Endo-β-mannanase activity during dormancy alleviation and germination of white spruce (Picea glauca) seeds

It is not known how embryos of seeds of the Pinaceae protrude from their enclosing tissues to complete germination. Prior to protrusion of the radicle there is an increase in endo-β-1,4-mannanase (EC 3.2.1.78) activity associated with weakening of the micropylar megagametophyte/nucellus from seeds of white spruce ( Picea glauca [Moench.] Voss). Mannanase activity is present as three isoforms (pI values 5.0, 4.8, 4.7) in both the embryo and surrounding structures (megagametophyte and nucellus) prior to and during imbibition. Activity of all the isoforms increases in the chalazal and micropylar megagametophyte during germination. Activity then declines after the testa splits, typically 1 day prior to radicle protrusion, due partially to its leaching from the seed into the surrounding water. Activity increases in the cotyledons and axis as the embryo commences elongation. Seeds from dormant seedlots exhibit a lower germination percentage, relative to seeds from nondormant seedlots, and the force necessary for the embryo to puncture the surrounding structures tends to be greater. Although similar mannanase activities are present in unimbibed seeds of dormant and nondormant seedlots, during germination, enzyme activity in seeds of dormant seedlots is lower. Moist chilling alleviates dormancy in the seeds of the Pinaceae and, during 3 weeks of this treatment, mannanase activity slowly increases. After 3 weeks of moist chilling and regardless of whether the seedlot was dormant or not prior to moist chilling, the force necessary to puncture the micropylar megagametophyte and nucellus is lower, and the speed of germination greater. Seeds from previously dormant seedlots also complete germination to a greater percentage, relative to unchilled seeds from dormant seedlots. Upon transfer to 25°C, mannanase activity in moist-chilled seeds decreases during germination of all seedlots regardless of their previous dormancy status.     

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of ¹⁴C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of ¹⁴C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.     

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.     

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

Summary We 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. NRCC no. 37345.     

Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds

The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca²⁺, Mg²⁺ and Mn²⁺; only the nucleases active at neutral pH were inhibited by Zn²⁺. The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture.     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

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.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Sucrose requirements and lipid utilization during germination of interior spruce (Picea glauca engelmannii complex) somatic embryos

Both somatic and excised zygotic embryos of interior spruce (Picea glauca engelmannii complex) required exogenous sucrose in the medium for germination in vitro. Over a period of 29 days on sucrose-containing medium germinants with roots and epicotyls developed from both kinds of embryo, and their content of linolenic acid (9,12,15-18:3) increased about six- to eightfold. Without added sucrose, embryos showed retarded growth or were necrotic, and the content of linolenic acid was barely detectable in their fatty acid profiles. Through¹⁴C-sucrose uptake studies, it was determined that germinants consumed only 25% of the sucrose available in a 1% (wt/vol) sucrose-containing medium. Since no radiolabelled fatty acids were detected, it appears that externally supplied sucrose was not used in the synthesis of lipids. Although sucrose was present during plantlet development, 72% of the initial lipids were consumed. To some extent, the plantlets appeared to be obligate storage lipid utilizers.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - FAMEs Fatty acid methyl esters - HPLC High-performance liquid chromatography     

Changs in pyrimidine nucleotide biosynthesis during germination of white spruce (picea glauca) somatic embryos

Summary Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-¹⁴C]uracil and [2-¹⁴C]uridine, intermediate metabolites of the salvage pathway and [6-¹⁴C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-¹⁴C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

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

Summary The 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.     

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.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - BA N⁶-benzyladenine - cDNA complementary deoxyribonucleic acid - Em early methionine-labelled - HSP heat-shock protein - LEA late embryogenesis abundant - PEG polyethylene glycol The authors thank Mr. Terry Bethune for his assistance, and Dr. Larry Pelcher, Mr. Barry Panchuk and Mr. Don Schwab for DNA sequencing and primer synthesis. This is National Research Council of Canada publication number 38929.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Understanding changes in black (Picea mariana) and white spruce (Picea glauca) foliage biomass and leaf area characteristics

Key message

Growth conditions related to inter-tree competition greatly influence black and white spruce foliage biomass and projected leaf area characteristics.

Abstract

Foliage characteristics such as biomass and area are important among other reasons because they can be related to tree growth. Despite their economic and ecologic importance, equations to characterize foliage biomass and projected area of black (Picea mariana (Miller) BSP) and white (Picea glauca (Moench) Voss) spruces are sparse. Total foliage biomass and projected leaf area, foliage biomass and leaf area density, and relative vertical distribution of black and white spruces foliage biomass and leaf area were modelled with linear and nonlinear mixed effect models. A total of 65 white spruces and 57 black spruces were destructively sampled at four different locations in Alberta, Québec, and Ontario, Canada. Our results show that for each species, total tree foliage biomass and projected leaf area is proportional to stem diameter, total height, and crown length. The addition of crown length in the equations improved the precision of the predictions of total foliage biomass for both species and diminishes greatly the site level random effect. An increase in DBH for black spruce and in the DBH to total height ratio for white spruce skewed the relative vertical foliage biomass distribution toward the base of the living crown. According to our results, growth conditions or tree development stage influence both foliage biomass and leaf area characteristics of black and white spruces. Our results emphasize the importance of inter-tree competition on foliage biomass characteristics.     

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C-labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐¹⁴C]cytidine, [2‐¹⁴C]deoxycytidine and [2‐¹⁴C]thymidine. The de-novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce-cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO₂ at all stages of embryo development.     

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter--glucuronidase-nopaline synthase 3 fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the -glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca x Picea engelmannii complex)

Putrescine, spermidine, and spermine levels during somatic embryogenesis of interior spruce (Picea glauca x Picea engelmannii complex) were quantified On abscisic acid supplemented growth medium putrescine and spermidine levels increased two-fold coinciding with maturation of the early somatic embryos to globular embryos. Polyclonal antibodies raised against Escherichia coli arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), following affinity purification specifically recognized spruce ADC and ODC, which corresponded to 85kD and 65kD bands on western blots of total protein extracts from embryogenic masses, Immunoassays using these antibodies showed increased ADC levels corresponding to embryo maturation while ODC levels remained the same. From these results it is concluded that polyamines are involved in the maturation of somatic embryos of interior spruce.Abbreviations ADC arginine decarboxylase - BSA bovine serum albumin - ODC ornithine decarboxylase - PBS phosphate buffered saline - PCA perchloric acid - SDS-PAGE sodium dodecyl sulfateporyacrylamide gel electrophoresis