Effects of calcium on embryogenic induction and the accumulation
of abscisic acid, and an early cysteine-labeled metallothionein gene
in androgenic microspores of
Triticum aesti
6
um
Thomas L. Reynolds *
Department of Biology,Uni6ersity of North Carolina Charlotte,9201Uni6ersity City Boule6ard,Charlotte,NC28223,USA
Received 8 July 1999; received in revised form 14 September 1999; accepted 14 September 1999
Abstract
A cloned cDNA to the wheat (Triticum aesti6um) early cysteine-labeled metallothionein (EcMt) has been identified as a molecular marker for pollen embryogenesis in this plant. This transcript is not detected in uninucleate microspores at the time of culture or in pollen at any stage during normal ontogeny; its mRNA does increase in embryogenic microspores within 6 h of culture, peaks at around 24 h, declines, then levels off through the next 21 days of development. Additionally, the accumulation of the embryoid-abundant EcMt gene transcript shows a direct and positive correlation with an increase in abscisic acid (ABA) in embryogenic microspores and developing pollen embryoids. External Ca2+is required for in vitro pollen morphogenesis, since reduced Ca2+ concentrations in the medium suppressed EcMt transcript accumulation and the ability of microspores to form embryoids; however, endogenous ABA levels were unaffected my manipulating calcium concentrations in the medium. The calmodulin antagonist, W-7 also suppressed androgenesis and EcMt levels in treated microspores without altering ABA concentrations. These results suggest that ABA alone cannot maintain sporophytic differentiation of microspores and that Ca2+ may play a role in signal transduction during pollen androgenesis in bread wheat. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Abscisic acid; Calcium; Metallothionein; Pollen embryogenesis;Triticum aesti6um
www.elsevier.com/locate/plantsci
1. Introduction
In previous investigations, it has been shown that when anthers of the bread wheat (Triticum aesti6um) cultivar, Pavon ‘76’ are cultured on a simple nutrient medium, some of the enclosed uninucleate microspores are capable of becoming functional zygotes and developing into haploid plants [1,2] through a process known as pollen embryogenesis or androgenesis. Later, it was demonstrated that genes specific for androgenesis were temporally expressed in microspores during the early stages of anther culture in this plant [3] and one of these genes was identified as a wheat metallothionein (MT) [4]. In general, the
metal-lothioneins are small proteins that have a high affinity for binding metal ions and whose synthesis is normally regulated by the concentration of these ions [5]. The wheat metallothionein is an early, cysteine-labeled (Ec) class II gene first character-ized by Kawashima et al. [6] and, although no metal responsive elements are present, it contains a core sequence in the 5%-flanking region known to be an abscisic acid-responsive element. Consistent with the presence of this core element, during normal germination of wheat zygotic embryos, there was no accumulation of EcMt mRNA; how-ever, if the embryos were germinated in 100 mM
ABA, EcMt gene expression was induced [6]. In wheat anther cultures, it was shown that the pres-ence of the EcMt transcript served as a molecular marker for pollen embryogenesis and that its accu-mulation was directly and positively correlated * Tel.: +1-704-547-3370; fax:+1-704-547-3279.
E-mail address:[email protected] (T.L. Reynolds)
with an increase in abscisic acid (ABA) in em-bryogenic microspores [4]. If treated with an in-hibitor of ABA biosynthesis, fluridone, ABA and EcMt mRNA accumulation were suppressed in differentiating microspores. These results have been used to suggest that ABA modulates the expression of an embryo-specific metallothionein gene in microspores of wheat during androgenesis [4].
Cytosolic Ca2+ is known to mediate plant
de-velopment [7,8] and has been implicated in the regulation of several diverse physiological pro-cesses, including those affected by ABA [9]. Cal-cium-mediated cell signaling appears to involve a transient increase in cytosolic calcium followed by Ca2+ binding to a protein. This Ca2+/protein
complex undergoes a conformational change that either stimulates its activity or allows it to interact with some other regulatory protein [10]. Calmod-ulin (CaM) has become widely recognized as the primary mediator of Ca2+ signaling in plants and
animals. Because of the possibility that Ca2+ and
CaM might serve in the signal pathway regulating pollen morphogenesis in vitro, this study was de-signed to determine the effects of Ca2+ and CaM
on the ability of wheat microspores to become embryogenic and accumulate ABA and the EcMt transcript.
2. Materials and methods
2.1. Plant material and anther culture
Bread wheat plants (T. aesti6um cv Pavon ‘76’)
were raised in a growth chamber as described previously [3]. Anther culture techniques were em-ployed to initiate pollen embryogenesis. Anthers containing late-stage uninucleate microspores, just prior to the first haploid mitosis, were plated aseptically onto liquid CHB-2 medium [11] con-taining 1 mg l−1 2,4-dichlorophenoxy-acetic acid and 1.5 mg l−1 kinetin; cultures were maintained in the dark at 28°C.
To monitor embryogenesis, anthers were col-lected at intervals, squashed and stained on a slide with acetocarmine, then viewed using bright-field microscopy. A total of 16 anthers (four replicates of four anthers) were sampled for each treatment. Anther efficiency was determined by scoring the number of anthers with at least one pollen
embry-oid, while microspore efficiency was evaluated by scoring 1000 microspores in an embryogenic an-ther for the number of pollen that had undergone at least two cell divisions. Cultures were coded, then randomized prior to scoring. Data were sub-jected to analysis of variance and the Student – Newman – Keuls multiple comparison test using
the INSTAT BIOSTATISTICS software package
(Graph-Pad, San Diego, CA).
2.2. Chemical treatments
All organic compounds were filter sterilized, and inorganic compounds autoclaved. Only deionized distilled water was used and all solu-tions were stored in sterile plasticware. To deter-mine the effect on pollen morphogenesis of various Ca2+ concentrations, anthers were floated
on Ca2+-free CHB-2 medium. In some
experi-ments, cultures were treated with the calcium channel blocker verapamil or the calcium ionophore A23187. A stock solution of verapamil (0.1 M) was prepared in 95% (v/v) ethanol then diluted in CHB-2 medium to give the various final concentrations. The ionophore A23187 was dis-solved in 0.1% (v/v) dimethyl sulfoxide (DMSO) in CHB-2 medium. The calmodulin antagonist, W-7 (N -(6-aminohexyl)-5-chloro-1-napthalenesul-fonamide), or W-5 (N -(6-aminohexyl-1-napthale-nesulfonamide), which is an inactive structural analog of W-7, were first dissolved in DMSO before being added to CHB-2 medium. Control experiments were performed to examine the ef-fects of ethanol and DMSO individually; at the concentrations employed in this study, these sol-vents were found to have no significant effects on any of the experimental data.
Glassware, if used, was washed in concentrated nitric acid to eliminate trace amounts of Ca2+.
The chemicals, A23187, verapamil, W-5, and W-7, were purchased from Sigma Chemical Company (St. Louis, MO).
2.3. ABA determinations
ABA was extracted from microspores as previ-ously described [4]. In brief, total unbound ABA was extracted in 80% (v/v) methanol (10 ml mg−1
in-soluble material was precipitated by centrifuga-tion and re-extracted in methanol as before. Ex-tracts were combined and diluted to 70% (v/v) methanol. ABA determinations were done using an antibody-linked enzyme-linked immunosorbent assay test kit (Sigma, St. Louis, MO).
2.4. RNA isolation and slot blot hybridization
RNA isolations were done using the RNeasy Plant Total RNA kit (Qiagen, Chatsworth, CA) following the manufacturer’s instructions. For slot blot hybridization, 5 mg RNA per well were
transferred to nylon membranes using a vacuum manifold. The uniformity of loading and the in-tegrity of the samples were determined by mea-suring the intensity of the 25S rRNA band by densitometry of ethidium bromide-stained repli-cate sample gels under ultraviolet light. Blots were probed using a wheat cDNA clone (pEMB222) that is equivalent to the cDNAII mRNA for the wheat EcMt protein [4]. The insert is 577 bp and codes for an 81 amino acid protein product that is identical to the wheat class II Ec metallothionein protein [6]. The probes were pre-pared by first isolating plasmids (pcDNAII; Invit-rogen, San Diego, CA.) using Magic miniprep columns (Promega, Madison, WI), followed by digestion with HindIII and XbaI to isolate the insert from the vector, and separation elec-trophoretically on a 0.7% agarose gel.
Insert cDNA was radioactively labeled by the random oligonucleotide-priming procedure [12]. Membranes were prehybridized for 12 h at 65°C in heat-sealed plastic bags containing 1% sodium dodecyl sulfate (SDS), 1 M NaCl, and 10% dex-tran sulfate. For hybridization, the [32P]-labeled probe ((1 – 3)×106 cpm ml−1) and denatured salmon sperm DNA (200 mg ml−1) were added
directly to the prehybridization solution. The filters were hybridized for 24 – 36 h at 65°C and the washed twice in 2× SSC, 0.1% SDS for 10 min at room temperature, once in 1× SSC, 0.1% SDS at 65°C for 15 min and, finally, in 0.2×
SSC, 0.1% SDS for 10 min at 65°C. Following hybridization, the membranes were exposed to preflashed X-ray film with an intensifying screen at −70°C. Results were quantified using a Bio-Rad (Hercules, CA) Model 620 video densitome-ter, and transcript abundance was standardized on a fresh weight (f.w.) basis.
3. Results
3.1. Effects of exogenous calcium on pollen embryogenesis and accumulation of the EcMt transcript and ABA
Previous studies have shown that embryogenic microspores of Pavon ‘76’ accumulated the EcMt transcript maximally between 24 and 48 h of cul-ture, after which transcript level declined to a basal, steady state by day 7 [4]. The increase in EcMt RNA coincided with a rise in endogenous ABA to approximately 0.82 pmol mg−1 f.w.
dur-ing the first 24 h and showed a similar decline through the later stages of culture, reaching a minimum concentration of 0.38 pmol mg−1 f.w.
for 21-day-old embryoids [4]. As such, 24 h after culture was selected as the time period to measure the effects of calcium on the presence of EcMt mRNA and ABA in embryogenic microspores. As shown in Fig. 1, exogenous Ca2+ was
re-quired for the accumulation of the EcMt tran-script but not for endogenous ABA in culture. Even in medium containing no calcium, the ABA content in cultured microspores was the same as the control. The calcium concen-tration that promoted control levels of the EcMt transcript only became equivalent to controls at a concentration above 1 mM. External calcium was also required for the induction of pollen embryos in culture; the threshold concen-tration of Ca2+ was 500
mM, while 2 mM was
optimal (Fig. 1).
3.2. Effects of the calcium channel blocker,
6erapamil, and the ionophore, A23187
To further investigate the role of Ca2+ on
pol-len androgenesis, anthers were treated with the calcium channel blocker, verapamil, in Ca2++
CHB-2 medium (2.5 mM Ca2+). As shown in Fig.
2A, increasing concentrations of verapamil sharply suppressed endogenous EcMt mRNA but had no significant effect on ABA concentrations. This compound also inhibited androgenesis and, at 50
mM, anther induction efficiency was less than 1%
100 mM, it did inhibit the expression of the EcMt
mRNA and suppress anther induction frequency (Fig. 2B).
Fig. 2. Effects of the calcium channel blocker, verapamil, (A) and calcium ionophore, A23187, (B) on pollen androgenesis and accumulation of the EcMt transcript and endogenous ABA inT.aesti6umcv. Pavon ‘76’. Anthers were treated with the indicated concentrations of verapamil or A23187 in CHB-2 medium containing CHB-2.5 mM Ca2+ for 24 h to measure the effects on EcMt mRNA and endogenous ABA concentra-tions. To determine the effects of these compounds on andro-genesis, anthers were collected after 14 days on CHB-2 medium supplemented with the indicated concentration of channel blocker or ionophore. Anther induction frequency was determined for each experimental treatment as a percent of the control (insets). As in Fig. 1, each experimental treat-ment was compared with the appropriate control and data are expressed as a percentage of the control value. Each point represents the mean9S.E. from three replicate samples. Fig. 1. Effects of Ca2+on pollen androgenesis and
accumula-tion of the EcMt transcript and endogenous ABA in T. aesti6umcv. Pavon ‘76’. In this and other figures, to monitor
changes in EcMt RNA, after 24 h, total RNA was isolated from experimental and control samples, blotted onto nylon membranes and hybridized to the pEMB222 cDNA. The autoradiograms were quantitated using a scanning densitome-ter and the mean relative optical density per microgram fresh weight of tissue for each experimental sample and control was determined. The results are expressed as the relative amount of EcMt transcript compared with the control (3 mM Ca2+) from three replicate samples9S.E. To monitor sample uni-formity, equivalent amounts of total RNA were separated electrophoretically and stained with ethidium bromide. Inten-sity of the 25S RNA was determined by densitometry and standardized on a fresh weight basis. Endogenous ABA accu-mulation was also determined from experimental and control samples after 24 h. Data are expressed as the relative amounts of endogenous ABA (pmolmg−1 f.w.) in each experimental
sample compared with the control (2.5 mM Ca2+). Each point represents the mean9S.E. To determine the effects of the various Ca2+ concentrations on androgenesis, anthers were collected after 14 days on CHB-2 medium supplemented with the indicated Ca2+ concentration. Anthers were col-lected, squashed, and stained on a slide with acetocarmine, then viewed using bright-field microscopy. A total of 16 anthers (four replicates of four anthers) were sampled for each treatment and compared with control cultures (14 days on CHB-2 medium containing 2.5 mM Ca2+). Anther induc-tion efficiency was determined by scoring the number of anthers with at least one pollen embryoid. Data are presented as induction frequency for each experimental treatment as a percent of the control (inset).
3.3. Role of calmodulin in wheat pollen embryogenesis
Since an external source of Ca2+ appears to be
Ca2+-modulating protein may be involved in
sig-nal transduction. To determine if inhibition of CaM activity had an effect on androgenesis, the CaM antagonist W-7 was included in Ca2++
medium. At concentrations of 10 mM and above,
W-7 significantly diminished the levels of EcMt mRNA in cultured microspores and their ability to become embryogenic, although even at the maximum concentration of W-7 employed in the study, ABA concentrations were reduced insignifi-cantly compared with controls (Fig. 3). The inac-tive analog of W-7, W-5 had no effect on any aspect of wheat androgenesis, even at 150 mM
(data not shown).
4. Discussion
In this study, an analysis was made of the ability of exogenous Ca2+ to induce and promote
pollen androgenesis in anther cultures of T. aes
-ti6um. Treatment of anthers with low Ca2+
con-centrations markedly limited the numbers of anthers producing pollen embryos relative to the numbers observed in anthers treated with 2.0 mM Ca2+. Additionally, an external supply of calcium
was necessary for the expression of the wheat metallothionein transcript in embryogenic mi-crospores. Although Ca2+ can be stored within
plant cells in organelles (see, for example, [13]), the major source of stored calcium in plants is the cell wall [14]. No attempt was made in this study to monitor changes in Ca2+ flux between the
apo-plast and cytoplasm of the microspores or other tissues of the anther, so it is not possible to consider whether or not these components acted as a source of Ca2+ during androgenesis. It is clear,
however, that if there is an endogenous source of calcium, it is not sufficient to mediate androgenic induction.
Androgenesis was also inhibited when the cal-cium channel blocker verapamil was added to the medium, again supporting the suggestion that Ca2+ movement across membranes is required for
pollen embryogenesis. The ionophore A23197 acts to increase intracellular free calcium by facilitating the movement of Ca2+ across membranes. Anther
cultures of Solanum carolinenseL. treated with 10
mM A23187 more than doubled the numbers of
pollen embryoids, compared with controls [15]; however, in wheat anther cultures, 10 mM A23187
had no significant effect on androgenesis and 100
mM A23187 decreased not only embryogenic
in-duction, but also the accumulation of EcMt mRNA. The reason for these differential effects of the ionophore is not clear but, since calcium gradi-ents across membranes are known to be important in regulating cellular differentiation or pattern for-mation [9], any dissipation of this gradient could affect the microspore’s capacity to become embryogenic.
As stated previously, calmodulin is a ubiquitous modulator of Ca2+ signal transduction in plants
and animals. Recently, Yang et al. [16] character-ized the CaM gene family in wheat and classified the genes into four subfamilies, each representing a series of homoallelic loci on homologous chro-mosomes of the three genomes of bread wheat. In a later study, it was shown that although these CaM genes exhibited developmentally regulated organ-, tissue-, and cell-specific expression pat-terns, the highest level of expression for all four subfamilies in developing seeds was observed in
Fig. 3. The effect of W-7, a calmodulin antagonist, on accu-mulation of EcMt mRNA, ABA, and pollen embryogenesis in T.aesti6um. Anthers were treated with the indicated
the embryo [17]. These results are consistent with many others that show high CaM expression in differentiating and dividing plants [18 – 21]. Al-though no effort was made to monitor changes in CaM expression in embryogenic microspores of bread wheat, the observation that increasing con-centrations of the CaM antagonist W-7 lead to a corresponding decrease in androgenesis in treated microspores suggests that this protein plays a role in pollen development in vitro. Although it is possible that W-7 might act in some nonspecific way to inhibit embryogenesis, this seems unlikely since the use of the inactive analog of this com-pound had no adverse effect.
The results presented in this study show that reducing the availability of external calcium to cultured microspores inhibited androgenic poten-tial and suppressed the expression of the EcMt transcript; however, there was no corresponding reduction of endogenous ABA. This finding is consistent with an earlier study in which wheat microspores irradiated with inhibitory wavelengths of light were still capable of synthesizing ABA but failed to accumulate the EcMt transcript and be-come embryogenic [22]. Taken together, the stud-ies make it clear that although a promoter of pollen embryogenesis in bread wheat, ABA alone cannot maintain sporophytic differentiation in vitro.
Although the relationships among Ca2+, CaM,
ABA accumulation, and EcMt transcript expres-sion during pollen embryogenesis in wheat are not yet defined, the results presented here fit within the framework of the current model for the induction of microspore embryogenesis. As reviewed earlier [23], there are two consistent physiological gener-alizations that hold for androgenic induction: (1) microspores must be at the appropriate stage of development (late uninucleate or early bicellular); and (2) the embryogenic response requires the application of some type of external cue that leads to a stress or starvation response, preventing mi-crospores from continuing their normal mode of gametophytic development and inducing sporo-phytic differentiation. The stimulus may be cold, heat, osmotic shock or wounding. In this context, it can be speculated that the physical shock brought on by anther excision and culture in wheat induces the synthesis of ABA. As an initial response, ABA accumulation triggers a secondary response such as an increase in the concentration
of cytosolic-free Ca2+. The role of Ca2+ as a
second messenger in ABA signaling pathways has been well established [24 – 27], although the origin of the Ca2+ required to elevate cytosolic calcium
in response to ABA is still unclear [26]. In maize coleoptile segments, however [24], ABA’s effect on growth is mediated by inducing changes in mem-brane properties and altering the movement of Ca2+ across those membranes. In a similar
man-ner, calcium may act as a second messenger in the hormonal regulation of ionic fluxes that determine embryogenic induction in wheat microspores. How the ABA-induced Ca2+ signals are decoded
and relayed in the transduction chain is again speculative; however, it could be argued that downstream signaling elements include a modula-tor protein such as CaM, which, when associated with Ca2+, alters the pattern of gene expression in
competent microspores. Since the EcMt gene tran-script is not expressed during normal pollen on-togeny, but only in microspore-derived embryoids [4] or developing zygotic embryos [6], this appears to be an example of a newly expressed sporophytic gene. It is expected that as additional androgenic-specific markers are identified, it will be possible to add to the framework of the transduction pathway linking the external signals affecting pollen em-bryogenesis, the genes regulated by these signals, and the developmental response of the microspores.
Acknowledgements
This work was supported in part by the North Carolina Biotechnology Center, the Foundation of the University of North Carolina Charlotte, and the state of North Carolina
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