Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh.

Mutant lines of Arabidopsis thaliana (L.) Heynh., which are characterized by symptoms of withering and the absence of seed dormancy, showed much lower levels of endogenous abscisic acid (ABA) in developing seeds and fruits (siliquae) than the wild type. Reciprocal crosses of wild type and ABA-deficient mutants showed a dual origin of ABA in developing seeds. The genotype of the mother plant regulated a sharp rise in ABA content half-way seed development (maternal ABA). The genotype of the embryo and endosperm was responsible for a second ABA fraction (embryonic ABA), which reached much lower levels, but persisted for some time after the maximum in maternal ABA. The onset of dormancy correlated well with the presence of the embryonic ABA fraction and not with the maternal ABA. Dormancy developed in both the absence and presence of maternal ABA in the seeds. In this respect maternal ABA resembled exogenously applied ABA which did not induce dormancy in ABA-deficient seeds. However, both maternal and applied ABA stimulated the formation of a mucilage layer around the testa, which could be observed during imbibition of the mature seeds. In the mature state, ABA-deficient seeds germinated in the siliquae on the plant, but only when the atmosphere surrounding the plant was kept at high relative humidity. In younger stages germination in siliquae occurred after isolation from the plants and incubation on wet filter paper. Therefore, it seems that limited access to water is the primary trigger for the developmental arrest in these seeds.     

Three zinc‐finger RNA‐binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions

Despite the increasing understanding of the stress‐responsive roles of zinc‐finger RNA‐binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2‐ or BrRZ3‐expressing Arabidopsis seeds was delayed compared with that of wild‐type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3‐expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ‐expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: VII. EFFECTS OF PARTIAL AND COMPLETE DESICCATION ON ABSCISIC ACID LEVELS AND SENSITIVITY IN RICINUS COMMUNIS L. SEEDS

During mid-development (25–40 d after pollination: DAP)of the castor bean seed the amount of abscisic acid (ABA) increasesin both the endosperm and the embryo, declining substantiallythereafter until there is little present in the mature dry (60DAP) seed. Premature desiccation of the seed at 35 DAP alsoleads to a major decline in ABA within the embryo and endosperm.Partial water loss from the seed at 35 DAP which, like naturaland premature desiccation, leads to subsequent germination uponreturn of the seed to full hydration, causes a much smallerdecline in ABA levels. In contrast, ABA declines substantiallyin the non-dried (hydrated) control at 35 DAP, but the seedsdo not germinate. Hence, a clear negative correlation betweenABA content and germinability is not observed. Both drying,whether natural or imposed prematurely, and partial drying decreasethe sensitivity of the isolated embryo to exogenous ABA by about10-fold. The protein synthetic response of the castor bean embryo exposedto 0.1 mol m^–3 ABA following premature desiccation exhibitssome similarity to the response of the non-dried developingembryo—in both cases the synthesis of some developmentalproteins is enhanced by ABA, and germination is suppressed.Germination of mature seeds is also suppressed by 0.1 mol m^–3ABA, but the same developmental proteins are not synthesized.In the cotyledons of prematurely-desiccated seed, some proteinsare hydrolysed upon imbibition in 0.1 mol m^–3 ABA, a phenomenonthat occurs also in the cotyledons of similarly treated matureembryos, but not in developing non-dried embryos. Hence theembryo exhibits an ‘intermediate’ response uponrehydration in 0.1 mol m^–3 ABA following premature desiccation;viz. some of the responses are developmental and some germinative.Following natural or imposed drying, the isolated embryo becomesrelatively insensitive to 0.01 mol m^–3 ABA: germinationis elicited and post-germinative reserve breakdown occurs inthe radicle and cotyledons. The reduced sensitivity of the embryoto ABA as a consequence of desiccation may be an important factorin eliciting the switch to germination and growth within thewhole seed. Key words: Abscisic acid, desiccation, astor bean endosperm, seed development, germination, protein synthesis, isolated embryos, hormone sensitivity     

The Role of Maturation Drying in the Transition from Seed Development to Germination: IV. PROTEIN SYNTHESIS AND ENZYME ACTIVITY CHANGES WITHIN THE COTYLEDONS OF RICINUS COMMUNIS L. SEEDS

Drying of immature seeds of Ricinus communis L. cv. Hale (castorbean) during the desiccation-tolerant phase of development causesthem to germinate upon subsequent rehydration. This desiccation-inducedswitch from development to germination is also mirrored by achange in the pattern of soluble and insoluble protein synthesiswithin the cotyledons of the castor bean. Following rehydrationof seeds prematurely dried at 40 d after pollination (DAP),cotyledonary proteins characteristic of development (e.g. storageproteins) are no longer synthesized; hydrolytic processes resultingin their degradation commence (after 12 h) in a manner similarto that observed following imbibition of the mature seed. Apattern of protein synthesis recognizable as germination/growth-associatedoccurs; premature drying has elicited a redirection in metabolismfrom a developmental to a germinative mode. Desiccation is alsorequired for the induction (within cotyledons of 35 DAP seeds)of enzymes involved in protein reserve breakdown (leucyl ß-naphthylamidase;LeuNAase) and lipid utilization (isocitrate lyase; ICL), anevent intimately associated with the post-germinative (growth)phase of seedling development. Thus, at a desiccation-tolerantstage of development, premature drying results in the suppressionof the developmental metabolic programme and a permanent switching-onof the germination/growth metabolic programme. Key words: Desiccation, metabolism, seed development, seed germination, castor bean, cotyledons     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Mechanical rigidity and lignine content in different developmental phases of winter rape siliqua

The lignine content and mechanical rigidity of siliquae ofBrassica napus L. ssp.oleifera f.biennis, cv. T?ebí?ská were studied. Experimental plants were harvested in five consecutive periods and developmental stages of siliquae were determined on the basis of the weight of 1000 seeds. During the development of siliquae a gradual accumulation of lignine takes place; the content reached its maximum in certain developmental phases and then decreased. The mechanical rigidity of siliquae had a similar tendency. There were statistically significant differences in both properties studied within individual developmental phases. A highly significant positive correlation was found between lignine content and mechanical consistency.     

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase(PG) gene previously isolated from the flower bud of Chinesecabbage (Brassica campestris L. ssp. chinensis Makino, syn.B. rapa ssp. chinensis). This gene was found to be expressedspecifically in tapetum and pollen after the tetrad stage ofanther development. Antisense RNA technology was used to studythe function of BcMF2 in Chinese cabbage. Scanning and transmissionelectron microscopy revealed that there were deformities inthe transgenic mature pollen grains such as abnormal locationof germinal furrows. In addition, the homogeneous pectic exintinelayer facing the exterior seemed to be overdeveloped and predominantlyoccupied the intine, thus reversing the normal proportionaldistribution of the internal endintine layer and the externalexintine layer. Since it is a continuation of the intine layer,the pollen tube wall could not grow normally. This resultedin the formation of a balloon-like swelling structure in thepollen tube tip in nearly 80% of the transgenic pollen grains.Premature degradation of tapetum was also found in these transgenicplants, which displayed decreased expression of the BcMF2 gene.BcMF2 might therefore encode a new PG with an important rolein pollen wall development, possibly via regulation of pectin'sdynamic metabolism. Key words: Brassica campestris, Brassica rapa, Chinese cabbage, intine, PG, polygalacturonase, pollen wall Received 28 August 2008; Revised 14 October 2008 Accepted 20 October 2008     

Regulation of seed germination and seedling growth by an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytocystatin isoform, <Emphasis Type="Italic">AtCYS6</Emphasis>

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin₄₊₇ (GA₄₊₇) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases.     

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.     

Pepper protein phosphatase type 2C,CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response

Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum A BA and D rought‐I nduced P rotein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1‐OX) exhibited an ABA‐hyposensitive and drought‐susceptible phenotype. We used a yeast two‐hybrid screening assay to identify CaRLP1 (Capsicum annuum R CAR‐L ike P rotein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1‐OX plants, CaRLP1‐OX plants displayed an ABA‐hypersensitive and drought‐tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress‐responsive genes relative to those of wild‐type plants. In CaADIP1‐OX/CaRLP1‐OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1‐induced ABA hyposensitivity during the germinative and post‐germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA‐dependent defense signalling response to drought stress.     

Gene transferability from transgenic Brassica napus L. to various subspecies and varieties of Brassica rapa

Gene transferability from transgenic rapeseed to various subspecies and varieties of Brassica rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 (Brassica napus) as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa (female) and the transgenic rapeseed Ms8 × Rf3 (B. napus) (male) ranged from 0.039 to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20 h when pollinated with the transgenic rapeseed Ms8 × Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10–70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.     

Assessment of the Germination Potential of Brassica oleracea Seeds Treated with Karrikin 1 and Cyanide,Which Modify the Ethylene Biosynthetic Pathway

The relationship between ethylene and cyanide (HCN) and karrikin 1 (KAR1) in dormancy release was studied in secondary dormant Brassica oleracea L. (Chinese cabbage) seeds. Freshly harvested seeds of Brassica oleracea usually have poor germination potential. Karrikin1 (KAR1) and cyanide (HCN) are able to stimulate seed germination. However, the stimulatory effects of these two chemicals depend on the activation of the ethylene biosynthesis pathway and on ethylene perception. In this study, KAR1 and HCN application increased the activity of ethylene and of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). KAR1 and HCN collectively promoted the accumulation of 1 aminocyclopropane-1-carboxylic acid (ACC). In the presence of NO (nitric oxide) and KAR1, ACS and ACO activities reached their maximum levels after 36 and 42 h, respectively. Ethylene inhibitors suppressed seed germination by approximately 55%, whereas the respiratory inhibitors SHAM and NaN3 inhibited seed germination by 5–10% in the presence of HCN and KAR1. KAR1 and HCN collectively reduced the abscisic acid (ABA) content in seeds, increased the gibberellic acid (GA) content and released seed dormancy. The expression of ethylene biosynthesis genes and ethylene receptor genes (BOACO1, BOACS1, BOACS3, BOACS4, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2) provided further evidence of the involvement of ethylene in KAR1 and HCN-induced germination. BOACO1, BOACS1, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2 genes were up regulated in the presence of KAR1 and HCN, while the remaining genes were down regulated. The expression of various ethylene biosynthesis and ethylene receptor genes suggested functional diversification and variations in seed sensitivity in the presence of KAR1 and HCN. Therefore, in the current study, KAR1 and HCN application effectively induced the germination of B. oleracea seeds (approximately 97% germination rate) after 6 days by modifying the ethylene biosynthetic pathway.

     

Expression of a Chinese cabbage cysteine proteinase inhibitor, BrCYS1, retards seed germination and plant growth in transgenic Tobacco plant

Phytocystatins are plant cysteine proteinase inhibitors that regulate endogenous and heterologous cysteine proteinases of the papain family. A cDNA encoding the phytocystatin BrCYS1 (Brassica rapa cysteine proteinase inhibitor 1 ) has been isolated from Chinese cabbage (B. rapa subsp.pekinensis) flower buds. In order to explore the role of this inhibitory enzyme, tobacco plants (Nicotiana tabacum L. cv. Samson) containing altered amounts of phytocystatin were generated by over-expressingBrCYS1 cDNA in either the sense or the antisense configuration. The resulting plants hadin vitro enzyme inhibitory activities that were over 10% of those detected in wild type plants. The transgenic plants exhibited retarded seed germination and seedling growth and a reduced seed yield, whereas these properties were enhanced in antisense plants. These data suggest that BrCYS1 participates in the control of seed germination, post-germination and plant growth by regulating cysteine peptidase activity.     

Toxicity of black nightshade (Solanum nigrum) extracts on Alternaria brassicicola, causal agent of black leaf spot of Chinese cabbage (Brassica pekinensis)

Root extracts of black nightshade (Solanum nigrum) were analyzed for activity against isolates ABA‐31 and ABA‐104 of Alternaria brassicicola, the causal agent of black leaf spot of Chinese cabbage (Brassica pekinensis). Preliminary results showed that dried root tissues of black nightshade extracted with 70% ethanol contained antifungal properties against A. brassicicola. Ethanol root extracts were used for further fractionations using ethyl acetate, n‐butanol and water. Among the three extracts, the n‐butanol fraction showed the strongest antifungal activity by its suppression of conidial germination of A. brassicicola. The n‐butanol extract of S. nigrum roots was fractionated further into six fractions (I–VI). Among the six fractions tested, fraction V showed a strong inhibitory effect on conidial germination of A. brassicicola and thereby suppressed lesion development of black leaf spot of Chinese cabbage at a concentration of 25 ppm or higher. Nuclear magnetic resonance analysis indicated that fraction V contained a mixture of saponins, and results of further bio‐guided fractionation and bioassay suggested that saponins in fraction V were key chemical components in the control of A. brassicicola. The potential of using black nightshade for developing natural products for the control of fungal plant diseases is discussed.     

Effect of ‘Mixtalol’ on growth,yield and yield components of Indian mustard (Brassica juncea)

Mixtalol (a mixture of long chain aliphatic alcohols varying in chain length from C₂₄ to C₃₂) applied to Brassica juncea plants as foliar spray caused an increase in secondary and tertiary branching with consequent enhancement in seed yield through increased number of inflorescences and siliquae per plant. The percentage of immature siliquae and shattering of siliquae decreased with this treatment. Mixtalol increased total dry matter of plants, partitioning coefficient and harvest index. The contents of starch, protein and oil were also higher in seeds from Mixtalol treated plants.