over three linkage groups, which individually accounted for 32%, 14% and 5% of the phenotypic variation in linolenic acid content. The rapeseed fad3 gene was mapped near the locus controlling 14% of the variation. The mode of inheritance appeared to be additive, and a QTL analysis showed that collectively the three loci explained 51% of the phenotypic variation within this population. PCR fragments for low linolenic acid ‘Apollo’ alleles (3% linolenic acid) were identified at all three loci. Simultaneous selection for low linolenic acid ‘Apollo’ alleles at each locus resulted in a group of DH lines with 4.0% linolenic acid. The use of these makers in the breeding programme will enhance the breeding of low linolenic acid B. napus cultivars for production in Canada. Received: 23 September 1997 / Accepted: 21 October 1997     

Characterization of the 12S storage protein of Brassica napus (cruciferin): Disulfide bonding between subunits

Cruciferin (12S globulin) is a large, neutral, oligomeric protein synthesized in rapeseed ( Brassica napus ) during the seed development. It is composed of six subunit pairs. Each pair consists of one heavy α chain (30 kDa) and one light β chain (20 kDa). Four different subunit pairs exist. In contrast to earlier studies, our investigations using two-dimensional electrophoresis showed, that the majority of α and β chains of each subunit are disulfide-linked. Analysis of subunit composition of cruciferin hexamers by ion-exchange chromatography suggested that a large array of hexamers exist, composed of mixed combinations of the four subunits.     

Enhancement of recombinant protein synthesis and stability via coordinated amino acid addition

In this work, effective feeding schemes that would minimize stress responses to cloned-protein overexpression are investigated. The cloned-protein (chloramphenicolacetyl-transferase, CAT) contains a high aromatic amino acid content, most notably a high phenylalanine content. Experiments performed on Escherichia coli RR1 [pBR329] (constitutive promoter) and E. coli JM105 [pSH101] (inducible promoter) demonstrated that phenylalanine addition increases the rate of synthesis and yield of CAT. A previous study correlating inducer strength with CAT expression in E. coli JM105 [pSH101] indicated that the highest expression rate was accompanied by the highest apparent rate of protein degradation. In this work, the combined addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) and phenylalanine at intermediate levels resulted in substantial increase of CAT synthesis and partial reduction of protein degradation. Furthermore, transmission electron micrographs verified the absence of inclusion bodies, which, along with proteases, were suspected to reduce protein activity. The research demonstrates that significant enhancement in production and stability of heterologous proteins is possible by designing feeding strategies that incorporate knowledge of the interaction between primary cellular metabolism and foreign protein expression. (c) 1993 John Wiley & Sons, Inc.     

Changes in protein and carbohydrate content during development of seeds and siliquas of rapeseed (Brassica napus L.)

Abstract

A study was made on the changes observed in the protein, starch and soluble sugar content during development of siliquas and seeds of rapeseed grown in central Italy.

Concentration of starch and soluble sugars in the seed increases to 75 per cent dry matter during the first few weeks of pod development and then drops to minimum values. The protein increases steadily until maturity, when a level of 0.85 mg per seed is reached, equivalent to 18 per cent dry matter. The protein and starch in the hull? decrease continuously during development, while in the initial stages the soluble sugars are accumulated until they account for 33 per cent dry matter, after which they decline towards maturity.     

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.     

A comparative study of the effect of abscisic acid and cAMP on protein synthesis in wheat caryopses under drought conditions

The effect of exogenous abscisic acid and cAMP on synthesis of soluble proteins in wheat caryopses in drought has been studied. Both compounds affected the formation of the polypeptides whose synthesis was stimulated by dehydration: they increased the incorporation of the label into polypeptides of 13, 15, and 26 kD and decreased the incorporation of the label into polypeptides of 14, 64, and 77 kD. Abscisic acid and cAMP increased the level of the incorporation of [¹⁴C]leucine into the low-molecular-weight polypeptides of 12, 17, and 19 kD whose synthesis was suppressed by drought. These data suggest that the cyclic adenylate signal system is probably involved in the effect of abscisic acid on protein synthesis in drought.     

油菜种子发育早期的油体发生与调控

以甘蓝型油菜( Brassica napus L.)品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示:油体出现在油菜胚胎发育早期,在授粉9 ~ 11 d后(球形胚时期)的胚体和胚柄中均存在直径小于0. 5 μm的油体;荧光定量实验结果表明,除 BnCLO3 的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因 Oleosins 、 Steroleosins 和 BnCLO1 的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子 BnLEC1 、 BnL1L 、 BnWRI1 和 BnFUS3 在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中 BnLEC1 最早, BnL1L 其次, BnWRI1 和 BnFUS3 较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。     

不同施肥水平对不同品种小麦籽粒蛋白质和地上器官游离氨基酸含量的影响

研究了小麦不同品种开花后各地上器官游离氨基酸含量的变化动态及其与籽粒蛋白质含量的关系,以及氮素营养的调节作用。结果表明,小麦叶片和颖壳十穗轴游离氨基酸含量均在开花后持续增加,至花后14d达到最大值,之后开始下降。茎和叶鞘游离氨基酸含量则在开花后上升较缓慢,至花后21d达最大值。籽粒游离氨基酸含量一般在开花后就持续降低。各地上器官游离氨基酸含量与成熟期籽粒蛋白质含量均呈显著或极显著正相关,说明源器宫中游离氨基酸供应充足,有利于籽粒蛋白质积累。增施氮肥能够提高各地上器官中的游离氨基酸含量,进而促进籽粒蛋白质的合成,提高籽粒蛋白质含量。品种之间籽粒蛋白质含量的差异,是由各地上营养器官向籽粒运输氨基酸的综合作用所造成的。     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

甘蓝型油菜钙依赖蛋白激酶6(BnaCPK6)的定位与互作蛋白分析

为了探究甘蓝型油菜中钙依赖蛋白激酶(calcium dependent protein kinase, CPK)在植物对逆境响应中的作用和机制,同时为油菜品质的升级改良发掘新的基因资源,开展了对于BnaCPK6研究的分子生物学试验。首先,通过在本氏烟草中瞬时表达BnaCPK6与GFP融合蛋白来检测它的亚细胞定位情况;其次,利用双分子荧光互补(Bimolecular fluorescence complementation)试验来检测BnaCPK6与ABA信号通路中转录因子BanABF1/3/4、BnaABI5、BnaAREB3的相互作用情况。结果显示BnaCPK6具有典型的钙依赖蛋白激酶特征,N端具有潜在的棕榈酰化和豆蔻酰化位点,并且与AtCPK6在进化上有着很高的同源性。亚细胞定位的结果发现BnaCPK6主要分布于细胞膜和细胞核中。同时,双分子荧光互补试验还发现BnaCPK6与调控ABA信号转导的关键转录因子BnaABF3/4、BnaABI5以及BnaAREB3之间存在相互作用。本研究为进一步研究BnaCPK6在ABA信号通路中的作用提供了依据。     

油菜种子发育早期的油体发生与调控

以甘蓝型油菜（Brassica napus L.）品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示：油体出现在油菜胚胎发育早期,在授粉9~11 d后（球形胚时期）的胚体和胚柄中均存在直径小于0.5 μm的油体;荧光定量实验结果表明,除BnCLO3的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因Oleosins、Steroleosins和BnCLO1的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子BnLEC1、BnL1L、BnWRI1和BnFUS3在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中BnLEC1最早,BnL1L其次,BnWRI1和BnFUS3较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。     

Promoter regions of the extA extensin gene from Brassica napus control activation in response to wounding and tensile stress.

To identify controlling cis acting promoter regions in the B. napus extA extensin gene, expression in transgenic tobacco of 5 –159, –433, –664, –789 and –940 bp promoter truncations linked to the uidA (B-glucuronidase) reporter coding sequence were analysed. The –159 and –433 bp truncations directed non specific expression in all cell types within the plant. An activator region which increased expression levels 10 fold in all cell types was located between –159 to –433 bp. A repressor region was found between –664 to –789 bp; removal of this region resulted in a 15 fold increase in expression. Histochemical analysis showed that transgenics containing the –664, –789 and –940 bp truncations directed expression of the fusion gene only in the phloem. A negative regulatory region located between –433 to –664 bp repressed expression in non-phloem cell types. In areas of the plant subject to tensile stress, the repression exerted by the negative regulatory region was overcome, allowing expression in all cell types. The quantitative repressor and activator regions which controlled absolute expression levels in all cell types were seperate from the negative regulatory region which controlled cell type specific expression in response to tensile stress. A wound responsive region was found to be located between –940 to –3500 bp. Thus, the extA gene is under complex control, being regulated by 4 sets of positively and negatively acting cis regions, which control wound inducibility, activation in response to tensile stress, and quantitative expression levels.