Differences in cell wall components and allocation of boron to cell walls confer variations in sensitivities of Brassica napus cultivars to boron deficiency

Aims

The cell wall is the main binding site of boron (B) in plants, and the differences in B requirements among different plant species are determined by pectic polysaccharide contents in the cell walls. The aim of this research was to illustrate the relationship between cell wall properties and allocation of B to cell wall and the differential sensitivity of Brassica napus cultivars to B deficiency.

Methods

Two cultivars with opposite B efficiency were used to analyse the relationship among cell wall pectin contents and glycosyl composition, B uptake and allocation, gene expression and cell wall ultrastructure.

Results

The Brassica napus B-efficient cultivar Qingyou 10 was more tolerant to B deficiency, exhibiting a higher biomass production, milder B deficiency symptoms and less cell wall thickening compared to the Brassica napus B-inefficient cultivar Westar 10. These differences were attributed to two factors; the first was that Qingyou 10 accumulated more B and distributed significantly higher proportion of it to the cell wall pectins than did Westar 10 under low B supply. Also, the cell walls of Qingyou 10 exhibited relatively less B-binding sites than those of Westar 10, which was indicated by the lower cell wall extraction rates, less pectin and glycosyl residue contents under the B-deficient and B-sufficient conditions. A comparison of the KDOPS gene expression levels in the two conditions suggests that Westar 10 had a higher potential for biosynthesizing B-binding substances than did Qingyou 10, regardless of B levels.

Conclusions

These results suggest that both higher cell wall pectin polysaccharide content, and limited accumulation and allocation of B to the cell walls contribute to the greater sensitivity of Westar 10 to B deficiency. These two physiological aspects may determine the differences in B deficiency tolerance between Brassica napus cultivars Qingyou 10 and Westar 10. Comparably, the difference in accumulation and allocation of B to cell wall plays a much more important role than cell wall components to sensitivity difference of Brassica napus cultivars to B deficiency.     

Differential response of oilseed rape (Brassica napus L.) cultivars to low boron supply

Three experiments were carried out on an alluvial sandy loam (Udifluvent) at Tonglu, Zhejiang Province, P.R. China from 1992 to 1995, to determine the genotypic range in boron (B) efficiency of 16 oilseed rape (Brassica napus L.) cultivars, to identify the B-efficient cultivars and to identify specific responses which can be utilised for selection in a breeding program. The 16 cultivars which included high-quality and conventional types differed significantly in survival, plant height, leaf area, shoot dry weight and seed yield; however, the ranking of the cultivars for their seed yield or other plant traits differed with B treatment. With severe B deficiency (CaCl2 extractable B < 0.26 mg/kg) and no boron applied, none of the cultivars exhibited significant B efficiency, with seed yield <300 kg/ha. With moderate B deficiency (CaCl2 extractable B 0.34 mg/kg or 0.17 kg B/ha applied), seed yield varied significantly among the cultivars from 397 to 1889 kg/ha in year 1 and from 616 to 1260 kg/ha in year 3. Zhongyou 821 and 92-13 were the most B-efficient and Wanyou 324, Huashuang 2 and Su 2051 were the most B-inefficient cultivars under moderate B deficiency. Significant differences were found among the cultivars in leaf B concentration; however, there was no close relationship between leaf B concentration and seed yield responses to B of oilseed cultivars. Of all the growth parameters measured, leaf area was the early indicator best correlated with subsequent seed yield and may be useful for evaluating the response of cultivars to low B supply. Contrary to current opinion, it was also found that high-quality oilseed rape cultivars were not all sensitive to low B supply nor were all conventional cultivars B-efficient.     

Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of six commercial Brassica napus winter cultivars is described. Two B. napus spring cultivars were analysed for comparison. Five strains of A. tumefaciens with different combinations of nopaline and octopine chromosomal backgrounds and virulence plasmids were used for cocultivation. Selection of putative regenerated transgenic plants was performed on kanamycin- or hygromycin-containing media. The scores of transgenic plants were calculated on the basis of GUS (-glucuronidase) activity, detected by the histochemical X-Gluc test. Target tissue derived from the cut surface of cotyledon petioles resulted in successful transformation with all the winter cultivars tested. Target tissue from hypocotyl segments resulted in a successful transformation with only one winter cultivar. The transformation rates for B. napus winter cultivars in this study were higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and in multiple copies and at multiple loci in the genome. The transgenic plants all grew normally and developed fertile flowers after a vernalization period. After self-pollination, Southern blot analysis of selected GUS active F1 plants revealed that introduced marker genes were stably inherited to the next generation. These data demonstrate that morphologically normal, fertile transgenic plants of B. napus winter cultivars can be achieved with both nopaline- and octopine-derived A. tumefaciens strains. This protocol should have a broad application in improvement of Brassica napus winter cultivars by introduction of foreign genes     

Identification of quantitative trait loci associated with low boron stress that regulate root and shoot growth in Brassica napus seedlings

Brassica napus (Brassicaceae) is among the most important oil crops and a promising biofuel. In the tropics and subtropics, boron (B) deficiency is a major factor limiting Brassica yields. The effect of B on the regulation of root and shoot growth in a doubled haploid (DH) population was evaluated in experiments that utilized hydroponic culture. Strong genetic variability for traits of interest at normal and low B concentrations was demonstrated. Quantitative trait loci (QTL) were analyzed for seven plant growth parameters: increment of primary root length (IPRL), shoot dry weight (SDW), root dry weight (RDW), ratio of RDW to SDW (R/S), shoot B accumulation (SBA), root B accumulation (RBA), and ratio of RBA to SBA [(R/S)BA] in the population. Twenty-seven QTL were detected at normal B levels: four for IPRL, seven for SDW, three for RDW, two for R/S, six for SBA, two for RBA, and three for (R/S)BA. At low B, 18 QTL were detected: four for IPRL, three for SDW, two for RDW, two for R/S, five for SBA, one for RBA, and one for (R/S)BA. Three QTL for adaptability were detected: one A_IPRL and two A_SDW. No putative QTL was detected at both low and normal B. B-related genes were mapped in silico and their locations compared with the QTL identified. The present analyses show the profound and varied effects of B on B. napus and studies on QTL related to B efficiency will help to locate candidate genes and elucidate possible functions of B-efficiency-related QTL.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Proteomics reveals the adaptability mechanism of Brassica napus to short-term boron deprivation

The application of pyrogenic carbon, biochar, to agricultural soils is currently discussed as a win-win strategy to sequester carbon in soil, thus improving soil fertility and mitigate global warming. Our aim was to investigate if biochar may improve plant eco-physiological responses under sufficient water supply as well as moderate drought stress. A fully randomized greenhouse study was conducted with the pseudo-cereal Chenopodium quinoa Willd, using three levels of biochar addition (0, 100 and 200?t ha^?1) to a sandy soil and two water treatments (60% and 20% of the water holding capacity of the control), investigating growth, water use efficiency, eco-physiological parameters and greenhouse gas (GHG) fluxes. Biochar application increased growth, drought tolerance and leaf-N- and water-use efficiency of quinoa despite larger plant?Cleaf areas. The plants growing in biochar-amended soil accumulated exactly the same amount of nitrogen in their larger leaf biomass than the control plants, causing significantly decreased leaf N-, proline- and chlorophyll-concentrations. In this regard, plant responses to biochar closely resembled those to elevated CO₂. However, neither soil- nor plant?Csoil-respiration was higher in the larger plants, indicating less respiratory C losses per unit of biomass produced. Soil-N₂O emissions were significantly reduced with biochar. The large application rate of 200?t ha^?1 biochar did not improve plant growth compared to 100?t ha^?1; hence an upper beneficial level exists. For quinoa grown in a sandy soil, biochar application might hence provide a win-win strategy for increased crop production, GHG emission mitigation and soil C sequestration.     

Molecular mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus.

Doubled haploid (DH) lines together with a cotyledon bioassay were employed for the molecular analysis of resistance to the blackleg fungus Leptosphaeria maculans in the Australian Brassica napus cultivars Shiralee and Maluka. We used bulked segregant analysis to identify 13 RAPD and two RFLP markers linked to the resistance phenotype and mapped these markers in the segregating DH population. Our data suggest the presence of a single major locus controlling resistance in the cultivar Shiralee, confirming our previous results obtained from Mendelian genetic analyses. In addition, preliminary mapping data for the cultivar Maluka also support a single locus model for resistance and indicate that the resistance genes from 'Shiralee' and 'Maluka' are either linked or possibly identical. The molecular markers identified in this study should be a useful tool for breeding blackleg resistant varieties using marker-assisted selection, and are the essential first step towards the map-based cloning of this resistance gene.     

Detection of QTL for phosphorus efficiency at vegetative stage in Brassica napus

Phosphorus (P) deficiency in soils is a major limiting factor for plant growth worldwide. Plants have developed adaptive strategies in response to P deficiency. The objective of this study was to map quantitative trait loci (QTL) for P efficiency using a recombinant inbred (RI) population consisting of 124 lines derived from a cross between Brassica napus P-inefficient cv. B104-2 and P-efficient cv. Eyou Changjia. Six traits (shoot dry weight, root dry weight, root/shoot ratio, P concentration, shoot P uptake and shoot P use efficiency) at vegetative stage were examined under high P (HP, 1 mM) and low P (LP, 5 ??M) conditions during three separate experimental trial periods. Their relative values (i.e., the ratio of a trait value under the LP condition to that under the HP condition) of these six traits were also determined. Eyou Changjia produced more biomass and acquired more P under the LP condition and, thus, had a higher relative dry weight and relative P uptake than B104-2, indicating Eyou Changjia was high P efficiency. A total of 71 QTL were detected on 13 linkage groups, including 28 QTL under the LP condition, 22 QTL under the HP condition and 21 QTL for relative traits. Nineteen and nine QTL were specific for the LP and HP conditions, respectively, suggesting that different mechanisms existed under the two P condition. Twelve of the twenty-one QTL for relative traits co-localized with QTL identified under the two P conditions. In addition, 18 orthologous genes involved in the P metabolic pathway of Arabidopsis were in silico mapped to the QTL confidence intervals identified in B. napus by comparative genomic analysis. These QTL and their corresponding candidate genes should be further investigated to better understand P efficiency in B. napus.     

Hormonal requirement and tissue competency for shoot organogenesis in two cultivars of Brassica napus

An investigation of the regeneration ability of explants taken from the floral stem of Brassica napus var. oleifera was performed in the winter cultivars Darmor and Bienvenu. Our purpose was to compare the regeneration ability of the two genotypes, to compare the competence of the different tissues of the stem and then to study histologically the regeneration of shoots. A strong genotypic effect was observed between the two cvs; Bienvenu had a poorer ability to produce shoots when cultured in the presence of benzyladenine: regeneration commenced later; the percentage of explants producing shoots and the number of shoots per regenerating explant were much lower. The comparison between the regeneration ability of different explants, i.e stem segments, internal stem segments, thin cell layer and peels, showed that the superficial tissues were able to regenerate roots but not shoots. Contrariwise, internal stem segments regenerated only shoots. The origin of shoots was investigated in stem segments of cv. Darmor. A kinetic histological analysis showed the basic role played by phloem and phloem-associated cells in shoot formation.     

中国甘蓝型油菜遗传多样性的RAPD分子标记

本文利用ＲＡＰＤ方法和统计学分析,对我国７省市和国外引进的总计４０份甘蓝型油菜品种的遗传多样性进行了研究。结果表明,４０个品种的甘蓝型油菜存在着广泛的遗传变异,根据ＲＡＰＤ指纹图谱,通过在ＤＮＡ分子水平上的聚类分析可以将它们分为３大类群,反映出这些品种之间的亲缘关系,并对如何引进甘蓝型油菜资源进行了初浅的讨论。     

Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars

Oil bodies were purified from mature seed of two Brassica napus crop cultivars, Reston and Westar. Purified oil body proteins were subjected to both 2-DE followed by LC-MS/MS and multidimensional protein identification technology. Besides previously known oil body proteins oleosin, putative embryo specific protein ATS1, (similar to caleosin), and 11-beta-hydroxysteroid dehydrogenase-like protein (steroleosin), several new proteins were identified in this study. One of the identified proteins, a short chain dehydrogenase/reductase, is similar to a triacylglycerol-associated factor from narrow-leafed lupin while the other, a protein annotated as a myrosinase associated protein, shows high similarity to the lipase/hydrolase family of enzymes with GDSL-motifs. These similarities suggest these two proteins could be involved in oil body degradation. Detailed analysis of the two other oil body components, polar lipids (lipid monolayer) and neutral lipids (triacylglycerol matrix) was also performed. Major differences were observed in the fatty acid composition of polar lipid fractions between the two B. napus cultivars. Neutral lipid composition confirmed erucic acid and oleic acid accumulation in Reston and Westar seed oil, respectively.     

Influence of high light and UV-B radiation on photosynthesis and D1 turnover in atrazine-tolerant and -sensitive cultivars of Brassica napus

An atrazine-tolerant mutant and an atrazine-sensitive cultivar of Brassica napus L. were grown under visible radiation (400 mumol m-2 s-1, photosynthetically active radiation, PAR) and then subjected to treatment conditions. These included short-term high PAR (1600 mumol m-2 s-1) which was given for 4 h either alone or in combination with an enhanced level of UV-BBE radiation (4.6 kJ m-2 h-1 biologically effective UV-B, 280-320 nm). Recovery from the radiation treatment was studied for 4 h under the light conditions for growth. Since it is known that the atrazine-tolerant mutant is susceptible to photoinhibition, one of the aims of the present study was to determine the effects of a supplemental, enhanced level of UV-B radiation with regard to the mutant. The results indicate an additive effect of UV-B radiation on Fv/Fm, photochemical yield and photosynthetic oxygen evolution during both exposure and recovery, and also a higher susceptibility of the mutant to photoinhibitory PAR conditions alone and in combination with UV-B, which may have implications in a changing environment. Both cultivars also showed a higher D1 turnover during the radiation stress than during recovery, as shown by immunoblotting and 35S-methionine incorporation measurements.     

用EST-SSR标记检测我国甘蓝型油菜品种的遗传多样性

杂交育种依然是我国油菜育种的主要方法,杂种优势的利用仍然是提高产量的重要途径.为了解我国甘蓝型油菜的遗传变异,采用16个EST-SSR标记对近年来推广的91个品种的遗传多样性进行了分析.共扩增到100个条带,其中84个多态性带,多态性比率为84%.平均每对引物扩增的带数和多态性带数分别为6.25个和5.25个.多态性信息含量(PIC)变化在0.022-0.926之间,平均为0.677,所揭示的基因型数变化于2-24之间,平均为12.44个.供试材料间遗传距离变幅较大(0.0530-0.7223之间),说明它们具有广泛的遗传变异.其中,杂交种和2000年以后育成品种的遗传基础较宽,遗传多样性分别明显高于常规品种和2000年以前育成的品种.按非加权成对平均数法(UPGMA)进行的聚类分析显示,在遗传距离为0.313处,参试材料可以分为三大类,其中,包含87份材料的第一大类在遗传距离为0.233处又可进一步分为10个亚类.聚类结果与系谱来源基本一致,比较真实反映了所用材料的遗传变异情况.     

Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus

Boron (B) is essential for vascular plants. Rapeseed (Brassica napus) is the second leading crop source for vegetable oil worldwide, but its production is critically dependent on B supplies. BnaA3.NIP5;1 was identified as a B-efficient candidate gene in B. napus in our previous QTL fine mapping. However, the molecular mechanism through which this gene improves low-B tolerance remains elusive. Here, we report genetic variation in BnaA3.NIP5;1 gene, which encodes a boric acid channel, is a key determinant of low-B tolerance in B. napus. Transgenic lines with increased BnaA3.NIP5;1 expression exhibited improved low-B tolerance in both the seedling and maturity stages. BnaA3.NIP5;1 is preferentially polar-localized in the distal plasma membrane of lateral root cap (LRC) cells and transports B into the root tips to promote root growth under B-deficiency conditions. Further analysis revealed that a CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 altered the expression level of the gene, which is tightly associated with plant growth and seed yield. Field tests with natural populations and near-isogenic lines (NILs) confirmed that the varieties carried BnaA3.NIP5;1^Q allele significantly improved seed yield. Taken together, our results provide novel insights into the low-B tolerance of B. napus, and the elite allele of BnaA3.NIP5;1 could serve as a direct target for breeding low-B-tolerant cultivars.     

Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus

Background and Aims

Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome A^rA^rC^cC^c) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation.

Methods

Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome AⁿAⁿCⁿCⁿ) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers.

Key Results

Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64·97 %) compared with D1-1 (32·23 %).

Conclusions

The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.     

甘蓝型特高含油量油菜种质资源及主栽品种指纹图谱构建

本研究选择特高含油量资源7份,与中国各油菜主产区具有代表性的主栽品种16份,利用SSR多引物组合法开展指纹图谱构建研究。选择多态丰富、图谱清晰稳定且来自不同连锁群的引物28对,对所有材料进行指纹图谱分析,共获SSR指纹条带302条,其中多态性条带为279条,每引物所获条带6-16条,平均10.79条,平均多态率92. 38%,通过指纹图谱将所有材料有效地区别开来。用非加权类平均法（UPGAM）聚类分析显示：高油材料之间以及高油材料与主栽品种之间遗传距离均有较大差异,在遗传距离0.171处可将23份材料分成9个类群,其中7份高油材料分处4个类群,遗传距离差异显著;而其他8份主栽品种被分别聚类在另外5个类群中;所有材料间皆具有丰富的遗传多样性,其中高油材料与主栽品种间遗传差异更大。     

Sodium and boron exclusion in two Brassica juncea cultivars exposed to the combined treatments of salinity and boron at moderate alkalinity

Salinity and high boron (B) concentrations are important co-limitations to crop production on naturally occurring alkaline soils in low rainfall regions of the world. Although the interactive effects of salinity and B toxicity on Brassica juncea growth have been reported in slightly acidic soils, very little is known about the interactive effects in alkaline soils. In the current study, a moderately tolerant (Vaibhav) and sensitive (Xinyou5) variety, were grown hydroponically for four weeks to assess mild salinity (50 mM NaCl) with or without high B (1 mM B) at moderate alkalinity (pH 8.5/5 mM NaHCO₃). The growth of the two varieties was more affected under the combined treatment than either salinity or high B alone. Although growth rate reduction was similar among the varieties, Vaibhav maintained a lower sodium (Na) and B and a higher potassium (K) concentration in the leaves than Xinyou5. In response to salinity, Vaibhav demonstrated essential tolerance mechanisms of partial exclusion and presumably compartmentalization of Na, leading to greater biomass than Xinyou5. Despite being able to better exclude B, Xinyou5 suffered a greater growth penalty, indicating higher B sensitivity than Vaibhav. In conclusion, screening for individual stresses is not necessarily the best strategy because plant responses to a single stress either salinity or high B may not always be the same as observed when both stresses are present together. Therefore, Brassica germplasm screening is essential for stresses in combination but not separately.