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CIPK3 is a member of CBL (calcineurin B-like)-interacting serine–threonine protein kinases which play an important role in many developmental and adaptation processes in Arabidopsis. Studies conducted on members of this family such as SOS2, PKS8 and PKS11 have provided insight into how these kinases interact with their target substrates in the signal-response process. Since SOS2, PKS8 and PKS11 have low enzymatic activities in vitro and their amino acid sequences are homologous to that of CIPK3, it was assumed that CIPK3 would have a low enzymatic activity. To enhance CIPK3 enzyme activity, a constitutively active form, CIPK3T183D, was generated by a Thr183 to Asp183 substitution in the activation loop. To obtain proteins for analysis, glutathione S-transferase (GST) fusion protein system was used. Although both CIPK3 and CIPK3T183D were successfully expressed, they were found in inclusion bodies with three truncated proteins. Since the truncated proteins had a similar affinity to the GST-Bind Resin as the target protein, the one-step affinity purification could no longer be used. As an alternative, His fusion protein expression system was employed for protein production. Although both His-CIPK3 and His-CIPK3T183D also accumulated in inclusion bodies, they were expressed as a single protein species. A method involving Sarkosyl was developed for isolating and purifying the His fusion proteins. His-CIPK3 and His-CIPK3T183D produced were highly purified and enzymatically active. In addition, a 9-fold increase in kinase activity in His-CIPK3T183D was observed, indicating that Thr183 to Asp183 substitution in the activation loop of CIPK3 had succeeded in enhancing the kinase activity.

Transcriptome profiling was conducted to detect genes whose expression is significantly changed in an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1) during early seedling development when mutant phenotypes could be clearly observed. A total of 2,040 differentially expressed genes were identified, representing approximately 6.7 % of the 30,385 DNA oligonucleotide targets on the microarray. Among these differential expressed genes, many were mapped to pathways essential to plant growth and development including those of primary, secondary and hormone metabolisms. A significant proportion of up-regulated genes encoded transposable elements which were mapped to the centromeric and pericentromeric regions of the Arabidopsis chromosomes that were analyzed. A number of down-regulated genes were found to be involved in root hair formation, which might have contributed to the root hair defective phenotype of the mutant. Analysis of genes encoding transposable elements and those associating with root hair development indicated that these genes were highly co-expressed during seedling development. Despite SAHH1 deficiency, the expression of genes encoding methyltransferase remained largely unchanged in the sahh1 mutant. Bisulfite sequencing analysis of the transposable elements and the FWA gene revealed that their sequences in the mutant were deficient of 5-methylcytosines. Analysis of mutant genomic DNA using restriction endonucleases that were unable to cut methylated DNA suggested a genome-wide hypomethylation had occurred in the mutant. These results indicated that SAHH1 plays a critical role in methyl homeostasis, and its deficiency is a major contributing factor to the change of global gene expression, metabolic pathways and activation of transposable elements in the sahh1 mutant.

Reactive oxygen species (ROS) are toxic by-products generated continuously during seed desiccation, storage, and germination, resulting in seed deterioration and therefore decreased seed longevity. The toxicity of ROS is due to their indiscriminate reactivity with almost any constituent of the cell, such as lipids, proteins, and DNA. The damage to the genome induced by ROS has been recognized as an important cause of seed deterioration. A prominent DNA lesion induced by ROS is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can form base pairs with adenine instead of cytosine during DNA replication and leads to GC→TA transversions. In Arabidopsis, AtOGG1 is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that is involved in base excision repair for eliminating 8-oxo-G from DNA. In this study, the functions of AtOGG1 were elaborated. The transcript of AtOGG1 was detected in seeds, and it was strongly up-regulated during seed desiccation and imbibition. Analysis of transformed Arabidopsis protoplasts demonstrated that AtOGG1-yellow fluorescent protein fusion protein localized to the nucleus. Overexpression of AtOGG1 in Arabidopsis enhanced seed resistance to controlled deterioration treatment. In addition, the content of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in transgenic seeds was reduced compared to wild-type seeds, indicating a DNA damage-repair function of AtOGG1 in vivo. Furthermore, transgenic seeds exhibited increased germination ability under abiotic stresses such as methyl viologen, NaCl, mannitol, and high temperatures. Taken together, our results demonstrated that overexpression of AtOGG1 in Arabidopsis enhances seed longevity and abiotic stress tolerance.

Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids. Although there is increasing evidence implicating their importance in plant stress responses, their functions in seeds remain to be further studied. In this study, we identified a heat-induced annexin, NnANN1, from the embryonic axes of sacred lotus (Nelumbo nucifera Gaertn.) using comparative proteomics approach. Moreover, the expression of NnANN1 increased considerably in response to high-temperature treatment. Quantitative real-time PCR (qRT-PCR) revealed that the transcripts of NnANN1 were detected predominantly during seed development and germination in sacred lotus, implicating a role for NnANN1 in plant seeds. Ectopic expression of NnANN1 in Arabidopsis resulted in enhanced tolerance to heat stress in transgenic seeds. In addition, compared to the wild-type seeds, transgenic seeds ectopically expressing NnANN1 exhibited improved resistance to accelerated aging treatment used for assessing seed vigor. Furthermore, transgenic seeds showed enhanced peroxidase activities, accompanied with reduced lipid peroxidation and reduced ROS release levels compared to the wild-type seeds. Taken together, these results indicate that NnANN1 plays an important role in seed thermotolerance and germination vigor.

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.

Seed development ends with a maturation phase that imparts desiccation tolerance, nutrient reserves, and dormancy degree. Here, we report the functional analysis of an Arabidopsis thaliana C2H2 zinc finger protein INDETERMINATE DOMAIN1 (IDD1)/ENHYDROUS (ENY). Ectopic expression of IDD1/ENY (2x35S:ENY) disrupted seed development, delaying endosperm depletion and testa senescence, resulting in an abbreviated maturation program. Consequently, mature 2x35S:ENY seeds had increased endosperm-specific fatty acids, starch retention, and defective mucilage extrusion. Using RAB18 promoter ENY lines (RAB18:ENY) to confine expression to maturation, when native ENY expression increased and peaked, resulted in mature seed with lower abscisic acid (ABA) content and decreased germination sensitivity to applied ABA. Furthermore, results of far-red and red light treatments of 2x35S:ENY and RAB18:ENY germinating seeds, and of artificial microRNA knockdown lines, suggest that ENY acts to promote germination. After using RAB18:ENY seedlings to induce ENY during ABA application, key genes in gibberellin (GA) metabolism and signaling were differentially regulated in a manner suggesting negative feedback regulation. Furthermore, GA treatment resulted in a skotomorphogenic-like phenotype in light-grown 2x35S:ENY and RAB18:ENY seedlings. The physical interaction of ENY with DELLAs and an ENY-triggered accumulation of DELLA transcripts during maturation support the conclusion that ENY mediates GA effects to balance ABA-promoted maturation during late seed development.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

Polyethylene glycol treatment induces secondary seed dormancy in Brassica napus L. cultivar ‘AC Excel’ (ACE), but not in ‘DH12075’ (DH). Gene expression, metabolite profiles, and hormone profiles were obtained from seeds of both cultivars following polyethylene glycol 8000 treatment. ACE seeds were more transcriptionally active: 28 genes were up-regulated in both cultivars and 10 and 158 genes were specifically up-regulated in DH and ACE, respectively. Nontargeted metabolite analyses combined with gene expression analyses showed significant differences in lipid, sugar, and phenylpropanoid metabolism between the cultivars. Abscisic acid (ABA) levels were higher and many ABA-inducible genes were expressed more in ACE. An association of ABA with secondary dormancy was supported by the observation that secondary dormancy was induced by polyethylene glycol 8000 in Arabidopsis wild-type seeds, but was reduced in ABA-deficient and ABA-insensitive mutants. Therefore, secondary dormancy appears to be realized through an active ABA-related mechanism that may involve changes in primary and secondary metabolism.

We have functionally characterized an Arabidopsis (Arabidopsis thaliana) gene AtHSD1 (At5g50600) that encodes a protein with homology to animal 11-beta-hydroxysteroid dehydrogenase (HSD). Transgenic Arabidopsis plants overexpressing AtHSD1 (designated AOHSD plants) under the control of the cauliflower mosaic virus 35S promoter showed increased growth and seed yield as well as increased tolerance of saline stress and reduced seed dormancy. In canola (Brassica napus), transgenic plants overexpressing AtHSD1 also outgrew wild-type plants. AOHSD phenotypes were similar to those of plants that overproduced brassinosteroids (BRs) or overexpressed the BR receptor gene BRI1. A loss-of-function hsd mutant produced by RNA interference displayed a semi-dwarfed phenotype with reduced sensitivity to BRs. In contrast, AOHSD plants were hypersensitive to BRs and exhibited increased catabolism of abscisic acid (ABA). Germination of AOHSD seeds was less sensitive to ABA, while hsd seed was more sensitive to ABA during germination. AtHSD transcription was rapidly induced by BR treatment in wild type and was expressed widely in aerial plant parts, especially vascular tissues. This study demonstrates that AtHSD1 is involved in regulating growth and development in plants and is likely to promote or mediate BR effects. The gene has significant potential for improving growth and yield of canola and other agricultural crops.

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T2 transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.

Sequences of three Arabidopsis thaliana and two Brassica napus cDNAs encoding squalene monooxygenase homologues (Sqp1 and Sqp2) are reported. Southern analysis confirmed that these cDNAs are derived from small gene families in both species. Expression analysis indicates that Sqp1 genes in B. napus are strongly expressed in leaves but not roots or developing seeds. Comparison of cDNA and genomic sequences indicate that the 3′ splice site of an intron in these genes has undergone junctional sliding. The evolutionary significance of this phenomenon is discussed.

Transformed white spruce cultures containing immature Stage I somatic embryos, were developed after particle bombardment of somatic embryos with pBI 426, carrying an expression cassette with a gus::nptII fusion gene. These Stage I cultures did not show tolerance to kanamycin concentrations above 3 to 5 mg l−1, although assays for GUS and NPTII showed that functional enzymes were present in the transgenic tissue. Embryonic liquid suspension cultures were initiated from this transformed tissue. After treatment on agar-solidified maturation medium with 48 μm (±)-ABA high numbers of Stage III (cotyledonary) somatic embryos were produced. When subjected to an embryogenesis re-induction process with 2,4-D and BA, these Stage III embryos produced a new generation of Stage I embryogenic tissues which could tolerate 5--10 mg l−1 kanamycin. Stage III somatic embryos could alternatively be placed onto germination medium for the development of somatic seedlings. When germinated in the presence of 20 mg l−1 kanamycin, 77% of inoculants were resistant. The stability of integration of the gus::nptII fusion gene in the genome of white spruce Stage III somatic embryos and somatic seedlings was confirmed through Southern blot analysis

Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. Although plant MTs have been intensively studied, their roles in seeds remain to be clearly established. Here, we report the isolation and characterization of NnMT2a, NnMT2b and NnMT3 from sacred lotus (Nelumbo nucifera Gaertn.) and their roles in seed germination vigor. The transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity, oxidative stresses and heavy metals. Analysis of transformed Arabidopsis protoplasts showed that NnMT2a-YFP and NnMT3-YFP were localized in cytoplasm and nucleoplasm. Transgenic Arabidopsis seeds overexpressing NnMT2a and NnMT3 displayed improved resistance to accelerated aging (AA) treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment. In addition, we showed that NnMT2a and NnMT3 conferred improved germination ability to NaCl and methyl viologen on transgenic Arabidopsis seeds. Taken together, these data demonstrate that overexpression of NnMT2a and NnMT3 in Arabidopsis significantly enhances seed germination vigor after AA treatment and under abiotic stresses.

Background MicroRNAs (miRNAs) are 20–21 nucleotide RNA molecules that suppress the transcription of target genes and may also inhibit translation. Despite the thousands of miRNAs identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant Brassica napus (canola) – especially in seeds. Results Using next-generation sequencing technologies, we performed a comprehensive analysis of miRNAs during seed maturation at 9 time points from 10 days after flowering (DAF) to 50 DAF using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at 4 selected time points. We identified more than 500 conserved miRNA or variant unique sequences with >300 sequence reads and also found 10 novel miRNAs. Only 27 of the conserved miRNA sequences had been previously identified in B. napus (miRBase Release 18). More than 180 MIRNA loci were identified/annotated using the B. rapa genome as a surrogate for the B.napus A genome. Numerous miRNAs were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. miRNA targets in B. napus were predicted and their expression patterns profiled using microarray analyses. Global correlation analysis of the expression patterns of miRNAs and their targets revealed complex miRNA-target gene regulatory networks during seed development. The miR156 family was the most abundant and the majority of the family members were primarily expressed in the embryo. Conclusions Large numbers of miRNAs with diverse expression patterns, multiple-targeting and co-targeting of many miRNAs, and complex relationships between expression of miRNAs and targets were identified in this study. Several key miRNA-target expression patterns were identified and new roles of miRNAs in regulating seed development are suggested. miR156, miR159, miR172, miR167, miR158 and miR166 are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. miR156 may regulate the developmental transition to germination.

The seed maturation programme occurs only during the late phase of embryo development, and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this programme in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants were made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Analysis of histone methylation status at the chromatin sites of a number of maturation loci revealed a synergistic effect of emf2 and sdg8 on the deposition of the active histone mark which is the trimethylation of Lys4 on histone 3 (H3K4me3). This is consistent with high expression of these genes and formation of somatic embryos in the emf2 sdg8 double mutants. Interestingly, a double mutant of sdg8 and vrn2 (vernalization2), a paralogue of EMF2, grew and developed normally to maturity. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis.

The seed maturation program only occurs during late embryogenesis, and repression of the program is pivotal for seedling development. However, the mechanism through which this repression is achieved in vegetative tissues is poorly understood. Here we report a microRNA (miRNA)–mediated repression mechanism operating in leaves. To understand the repression of the embryonic program in seedlings, we have conducted a genetic screen using a seed maturation gene reporter transgenic line in Arabidopsis (Arabidopsis thaliana) for the isolation of mutants that ectopically express seed maturation genes in leaves. One of the mutants identified from the screen is a weak allele of ARGONAUTE1 (AGO1) that encodes an effector protein for small RNAs. We first show that it is the defect in the accumulation of miRNAs rather than other small RNAs that causes the ectopic seed gene expression in ago1. We then demonstrate that overexpression of miR166 suppresses the derepression of the seed gene reporter in ago1 and that, conversely, the specific loss of miR166 causes ectopic expression of seed maturation genes. Further, we show that ectopic expression of miR166 targets, type III homeodomain-leucine zipper (HD-ZIPIII) genes PHABULOSA (PHB) and PHAVOLUTA (PHV), is sufficient to activate seed maturation genes in vegetative tissues. Lastly, we show that PHB binds the promoter of LEAFY COTYLEDON2 (LEC2), which encodes a master regulator of seed maturation. Therefore, this study establishes a core module composed of a miRNA, its target genes (PHB and PHV), and the direct target of PHB (LEC2) as an underlying mechanism that keeps the seed maturation program off during vegetative development., Author Summary Seed development can be conceptually divided into two phases: namely the morphogenesis phase, in which cell division is active and all the major organs are formed, and the maturation phase, in which cells enlarge and storage reserves are synthesized and accumulated. Expression of the seed maturation program is tightly controlled such that it only occurs during the late phase of seed development. To uncover the molecular mechanisms underlying the repression of seed genes during vegetative development, we performed a reporter-assisted genetic screen, and one mutant identified is a weak allele of ARGONAUTE1 (AGO1) that displays ectopic seed gene expression. We then performed a series of transgenic and genetic analyses to search for the molecular mechanisms underlying the mutant phenotype. We first demonstrate that the decrease in miR166 in ago1 is a major cause of the mutant phenotype. Further, we show that the targets of miR166, type III HD-ZIP transcription factors PHB and PHV, are sufficient for derepressing seed maturation genes in seedlings, likely by binding directly to the promoter of a master regulator gene of maturation. Thus, this work establishes a miRNA–mediated pathway that represses the embryonic program and also establishes PHB/PHV as direct activators of the maturation program.

Immature embryos (stage I) and cotyledonary somatic embryos (stage III) of black spruve [Picea mariana (Mill) B.S.P.] were bombarded with tungsten particles coated with a gene construct containing the fusion of gus::nptll. GUS (β-glucuronidase) activity was monitored histochemically with X-gluc giving a blue colour where transient gene expression was detected in the bombarded tissues. A high transient expression of gus was observed in stage I embryo cultures 2 d after bombardment (202 GUS foci per 300 g tissue)

Peanut (Arachis hypogaea) allergy is one of the most serious food allergies. Peanut seed protein, Ara h 3, is considered to be one of the most important peanut allergens. Little is known about the temporal and spatial regulation mechanism of Ara h 3 during seed development. In this study, chromatin structure of the Ara h 3 promoter was analyzed to examine its transcriptional regulation. Analysis of transgenic plants of Arabidopsis thaliana expressing Arah3: GUS showed that the Ara h 3 promoter could efficiently direct the seed-specific expression of the GUS reporter gene. Chromatin immunoprecipitation revealed that nucleosomes were depleted at the core promoter of the Ara h 3 upon full activation in the late stage of embryo maturation, which was accompanied by a dramatic decrease of histone acetylation. However, in the early stage of embryo maturation, histone H3 hyperacetylation at the core promoter of Ara h 3 was detected. A decrease of histone H3-K9 dimethylation levels at core promoter of Ara h 3 was also observed with concomitant repression of Ara h 3 in the vegetative tissues. Our results suggest that the histone modification status of Ara h 3 undergoes targeted changes including the increase of histone H3 acetylation and decrease of histone H3-K9 dimethylation in early maturation embryos. In addition, the loss of histone H3 from the proximal promoter of Ara h 3 is associated with its high expression during late embryo maturation.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

Brassica species represent several important crops including canola ( Brassica napus ). Understanding of genetic elements that contribute to seed-associated functions will impact future improvements in the canola crop. Brassica species share a very close taxonomic and molecular relationship with Arabidopsis thaliana. However, there are several subtle but distinct seed-associated agronomic characteristics that differ among the oil seed crop species. To address these, we have generated 67 535 ESTs predominately from Brassica seeds, analyzed these sequences, and identified 10 642 unigenes for the preparation of a targeted seed cDNA array. A set of 10 642 PCR primer pairs was designed and corresponding amplicons were produced for spotting, along with relevant controls. Critical quality control tests produced satisfactory results for use of this microarray in biological experiments. The microarray was also tested with specific RNA targets from embryos, germinating seeds, and leaf tissues. The hybridizations, signal intensities, and overall quality of these slides were consistent and reproducible. Additionally, there are 429 ESTs represented on the array that show no homology with any A. thaliana annotated gene or any gene in the Brassica genome databases or other plant databases; however, all of these probes hybridized to B. napus transcripts, indicating that the array also will be useful in defining expression patterns for genes so far unique to Brassica species.

The inability of superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1)-deficient mutants of Escherichia coli to grow aerobically on minimal medium can be restored by functional complementation with a heterologous SOD-encoding sequence. Based upon this property, a phenotypic selection system has been developed for the isolation of clones containing eukaryotic SOD cDNAs. cDNA expression libraries from both Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed into a SOD-deficient E. coli strain by electroporation, and clones containing functional SODs were selected by growth on minimal medium. Analysis of these clones revealed the identity of cDNAs encoding the iron form of superoxide dismutase (FeSOD)--the first SODs of this type to be cloned from eukaryotes. The presence of this enzyme in these two divergent plant species challenges previous ideas that FeSOD is found in only a few plant families. In addition, these results show the potential for shotgun cloning of eukaryotic genes by complementation of bacterial mutants, particularly when it is combined with a highly efficient transformation method, such as electroporation.

Gene expression in two cultivars of Brassica napus (AC Excel and DH12075) has been compared at the full-size embryo, desiccation, and mature stages of seed development. Seed of these cultivars differ in their potential to exhibit secondary dormancy following environmental stress; Excel has high potential and DH12075 has low potential. A majority of genes were down-regulated during maturation in both cultivars but a significant number of differences in gene expression between the cultivars were apparent in the transition from full-size embryo to mature seed. However, most differences were apparent in the desiccation stage and some of the differences were in genes related to signaling processes and protein biosynthesis. We suggest that the propensity of Brassica seeds to manifest secondary dormancy may be determined by changes in gene expression that occur during late seed development.

Using an Arabidopsis microarray, we compared gene expression between germinating Brassica napus seeds and seeds in which germination was inhibited either by polyethylene glycol (PEG) or by the abscisic acid (ABA) analog PBI429, which produces stronger and longer lasting ABA-like effects. A total of 40 genes were induced relative to the germinating control by both treatments. Conspicuous among these were genes associated with late seed development. We identified 36 genes that were downregulated by both PEG and PBI429. Functions of these genes included carbohydrate metabolism, cell wall-related processes, detoxification of reactive oxygen, and triacylglycerol breakdown. The PBI429 treatment produced an increase in endogenous ABA and increased ABA catabolism. However, PEG treatment did not result in similar effects. The transcription factor ABI5 was consistently upregulated by both treatments and PKL was downregulated. These results suggest a greater importance of ABA signaling and reduced importance of GA signaling in nongerminating seeds.

Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the alpha-subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively. Sequence analyses of the genomic DNA flanking the deletion sites have demonstrated that both deletions occurred at the genomic level. These two mutants are referred to as CRBDelta12 and CRCDelta13 with the delta sign indicating a deletion and the number indicating amino acids deleted. Alignment of these two mutant sequences with that of soybean A3B4 subunit, for which the crystal structure was determined recently, have revealed that the CRCDelta13 deletion is located in a hypervariable/disordered region, and will probably not affect the structure of the hexameric globulin. The CRBDelta12 deletion, however, is located in a binding region that is thought to be important for the hexamer formation. However, CRBDelta12 appears to accumulate normally as judged by its band intensity relative to the other SSP subunits on the protein gels. Thus it seems that the seed can, to a certain extent, tolerate some mutations in its storage proteins.

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T2 transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.

Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus.

A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S-GT gene were present in B. napus. The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.

The Biolistic® microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the β-glucuronidase gene.

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.