Isolation and characterization of LEAFY COTYLEDON 1-LIKE gene related to embryogenic competence in Citrus sinensis

A member of the LEAFY COTYLEDON gene family encoding a HAP3 (heme activated protein 3) subunit of the CCAAT box-binding factor was isolated and termed as Citrus sinensis LEAFY COTYLEDON 1-LIKE (CsL1L). The deduced amino acid sequence shared a high similarity with LEAFY COTYLEDON 1-LIKE (L1L) in Arabidopsis thaliana, Phaseolus coccineus, Theobroma cacao, and Helianthus annuus. Quantitative RT-PCR results indicated that CsLIL was highly expressed in embryogenic callus, somatic embryos and immature seeds, but was rarely detected in non-embryogenic callus, vegetative and floral tissues. Ectopic expression of CsL1L in vegetative tissues could induce embryo-like structures, suggesting that CsL1L has the capability to transit cells from vegetative to embryogenic phase. Comparison of CsL1L expression in the newly formed and long-term subcultured embryogenic calli of W. Murcott tangor (C. sinensis × C. reticulata) and Hongkong kumquat (Fortunella hindsii Swingle) revealed that the potency of embryogenesis was related to the level of CsL1L expression. Sub-cellular localization analysis indicated that CsL1L was a nuclear protein in plant. A microsatellite in CsL1L was verified with polymorphism among the citrus species.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing

Peroxidase activity in isolated thylakoids from degreening canola ( Brassica napus cv. Westar) seeds was demonstrated. The enzyme catalyzes the degradation of thylakoid-bound pigments in the presence of H₂O₂ and 2,4-dichlorophenol. Peroxidase activity is related to degreening, with periods of rapid degreening associated with high enzyme activity. Both de novo synthesis and substrate availability appear to control enzyme activity. Peroxidase is initially inhibited and then stimulated by sublethal freezing. Therefore, inhibition of peroxidase activity following sublethal freezing may be responsible, in part, for a failure of the seed to degreen.     

The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase 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.     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

Confirmation of QTL controlling seed yield in spring canola (Brassica napus L.) hybrids

Allelic effects observed in QTL discovery experiments must be confirmed to be useful in subsequent breeding efforts. Two QTL affecting seed yield of spring hybrid canola (Brassica napus L.) were previously identified in two populations of inbred backcross lines (IBLs) containing germplasm introgressed from a winter cultivar. The effects of favorable alleles at these QTL were retested by crossing two selected IBLs (M5 and M31) to three spring canola lines having different genetic backgrounds. Doubled haploid (DH) lines derived from each F₁ were genotyped with RFLP markers flanking the QTL and grouped into the four possible QTL genotypes. For the first field experiment, DH lines derived by crossing the M5 line to one spring line were crossed to two female testers and evaluated as individual testcross progenies in one environment. QTL genotypes had large variances and were not significantly different. A second field experiment was conducted using the DH lines from the first experiment and two other sets of DH lines derived from the M31 line crossed to two different spring canola lines. Individual lines within each QTL genotype of each set were bulked and crossed to the same testers used in Experiment 1. Bulked hybrid seeds of each QTL genotype were planted in a split-split plot randomized block design and 12 replicates. QTL genotypes had smaller variances in this experiment, and the effects of one QTL were confirmed in some genetic backgrounds. These results suggest that bulking of QTL genotypes and use of an appropriate experimental design with many replicates are needed to detect small differences between QTL genotypes.     

LEAFY COTYLEDON 2 activation is sufficient to trigger the accumulation of oil and seed specific mRNAs in Arabidopsis leaves

LEAFY COTYLEDON 2 (LEC2) is a key regulator of seed maturation in Arabidopsis. To unravel some of its complex pleiotropic functions, analyses were performed with transgenic plants expressing an inducible LEC2:GR protein. The chimeric protein is functional and can complement lec2 mutation. Interestingly, the induction of LEC2 leads to the accumulation of storage oil in leaves. In addition, short-term induction and use of translation inhibitors allowed to demonstrate that LEC2 can directly trigger the accumulation of seed specific mRNAs. Consistent with these results, the expression of three other major seed regulators namely, LEC1, FUS3, and ABI3 were also induced by LEC2 activation.     

Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed

Chlorophyllase and peroxidase activities were measured in relation to seed maturation and degreening in canola ( Brassica napus cvs Westar and Alto) and mustard ( Brassica juncea cvs Cutlass and Lethbridge 22A). Samples of seed collected at the same moisture content were pooled, then divided and used for each assay. During maturation the green pigment (chlorophyll and related pigments) content of canola seed decreased linearly and was lower than that measured in mustard at all moisture contents studied, except for the highest and lowest moisture contents. Chlorophyllides and pheophorbides were detected in canola and were essentially absent in mustard. This difference in accumulation of dephytylated pigments infers differences in the pigment degradation pathways in Brassica species. Interspecific differences in the enzymology of degreening were found. Green pigment degradation was associated with increased chlorophyllase activity and low peroxidase activity in canola and low Chlorophyllase and high perosidase activity in mustard. The possible role of ethylene in seed degreening is discussed.     

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.     

Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus

Rapeseed (Brassica napus) is one of the most important edible oilseed crops in the world and is increasingly used globally to produce bio-diesel. Therefore, increasing oil content of oilseed corps is of importance economically in both food and oil industries. The wri1 genes are differentially expressed in B. napus lines with different oil content. To investigate the effects of B. napus WRI1 (BnWRI1) on oil content, two Bnwri1 genes with different lengths, Bnwri1-1 and Bnwri1-2, were identified and sequenced. Homology analysis shows 80% amino acids of Bnwri1s are homologous to Arabidopsis thaliana WRI1 (AtWRI1). Overexpression of Bnwri1 cDNAs driven by cauliflower mosaic virus 35S-promoter in 51 transgenic A. thaliana lines resulted in 10–40% increased seed oil content and enlarged seed size and mass. Detailed analysis on transgenic embryos indicates an increased cell size other than cell number. In addition, Bnwri1 sequence polymorphism is highly related to oil content (p < 0.001). Taking together, Bnwri1 has potential applications in food and oil industries and in rapeseed breeding.     

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。     

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。