Summary We examined endogenous abscisic acid [(+)-ABA] amounts and mRNA populations in white spruce (Picea glauca (Moench) Voss) somatic embryos in liquid suspension culture during a pretreatment prior to induction of maturation with exogenous (+)-ABA. The pretreatment consisted of re-moval of 2,4-dichlorophenoxyacetic acid (2,4-D) from the multiplication medium for 7 days. The removal of 2,4-D re-sulted in a slight increase in endogenous ABA in pretreated tissues compared to nonpretreated tissues at the end of the 7-day pretreatment period (42.4 versus 20.0 pg mg−1 lyophil-ized tissue). Altered gene expression patterns were observed as early as one day after the start of the pretreatment, with more than 17 new polypeptides found in pretreated tissues. The influence of pretreatment continued to be observed after tissues were transferred to ABA-containing maturation medium. By comparing mRNA populations in pretreated and nonpretreated tissues cultured on either ABA-containing or ABA-free matu-ration medium, at least 12 mRNAs were observed to be induced by ABA, among which three polypeptides were ABA inducible only in pretreated tissues.
Keywords: abscisic acid, 2,4-dichlorophenoxyacetic acid, em-bryogenic cultures, gene expression.
Introduction
In white spruce, somatic embryo maturation can be promoted by transferring immature embryos in culture from a medium supplemented with benzyl adenine (BA) and 2,4-dichloro-phenoxyacetic acid (2,4-D) to a medium supplemented with racemic abscisic acid [(±)-ABA] (Dunstan et al. 1995). Several factors that influence the maturation process in culture have been identified, including the concentration of ABA (Dunstan et al. 1988, Hakman and von Arnold 1988, Attree et al. 1990, Dunstan et al. 1991, Attree et al. 1992), nutrient formulation (Tautorus et al. 1991), the use of polyethylene glycol (Attree et al. 1991, 1992), and the culture source (age of culture and genotype) (Jalonen and von Arnold 1991, Dunstan et al. 1993, 1995). Although such studies have lead to considerable
im-provements in the promotion of somatic embryo maturation in vitro, technical difficulties are still encountered, including low maturation efficiency, variability in response associated with culture age and maintenance condition (Dunstan and Bethune 1996), and aberrant embryo production (Tremblay 1990).
In an earlier study, we found that a pretreatment during which 2,4-D was not used one week before transfer of somatic embryos to maturation-inducing conditions (Dunstan et al. 1993) led to a significant improvement in production of mor-phologically normal cotyledonary somatic embryos (37.1 ± 4.2 in pretreated culture compared to 8.9 ± 1.4 in nonpretreated culture per 150 mgfw embryogenic culture inoculum). In
addi-tion, subsequent germination was improved from 59% in non-pretreated cultures to 86% in non-pretreated cultures. A reduction in the aberrant embryo population was also observed (11% of cotyledonary embryos in the pretreated population versus 71% in the control population).
Because of the important role that ABA plays in white spruce somatic embryo maturation, we investigated the possi-bility that pretreatment resulted in an increase in amount of tissue (+)-ABA. We also investigated the effects of pretreat-ment on gene expression, by analysis of polypeptides pro-duced in tissues during suspension culture and in the first seven days after tissues were transferred to medium with or without exogenous ABA.
Materials and methods
Induction of somatic embryo maturation
An embryogenic suspension culture of white spruce (Picea glauca (Moench) Voss) was initiated from zygotic embryos following the methods of Hakman and Fowke (1987). The culture was maintained in liquid suspension through weekly transfer of 10-ml aliquots to 250-ml DeLong flasks, each containing 50 ml of fresh 0.5 × LM liquid medium (Litvay et al. 1981) supplemented with 1.5% (w/v) sucrose, 1.8 g l−1 casein hydrolysate, 0.9 g l−1 glutamine, 9 µM 2,4-D and 4.4 µM BA (pH 5.6). The cultures were agitated at 150 rpm on a gyratory shaker at 25 °C in the dark.
Influences of altered phytohormone use on endogenous ABA and
mRNA populations during white spruce (Picea glauca) somatic
embryo culture
JIN-ZHUO DONG, CHERYL A. BOCK and DAVID I. DUNSTAN
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada
Received November 9, 1995
Before induction of somatic embryo maturation, half of the cultures were exposed to a pretreatment. For the pretreatment, 7-day-old suspension cultures were harvested by vacuum fil-tration and washed twice with 0.5 × LM medium lacking 2,4-D. Cultures were resuspended in 0.5 × LM medium con-taining only BA (4.4 µM) and cultured as described previously. Pretreated and nonpretreated tissues were collected on Days 1 and 6 of culture for investigation of gene expression. Part of the Day 6 harvest was also used to study gene expression during induction of somatic embryo maturation.
Induction of somatic embryo maturation was performed as described by Dunstan et al. (1988, 1993). Briefly, pretreated or nonpretreated tissues were collected on Day 6 by filtration and resuspended in 20% (w/v) 0.5 × LM medium lacking phyto-hormones. A 0.75-ml aliquot of suspension (about 0.15 gfw
tissue) was dispensed onto gridded black filter discs (No. AABG047S0, Millipore, Bedford, MA) placed on 25 ml agar-solidified (0.5% w/v) 0.5 × LP medium (von Arnold and Eriksson 1981) in the presence or absence of 15 µM natural (+)-ABA (pH 5.6) in sterile disposable petri dishes (100 × 15 mm). The cultures were incubated in low light providing photosynthetically active radiation (1 µmol m−2 s−1) for 7 days (Krizek 1982).
ELISA assay for endogenous ABA
Endogenous abscisic acid in pretreated and nonpretreated tis-sues was determined daily during pretreatment by an indirect, competitive immunoassay as per Walker-Simmons (1987) with the following modifications. Pretreated and nonpretreated tissues (about 200 mgfw per sample) were lyophilized
over-night and then ground. Abscisic acid was extracted overover-night with 1 ml of extraction buffer containing 0.05% (w/v) citric acid monohydrate in methanol. Following centrifugation, the supernatant was filtered through a 0.2 µm nylon filter and evaporated by vacuum centrifugation. After the residue was resuspended in 100 µl methanol, 900 µl TBS (Tris-buffered saline: 6.05 g Tris base, 0.20 g MgCl.6H2O and 8.80 g NaCl l−1) was added. This solution was transferred to the upper reservoir of a Centricon 3 microconcentrator (Amicon Canada Ltd, Oakville, ON, Canada), and centrifuged at 7,500 g until only a thin film of liquid remained in the upper reservoir. There were three replicates per treatment and each sample was assayed twice.
Extraction of RNA and in vitro translation
Total RNA was extracted as described by Shirzadegan et al. (1991). Poly A-tailed mRNA was obtained from total RNA using biotinylated oligonucleotides (dT) bound to paramag-netic particles (Promega, Ottawa, ON, Canada). The mRNAs were translated with a rabbit reticulocyte lysate cell-free trans-lation system (Boehringer Mannheim, Laval, Québec, Canada) in the presence of 35S-methionine (Amersham, Oakville, ON, Canada). The translated products were precipitated and dis-solved in UKS buffer (9.5 M urea, 5 mM K2CO3, 1.25% (w/v)
sodium dodecyl sulfate (SDS), 0.5% (w/v) dithiothreitol (DTT), 6% Triton X-100, 1.6% (v/v) Ampholytes 5--7 and
0.4% (v/v) Ampholytes 3--10) (Granier 1988) and then ana-lyzed by two-dimensional SDS-polyacrylamide gel electro-phoresis (SDS-PAGE). Radioactivity of labeled proteins was determined by liquid scintillation spectrometry.
Two-dimensional SDS-polyacrylamide gel electrophoresis In vitro-translated proteins were separated by two-dimensional SDS-PAGE based on the method of O’Farrell (1975) with some modifications. The gel solution used for isoelectric fo-cusing (IEF) consisted of 8 M urea, 3.5% acrylamide/bisacry-lamide (36.5/1 w/w), 2% NP-40, 1.6% (v/v) Ampholytes 5--7 and 0.4% (v/v) Ampholytes 3--10. The gel overlay buffer contained 8 M urea, 5% 2-mercaptoethanol, 1.6% Ampholytes 5--7 and 0.4% Ampholytes 3--10. A protein sample with ap-proximately 106 cpm was loaded on each gel tube (1.5 mm diameter). For IEF in the first dimension, an anodic buffer (0.06% phosphoric acid) and a cathodic buffer (0.1 M sodium hydroxide) were used at 200 V for 2 h, 400 V for 15 h and then 800 V for 2 h. The IEF gels were equilibrated with SDS-reduc-ing buffer (62.5 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 0.05% (v/v) 2-mercaptoethanol and 0.01% (w/v) bromophenol blue) for 10 min before development of the second dimension in SDS-PAGE. Slab gel electrophoresis methods were as described by Laemmli (1970). The stacking gels contained 5% (w/v) polyacrylamide/bisacrylamide (36.5/1 w/w), and the separating gels contained 12% (w/v) polyacry-lamide/bisacrylamide (36.5/1 w/w). A Protean II electrophore-sis system (Bio-Rad, Richmond, VA) was used for first- and second-dimension gel electrophoresis. The 14C-labeled protein standards (GIBCO BRL, Burlington, ON, Canada) were lysozyme (14.3 kDa), β-lactoglobulin (18.4 kDa), carbonic anhydrase (29.0 kDa), ovalbumin (43.0 kDa), bovine serum albumin (68.0 kDa), phosphorylase B (97.4 kDa) and myosin (H-chain, 200.0 kDa). Slab gels were fixed in a solution of 25% (v/v) isopropanol and 10% (v/v) acetic acid for 30 min with gentle shaking. The fixation solution was replaced with Am-plify Amersham, Oakville, ON, Canada), and the gels were agitated for another 30 min, and then vacuum dried at 65°C. X-Ray film (Kodak Diagnostic Film, O-Omat RP, Eastman Kodak, Rochester, NY) was exposed to dried gels for 5--7 days at −70 °C for fluorography of radio-labeled proteins. Three experiments were conducted for each treatment and two two-dimensional SDS-PAGE gels were run for each sample.
Results
Endogenous ABA amounts in pretreated and nonpretreated tissues
pretreated tissues (42.4 ± 13.0 pg mg−1 lyophilized tissues, P < 0.05).
Effect of pretreatment on mRNA population during the pretreatment
To determine the principal differences in gene expression pro-files between pretreated and nonpretreated tissues, mRNA isolated from both tissues on Days 1 and 6 of pretreatment was translated in vitro. Translation products were separated by two-dimensional SDS-PAGE as shown in Figure 2. More than 10 new polypeptides were found (circles) and another two polypeptides increased in abundance (arrowheads) on Day 1 in pretreated tissues compared with nonpretreated tissues. Five polypeptides were detected in nonpretreated tissues but not in pretreated tissues (squares).
On Day 6, approximately seven polypeptides were found only in pretreated tissues (circles), whereas three polypeptides (squares) were found only in nonpretreated tissues. An addi-tional polypeptide (arrow) was much less abundant in pre-treated cells than in nonprepre-treated cells.
Effect of pretreatment on mRNA population during culture on maturation medium
Seven days after pretreated and nonpretreated tissues were placed on ABA-containing maturation medium, differences in gene expression patterns were evident (Figure 3). The main differences included at least nine polypeptides that were unique to pretreated tissues (circles) and four polyeptides that were more abundant (arrowheads) in pretreated tissues than in nonpretreated tissues. Two polypeptides were detected in non-pretreated tissues but were not found in non-pretreated tissues (squares).
mRNA Population regulated by ABA
To observe ABA effects on gene expression in pretreated tissues, mRNAs were isolated from cultures growing on ABA-containing and ABA-free maturation medium. Polypeptide products from in vitro-translation were fractionated by two-di-mensional SDS-PAGE (Figure 4). After 7 days of culture on maturation medium, approximately 12 new polypeptides (cir-cles) were induced by ABA (Figure 4), including three (a, b, c) that had previously been identified as ABA inducible only in pretreated tissues (cf. Figures 3 and 4). Six polypeptides (Fig-ure 4; squares) detected in tissues cult(Fig-ured on ABA-free me-dium were not observed in ABA-treated tissues. Seven polypeptides (arrowheads) were more abundant in tissues cul-tured on ABA-containing medium than in tissues culcul-tured on ABA-free medium, whereas another polypeptide (arrow) was
Figure 1. Endogenous ABA in pretreated and nonpretreated embryo-genic tissues of white spruce. The amount of (+)-ABA was measured by ELISA. Three replicates were conducted for each treatment and each sample was assayed twice. The vertical bars show the standard deviation of the mean.
less abundant in tissues cultured on ABA-containing medium than in tissues cultured on ABA-free medium.
Discussion
Although several improvements have been made to the meth-ods for white spruce somatic embryo maturation (Dunstan et al. 1995), difficulties are still encountered. These include low maturation efficiency, variability in response associated with
culture age and maintenance condition, and aberrant embryo production. A one-week pretreatment period, in which 2,4-D was removed from the multiplication medium immediately before transfer of cultures to ABA-containing maturation me-dium, improved the maturation characteristics of white spruce somatic embryo cultures (Dunstan et al. 1993). Pretreatment led to a significant improvement in production of morphologi-cally normal cotyledonary somatic embryos and germinants, and there was an associated reduction in aberrant embryos.
Figure 3. Autoradiograph after two-dimensional SDS-PAGE of in vitro-translated products from pretreated and nonpretreated white spruce em-bryogenic tissues cultured on ABA-containing (15 µM (+)-ABA) maturation medium for 7 days. The symbols indicate polypeptide syn-thesis changes due to pretreatment:
s = new polypeptides present only in pretreated tissues; u = polypep-tides present only in nonpretreated tissues; = polypeptides with in-creased synthesis in pretreated tis-sues.
Figure 4. Autoradiograph after two-dimensional SDS-PAGE of in vitro-translated products from pretreated white spruce embryogenic tissues cultured on maturation medium for 7 days in the presence or absence of (+)-ABA (15 µM). Symbols indi-cate polypeptide synthesis changes resulting from exposure to ABA: s
Phytohormones serve as signal molecules in induction of embryogenesis and subsequent morphological changes (Qua-trano 1986, Schmidt et al. 1994). Removal or addition of phytohormones, or changes in culture conditions that modify the concentrations of endogenous phytohormones, are there-fore likely to alter embryo development. For example, the pretreatment used in this study altered the relative availability of 2,4-D and BA. 2,4-Dichlorophenoxyacetic acid, which is known to inhibit carrot somatic embryo maturation at high concentrations (de Vries et al. 1988), decreases in tissues when cultures are grown on 2,4-D-free medium (Michalczuk et al. 1992). In white spruce, the consequence of the pretreatment was a gradual increase in the amount of endogenous ABA in pretreated tissues compared with nonpretreated tissue between Days 4 and 6 of pretreatment, and altered mRNA populations both during pretreatment and during subsequent culture on maturation medium. There were at least 26 polypeptides whose expression was affected by pretreatment. Although two-dimensional SDS-PAGE has limited resolution, the differences detected in expression of polypeptides can serve as a guide for understanding developmental processes (Borkird et al. 1988, de Vries et al. 1988, Wilde et al. 1988). The altered mRNA populations may be directly associated with improvements in maturation, but even if the effect is less direct, it is commensu-rate with improved maturation characteristics. Of the 12 polypeptides that were induced when tissues were cultured on ABA-containing maturation medium, three polypeptides were induced only in tissues that had been pretreated. The effects of pretreatment on gene expression patterns provide a means of studying some of the critical early events in white spruce somatic embryo maturation.
Acknowledgments
This is NRCC No. 38926.
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