Cheryl A. Bock

Metabolism of (+)- and (−)-abscisic acid by somatic embryo suspension cultures of white spruce

Abstract Somatic embryo suspension cultures of white spruce in medium containing (+)-abscisic acid [(+)-ABA], at an initial concentration of 15 μM, metabolized the ABA essentially completely within seven days. The metabolites accumulated in the liquid medium. The (+)-ABA was converted almost quantitively to phaseic acid, with little further transformation into dihydrophaseic acid. The sum of the concentrations of the two metabolites in the medium closely approximated the concentration of (+)-ABA initially supplied. (−)-ABA remained essentially unchanged under the same culture conditions, and when the cells were supplied with racemic (±)-ABA, only the (+) enantiomer was metabolized. When (+)- or (±)-ABA was present in the medium, the size of the embryos and their suspensors increased during the culture period, a development consistent with somatic embryo maturation. Embryos in control cultures lacking exogenously supplied ABA, and embryos in cultures provided with only (−)-ABA, did not increase in size. The early disappearance of the (+)-ABA from the suspension medium, and the completeness of its conversion to phaseic acid, raises the question of the relative roles of ABA and phaseic acid in the maturation of conifer somatic embryos.

Over‐expression of Brassica napus phosphatidylinositol‐phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositol-specific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca(2+) modulator which induces distinctive oscillating bursts of cytosolic Ca(2+), resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stress-related genes such as glutathione S-transferase, hormone-regulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)—comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus

The cloning and identification of full-length cDNA fragments coding for the Brassica napus phosphatidylinositol-specific phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase (BnPtdIns S1) is described. In addition, two complementary fragments (120 nucleotides long) corresponding to Arabidopsis PtdIns 4-kinase (PtdIns 4-K) and PtdIns-4-phosphate 5-kinase (PtdIns4P 5-K) sequences were chemically synthesized. These, as well as the cDNA clones, were used as probes to study the corresponding steady state mRNA levels in different tissues and developmental stages of B. napus, as well as in response to different environmental conditions. Transcripts corresponding to BnPLC2, BnPtdIns S1, BnVPS34 and PtdIns 4-K were found constitutively expressed at different levels in most tissues, with young leaves, siliques, and developing seeds showing the lowest levels. No detectable PtdIns4P 5-K transcripts were found in buds or flowers. Up-regulation of BnPLC2 was seen in response to low temperature stress, which was notably accompanied by a parallel down-regulation of BnPtdIns S1, while BnVPS34 and PtdIns 4-K remained at control levels. A moderate increase in PtdIns4P 5-K levels was noted. In high salinity conditions BnPtdIns S1, BnVPS34 and BnPLC2 transcripts had similar responses but at different levels, with no major changes detected for PtdIns 4-K or PtdIns4P 5-K. Significantly, all five transcripts increased under drought stress conditions and all stressed plants clearly showed relatively higher levels of total inositol trisphosphate.

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

SummaryThe production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (±)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.

Perturbing the metabolic dynamics of myo-inositol in developing Brassica napus seeds through in vivo methylation impacts its utilization as phytate precursor and affects downstream metabolic pathways

Backgroundmyo-Inositol (Ins) metabolism during early stages of seed development plays an important role in determining the distributional relationships of some seed storage components such as the antinutritional factors, sucrose galactosides (also known as raffinose oligosaccharides) and phytic acid (PhA) (myo-inositol 1,2,3,4,5,6-hexakisphosphate). The former is a group of oligosaccharides, which plays a role in desiccation at seed maturation. They are not easily digested by monogastric animals, hence their flatulence-causing properties. Phytic acid is highly negatively charged, which chelates positive ions of essential minerals and decreases their bioavailability. It is also a major cause of phosphate-related water pollution. Our aim was to investigate the influence of competitive diversion of Ins as common substrate on the biosynthesis of phytate and sucrose galactosides.ResultsWe have studied the initial metabolic patterns of Ins in developing seeds of Brassica napus and determined that early stages of seed development are marked by rapid deployment of Ins into a variety of pathways, dominated by interconversion of polar (Ins phosphates) and non-polar (phospholipids) species. In a time course experiment at early stages of seed development, we show Ins to be a highly significant constituent of the endosperm and seed coat, but with no phytate biosynthesis occurring in either tissue. Phytate accumulation appears to be confined mainly within the embryo throughout seed development and maturation. In our approach, the gene for myo-inositol methyltransferase (IMT), isolated from Mesembryanthemum crystallinum (ice plant), was transferred to B. napus under the control of the seed-specific promoters, napin and phaseolin. Introduction of this new metabolic step during seed development prompted Ins conversion to the corresponding monomethyl ether, ononitol, and affected phytate accumulation. We were able to produce homozygous transgenic lines with 19% - 35% average phytate reduction. Additionally, changes in the raffinose content and related sugars occurred along with enhanced sucrose levels. Germination rates, viability and other seed parameters were unaffected by the IMT transgene over-expression.ConclusionsCompetitive methylation of Ins during seed development reduces seed antinutritional components and enhances its nutritional characteristics while maintaining adequate phosphate reserves. Such approach should potentially raise the canola market value and likely, that of other crops.

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

SummaryWe investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation.

Metabolomic shifts in Brassica napus lines with enhanced BnPLC2 expression impact their response to low temperature stress and plant pathogens

Phosphatidylinositol-specific phospholipase C2 (PLC2) is a signaling enzyme with hydrolytic activity against membrane-bound phosphoinositides. It catalyzes the cleavage of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2) into two initial second messengers, myo-inositol-1,4,5-trisphosphate (InsP 3) and diacylglycerol (DAG). The former, as well as its fully phosphorylated derivative, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP 6), play a major role in calcium signaling events within the cell, while DAG may be used in the regeneration of phospholipids or as a precursor for phosphatidic acid (PA) biosynthesis, an important signaling molecule involved in both biotic and abiotic types of stress tolerance. Overexpression of the gene for Brassica napus phospholipase C2 (BnPLC2) in Brassica napus has been shown to enhance drought tolerance, modulate multiple genes involved in different processes and favorably affect hormonal levels in different tissues. We, therefore, undertook the current study with a view to examining, at the metabolome level, its effect on both abiotic (low temperature) and biotic (stem white rot disease) types of stress in canola. Thus, while transgenic plants exhibited a significant rise in maltose levels and a concomitant elevation in some unsaturated free fatty acids (FFAs), glycerol, and glycerol 3-phosphate under subzero temperatures, they accumulated high levels of raffinose, stachyose and other sugars as well as some flavonoids under acclimatization conditions. Collectively, overexpression of BnPLC2 appears to have triggered different metabolite patterns consistent with its abiotic and, to a limited extent, biotic stress tolerance phenotypes.

Abscisic acid [(+)-ABA] content in white spruce somatic embryo tissues related to concentration of fed ABA

Summary The availability of ABA and tissue content of (+)-ABA were investigated, when using different starting concentrations of (±)-ABA to stimulate white spruce somatic embryo maturation. ELISA analysis of tissue (+)-ABA in white spruce somatic embryos during maturation showed that tissue content of (+)-ABA rose proportionally to the concentration of fed ABA (between 12 and 60μmol·L −1 (±)-ABA). Irrespective of the starting concentration of fed (±)-ABA, peak values in tissue (+)-ABA were obtained after 3–7d. Following this there was a steady decline from approximately 12 to 28 d, after which tissue (+)-ABA remained at relatively constant low levels. The levels of tissue (+)-ABA under each starting concentration of (±)-ABA were very similar to one another by 28 d. There was no significant difference in tissue (+)-ABA content between embryos and embryo-free tissue fractions. Metabolism of fed (±)-ABA followed a similar pattern to (+)-ABA content, being proportional to the initial concentration. Metabolites of (+)-ABA were present in the medium in the first 6d, metabolism of (+)-ABA is likely to have depleted it from the medium between 12 to 28 d, irrespective of the initial concentration of (±)-ABA in the medium. These data indicate that the high levels of exogenous (±)-ABA that are needed to stimulate white spruce somatic embryo maturation, most likely have their major stimulatory effect on maturation during the first 12 d of the 6 to 8 wk culture period, there being no substantive difference among the concentrations in the period of (+)-ABA availability. To improve the yield of mature somatic embryos it will either be necessary to increase the abundance of responsive immature somatic embryos during this 12 d period, or to extend the period of availability of (+)-ABA to the embryo culture through periodic addition of (±)-ABA.

ABA content and lipid deposition in interior spruce somatic embryos

SummaryInterior spruce (Picea glauca engelmannii complex) somatic embryos grown on 48 µmol (±)-ABA per L over a period of 42 d without transfer underwent precocious germination by 49 d. Those transferred at 28 d to fresh medium with 48 µmol (±)-ABA continued embryo development until harvested at 56 d; the transfer at 28 d resulted in an increase in embryo lipid content after 42 d. Somatic embryos grown under this condition contained 181.4±41.2, 116.0±42.4, and 91.8±33.6 ng (+)-ABA per mg of lyophilized tissue at 42, 49, and 56 d, respectively. By comparison, embryos grown without the transfer at 28 d had 86.8±25.4 ng (+)-ABA per mg of lyophilized tissue at 42 d, just prior to precocious germination. After 3 weeks’ storage in a drying chamber under high humidity, the (+)-ABA content of 56-d-old transferred embryos decreased to 15.4 ± 4.4 ng (+)-ABA per mg of lyophilized tissue. The increased lipid content resulting from embryo transfer and the reduction in internal (+)-ABA content during storage are factors which will contribute to improved conversion of somatic embryos to plantlets.

Faba Bean: Transcriptome Analysis from Etiolated Seedling and Developing Seed Coat of Key Cultivars for Synthesis of Proanthocyanidins, Phytate, Raffinose Family Oligosaccharides, Vicine, and Convicine

Faba bean (Vicia faba L.) has been little examined from a genetic or genomic perspective despite its status as an established food and forage crop with some key pharmaceutical factors such as vicine and convicine (VC), which provoke severe haemolysis in genetically susceptible humans. We developed next‐generation sequencing libraries to maximize information to elucidate the VC pathway or relevant markers as well as other genes of interest for the species. One selected cultivar, A01155, lacks synthesis of the favism‐provoking factors, VC, and is low in tannin, while two cultivars, SSNS‐1 and CDC Fatima, are wild‐type for these factors. Tissues (5‐ to 6‐d‐old root and etiolated shoot and developing seed coat) were selected to maximize the utility and breadth of the gene expression profile. Approximately 1.2 × 106 expressed transcripts were sequenced and assembled into contigs. The synthetic pathways for phosphatidylinositol or phytate, the raffinose family oligosaccharides, and proanthocyanidin were examined and found to contain nearly a full complement of the synthetic genes for these pathways. A severe deficiency in anthocyanidin reductase expression was found in the low‐tannin cultivar A01155. Approximately 5300 variants, including 234 variants specific to one of the three cultivars, were identified. Differences in expression and variants potentially related to VC synthesis were analyzed using strategies exploiting differences in expression between cultivars and tissues. These sequences should be of high utility for marker‐assisted selection for the key traits vicine, convicine, and proanthocyanidin, and should contribute to the scant genetic maps available for this species.