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.     

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.     

The impact of freezing during maturation on storage products in canola seeds

Mature canoia ( Brassica napus cv. Westar) seeds contain large quantities of the storage proteins cruciferin and napin and storage lipids rich in C18: 1 and C18:2 fatty acids. Both the quantity and quality of these products are altered by freezing during development. Further, the response to freezing changes during seed development. The effects include decreased fatty acid chain elongation, altered fatty acid unsaturation, higher lipid levels and lower protein levels. In addition, seeds in the pivotal moisture range (55%) may be predisposed to precocious germination, which is then inhibited by a lack of adequate seed moisture. The results indicate that freezing imparts its effect in two ways. Initially, there is a freezing (low temperature) component and this is followed by rapid desiccation of the seeds. Although most responses probably result from a combination of the stresses, it appears that inhibition of fatty acid chain elongation is caused by the freezing component and the gradual inhibition of storage protein accumulation is a result of accelerated seed desiccation.     

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.     

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.     

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8 days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8‐DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1‐altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over‐representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage‐specific and SHB1‐induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.     

Effects of seeding date and canola species on seedling damage by flea beetles in three ecoregions

Flea beetles ( Phyllotreta striolata (Fabricius), Phyllotreta cruciferae (Goeze) and Psylliodes punctulata Melsheimer) (Coleoptera: Chrysomelidae) are chronic threats to canola seedling establishment throughout the Northern Great Plains of North America. We conducted field studies in AB, Canada, from 2001 to 2004 at Lethbridge, Lacombe and Beaverlodge in the southern, central and northern regions of the province, respectively, to assess the impacts of seeding date (fall, April, May), and canola species ( Brassica rapa L., Brassica napus L.) on flea beetle damage to canola. Flea beetle damage was generally low at Lacombe but reached economic threshold levels at other sites. Although our results were not consistent in all years, seeding date had opposite effects on flea beetle damage to canola seedlings at the southern and northern sites. In the south, both canola species planted in April escaped flea beetle damage unlike the May-seeded plots, which required insecticide spraying in 2003. The numbers of flea beetles on sticky cards sampled during the seedling stage of canola were also lower in early-planted plots than in those planted in May. In the north, flea beetle damage was lower in the May-planted plots compared with those planted earlier. To maximize yields, canola growers need to plant as early as possible in all agro-ecoregions; our study showed that crops in the central and northern regions will be at greater risk of flea beetle damage than in southern regions.     

An analysis of seed development in Pisum sativum. VII. Embryo development and precocious germination in vitro

Three different culture media have been examined for their ability to support growth in culture of embryos of two pea lines near-isogenic except for the r-locus. Embryos showed a greater increase in fresh weight on a medium containing 10% sucrose and a high level of a mixture of amino acids than on either one containing an equivalent amount of glutamine as the sole nitrogen source or one containing both inorganic nitrogen and a low level of glutamine. Small embryos (up to 10 mg fresh weight) showed the greatest relative increase in fresh weight. Decreasing the osmotic pressure of an agar medium by lowering the sucrose content to 2% and reducing the concentration of amino acids induced precocious germination. Shoot growth was more sensitive than root growth to increasing sucrose concentrations and optimum development was obtained when embryos were cultured in liquid culture at a high osmotic pressure followed by growth on an agar medium at low osmotic pressure. Alternatively, precocious germination could be induced by removing the cotyledons. Embryos of all sizes and of both genotypes of pea responded in a similar manner.     

Water stress injuries and tolerance as related to potassium efflux from winter rape hypocotyls

Desiccation- or immersion-induced injuries in isolated winter rape ( Brassica napus L. var. oleifera cv. Górczański) hypocotyls were highly correlated with potassium efflux and were reversible if the tissue damage was not severe (i.e. when less than 20% of electrolytes leaked from stressed tissue). The time-course of leakage of potassium and other electrolytes showed that increased membrane permeability might result from stress-induced disturbances in membrane integrity. Increased stress tolerance in hypocotyls predehydrated to a water saturation deficit of about 60% seemed to be due to stabilization of membrane structure rather than to promotion of a repair mechanism. The hardening effect of predehydration was more effective for potassium efflux than for efflux of other electrolytes.     

Changes in starch and soluble sugars in relation to the acquisition of desiccation tolerance during maturation of Brassica campestris seed

Abstract. We investigated the onset of desiccation tolerance in developing embryos of Brassica campestris seeds and possible correlated ultrastructural modifications in the radiele cells. Since the acquisition of desiccation tolerance is a long asynchronous process which took 9 d to be achieved, we determined criteria allowing us to separate freshly intact harvested seeds into desiccation intolerant and desication tolerant batches that differed in age by only 2 d. No particular structural modifications were found except a strong depletion of intraplastidial starch (-90%) coincident with the appearance of stachyose and an increase of sucrose (+30%) on the acquisition of desiccation tolerance. As we did not observe an increase of lipid reserves as a consequence, we suggest that these metabolic events can be a key factor towards the acquisition of desiccation tolerance.     

Greenhouse and field evaluations of transgenic canola against diamondback moth, shape Plutella xylostella, and corn earworm, shape Helicoverpa zea

Canola (Brassica napus L.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis (Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea (Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and 90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and 95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995–1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non-transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.     

Canola seed germination and seedling emergence from pre-hydrated and re-dried seeds subjected to salt and water stresses at low temperatures

Low soil temperatures and low water potentials reduce and delay the seed germination of canola (Brassica rapa L., B. napus L.) in western Canada. Germination is also very sensitive to the salinity effects of nitrogen fertiliser placed with the seed, especially when the seed bed is relatively dry. The effects of pre-hydration and re-drying treatment on canola (Brassica rapa L. cv. Tobin) seed germination and seedling emergence at 10°C subjected to either a water or salt stress were determined. Low water potentials, induced by polyethylene glycol (PEG 8000), low soil moisture, or high concentrations of salts, reduced both germination and seedling emergence, and increased the time to 50% germination and emergence of seeds at 10°C. At equal osmotic potentials, Na₂SO₄ was less inhibitory on low temperature germination than either NaCl or PEG, suggesting that the sulphate ion partially alleviated the inhibitory effects of low water potential. Solutions of NaCI produced more abnormal seedlings compared to Na₂SO₄, suggesting that NaCl was more toxic than Na₂SO₄ during seedling development. Pre-hydration and re-drying partially overcame the inhibitory effects of both low water potential and salts on seed germination and seedling emergence at 10°C. The seed treatment increased the germination rate in Petri dishes and seedling emergence from a sandy loam soil. Water potentials or soil water contents required to inhibit 50% germination or emergence at 10°C were lower for treated seeds compared to control seeds. Salt concentrations inhibiting 50% emergence were higher for treated seeds than control seeds. Neither treated nor control seeds produced seedlings which emerged if the soil water content was lower than 9% or when the soil was continuously irrigated with salt solutions of 100 mmol kg^-1 of NaCl or 50 mmol kg^-1 of Na₂SO₄. These results suggest that the pre-hydration and re-drying treatment did not lower the base water potentials at which seedling emergence could occur. Abnormal seedlings were observed in both treated and control seeds, particularly if the soil was watered with NaCl solutions; however, the seed treatment reduced the number of abnormal seedlings.     

壳聚糖包衣对油菜种子萌发及幼苗耐盐性影响

以不同浓度的壳聚糖对油菜种子进行包衣处理,考察其对油菜种子萌发及幼苗耐盐性的影响,并在不同盐浓度胁迫条件下对种子萌发时的发芽势、发芽率、生物量（鲜重、干重、根长、芽长）等指标进行测定,同时分析油菜幼苗叶绿素含量、可溶性蛋白及可溶性糖含量的变化。结果表明,一定浓度的壳聚糖包衣处理可提高油菜种子发芽率、发芽势、生物量、幼苗的耐盐指数、叶绿素含量、可溶性蛋白及可溶性糖的含量,其中浓度为0.25 g·L^-1壳聚糖包衣处理对油菜种子萌发的促进效果较好,而浓度为0.50 g·L^-1壳聚糖包衣处理对提高油菜幼苗耐盐性具有较好的促进作用。     

重金属对油菜种子萌发和胚根生长的影响

分析了Hg2 、Cd2 、Ni2 、Co2 、Zn2 5种重金属离子对油菜种子萌发和胚根伸长的影响,以及金属离子K 、Mg2 和Ca2 与重金属的交互作用。结果表明:(1)重金属对油菜种子萌发的抑制作用依次为Hg2 >Cd2 和Co2 >Ni2 >Zn2 ,而对胚根生长的毒害作用依次为Hg2 >Cd2 >Co2 >Ni2 >Zn2 。(2)萌发率为40%以上时,K 和Ca2 可以提高Ni2 、Zn2 和Co2 胁迫下油菜种子的萌发率,却进一步降低了Hg2 、Cd2 胁迫下种子的萌发;Mg2 可以提高Ni2 、Zn2 、Cd2 和Co2 胁迫下种子的萌发率,但对Hg2 毒害却没有缓解。(3)胚根伸长率达到60%以上时,K 和Mg2 增强了Ni2 、Hg2 、Cd2 和Co2 对胚根生长的抑制,而Ca2 则缓解了Zn2 、Ni2 和Co2 对胚根生长的抑制作用。研究结果对于重金属复合污染土壤中植物种子的萌发和定植具有理论和实践意义。     

Protective mechanism of desiccation tolerance in Reaumuria soongorica: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was char-acterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then de-clined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(ΦPSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dis-sipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than –21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon re-watering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation.     

Protective mechanism of desiccation tolerance in Reaumuria soongorica: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was characterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then declined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(Φ_PSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dissipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than −21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon rewatering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation. Supported by the Program of the Research of Vegetation Restoration in Arid Areas of Lanzhou (Grant No. 03-2-27) and the National Natural Science Foundation of China (Grant No. 30270243)     

Photoinhibition of radish (Raphanus sativus L) seed germination: control of growth potential by cell-wall yielding in the embryo

The biophysical mechanism underlying photoinhibition of radish (Raphanus sativus L.) seed germination was investigated using three cultivars differing in sensitivity to continuous irradiation with far-red light (high-irradiance reaction of phytochrome). Sensitivity of germination to the inhibitory action of light was assessed by probing germination under osmotic stress (incubation in media of low water potentials adjusted with polyethylene glycol 6000) and expressed in terms of ‘germination potential’ (positive value of the water potential at which germination is inhibited by 50%). Far-red light decreases the germination potential to various degrees in the different cultivars, reflecting the light-sensitivity of germination in water. Removal of the seed coat increases the germination potential by a constant amount in darkness and light. It is concluded that germination depends on the expansive force of the embryo which can be drastically diminished by far-red light. Seed-coat constraint and expansive force of the embryo interact additively on the level of the germination potential. Photoinhibition of germination was accompanied by an inhibition of water uptake into the seed. Analysis of seed water relations showed that osmotic pressure and turgor assumed higher levels in photoinhibited seeds, compared to seeds germinating in darkness, while the water potential was close to zero under both conditions. Far-red light produced a shift (to less negative values) in the curve relating water-uptake rate to external water potential, i.e. a reduction in the driving force for water uptake. It is concluded that photoinhibition of germination results from the maintenance of a high threshold of cell-wall extensibility in the embryo.     

Dynamics of carbohydrates in the embryo axes of horse chestnut seeds during their transition from dormancy to germination

It was shown that the content of carbohydrates and their composition in embryo axes of horse chestnut seeds changed as seeds acquired a capability of dormancy release and germination. Sucrose prevailed among carbohydrates, comprising to 150–160 mg/g dry wt. During the first half of the seed imbibition time, oligosaccharides, namely raffinose and stachyose, degraded, whereas the contents of glucose and fructose were very low. The second half of the imbibition period (until radicle protrusion) was characterized by a cessation of oligosaccharide breakdown and accumulation of monosaccharides. Carbohydrate balance showed that the contribution of oligosaccharide breakdown to sucrose and monosaccharide accumulation was rather small, and monosaccharides accumulated mostly at the expense of sucrose gradually coming from cotyledons during imbibition. The trend of carbohydrate metabolism in imbibing axial organs was similar during the entire period of a seed dormancy release in the course of stratification. A readiness for the commencement of these processes during the entire dormancy period implies that carbohydrate conversions in embryo axes are not a trigger for a dormancy release. Monosaccharide accumulation in embryo axes before radicle protrusion produces an increase in the osmotic pressure, as compared to that provided by sucrose, by approximately 20%. Recalcitrance of the horse chestnut seeds is discussed in relation to the role of carbohydrates and other endogenous osmotica in the establishment of osmotic properties.     

Seed dormancy in Acer: The role of abscisic acid in the regulation of seed development in Acer platanoides L.

Germinability of isolated embryos from developing fruits of Acer platanoides was high at the earliest developmental stage assessed (90 dpa), but fell subsequently and at seed maturity was very low. These observations showed an inverse correlation with changes in endogenous free abscisic acid (ABA) levels in the embryo, which were low during early ontogeny, but reached maximum levels late in development (150–160 dpa). These observations suggest the possibility that dormancy may be induced during development as a result of ABA accumulation in the embryo, an argument strengthened by the obvious inhibitory effect of added ABA on the germinability of isolated embryos. The cotyledons appear to exert an inhibitory influence on embryo germinability that may result from their free ABA content although the embryonic axis itself possesses an innate dormancy that may reflect its own free ABA content. The increased germinability of isolated embryos resulting form added kinetin serves only to emphasise the complexity of the system and the dangers of simplistic interpretation.The correlation between germinability and ABA content is not complete, however, since much of the reduction in germinability had occurred before any appreciable increase in free ABA levels in the embryo was observed. Indeed the failure of the intact seed to respond to endogenous changes in embryonal ABA levels suggests that even though free ABA in the embryo may influence embryo germinability, it has little effect in the intact seed, where the presence of an intact testa may be a more important factor.The absence of a desiccation phase in the embryo during the late stages of development suggests that the large increases in endogenous free ABA did not cause dormancy by inhibiting water uptake, nor did they result from water stress in the embryonal tissues.     

Procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on the seed and vegetative plant

The procera (pro) mutant of tomato exhibits a well-characterizedconstitutive gibberellic acid (GA) response phenotype. The tomatoDELLA gene LeGAI in the pro mutant background contains a pointmutation that results in an amino acid change in the conservedVHVID putative DNA-binding domain in LeGAI to VHEID. This samepoint mutation is in four different genetic backgrounds exhibitingthe pro phenotype, suggesting that this mutation co-segregateswith the pro phenotype. Complementation of the mutant with aconstitutively expressed wild-type LeGAI gene sequence was notconclusive due to the infertility of transgenic plants. Thepro mutation alters tomato branching architecture through differentialsuppression of axillary bud development, indicating a role forDELLA proteins in the regulation of plant structure. Isolatedgib-1 pro double mutant embryo axes, which are unable to synthesizeGA, germinate faster than their wild-type counterparts, andexert greater embryo growth potential. The pro mutation is thereforeregulating GA responses within the tomato embryo. Transientexpression of a LeGAI–GFP (green fluorescent protein)fusion protein in onion epidermis results in its location tothe nucleus, and this protein is rapidly degraded by the proteasomein the presence of GA. Key words: Branching pattern, DELLA, embryo growth potential, tomato seed germination Received 12 October 2007; Revised 27 November 2007 Accepted 28 November 2007