Jin-Zhuo Dong

Endochitinase and β-1,3-glucanase genes are developmentally regulated during somatic embryogenesis inPicea glauca

Two cDNAs isolated from white spruce [Picea glauca (Moench) Voss] somatic embryos, are predicted to encode a basic class IV chitinase and a β-1,3-glucanase, respectively corresponding to genesPgChi-1 andPgGlu-1. Each represents a multigene family in spruce. Transcripts homologous toPgChi-1 orPgGlu-1 genes were highly abundant in embryogenic tissues and gradually decreased after tissues were placed on abscisic acid-containing maturation medium, with lowest abundance in globular embryos. Transcripts related toPgGlu-1 became highly abundant again in early cotyledonary embryos but decreased thereafter, whereas transcripts related toPgChi-1 were also highly abundant in late cotyledonary embryos and plantlets in vitro; transcripts were either low (PgChi-1) or were not detectable (PgGlu-1) in needles. Wounding, drying and flooding stresses enhancedPgChi-1-andPgGlu-1-related gene expression. Fungal cell wall suspension enhancedPgGlu-1-related transcript accumulation, but reducedPgChi-1-related transcript abundance within 24 h.PgChi-1 andPgGlu-1 and their homologues may have roles in plant defense, and possibly developmental roles during spruce somatic embryo maturation.

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss].

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N6-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.

Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce [Picea glauca (Moench) Voss]

Three cDNAs (PgEMB22, 27 and 29) predicted to encode low-molecular-weight (LMW) heat-shock proteins (HSPs) were cloned and characterized from white spruce [Picea glauca (Moench) Voss] somatic embryo tissues by differentially screening a cotyledonary embryo cDNA library. Clone PgEMB22 is predicted to encode a putative mitochondria-localized LMW HSP, and PgEMB27 and 29 are predicted to encode different cytoplasmic class II LMW HSPs, although they share 84.7% identity within DNA coding regions and 83.0% identity for predicted proteins. They are developmentally regulated during somatic embryo development and subsequent embryo germination, in addition they show strong response to heat-shock stress. Transcripts of the two kinds of hsp genes could be detected in embryogenic tissues before induction of embryo maturation, but subsequently increased, being most abundant at late embryo stages. Gene expression levels were very low or not detectable in germinated plantlets or needle tissues from older plants. Abscisic acid and polyethylene glycol, stimulators for spruce embryo maturation, could also induce the hsp genes.

A reliable method for extraction of RNA from various conifer tissues

SummaryA simple and efficient procedure suitable for extraction of high-quality RNA from cultured conifer tissues, somatic embryos, zygotic embryos, needles, stem and root tissues was developed. It produced from 100 μg up to 700 μg total RNA per gram tissue dependent on the types of tissues used. RNA quality was estimated by spectrophotometry, agarose gel electrophoresis, in vitro translation of mRNA, cDNA synthesis and Northern blot analysis. The method also worked well with Arabidopsis thaliana and tobacco tissues.

Characterization of cDNAs representing five abscisic acid-responsive genes associated with somatic embryogenesis in Picea glauca, and their responses to abscisic acid stereostructure

Abstract. Five abscisic acid (ABA)-responsive cDNAs that were identified by differential screening of a white spruce [Picea glauca (Moench) Voss] cotyledonary somatic embryo cDNA library are characterized. Three of the cDNAs (PgEMB12, 14, 15) are predicted to encode homologues of different late-embryogenesis-abundant (LEA) proteins, a further two (PgEMB 5 and 23) share little similarity to any known DNA or protein sequences. When suspension cultures were fed 1.5 × 105 M S-(+)-ABA and assessed for transcript abundance over 48 h, gene expression corresponding to each lea-like gene was inducible and was evident during the 48-h period. Conversely, transcripts were at a very low abundance when suspensions were fed R-(−)-ABA. When suspensions were treated with a range of S-(+)-ABA and R-(−)-ABA concentrations (10−7–10−4 M), expression was induced at S-(+)-ABA concentrations of 10−5 M or above. Expression corresponding to PgEMB5 and PgEMB23 indicates less specificity for ABA stereostructure, with similar expression patterns being observed for either enantiomer during the 48 h after feeding. The two corresponding genes also appear more responsive to ABA concentration than the predicted lea genes, within the range examined (10−7–10−4 M).

events following ABA treatment of spruce somatic embryos

SummaryAbscisic acid (ABA) is involved in various physiological processes in plant growth and in the development of embryos and the maturation of seed. There is still much to learn about the influence of ABA on regulation of gene expression during plant and seed development. Perhaps not surprisingly, ABA has a major role in the stimulation of somatic embryo maturation in several conifer species, especially spruces. In spite of this, our knowledge of the effects of exogenous ABA is incomplete, for example the effect of ABA concentration on its uptake and fate has rarely been investigated during somatic embryo culture, and our knowledge of molecular events in conifer somatic embryo development is very scant. The intent of this review is to summarize some of the recent research in spruce somatic embryo development related to the use of ABA and to the consequence of its use.

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.

GROWTH PARAMETERS, PROTEIN AND DNA SYNTHESIS OF AN EMBRYOGENIC SUSPENSION CULTURE OF WHITE SPRUCE (PICEA GLAUCA)

Summary Growth parameters of a white spruce ( Picea glauca ) embryogenic suspension culture were characterized. Tissue fresh mass, dry mass, number of immature somatic embryos (stage 1), intracellular protein content, and total DNA accumulation showed similar growth curves over a 21-d culture period. Extracellular proteins gradually accumulated during the first 9 d, then dramatically increased. Immature embryo number showed a positive correlation to intracellular protein and DNA accumulation, correlation coefficients for these parameters were higher than for fresh and dry masses. The results from in vivo pulse-labelling with 35 S-methionine showed a peak for incorporation into proteins at day 7. The protein profiles of intracellular proteins were greatly different Erom those of extracellular proteins.