expression increases during cold-temperature stress (Dunn et al., 1993) and after prolonged hypoxia .... Patricia Welch. Received March 2, 1994; accepted July ...
Plant Physiol. (1 994) 106: 897-903
,
Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit a! upon Wounding in Potato Tubers' James K. Morelli, Christine K. Shewmaker, and Michael E. Vayda*
Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, Maine 04469-5735 (J.K.M,M.E.V); and Calgene Inc., 1920 Fifth Street, Davis, California 95616 (C.K.S)
genes and a general stimulation of translational activity (reviewed by Davis et al., 1990). Increased protein synthesis appears integral to the wound response: wounding prior to bacterial inoculation reduces the severity of bacterial rot, and, conversely, hypoxic stress or chemical agents that block tuber protein synthesis increase the severity of bacterial disease (Vayda et al., 1992). One function of the wound response is formation of a physical bamer to bacterial entry. Mechanical wounding rapidly elicits expression of genes encoding enzymes involved in phenylpropanoid metabolism (Vayda and Schaeffer, 1988; Bevan et al., 1989; Dyer et al., 1989; Butler et al., 1990; Crosby and Vayda, 1991; Ishizuka et al., 1991; Vayda et al., 1992), sesquiterpenoid metabolism (Oba et al., 1985; Stermer and Bostock, 1987; Yang et al., 1991; Choi et al., 1992), and suberization (Lojkowska, 1988; Roberts et al., 1988). Also expressed in a later time frame, 12 to 24 h after wounding, are genes whose products are involved in cell division and cell wall biosynthesis, including histones and the cell wall proteins HRGP and GRP (Butler et al., 1990; Rumeau et al., 1990). Although it has not been demonstrated
that any of these gene products contribute to disease resistance, their appearance suggests that two major roles of the wound response are suberization and formation of a wound periderm. Stimulation of translational activity appears to coincide with the expression of these gene products. The mechanism by which translational activity is stimulated is unknown. The increase in translational activity is most likely the result of transition from the slow respiration of the mature tuber after harvest to a more active metabolic state upon wounding; the respiration of tubers is known to increase upon wounding (Kahl, 1978). This increase in protein synthesis cannot be due simply to an increase in mRNA. Indeed, there is no significant increase in the total poly(A)+ RNA content upon wounding, although the abundance of specific mRNA species does change considerably (Butler et al., 1990). Polysomes isolated from wounded tubers are severalfold more active in translational run-off than polysomes isolated from mature resting tubers (Crosby and Vayda, 1991), suggesting a recruitment of ribosomes into polysomes or a stimulation of the protein synthetic machinery. Increases in transcription of ribosomal RNA (Belknap and Rickey, 1990) and at least one ribosomal protein gene (Garbarino et al., 1993) have been reported upon wounding of potato tubers. Similarly, a 32-kD polypeptide accumulates in the polysome fraction of wounded tubers within minutes after wounding, paralleling the presence of wound-response mRNA species (Crosby and Vayda, 1991). However, it is not clear whether this polypeptide is involved in activation of translational processes or is simply associated with wound-response mRNAs. In a wide variety of organisms, an increase in translational activity is accompanied by an increase in the abundance of EF-la, whose function is to present amino acyl-tRNAs to 80s ribosomes (Webster, 1985; Cavallius et al., 1986; Brady, 1988; Ursin et al., 1991). These observations indicate that the stimulation of protein synthesis is likely to involve activation of components of the translational apparatus. Genes encoding EF-la are constitutively expressed in plant
This work was supported by U S . Department of Agriculture/ National Research Initiative Competitive Grants Program grant 9137100-6622 and Maine Agricultural Experiment Station project ME38402. This is publication No. 1844 of the Maine Agricultural and Forest Experiment Station. * Corresponding author; fax 1-207-581-2801.
Abbreviations: EF-la, translation elongation factor 1 subunit a; GRP, the Gly-rich cell wall protein; GUS, fl-glucuronidase; HRGP, the Hyp-rich protein extensin; 4-MU, 4-methyl umbelliferone; 4-MUG, 4-methyl umbelliferyl glucuronide; PAL, Phe ammonialyase; PVDF, polyvinylidene difluoride; X-Gluc, 5-bromo-4-chloro3-indolyl-fl-~-glucuronic acid.
Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. l h e response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit u (EF-lu) parallels translational activity. Changes in the steady-state level of EF-lu mRNA, and expression of a chimeric E F - l u promoter/ @-glucuronidaseconstruct in transgenic potato tubers, indicatethat the gene encoding E F - l a is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors.
Wounding elicits a rapid and complex response in potato
(Solanum tuberosum)tubers that includes induction of specific
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cells (Axelos et al., 1989; Liboz et al., 1989; Perlick and Puhler, 1993) but expression varies with organ type and age. Specifically, expression of EF-1a is increased in tissues exhibiting high levels of protein synthesis such as meristems, somatic embryos, and auxin-treated tissues (Pokalsky et al., 1989; Ursin et al., 1991; Kawahara et al., 1992; Curie et al., 1993). EF-la appears to be encoded by a small multigene family. For example, in Arubidopsis there are four EF-la genes (Liboz et al., 1989). However, individual EF-la promoter/ GUS constructs are active in various tissues of transgenic tobacco and Arabidopsis plants (Ursin et al., 1991; Curie et al., 1993). These observations indicate that single EF-la promoters have the ability to respond to both developmental and environmental cues. Further, Curie et al. (1992) have demonstrated that transcription factors that interact with the Arubidopsis EF-la promoter are present in nuclear extracts of several dicot and monocot species. Here we report that a tomato EF-la promoter/GUS transgene that is transcribed in transgenic tobacco tissues active in protein synthesis (Ursin et al., 1991) is also expressed in transgenic potato tubers and is induced by wounding in a biphasic manner that parallels the observed increase in translational activity in vivo. MATERIALS AND METHODS Plant Material
Mature potato tubers (Solanum tuberosum cv Russet Burbank) were obtained from the Maine Agricultural Experiment Station, purchased in a local market, or harvested from greenhouse-grown plants at the University of Maine. After harvest, tubers were stored for 2 weeks at room temperature, then stored at 4OC prior to use. Transgenic plants containing the tomato EF-la promoter/GUS construct pCGN2158 (described by Ursin et al., 1991)were obtained by Agrobacteriummediated transformation using the binary vector technique (McBride and Summerfelt, 1990). Transgenic plants containing the patatin promoter/GUS construct (described by Wenzler et al., 1989) were generously provided by Dr. W. Park. In all cases, tubers were rinsed briefly with warm water and surface sterilized with 5 % hypochlorite (v/v), then dried and allowed to equilibrate at room temperature for 16 to 24 h before use. Tubers were wounded using a I'-200 micropipette tip as described previously by Vayda and Schaeffer (1988).
Plant Physiol. Vol.. 106, 1994
(Markson Scientific, Phoenix, AZ),then thawed in 5 mL of homogenization buffer containing 40 m Hepes, pH 7.6, 1 m Mg acetate, 100 m K acetate, 2 m CaC12, and 4 II~M DTT. A 40-pL aliquot of clarified supematant (lfl,OOOg) was assayed for translational activity in a 50-pL reacti,m containing 40 n w Hepes, pH 7.5, 1.2 m ATP, 0.08 m~ GTP, 9.6 m creatine phosphate, 0.25 unit of creatine phosphokinase, 40 units of human placental RNase inhibitor (Boehringer Mannheim), 0.8 m spermine, 1.6 m DTT, 20 1x-m each of the amino acids except Met, and 0.2 pCi of [35S]Met.Either 2 pg of brome mosaic virus RNA or 5 pg of total tuber RNA was added as template. After a 30-min reaction at 22OC, extracts were treated with RNase A and incorporation was measured as TCA-precipitable cpm. lmmunoblot Analysis of EF-la
Total proteins were extracted from potato tubers as described by Vayda and Schaeffer (1988). Proteins were fractionated by selective precipitation with 40, 70, or 100% ammonium sulfate as described previously (Lax emt al., 1986; Browning et al., 1990; Webster et al., 1991). Proteins were resolved by SDS-PAGE and electroblotted to P " F paper (Immobilon-CD, Millipore, Bedford, MA). Blots were incubated with anti-EF-la antiserum prepared against either wheat germ EF-la (generously provided by Dr. K. Browning) or Artemia EF-la (generously provided by Dr. \V. Moller). Immunoblots were developed using the ECL che~niluminescent detection system (Amersham). Analysis of GUS Activity
Fluorometric analysis of GUS activity was performed as described by Jefferson et al. (1987) using 100 mg of tuber material or 50 mg of leaf tissue. Samples were frozen in liquid nitrogen, ground to a powder, and thawed in prol ein extraction buffer containing 50 m NaH2P04, pH 7.0, 10 m EDTA, 0.1% Triton X - l O O , O . l % sodium lauryl sarcosine, and 10 m 8-mercaptoethanol. GUS activity was determined from the rate of 4-MU production upon hydrolysis of 4-MUG as described by Jefferson et al. (1987). Histochemical localization of GUS activity was performed on 2-mm-thick tuber slices using X-Gluc as substrate, as described by Jefferson et al. (1987).
Assessment of Protein Synthesis in Vivo and in Vitro
RNA Gel Blot Hybridization
Tubers were labeled in vivo by presenting 0.5 mCi of [35S]Met (New England Nuclear) to a tuber wound at the time of wounding or at the times indicated after wounding. After a 15-min incubation at 22OC, a 2-g core of tuber tissue surrounding the wound was extracted as described previously (Vayda and Schaeffer, 1988). A 30-min digestion with 100 pg/mL RNase A was included to degrade amino acyl-tRNAs. Protein synthesis was measured as TCA-precipitable counts per g fresh weight of tuber tissue. Crude extracts of potato tubers were prepared by a modification of the procedure of Erickson and Blobel (1983). Ten grams of tubers, either before wounding or 4 or 16 h after wounding, were frozen in liquid nitrogen and ground to a fine powder using a Miracle Mill
RNA was isolated from fresh tissue that was frozen in liquid nitrogen, ground to a fine powder using a Miracle Mill, and immediately extracted using the phenol method described previously (Butler et al., 1990). Total RNA was resolved by formaldehyde agarose gel electrophoresis and gel blot hybridization, using the methods described previously (Butler et al., 1990; Crosby and Vayda, 1991). The tomato EF-la coding sequence probe pCGN666 is described by Pokalsky et al. (1989). The insert DNA was isolated from agarose gels using the Geneclean I1 system (BiolOl, Inc., Mount Prospect, IL) after digestion of pCGN666 with EcoRI and XbuI. The insert encoding a segment of potato 18s rDNA (Butler et al., 1990; Davis et al., 1990) was isolated after
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Biphasic Induction of Elongation Factor 1 Subunit a upon Wounding
digestion of the construct pMlOl with EcoRl. The probes were labeled using [a-32P]-dCTP by the random primer method (Boehringer Mannheim).
Table I. Incorporation in vitro of ^SJMet by cell-free extracts prepared from nonwounded and wounded potato tubers Extract
RESULTS
No RNA
Nonstressed 653 ± 83 4-h wounded 738 ±216 16-h wounded 612 ± 176
Stimulation of Translation in Vivo Is Biphasic
Figure 1 shows the incorporation of [35S]Met by potato tuber tissue in a 15-min reaction, with label presented at the times indicated after wounding. There is a 5-fold increase in the amount of label incorporated by 2-h-wounded tubers relative to those same tubers when label is presented at the time of wounding. Incorporation by 24-h-wounded tubers is 10-fold greater than by the nonstressed sample. However, rather than a continuous increase in the incorporation as a function of time after wounding, tubers reproducibly exhibit a biphasic induction of protein synthetic activity. The initial phase begins within 30 min after wounding and peaks 2 to 4 h after wounding. The second induction is evident by 12 h after wounding and is maximal 24 to 36 h after wounding. During the interval between phases, tubers exhibit a slight decrease in the incorporation during the 15-min labeling reaction. This biphasic stimulation of translational activity upon wounding is consistent with the previous observation that there are two classes of potato tuber wound-response mRNAs: one class represented by PAL accumulates to maximal levels within 1 to 4 h after wounding, the other class represented by HRGP accumulates 12 to 24 h after wounding (Butler et al., 1990; Davis et al., 1990; Crosby and Vayda, 1991).
Brome Mosaic RNA
Specific Activity* Potato RNA
1,763 ±160 3,785 ±216 10,283 ± 1920
1244 ±35 3246 ± 408 8264 ± 271
' Specific activity expressed as TCA-precipitable cpm incorporated in a 30-min reaction at 22°C. Activity is the mean ± SE for three independent trials.
RNA or 5 jig of total tuber RNA as template, wounded tuber extracts exhibited a greater ability to incorporate [35S]Met into TCA-precipitable products than extracts from those same tubers made prior to wounding. The increased activity is comparable to the increased incorporation of label observed in vivo (Fig. 1), i.e. 4-h- and 16-h-wounded tuber extracts exhibit approximately 2- and 6-fold greater incorporation, respectively, than nonstressed tuber extracts. Thus, increased translational activity upon wounding is due, at least partially, to a stimulation of the translational machinery and not to an increase in available mRNA. Abundance of EF-1a Increases upon Wounding
The immunoblot shown in Figure 2 demonstrates that the amount of total cellular EF-la increases upon wounding in parallel with the observed increase in translational activity. Antiserum raised against purified wheat germ EF-la (Brown-
Increased Translational Activity Is Evident in Vitro
The data presented in Table I indicate that the translational apparatus of wounded tubers is more active than that of mature, nonstressed tubers. Cell-free extracts were prepared from individual tubers prior to stress and 4 or 16 h after wounding. When provided with 2 /ig of brome mosaic virus
A.
a
Hours Wounded
1 2 4 6 8 12 18 24
0
2
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6
8
10
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14
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18
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Time After Wounding (h)
Figure 1. Translational activity of potato tubers in response to wounding. [35S]Met (0.5 mCi) was presented to a tuber wound at the time of wounding (time 0) or at the times indicated after wounding. After a 15-min incubation at 22 °C, the 2-gcore of tuber tissue surrounding the wound was extracted as described previously (Vayda and Schaeffer, 1988). Incorporation is measured as TCAprecipitable cpm per g fresh weight. Results shown are the mean ± SE of three trials.
Figure 2. Accumulation of the EF-1a polypeptide upon wounding. A, Total soluble proteins from a 2-h-wounded potato tuber were fractionated by precipitation with 0 to 40% (lane a), 40 to 70% (lane b), or 70 to 100% (lane c) ammonium sulfate. Twenty micrograms of each fraction was separated by SDS-PACE and electrophoretically transferred to a PVDF membrane (Millipore). EF-la was detected using an antiserum raised against wheat germ EF-1 a (kindly provided by K. Browning). B, Polypeptides present in 20 /ig of the 40 to 70% ammonium sulfate fraction of a nonstressed tuber (lane 0) and samples of that same tuber 1, 2, 4, 6, 8, 12, 18, or 24 h after wound induction were resolved by SDS-PACE, blotted to PVDF, probed using the anti-wheat germ EF-1« antiserum, and visualized using the ECL system (Amersham). Arrows indicate the immunoreactive polypeptide with a mobility of 48 kD relative to known polypeptide molecular mass markers (Bio-Rad).
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ing et al., 1990) specifically recognizes a 48-kD protein in the 40 to 70% ammonium sulfate precipitate of potato tuber extracts (Fig. 2A). This species is also recognized by an antiserum prepared against Artemia EF-la (data not shown). The electrophoretic mobility of the immunoreactive polypeptide is consistent with that of EF-la of other species (Browning et al., 1990) contained in the 40 to 70% ammonium sulfate fraction (Lax et al., 1986; Webster et al., 1991). Thus, we conclude that these antisera efficiently detect potato EFla. The abundance of this immunoreactive 48-kD polypeptide increased in tubers throughout the initial 4 h after wounding (Fig. 2B). After a decrease during the period 6 to 8 h after wounding, the abundance of this polypeptide increased greatly during the interval 12 to 24 h after wounding (Fig. 2B). This observation suggests that EF-la increases in potato tuber tissue in parallel with the observed biphasic increase in translational activity. Accumulation of EF-la upon Wounding Is Due to
Increased Transcription
A transgenic potato clone (cv Russet Burbank) containing a chimeric gene construct employing the tomato EF-la promoter to drive expression of the GUS coding sequence (described by Ursin et al., 1991) was used to test whether the observed accumulation of EF-la was due to increased transcription or increased stability of the protein. It was previously shown that this construct was expressed in transgenic tobacco tissues that display high translational activity (Ursin et al., 1991). Transformed potato plants containing this construct exhibit a similar pattern of expression in shoot tissues (data not shown). Nonstressed tubers grown from these plants have residual GUS activity, indicating expression of this transgene during tuberization. Figure 3 shows that GUS activity increased during the first 2 h after wounding, followed by a subsequent increase during the period 12 to 24 h
0
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6
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10
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14
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18
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22
24
Time After Wounding (h)
Figure 3. Expression of a chimeric tomato EF-1« promoter/CUS gene construct in wounded potato tubers. Transgenic tubers were obtained from potato clones containing either the EF-1a/CUS construct (•) described by Ursin et al. (1991) or the patatin/CUS construct (•) described by Wenzler et al. (1989). CUS activity in 100 mg of nonstressed tuber tissue (time 0) or 100 mg from the same tuber removed at the times indicated after wounding was determined by the rate of hydrolysis of 1 mM 4-MUC, with spectrofluorometric determination of fluorescence relative to 4-MU standards. Results shown are the mean ± SE of three trials.
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01
Hours Wounded
2
4 6 8 12 24
if*
EF-1a
18SrDNA
Figure 4. Accumulation of EF-1a mRNA in wounded potato tubers. Total RNA (Butler et al., 1990) was isolated from a potato tuber prior to wounding (lane 0) and fractions of that same tuber were isolated 1, 2, 4, 6, 8, 12, and 24 h after wounding. The RNA gel blot was hybridized to 32P-labeled EF-1a cDNA (Pokalsky et al., 1989) then reprobed with 32P-labeled potato 18S rDNA (Butler et al., 1990).
after wounding. An increase in GUS activity was not observed (Fig. 3) upon wounding of control tubers containing a patatin promoter/GUS reporter construct (Wenzler et al., 1989). The GUS activity of the latter tubers is low relative to that reported previously (Wenzler et al., 1989), presumably because transcription of patatin ceases after harvest and there is significant turnover of the GUS protein. From the data in Figure 3, we conclude that expression of the tomato EF-la/ GUS construct in potato tubers shows a biphasic induction upon wounding. Figure 4 shows that steady-state levels of mRNA encoding endogenous EF-la fluctuate in a manner similar to expression of the tomato EF-la/GUS transgene. RNA gel blot hybridization using the tomato EF-la coding sequence (Pokalsky et al., 1990) demonstrates that EF-la mRNA accumulated during two distinct periods after wounding: 1 to 2 h after wounding and 12 to 24 h after wounding. Hybridization of this same blot to a ribosomal rDNA probe confirms that equal amounts of total RNA were loaded in each lane. The biphasic accumulation of EF-la mRNA upon wounding (Fig. 4) is distinct from previously observed changes in steady-state levels of other mRNA species (Butler et al., 1990; Davis et al., 1990). Wound-induced mRNAs such as PAL and HRGP show single increases with highest abundance at 4 and 24 h, respectively, whereas the abundance of noninduced mRNAs, such as actin, remain unchanged (Butler et al., 1990; Davis et al., 1990). The clear decrease in EF-la mRNA during the period 6 to 8 h after wounding and the absence of increased GUS activity during this same interval suggest that induction of EF-la in response to wounding is due to two distinct bursts of transcriptional activity coinciding with the observed increase in translational activity in vivo. Histochemical staining of wounded EF-la/GUS minitubers for GUS activity shows that expression of the transgene is localized to within 3 to 5 mm of the wound site (Fig. 5). Tubers assayed within the first 8 h after wounding exhibited intense blue staining limited to the immediate vicinity of the wound. In 12-h- to 24-h-wounded tubers, GUS activity was
Biphasic Induction of Elongation Factor 1 Subunit a upon Wounding
24
NW
Figure 5. Histochemical localization of EF-la/CUS expression in potato tubers after wounding. Transgenic potato tubers were wounded by single insertion of a P-200 pipette tip (position and direction of wound indicated by arrows). Nonwounded tubers (NW) and tubers wounded for either 2, 4, 6, 8, 12, or 24 h were sliced through the plane of the wound with a razor blade and incubated in 1 HIM X-Cluc for 4 h (Jefferson et al., 1987), then stored in 70% ethanol prior to photography. Bar indicates 5 mm.
apparent up to 5 mm from the wound site. This observation is consistent with the previous observation that expression of wound-induced mRNAs does not occur throughout the tuber but is confined to the immediate vicinity of the wound (Butler et al., 1990; Davis et al., 1990). Thus, the induction of EF-la apparent in Figures 2, 3, and 4 is likely to underestimate the true level of induction in the responding cells. DISCUSSION
Wounding of potato tubers causes a stimulation of translational activity that occurs in two distinct phases. The increased translational activity is apparent in vitro. Most significantly, increases in protein synthesis are accompanied by increased expression and accumulation of EF-la. Although EF-la expression is unlikely to be the sole component necessary for translational activation, our data demonstrate that expression of EF-la is a good indicator of translational stimulation. These observations allow us to draw several conclusions regarding the nature of the potato tuber wound response and the induction of protein synthesis. Figure 1 shows that wounding triggers a biphasic response in potato tubers in a time frame that correlates with the expression of the two classes of wound-response genes noted previously (Butler et al., 1990; Davis et al., 1990). The initial phase of the wound response includes induction of PAL (Butler et al., 1990) and other gene products involved in suberization to prevent dehydration and limit pathogen entry (Davis et al., 1990). The second phase includes synthesis of HRGP and other gene products involved in the formation of a wound periderm (Barkhausen, 1978; Butler et al., 1990). The biphasic induction of protein synthesis suggests that these two phases may be triggered by distinct signaling events: one responding to the immediate injury and the second responding to a mitotic signal. Both jasmonic acid
901
(Farmer and Ryan, 1990) and ABA (Pena-Cortes et al., 1989; Hildmann et al., 1993) have been implicated as mediators of the wound response in plant shoot tissues. Auxin treatment has been shown to elicit expression of EF-la (Ursin et al., 1991) and ribosomal protein S15a (Bonham-Smith et al., 1992), but the action of this hormone may be indirect, due to its effect on stimulating cellular elongation. The signal molecules mediating the tuber wound response have not yet been determined. Whatever those signals are, they are recognized by the tomato EF-la promoter in transgenic tubers. This same promoter is expressed in a variety of tissues during plant development (Ursin et al., 1991), indicating an ability to respond to a variety of stimulators, or alternatively, to respond to a general signal common to several conditions. Indeed, EF-la expression increases during cold-temperature stress (Dunn et al., 1993) and after prolonged hypoxia (M. Vayda and J. Morelli, unpublished observations), but decreases upon fungal infection (Mahe et al., 1992). Similarly, the biphasic induction of translational activity may occur in response to other environmental stress conditions. For example, EckeyKaltenbach et al. (1994) have reported the sequential induction of PAL and HRGP mRNAs in parsley leaves upon ozone treatment, in a pattern and time frame similar to their induction in potato tubers upon wounding. Thus, the biphasic response we have observed may be a general reaction to physical stress. The wound response of potato tubers differs from that of shoots. Histological analysis of EF-la/GUS construct expression (Fig. 5) confirms previous observations that the tuber response to wounding is very localized (Bevan et al., 1989; Butler et al., 1990). The tuber wound response differs from the wound response of leaves, which exhibit systemic induction. Wounded tubers fail to express several genes induced in leaves upon wounding, for example chalcone synthase, chalcone isomerase, proteinase inhibitor I, and proteinase inhibitor II (Butler et al., 1990; Davis et al., 1990). Indeed, the proteinase inhibitor II mRNA that accumulates to high levels during tuberization (Keil et al., 1989) is displaced from polysomes and degraded when tubers are wounded (Butler et al., 1990). The degradation of ruberizanon-associated mRNAs upon wounding may correspond to the dissociation of polysomes that occurs concurrently with an immediate, but transient, inhibition of protein synthesis in pea shoots upon wounding (Ramaiah and Davies, 1985; Davies et al., 1986). A similar response during the 15-min labeling in vivo, or extract isolation periods, would explain the low incorporation by nonwounded tubers in Figure 1, and the low activity in vitro of non wounded tuber extracts in Table I. But if wounding causes an immediate inhibition of protein synthesis, the recovery from this state is rapid compared to the recovery in pea shoots; Figure 1 shows that tubers greatly increase translational activity in the interval 15 to 30 min after wounding. Conversely, steady-state levels of EF-la protein and mRNA are extremely low in nonwounded tubers (Figs. 2 and 4, respectively). Further, nonstressed, mature tubers lack significant EF-la transgene expression (Figs. 3 and 5). These observations lead us to conclude that, unlike shoot tissues, the translational activity of postharvest tubers is truly low prior to wounding.
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Morelli et al.
The tumover of EF-la gene products is very rapid. Steadystate levels of both EF-la mRNA (Fig. 4) and protein (Fig. 2) decrease during the period 6 to 8 h after wounding, when transcription of the transgene construct is minimal (Fig. 3). If all members of the endogenous EF-la gene family are transcriptionally inactive during this interval, the half-life of EFla mRNA is approximately 2 h. The decrease in EF-la polypeptide is less dramatic, presumably because the mRNA continues to be translated during this period. However, we have repeatedly, although inconsistently, observed two immunoreactive polypeptides with apparent mobilities of 45 and 42 kD that may be degradation products of EF-la. The rapid tumover of EF-la gene products indicates that both EF-la transcription and protein abundance correlate with increased translational activity. EF-la plays an essential role in protein synthesis but may contribute to other cell functions as well. EF-la is one of the most abundant translation factors (Browning et al., 1990), yet its expression is increased by several stimuli. Although it is clear why at least one functional EF-la gene is required for cell viability (Cottrelle et al., 1985), it is less evident why increased expression of EF-la appears to contribute to longevity (Shepard et al., 1989; Silar and Picard, 1994) whereas decreased expression correlates with senescence (Webster, 1985; Cavallius et al., 1986). Yang et al. (1993) have recently characterized a cytoskeleton-associated factor from carrot cells that is more than 90% homologous with Arubidopsis and tomato EF-la, exhibits EF-la activity, has actin-bundling activity, and serves as an activator of phosphatidylinositol 4kinase. Thus, EF-la could play some role in signal transduction within the cell or assist in cytoskeletal attachment of polysomes. Finally, the observed increases in translational activity parallel previously reported increases in transcriptional activity. However, all mRNAs present in the tuber tissue are not translated in vivo (Butler et al., 1990). This suggests that the increase in macromolecular metabolism is either coordinated with changes in transcriptional activity or occurs only in specific cell types. ACKNOWLEDGMENTS
The authors sincerely thank Dr. W. Moller and Dr. K. Browning for generously providing anti-EF-la antisera and Dr.W. Park for providing transgenic plants expressing the patatin/GUS construct. The authors also express their gratitude for the technical support of Dr. Shengyou Zheng, Deena Small-Barry, Kenneth Simpson, and Patricia Welch. Received March 2, 1994;accepted July 30, 1994. Copyright Clearance Center: 0032-0889/94/106/0897/07 LITERATURE CITED
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