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.     

Ethylene Production during Development of Mustard (Brassica juncea) and Canola (Brassica napus) Seed

An open, continuous flow system was used to investigate ethylene production during degreening of maturing seed of mustard (Brassica juncea cv Cutlass and cv Lethbridge 22A) and canola (Brassica napus cv Westar and cv Alto). Isolated mustard seed evolved higher amounts of ethylene than those of canola, and this was particularly evident both early in embryogeny and later during the desiccation phase of seed maturation. The silique walls produced negligible amounts of ethylene in both species. The concentrations of ethylene surrounding seed as they matured within siliques were significantly higher in mustard than in canola, and this interspecies difference was greatest during the seed desiccation phase. In mustard, a 4-fold increase in silique internal ethylene levels was apparent during desiccation. In comparison, only a moderate increase in silique-derived ethylene occurred in canola.     

Relationship of chlorophyll, seed moisture and ABA levels in the maturing Brassica napus seed and effect of a mild freezing stress

Chlorophyll (Chl) retention by mature seed of canola as the result of an early frost or other environmental factors (the "green seed problem") causes serious economic losses. The relationship of seed degreening to rate of moisture loss by seed and silique and the role of ABA in this process were investigated as a function of developmental age. During the normal predesiccation stage (28–45 days after pollination), seed of Brassica napus (cv. Westar) loses Chl rapidly but seed moisture slowly. After a mild freezing stress, there is a rapid loss of moisture from silique walls, followed by accelerated loss of seed moisture. Chl degradation ceases at 35–45% seed moisture. ABA levels in silique walls of frozen plants (determined by enzyme‐linked immunosorbant assay) increased after freezing, apparently in response to moisture loss. In contrast, ABA levels in the seed increased dramatically 1 day after freezing, then decreased to control levels. The influence of the rate of seed moisture loss on Chl degradation was investigated by fast and slow drying of isolated seed under controlled humidity conditions. Seed dried rapidly at 22% RH retained most of its Chl, whereas seed dried slowly at 86% RH lost Chl as fast or faster than seed on control (unfrozen) plants. In all treatments, Chl loss stopped at about 40% seed moisture.     

Non-lethal freezing effects on seed degreening in Brassica napus

The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28–44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.     

Biochemical Changes Associated With Brassica juncea Seed Development. IV. Acid and Alkaline Phosphatases

Changes in acid and alkaline phosphatase activities in cytoplasmic and wall-bound fractions of developing mustard (Brassica juncea) seed were studied. Growth was measured by seed dry weight and water content. Seed dry weight data were fitted to a cubic polynomial equation. Seed water content and dry matter accumulation was significantly correlated. Cytoplasmic acid and alkaline phosphatase activities were substantially less in the cytoplasmic fraction than the wall-bound fraction. Wall-bound acid phosphatase activity was low initially, but high levels were maintained after day 25, indicating a relationship with dry matter accumulation. The results suggest that acid phosphatase plays an important role during mustard seed development. Received February 19, 1998; accepted May 6, 1999     

Gene transfer between canola (Brassica napus L. and B. campestris L.) and related weed species

Brassica species are particularly receptive to gene transformation techniques. There now exists canola genotypes with transgenic herbicide resistance for glyphosate, imidazolinone, sulfonylurea and glufosinate herbicides. The main concern of introducing such herbicide resistance into commercial agriculture is the introgression of the engineered gene to related weed species. The potential of gene transfer between canola (Brassica napus and B. campestris) and related weed species was determined by hand pollination under controlled greenhouse conditions. Canola was used as both male and female parent in crosses to the related weed species collected in the Inland Northwest region of the United States. Weed species used included: field mustard (B. rapa), wild mustard (S. arvensis) and black mustard (B. nigra). Biological and cytological aspects necessary for successful hybrid seed production were investigated including: pollen germination on the stigma; pollen tube growth down the style; attraction of pollen tubes to the ovule; ovule fertilisation; embryo and endosperm developmental stages. Pollen germination was observed in all 25 hybrid combinations. Pollen tubes were found in the ovary of over 80% of combinations. About 30% of the hybrid combinations developed to the heart stage of embryo development or further. In an additional study involving transgenic glufosinate herbicide resistant B. napus and field mustard it was found that hybrids occurred with relatively high frequency, hybrids exhibited glufosinate herbicide resistance and a small proportion of hybrids produced self fertile seeds. These fertile plants were found to backcross to either canola or weed parent.     

Genetic analysis of agronomic and quality traits in mustard (Brassica juncea)

To develop an efficient mustard (Brassica juncea) breeding programme, a better knowledge of the genetic control and relationships of the main selected characters is needed. Thus, doubled haploid (DH) lines were evaluated over 2 years in the field. Days to flowering, plant height, thousand-seed weight, fatty acid composition, seed oil content, sinigrin, gluconapin and total glucosinolate contents were determined in the DH population. The influence of seed coat colour was estimated. Results showed significant differences between yellow and brown seeds only for oil and fatty acid contents. Molecular analysis revealed that seed coat colour is associated with two Mendelian trait loci: Bjc1 [on linkage group (LG) 3] and Bjc2 (on LG6). The quantitative trait loci associated with characters—detected by composite interval mapping—were not co-localised and revealed a genetic independence. The results obtained in this study show that the most important agronomic and quality traits of brown mustard could be bred independently. Correlation between the studied traits is also discussed.     

The use of haploidy to develop plants that express several recessive traits using light-seeded canola (Brassica napus) as an example

Summary The use of haploidy to introgress recessive traits into Brassica napus canola is illustrated by describing the properties of doubled haploids obtained by microspore culture from crosses between a yellow-seeded rapeseed line (low erucic acid, high glucosinolate) and black-seeded canola. Of the 99 doubled haploid lines that were produced, 3 were yellow-seeded canola lines. This result was not significantly different than the predicted frequency of 1 in 64 for the homozygous recessive phenotype in a doubled haploid population segregating for six recessive genes. Thus, the study supports previous models of inheritance determined for yellow seededness and glucosinolate content in Brassica napus. Also, since the chances of obtaining a plant with the same characteristics in a F₂ population are 1 in 4,096, the underscore results the advantages of using haploidy to introgress recessive traits into Brassica napus canola.     

Biochemical Changes Associated with Brassica juncea Seed Development, II: Glycosidases

Changes in α- and β-galactosidase, glucosidase, and α-mannosidase and β-xylosidase activities were analyzed in developing mustard (Brassica juncea) seed. A cubic polynomial described the seed dry weight data adequately. A close parallelism between the water content and dry matter accumulation was shown (r= 0.991). Glycosidase activities were detected in both cytoplasmic and wall fractions. The trend was similar in both of the fractions, but the activity of α-mannosidase and α-galactosidase was considerably greater in the wall-bound fraction. The water content and the activity of glycosidic enzymes showed a significant linear correlation (p < 0.001). The results suggest that glycosidases have an important role in cell wall loosening during mustard seed development. Received July 10, 1997; accepted January 28, 1998     

Pronounced effects of slug herbivory on seedling recruitment of Brassica cultivars and accessions,especially those with low levels of aliphatic glucosinolates

In demographic studies on Brassica cultivars and accessions we observed large genotypic variation in the ability of seedlings to establish. Here we quantify the role of slugs during establishment by including and excluding slug herbivory.Slug exclusion during the first 10 days after germination led to 26.5 times more surviving seedlings in modern canola varieties of Brassica napus, 4.3 times more in feral B. napus and 1.9 times more in wild Brassica rapa. While seed and seedlings of canola were low in aliphatic glucosinolates (AGS), feral B. napus and wild B. rapa had much higher AGS levels. Consequently, we find a positive correlation between the survival of young seedlings and AGS concentration. Concentrations of indole glucosinolates (IGS) in seeds were much lower than those of AGS and did not correlate significantly with survival. Subsequently, we exposed 10-day-old seedlings to herbivory. In the 4 days following exposure, slug herbivory was negatively correlated to AGS concentration but not to IGS.In choice experiments in the lab, the preference of the slug Arion lusitanicus also correlated negatively with AGS content. A. lusitanicus preferred to feed on B. napus rather than on wild B. rapa, but had no significant preference when presented with plants similar in AGS content.Slugs can be a limiting factor for seedling recruitment in populations of B. napus, especially for modern canola cultivars with a low AGS content.     

Antioxidants from defatted Indian Mustard (Brassica Juncea) protect biomolecules against in vitro oxidation

Indian mustard seeds were defatted by distillation with hexane and the residue extracted with methanol was analyzed for potential antioxidants; ascorbate, riboflavin, and polyphenols. Gallic acid (129.796 μg), caffeic acid (753.455 μg), quercetin (478.352 μg) and kaempferol (48.060 μg)/g dry seeds were identified by HPLC analysis of the extract. DPPH free radical scavenging activity and protection of lipids, proteins and DNA against metal induced oxidation was examined. Defatted mustard seed remnant had excellent free radical scavenging activity and protects biomolecules with IC₅₀ value 2.0–2.25 mg dry seed weight. Significant content of polyphenols in methanol extract of defatted seeds accounts for high antioxidant potential. We are the first to report the detailed analysis of antioxidant composition and protection of biomolecules against oxidative damage by methanol extract of mustard seed remnant after oil extraction.     

Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity subjected to cadmium stress

Photosynthetic performance, contents of chlorophyll and associated pigments, cellular damage and activities of antioxidative enzymes were investigated in two mustard (Brassica juncea L.) cultivars differing in photosynthetic capacity subjected to cadmium (Cd) stress. Exposure to Cd severely restricted the net photosynthetic rate (P(N)) of RH-30 compared to Varuna. This corresponded to the reductions in the activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase (Rubisco) in both the cultivars. Decline in chlorophyll (Chl) (a+b) and Chl a content was observed but decrease in Chl b was more conspicuous in Varuna under Cd treatments, which was responsible for higher Chl a:b ratio. Additionally, the relative amount of anthocyanin remained higher in Varuna compared to RH-30 even in the presence of high Cd concentration, while percent pheophytin content increased in RH-30 at low Cd concentration. A higher concentration of Cd (100 mg Cd kg(-1) soil) resulted in elevated hydrogen peroxide (H(2)O(2)) content in both the cultivars. However, Varuna exhibited lower content of H(2)O(2) in comparison to RH-30. This was reflected in the increased cellular damage in RH-30, expressed by greater thiobarbituric acid reactive substances (TBARS) content and electrolyte leakage. The enhanced activities of antioxidative enzymes, ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) and also lower activity of superoxide dismutase (SOD) in Varuna alleviated Cd stress and protected the photosynthetic activity.     

Cytochemical Localization of Phenolic Compounds in Columella Cells of the Root Cap during Maturation of Seeds of Brassica napus L.

Abstract: Ultrastructural localization of phenolic compounds in the columella of the root cap of a canola embryo in Brassica napus L. during subsequent seed maturation stages was investigated. In bright green seeds the first signs of maturation and appearance of the first phenolic structures in the cell nuclei and cytoplasm in the area of the initial centre were found. Phenolics spread acropetally, gradually covering the whole columella in the dark green seeds. An increase in the number of phenolics occurred, together with an increase in the number of endoplasmic reticulum structures and in storage materials. In brown seeds, together with ultrastructural degradation and the end of production of storage substances, the number of phenolic deposits in the nucleus and cytoplasm decreases until their disappearance in black dormant seeds. Simultaneously, numerous phenolic deposits appear between the plasmalemma and the cell walls in the outer layer of the cap columella.     

聚乙二醇（PEG）浸种对茎瘤芥老化种子活力及其幼苗生长的影响

茎瘤芥品种‘永安小叶’老化种子经聚乙二醇（PEG）浸种后,其种子活力和幼苗生长与PEG浓度有一定的相关性,不同浓度PEG浸种后的茎瘤芥种子膜透性均降低,种子发芽率、发芽势、发芽指数和活力指数增大,幼苗根干重、茎干重和根长也均增大,幼苗叶片中丙二醛（MDA）含量降低。幼苗生长与种子活力的变化趋势一致,PEG浓度小于30%时呈增大趋势,大于30%时呈减小趋势,但均大于不做PEG处理的;幼苗叶片中MDA含量与种子膜透性变化趋势一致,PEG浓度小于25%时,呈降低趋势,大于25%时则呈升高趋势,但这些指标均低于不做PEG处理的。     

Reproductive rate of the Indian mustard aphid (Lipaphis erysimi pseudobrassicae) on different Brassica oilseeds: colmparisons with Swedish strains of mustard (Lipaphis erysimi erysimf) and cabbage aphid (Brevicoryne brassicae)

The reproductive rate of the Indian mustard aphid (Lipaphis erysimi pseudobrassicae), a serious pest on Brassica oilseeds in India, was compared with that of Swedish strains of mustard aphid (L. e. erysimi) and cabbage aphid (Brevicoryne brassicae) on six common Indian and Swedish Brassica oilseed cultivars. There were clear differences between L. e. erysimi and L. e. pseudobrassicae in the relative suitability of the cultivars, emphasisipg the biological differences between these aphids. B. brassicae had similar reproductive rates on all cultivars.
In addition, the reproductive rate of L. e. pseudobrassicae was measured on both young and old plants of the six Brassica oilseed cultivars and 14 Indian rai (B. juncea) cultivars. Among the six Brassica cultivars, the reproductive rates were highest on turnip rape and toria (B. campestris), and lowest on yellow sarson (B. campestris) and rai (B. juncea). The ranking of the cultivars was similar for young and old plants. There were no significant differences between the rai (B. juncea) cultivars tested, neither among young nor among old plants. These results from laboratory studies are compared with field data from India on the relative susceptibility of different Brassica oilseed cultivars to L. e. pseudobrassicae.     

硼对大白菜生长发育及若干生理生化指标的影响

研究了不同水平的硼对大白菜生长发育及若干生理生化指标的影响。结果表明,缺硼明显抑制大白菜植株生长、抽薹和开花,导致花粉活力下降、叶绿素含量降低、可溶性糖含量增加、膜脂过氧化产物丙二醛(MDA)含量增加、超氧化物歧化酶(SOD)活性下降、过氧化物酶(POD)活性上升,最终导致植株代谢紊乱,生长发育受抑制。