Differential Expression and Turnover of Polyphenol Oxidase Gene Family during ... · tive...

Plant Physiol. (1 997) 1 13: 707-71 8

Differential Expression and Turnover of the Tomato Polyphenol Oxidase Gene Family during Vegetative and

Reproductive Development'

Piyada Thipyapong, Daniel M. Joel, and John C. Steffens*

Department of Plant Breeding and Biometry, 252 Emerson Hall, Cornell University, Ithaca, New York 14853- 1901 (P.T., J.C.S.); and Department of Weed Research, Agricultural Research Organization, Newe-Yaár Research

Center, Ramat-Yishay 30095, Israel (D.M.J.)

Polyphenol oxidases (PPOs) are encoded by a highly conserved, seven-member gene family clustered within a 165-kb locus on chromosome 8 of tomato (Lycopersicon esculentum). Using gene- specific probes capable of differentiating between PPO A/C, PPO B, PPO D, and PPO E/F, we examined the spatial and temporal expression of this gene family during vegetative and reproductive development. RNA blots and in situ hybridization using these probes showed that although PPO expression i s primarily confined to early stages of development, the steady-state mRNA levels of these genes are subjed to complex patterns of spatial and temporal regulation in vegetative and reproductive organs. Young tomato leaves and flowers possess the most abundant PPO transcripts. PPO B i s the most abundant in young leaves, whereas in the inflores- cence PPO B and E/F transcripts are dominant. Differential expression of PPOs i s also observed in various trichome types. PPO A/C are specifically expressed in type I and type IV trichomes. I n contrast, PPO D is only expressed in type VI trichomes. Type I, IV, and VI trichomes possess PPO E/F transcripts. lmmunolocalization ver- ified the translational activity of PPOs identified by in situ hybridization and suggested cell-type-specific, developmentally programmed PPO turnover. I n addition, immunolocalization demonstrated the accumulation of PPO in specific idioblast cells of stems, leaves, and fruits.

~ _____

PPOs (EC 1.14.18.1 or EC 1.10.3.2) catalyze the 0,- dependent oxidation of phenols to quinones, reactive species that can covalently modify and cross-link a variety of cellular nucleophiles via a 1,4 addition mechanism and/ or undergo reverse disproportionation to semiquinone radi- cais. The secondary reactions of quinones lead to formation of polymeric brown or black pigments. The discoloration of wounded plants and of plant extracts are not only familiar to plant scientists, but are also responsible for significant postharvest losses of fruits and vegetables (Vámos- Vigyázó, 1981). Although PPOs are ubiquitous among an-

' This work was supported by grants from the U.S. Department of Agriculture National Research Initiative (grant no. 91-37301- 6571) and the Binational Agricultura1 Research and Development Fund (grant no. US 1870-90) to J.C.S., and by fellowships from the Cornell National Science Foundation/Department of Energy /U.S. Department of Agriculture Plant Science Center to P.T.

* Corresponding author; e-mail [email protected]; fax 1-607- 255-6683.

707

giosperms, occur in a variety of vegetative and reproductive organs, and have been studied intensively, the biological role of PPO remains obscure (Mayer, 1987; Sher- man et al., 1991). A defensive role for PPO has frequently been suggested due to the conspicuous appearance of PPO reaction products upon wounding or pathogenesis and on the basis of its wound inducibility (Mayer and Harel, 1979; Constabel et al., 1995; Thipyapong et al., 1995). PPO is presumed to function in defense in severa1 ways. The highly abundant PPO in glandular trichomes of many Ly- copersicon and Solanum species is responsible for the oxidative polymerization of trichome exudate and entrapment of small-bodied insects (Kowalski et al., 1992; Yu et al., 1992). Alternatively, covalent modification of nucleophilic amino acids by quinones has been proposed to constitute an antinutritive strategy directed against insects and pathogens (Duffey and Felton, 1991). In addition, direct toxicity of quinones against pathogens has been proposed (Mayer and Harel, 1979). Recently, we showed that antisense expression of a Solanum tuberosum PPO cDNA down-regulated every member of the tomato (Lycopersicon esculentum Mill.) PPO gene family, resulting in pathogen hypersusceptibility in the apparent absence of other effects on growth, development, or reproduction (M.D. Hunt, P. Thipyapong, J.C. Steffens, unpublished data). This suggests that PPO may possess a key role in plant defensive systems.

In contrast, the localization of PPO in mesophyll thylakoid membranes and high K , for O, have led to the prop- osition that PPO may function in pseudocyclic photophos- phorylation (ATP production with oxygen rather than NADP+ as the terminal electron acceptor) and regulation of plastidic oxygen levels (Vaughn and Duke, 1984; Trebst and Depka, 1995). Co-fractionation of Vicia faba PPO with PSII suggests that PPO may also be involved in some aspect of chloroplast metabolism (Lax and Vaughn, 1991). In addition, PPO may interact with PSII through competi- tion between PPO and the 23-kD protein of the oxygen- evolving complex for transport into the lumen (Hind et al., 1995).

In tomato, PPOs are encoded by a seven-member (PPO A, A', B, C, D, E, and F) multigene family clustered within a 165-kb locus on chromosome 8 (Newman et al., 1993).

Abbreviation: PPO, polyphenol oxidase.

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708 Thipyapong et al. Plant Physiol. Vol. 11 3, 1997

Similar to recently described resistance genes (Martin et al., 1993; Sudupak et al., 1993; Ellis et al., 1995), the PPO gene family appears to have arisen through duplication of an ancestral single-copy gene through unequal crossing over or other localized processes (Maeda and Smithies, 1986). A11 seven PPOs possess similar plastidic transit peptides that have been shown in in vitro uptake and processing studies to direct the preprotein to the thylakoid lumen (Sommer et al., 1994). In addition, PPO A, A', and C also possess hydrophobic, potentially membrane-spanning C-terminal domains (Newman et al., 1993; Sommer et al., 1994). Although PPO sequences are highly conserved within coding regions, the 5' flanking regions of severa1 PPOs are highly divergent, leading to the speculation that their expression may be differentially regulated or, further, that individual members of the gene family possess phys- iologically different roles (Newman et al., 1993). Determi- nation of cell-type-specific expression patterns may provide additional insight into the roles of individual PPO genes, as well as an increased understanding of the limits to which PPO expression may be manipulated. In this communication we use in situ hybridization, RNA blot analysis, and immunogold localization to document the differential temporal and spatial expression and accumulation of PPOs in vegetative and reproductive tissues of tomato.

MATERIALS AND METHODS

Tomato (Lycopeusicon esculentum Mill. cv VFNT Cherry) plants were grown under greenhouse conditions with a 16-h photoperiod. Leaf, stem, root, flower, and fruit tissues at different developmental stages of 10-week-old plants were collected for in situ hybridization, RNA analysis, and immunolocalization. In addition, apical root tissues were also harvested from 2-week-old plants grown hydroponi- cally in Hoagland solution. For RNA analysis, plant tissues were immediately frozen in liquid nitrogen and stored at -70°C until used. For in situ hybridization and immunolocalization, plant tissues were fixed in 2.5% (v/v) glutar- aldehyde, 2% (w/v) paraformaldehyde in 0.1 M sodium phosphate, pH 7.2, at 4°C for 4 to 12 h with occasional mild degassing. The tissues were dehydrated with a graded ethanol series. Ethanol was replaced by xylene and then by liquid paraffin, and tissues were embedded in paraffin blocks that were cut into 8- to 12-pm sections that were mounted on Superfrost / Plus microscopic slides (Fisher). Consecutive seria1 sections were spread onto different slides, and each slide contained a collection of sections from different organs. In this manner we could treat sections from each particular tissue using different probes and staining procedures and still compare different tissues on the same slide.

RNA lsolation and Analysis

Total RNA was isolated from frozen tissues as described by Verwoerd et al. (1989). The contaminating polysaccharides were then removed by dissolving RNA samples in high-salt (2 M NaCl) solution followed by isopropanol pre-

cipitation (Fang et al., 1992). Total RNA was transferred to nylon filters (GeneScreen, NEN) using a slot-blot apparatus (Bio-Rad). The filter was hybridized with 0.5 X 106 cpm mL-l to 1 X 106 cpm mL-l 32P-labeled gene-specific probes. Differences in the 3' nontranslated sequences among PPO genes were utilized to make gene-specific probes. Because PPO A and A' have identical sequences, PPO A, A', and C have identical 3' nontranslated sequences, and PPO E and F have 80% 3' nontranslated sequence homology, we synthesized four 32P-labeled gene- specific antisense riboprobes corresponding to PPO A/ C, PPO B, PPO D, and PPO E / F, and a PPO D sense riboprobe as a control. PPO B and PPO E cDNAs and DNA fragments containing genomic 3' nontranslated sequences of PPOs A and D, which were cloned in Bluescript SK- phagemid, were used as DNA templates. These DNA templates were cut with specific restriction enzymes at restriction sites located about 200 to 270 bp downstream of T3 or T7 promoter. Riboprobes were then transcribed using either T3 or T7 RNA polymerase (Stratagene transcription system). Northern hybridization of these gene-specific antisense RNA probes to mRNA transcribed from each cloned PPO gene confirmed the specificity of these probes. The filters were washed under the following conditions: two 10- to 15-min washes in 2x SSC and 0.5% (w/v) SDS at room temperature; two 10- to 15-min washes in l x SSC and 0.5% (w/v) SDS at 45°C; and two 10- to 15-min washes in 0.5X SSC and 0.5% SDS (w/v) at room temperature (1 X SSC = 150 mM NaC1, 15 mM sodium citrate, pH 7.0).

In Situ Hybridization

In situ hybridization was performed primarily according to the method of Smith et al. (1987) with the following modifications. During the prehybridization step, sections mounted on slides were incubated with proteinase K for 15 to 45 min, depending on tissue types. To 32P- or 35S-labeled gene-specific antisense or sense riboprobes, yeast tRNA was added to 150 pg mL-' and DTT was added to 1 mM, and the mixture was heated at 80°C for 5 min before hybridization. The hybridization solution consisted of 50% deionized formamide, 200 mM NaCl, 10 mM Tris-HC1, pH 8.0, 1 mM Na,EDTA, 10% (w/v) dextran sulfate, 10 mM DTT, 1 X Denhardt's solution, 150 pg mL-' yeast tRNA, and 100 pg mL-l salmon sperm DNA. The slides were hybridized at 45°C for 14 to 16 h. Sections were washed with gentle agitation for 2 h each in 1.5X SSPE at 50°C, followed by 0.75X SSPE at 50°C ( 1 X SSPE = 180 mM NaC1, 1 mM Na,EDTA, 10 mM sodium phosphate, pH 7.5). After posthybridization washes and autoradiography, the slides were stained with toluidine blue, dehydrated with a graded ethanol series, permanently mounted, and viewed under dark- and bright-field light microscopy. The control (PPO D sense riboprobe) showed no detectable signal above background level.

lmmunogold Localization

The sections mounted on slides were dewaxed in xylene rehydrated through a graded ethanol series, and rinsed in



Polyphenol Oxidase Expression and Turnover 709

IBS (50 mM Tris-HCl, pH 7.5, I mM MgCl2/ 10 mM NaCl).Sodium m-periodate was added to 140 mM and incubatedfor 20 min to eliminate reactivity of antisera to glycosidicepitopes. The sections were then incubated in 0.5 mg mL^1

sodium borohydride for 10 min to block the reactive alde-hyde groups. Nonspecific adsorption sites in all sectionswere blocked with 5% (v/v) normal goat serum (Sigma) inTBS for 1 h. Primary antibody (polyclonal rabbit anti-Solanum berthaultii 59-kD trichome PPO [Kowalski, 1989])diluted 1:100 in the latter blocking solution was added tothe sections in a moist chamber for 1 to 1.5 h. After threerinses of 5 min each in washing solution (1% [w/v] BSA,1% [v/v] goat serum, and 0.01% Tween 20 in TBS), second-ary antibody (goat anti-rabbit 5-nm colloidal gold conju-gate [Sigma]) diluted 1:100 in 1% (w/v) BSA and 0.1%(v/v) Tween 20 in TBS was added, incubated for 30 to 45min, and washed as in the previous step. The colloidal goldlabels were enlarged using a silver-enhancer kit (Sigma)according to the manufacturer. The sections were thencounterstained with methylene green, mounted, and visu-alized by light microscopy. The control (nonimmune serumand omission of the primary antibody) did not show silverdeposition above background level.

Histochemical Test for Polyphenols

A histochemical test for polyphenols was performed ac-cording to the method of Reeve (1951) using 10% (w/v)sodium nitrite, 20% (w/v) urea, 10% (v/v) acetic acid, and2 N sodium hydroxide. Leaf, stem, flower, and fruit tissueswere collected from 3-month-old tomato plants, and roottips were collected from tomato seedlings germinating onmoist filter paper.

RESULTS

Gene-Specific Probes Demonstrate DifferentialPPO Expression

Spatial and temporal differences in expression patternsof individual PPO genes were evaluated using gene-specific antisense RNA probes. Based on 3' nontranslatedsequence divergence among PPO genes, four gene-specificantisense probes capable of discriminating between PPO B,D, and the PPO E/F and A/C gene pairs, respectively,were constructed. The specificity of these probes was con-firmed as shown in Figure 1. Using these gene-specificprobes, RNA blot analysis was employed to determine theexpression patterns of individual PPOs in young leaves(apical nodes 1-3), older leaves (node 5), stems (internodes3-4), roots, flower buds, and fruits (2-5 mm diameter [3-7d postanthesis]) of 10-week-old tomato plants (Fig. 2). Inmost organs, PPO B and E/F transcripts are the mostabundant and are particularly abundant in young leavesand inflorescences. In young leaves, PPO B is most abun-dant, but PPO E/F is slightly more abundant in inflores-cences. The abundance of PPO B and E/F transcripts de-clines in older leaves (node 5). PPO B and E/F transcriptsare present at a relatively low level in other organs, includ-ing stems, fruits, and roots. PPO D is expressed at a sig-nificantly lower level in all tissues. No hybridization was

PPO A/C

PPOB

PPOD

PPO E/F

*1f

Figure 1. Specificity of PPO A/C, B, D, and E/F gene-specific probes.mRNA was transcribed from cloned PPO genes (pPPO A/C, pPPO B,pPPO D, and pPPO E/F, respectively). Slot-blot analysis was per-formed with 1 ng of transcribed RNA per slot and hybridized toJ2P-labeled antisense 3' nontranslated PPO A/C, B, D, and E/F RNAprobes, respectively.

observed for PPO A / C in all organs examined, suggestingthat these genes are either expressed at a very low level orare expressed in only a small population of cells.

The steady-state accumulation of PPO A/C, B, D, andE/F transcripts was further investigated at the cellularlevel by in situ hybridization using these gene-specificprobes. To examine the relationship between the presenceof PPO mRNA and accumulation of PPO at various stagesof development, immunolocalization of PPO was also per-formed. The results of immunolocalization and in situhybridization are discussed below and summarized inTable I.

PPO Expression and Localization in Vegetative Organs

Root

In root tips, PPO B (Fig. 3A) and E/F transcripts areexpressed at a high level in the apical meristem and at avery low level in the root cap. PPO D (Fig. 3B) and PPOA/C are not expressed in the root tip. However, lateral rootprimordia exhibit a different pattern of expression: PPO D(Fig. 3D) is expressed at the highest level, PPO E/F isweakly expressed, and expression of PPO A/C and B (Fig.3C) is not detectable. As the primary root grows, a cell-specific pattern of PPO expression is established in phloem,in which the exclusive presence of PPO D and E/F tran-scripts probably accounts for the PPO accumulated in thesecells (not shown).

Stem

In contrast to the root phloem pattern of PPO expression,PPO A/C, B, D, and E/F are expressed in stem phloem cells



710 Thipyapong et al. Plant Physiol. Vol. 113, 1997

Figure 2. Expression of PPO A/C, B, D, and E/Fin various organs of tomato. Total RNA wasisolated from nodes 1 to 3 and node 5 leaves,internode 3 and 4 stems, flower buds, youngfruits (2-5 mm diameter [3-7 d postanthesis]),and roots. Slot-blot analysis was performed with25 (j,g of total RNA per slot and hybridized to32P-labeled PPO A/C, B, D, and E/F gene-specific probes, respectively.

Leaf nodes 1 to 3[Leaf node 5L Stem

Flower budFruitRoot

(Fig. 3J). PPOs are abundantly accumulated in both theinternal and the external primary phloem (Fig. 3, G and I) aswell as in the secondary phloem. In contrast to RNA blotanalysis (which failed to detect PPO A/C transcripts instems), in situ hybridization showed a low level of PPO A/Ctranscripts in stem phloem cells. In addition, PPO is also

abundant in trichomes and idioblasts of the stem (Fig. 3, Hand I). The idioblasts (Fig. 3, H, see also M) are solitary cellsclearly distinguishable from other mesophyll cells in theirisodiametric shape and high PPO and phenolic content.

In addition to expression in stem phloem, PPO A/C arealso specifically expressed in type I (Fig. 3E) and type IV

Table I. Expression of the PPO gene family and accumulation of PPO in tomato

Organs/CellsPPO Transcripts'1

A/C B D E/FPPO

Young leaf (node 1)Mesophyll

Mature leaf (node 5)Mesophyll

StemPhloem cell

TrichomeType VIType I and IV

RootApical meristemLateral root meristemPhloem cell

FlowerFloral meristemStamen (before meiosis)

Tapetum cellPollen mother cell

Pollen tubeCarpel (before anthesis)

StyleOvulePlacenta

Fruit (5 mm diameter [7 d postanthesis])OvuleEmbryo sac

Mature seedEmbryoEndosperm

NANA

+ ± +- + -

a Levels of PPO transcripts and PPO range from ± (detectable but not abundant) to ++ (maximalabundance); -, not detectable; NA, not available.



Polyphenol Oxidase Expression and Turnover 71 1

(not shown) trichomes (trichome classification, shown in Figure 4, is from the method of Luckwill [1943]). Both type I and type IV trichomes also contain PPO E / F transcripts, although at a lower level (not shown). In contrast, the head cells of type VI trichomes primarily contain PPO D (Fig. 3F) and E / F transcripts (not shown). This trichome expression pattern was also observed in other organs such as leaves and sepals (eg. Fig. 3L). Although no PPO is observed in epidermis, the adjacent two to three cell layers of cortex appear to accumulate a low amount of PPO at this developmental stage (Fig. 3, G and H).

Leaf

At early developmental stages of leaves and stems, PPO is predominantly accumulated in epidermal cells and trichomes (not shown). Both PPO B and E/F transcripts are detected in epidermal and interna1 tissue (not shown). However, leaves at node 1 accumulate PPO in the differentiating mesophyll (in both spongy and palisade cells), in the phloem, and in trichomes, but not in the epidermis (Fig. 3K), suggesting that a developmental-stage-dependent turnover of PPO exists in the epidermis. At this stage, in situ hybridization revealed that PPO B, D, and E/F genes are expressed in the leaf mesophyll (Fig. 3L). In contrast, PPO A / C transcripts are detectable only in type I and type IV trichomes. Mature leaves (node 5) contain lower levels of PPO transcripts in the mesophyll (not shown), consistent with the RNA blot analysis (Fig. Z), with immunocyto- chemical results showing lower levels of PPO in these cells, and also with immunoblot analyses (Steffens et al., 1994). Mature leaves also possess idioblasts in the mesophyll, which accumulate high amounts of PPO (Fig. 3M).

PPO Expression and Localization in the Flower

During early flower initiation, accumulation of PPO is conspicuous in meristematic cells in the flower bud. How- ever, no PPO was found in the "ground tissue" of the flower stalk and receptacle. The bulk of PPO expression in the floral meristem is contributed by PPO B and E / F transcripts, which are present throughout the flower meristem (not shown). Further development results in cell-specific expression and accumulation of PPOs in sepals and petals, stamens, and styles and ovules, as described below.

Sepal and Petal

PPO is temporally regulated in sepals; PPO B and E/F transcripts are detectable only in sepal primordia (not shown). Strong accumulation of PPO in sepal primordia is shown in Figure 5A. However, at later stages of development PPO mRNA is not detectable in sepals and PPO is found only at low abundance in sepal mesophyll (Fig. 5, B and C). Concomitantly, the level of PPO in the epidermis subsequently declines and can be detected only at low levels in the adaxial epidermis before and -during pollen grain differentiation (Fig. 5, A, C, and G). After pollen grain differentiation, epidermal PPO accumulation is no longer detectable (not shown). Similar to sepals, petal primordia

express PPO B and E/F transcripts (not shown). In contrast to sepals, petals of very young flowers accumulate a high level of PPd in the parenchyma (Fig. 5B), which remains detectable, albeit a ta lower level, during (Fig. 5G) and after (not shown) pollen grain differentiation. In contrast to the rapid loss of PPO in leaf and sepal epidermis, PPO persists in petal epidermis from the petal primordia stage to after pollen grain differentiation (Fig. 5, A-C, G, and not shown). However, at anthesis PPO appears to be selec- tively lost in the abaxial petal epidermis, whereas it remains detectable in adaxial epidermal cells (not shown). In addition, PPO appears to accumulate preferentially at the tip of petals. Sepals and petals at anthesis contain no levels or low levels of PPO in the parenchyma (not shown).

Stamen

Following mitotic division of the primary sporogenous layer to form pollen mother cells and differentiation of tapetum and endothecium from the primary parietal layer, distinct patterns of PPO expression in stamen become evident. Pollen mother cells express PPO D and PPO E/F (Fig. 5F). In contrast, PPO B expression is primarily restricted to tapetum cells (Fig. 5E). Neither cell type contains PPO A/ C transcripts (Fig. 5D). Immunogold localization of the same-stage flowers shows that PPO is abundant in the pollen mother cells, whereas only low levels are observed in tapetum, endothecium, and connective cells (Fig. 5C).

Shortly after pollen grain differentiation, accumulation of PPO in tapetum cells becomes obvious (Fig. 5H). PPO accumulation in connective cells, endothecium, and the outer epidermal layer of anthers becomes evident at the earlier tetrad stage (Fig. 5G). This accumulation subsequently declines in connective cells and in the endothecium, whereas PPO remains detectable in the outer epidermal layer at anthesis (Fig. 51), again suggesting that PPO may be differentially turned over in a developmentally programmed, cell-specific manner. Developmentally regulated PPO turnover is also evident during the development of the male gametophyte. Tetrads and young differentiated pollen grains contain PPO (Fig. 5, G and H), but PPO is not detectable in mature pollen grains from flowers at anthesis (Fig. 51).

Style

PPO B mRNA is specifically accumulated in the connective cells surrounding transmitting tissue of developing styles and is accompanied by abundant accumulation of PPO in this tissue (Fig. 5, J and K). This expression, however, is transient and is no longer detectable in styles of postanthesis flowers (Fig. 5M). In this latter stage, immu- nolabeling shows PPO in pollen tubes inside the style (Fig. 5L) and in situ hybridization identifies this as originating from PPO B transcripts (Fig. 5M). In vitro pollen germina- tion confirms induction of PPO B and accumulation of PPO in pollen germ tubes (P. Thipyapong, J.C. Steffens, unpublished data).




Figure 3. Localization of PRO A/C, B, D, and E/F transcripts and PRO in vegetative organs of tomato. Dark-field microscopywas used in A, B, C, D, J, and L, in which hybridization signals are visible as white dots. Bright-field microscopy was usedin E, F, G, H, I, K, and M, in which hybridization signals are visible as brown dots and immunolabeled signals are in brownto black color. A and C, Expression of PRO B in root apex (A) and mature root with lateral root primordia (C). Longitudinalsections were hybridized with PRO B antisense RNA probe. PPO B is expressed only in the axial root tip. Magnification X50.

(Legend continues on facing page.)




n

Figure 4. Trichome classification in tomato. Roman numbers show various types of trichomes that are described in this study. Trichome type classification follows the work of Luckwill (1 943), except type IV, which are nonglandular.

Ovule

Expression of PPO D and especially PPO E/F is evident in the developing ovules of flowers prior to and at anthesis (Fig. 5, O and P). Placenta, in contrast, contains only PPO D transcripts (Fig. 5, N-P). Expression of PPO A / C was not detected in developing ovules and placenta (Fig. 5N).

PPO Expression and Localization in Fruit and Seed

The temporal expression patterns of PPO in ovules appears to be gene-specific. The accumulation of PPO D and E / F transcripts in ovules continues from flowering through early fruit development, and up to at least approx- imately 5-mm-diameter (7 d postanthesis) fruits (Fig. 6, B, E, and F). After the fruit is about 2 mm in diameter (3 d postanthesis), PPO B transcripts accumulate in ovules (not shown). PPO A / C transcripts were not found at this stage but are detectable later in the ovules of 5-mm fruit (not shown).

In 5-mm-diameter fruits accumulation of PPO is detected in the embryo sac and epidermis (Fig. 6D). PPO E/F transcripts are not present during early seed development (Fig. 6F), whereas PPO D is the only transcript present in the

embryo sac and is also expressed at a high level in the outer epidermis of ovules (Fig. 6E). PPO D mRNA subsequently declines to very low levels and is restricted to the apical root meristem in the embryo of mature seeds (not shown). In mature seeds PPOs are accumulated not only in the embryo but also in endosperm tissues (Fig. 61). Both PPO B and E/F are expressed in the embryo of mature seeds, but only PPO E / F is expressed in endosperm. Figure 6K shows expression of PPO E/F in both embryo and endosperm of mature seed. PPO A / C transcripts were not detected in mature seeds (Fig. 6J).

The accumulation of PPOs in developing ovules and embryos parallels PPO transcript appearance (Fig. 6, A and D). In 5-mm-diameter fruits PPO is found more abundantly in the outer epidermis of the ovule and in the inner integument epidermis surrounding the developing endosperm (Fig. 6D). In addition, PPO accumulates preferentially in the outer epidermis of the pericarp, in carpellar phloem cells, and in placental idioblasts (Fig. 6, A, G, and H). Accumulation of PPO mRNAs and PPOs in developing ovules coincides with the accumulation of nitroso-positive compounds corresponding to phenols (Fig. 6C).

PPO Accumulation in ldioblasts

In many tomato organs, immunogold localization revealed the presence of specific solitary parenchyma cells (idioblasts) that accumulate very high levels of PPO. These idioblasts are relatively large isodiametric cells scattered throughout stem cortex, mature leaf mesophyll, pericarp, and placental tissues (Fig. 3, H and M, and Fig. 6, A and H, respectively). PPO accumulation in idioblasts is accompanied by accumulation of nitroso-positive phenolics (not shown). The presence of phenolics at high levels in idioblasts may imply a functional relationship, i.e. co- accumulation of PPO and phenolics suggests that a primary PPO function in these cells is the generation of quinones as a defense response to cell disruption.

DISCUSSION

The results presented here show a complex regulation of steady-state PPO mRNA at a temporal and spatial level, as well as provide evidence for the developmentally programmed turnover of PPO. RNA blot analysis shows that the most abundant PPO transcripts are found in young tomato leaves and flowers. This result is consistent with

(Continued from facing page.) B and D, Expression of PPO D in root apex (B) and mature root with lateral root primordia (D). Longitudinal sections were hybridized with PPO D antisense RNA probe. PPO D expression was seen only in lateral root primordia. Magnification X50. E, Localization of PPO A/C transcripts on type I trichome. Magnification X300. F, Localization of PPO D transcripts on type VI trichome. Magnification X300. G, PPO in sieve elements and in two to three cell layers of cortex underlying epidermis (arrowhead) in a longitudinal section of young stem (internodes 1 and 2). Magnification ~ 2 0 0 . H, PPO in internal phloem, idioblasts, and in two to three cell layers of cortex underlying epidermis (arrowhead) in a cross-section of internode 3 and 4 stem. Magnification X50. I, PPO in internal and external phloem in a cross-section of internode 1 and 2 stem. Magnification X50. J, Localization of PPO B transcripts in external phloem o n stem cross-section (internodes 2-3). Magnification X50. K, Accumulation of PPO in mesophyll, phloem, and type VI trichomes of young node 1 leaf. Magnification X50. L, Expression of PPO E/F in young node 1 leaf. Magnification X75. M, Localization of PPO in idioblast of mature leaf (node 5). Magnification X120. Ep, Epidermis; Ib, idioblast; LR, lateral root primordium; Ph, phloem; ePh, external phloem; iPh, internal phloem; Ps, palisade cell layer; SE, sieve element; Sg, spongy cell layer; ITc, type I trichome; IIITc, type 111 trichome; VITc, type VI trichome; XI, Xylem.




Figure 5. Localization of PRO and PRO A/C, B, D, and E/F transcripts during flower development in tomato. Dark-fieldmicroscopy was used in D, E, F, K, M, N, O, and P, in which hybridization signals are visible as white dots. Bright-fieldmicroscopy was used in A, B, C, G, H, I, ), and L, in which immunolabeled signals are in brown to black color. A,Longitudinal section of flower bud showing accumulation of PRO in sepal and petal primordia. Magnification X50. B,Immunogold localization of PRO on a cross-section of young flower bud after initiation of sepal, petal, and stamendevelopment. Magnification X50. C, Localization of PRO in microspore mother cell. Magnification X50. D to F, Cross-

(Legend continues on facing page.)




abundant PPO transcripts previously reported in flowers (Shahar et al., 1992; Hunt et al., 1993), but additionally demonstrates that PPO synthesis in tomato inflorescences is primarily based on the expression of the E/F and B members of the PPO gene family. The abundance of PPO transcripts in young leaves and the relatively low level in mature tomato roots is consistent with the observations in potato (Hunt et al., 1993). PPO B is the most abundant PPO mRNA in young tomato leaves (nodes 1-3); levels of this transcript subsequently decline as leaves mature (node 5 ) . The presence of PPO transcripts primarily at early developmental stages has been observed previously in tomato, potato, and grape berries (Rathjen and Robinson, 1992; Shahar et al., 1992; Hunt et al., 1993; Thygesen et al., 1995). In contrast, broad bean PPO transcripts are abundant in mature leaves (Cary et al., 1992). Differential regulation of PPO at different developmental stages or between different plant taxa is also suggested by observation of PPO accumulation in sorghum mesophyll and its absence in bundle- sheath and guard cells (Vaughn and Duke, 1981), and by its presence in bundle-sheath, vascular parenchyma, and idioblasts in water hyacinth (Martyn, 1977).

At a cellular level, leaves expressing primarily PPO B and E / F exhibit differential spatial and temporal patterns of PPO accumulation. Very young leaves near the apical meristem accumulate PPO predominantly in epidermal cells and trichomes, whereas leaves at node 1 accumulate PPO in the differentiated mesophyll, phloem, and trichomes. The transition from leaf differentiation to ex- pansion growth is accompanied by a loss of PPO B and E / F transcripts in the epidermis, with a drastic decline in epidermal PPO, suggesting that expression of PPO B and E / F in nonepidermal layers is coordinately regulated by both down-regulation of these genes and turnover of their prod- uct in the epidermal layer.

PPO A, A', and C are distinguished from other members of the tomato PPO gene family by their possession of hydrophobic C-terminal extensions, which may act as transmembrane domains (Newman et al., 1993). Whether these differences in primary structure are related to functional differences is unknown. PPO A/ C mRNAs were not detected by RNA blots of tomato leaves, stems, roots, flowers, and fruits, suggesting that these genes are expressed either at a low level or in a restricted population of cells. In

situ hybridization demonstrates that PPO A/ C is expressed at a low level in ovules and in phloem cells of stems. In addition, PPO A / C are specifically expressed at a high level in type I and type IV trichomes that develop on leaves, stems, and sepals.

Other PPO genes are also differentially expressed in various trichome types. In addition to PPO A/ C, type I and type IV trichomes also possess PPO E/F transcripts. In contrast, type VI (tetralobulate) trichomes express PPO D and PPO E / F transcripts and do not show expression of PPO A/C. PPO frequently constitutes >50% of total cell protein in type VI trichomes of Lycopersicon and Solanum species (Yu et al., 1992). In this trichome type, PPO is sequestered in protein bodies within the thylakoid lumen of trichome leucoplasts (Hunt, 1995), whereas phenolic substrates are stored in vacuoles within the tetralobulate trichome head cells (Beckman et al., 1972). Rupture of type VI trichome cells leads to oxidative polymerization of trichome exudate, where small-bodied insects are en- trapped (Kowalski et al., 1992). The function of PPO in type I and IV trichomes requires further investigation, as does the differential expression of the PPO gene family in these tissues. Sepals and petals resemble vegetative leaves in their expression of PPO B and E / F genes, consistent with the results of Shahar et al. (1992), who described the occurrence of PPO and PPO transcripts in tomato sepal primordia. Sepals also follow the vegetative pattern of PPO A / C expression in type I and type IV trichomes.

PPO expression in tomato roots is primarily confined to meristems. Whereas PPO B and E/F are expressed in the root apical meristem, lateral root primordia express high levels of PPO D, along with lower levels of PPO E/F mRNAs. Because lateral root formation involves rupture of the root cortex, it is possible that PPO induction is due to localized wounding signals and that expression at this site is an adaptation to minimize adventitious infection at this wound site. It is interesting to note that PPO D is also expressed in the apical meristem of the radicle in the seed. Although Shahar et al. (1992) concluded that PPO is up- regulated only in the dermal tissue of young tomato roots, and that little PPO mRNA accumulates in the other root meristematic layers, the figure they present as a reference appears to be of an oblique section in a maturing root rather than of the root apex itself.

(Continued from facing page.) sections of flower bud at the same developmental stage as C, hybridized with PPO A/C, B, and E/F antisense RNA probes, respectively. D, No detectable expression of PPO A/C. Apparent signal i s comparable to that of sense control probe. Magnification X50. E, PPO B expression in tapetum (arrowhead). Magnification X50. F, PPO E/F expression in pollen mother cells (arrowhead). Magnification X50. G to I, Cross-sections of anthers showing PPO accumulation sites. G, Tetrad stage. Magnification X70. H, Pollen maturation stage. PPO in tapetum. Magnification X 120. I, Anthesis. PPO in papillate abaxial epidermis. Magnification X50. J and K, Cross-sections of flower bud style at the same developmental stage as C to F. J, Section was immunolabeled with anti-PPO. Magnification X220. K, Section was hybridized with PPO B antisense RNA probe. Magnification X220. L and M, Cross-section of style postanthesis. L, Section was immunolabeled with anti-PPO. PPO in pollen tubes (arrowheads). Magnification X l 2 5 . M, Section was hybridized with PPO B antisense RNA probe. PPO B expression in pollen tubes (arrowheads). Magnification X125. N to P, Cross-sections of ovary hybridized with PPO A/C, E/F, and D antisense RNA probes, respectively. N, N o detectable expression of PPO A/C. Apparent signal i s comparable to that of sense control probe. Magnification X50. O, PPO E/F expression in ovules. Magnification X50. P, PPO D expression in ovules and placenta. Magnification X50. An, Anther; C, connective cell; En, endothecium; Ep, epidermis; MMC, microspore mother cell; Ov, ovule; Pc, placenta; PI, pollen grain; Pt, petal; Sp, sepal; IVTc, type IV trichome; VITc, type VI trichome; Tp, tapetum cell; TT, transmitting tissue.




V-T '•kf-i-'-^ A -t- IM? E- •,-.

Figure 6. Localization of PRO A/C, B, D, and b/h transcripts and PPO in fruit and seed ot tomato. Dark-field microscopy wasused in B, E, F, J, and K, in which hybridization signals are visible as white dots. Bright-field microscopy was used in A, C,D, G, H, and I, in which immunolabeled signals are in brown to black color. A, Immunogold localization of PPO in pericarpepidermis and idioblasts, and in ovules of a 2-mm-diameter (3 d postanthesis) fruit. Magnification X50. B, Localization ofPPO E/F transcripts in ovules of a 2-mm-diameter fruit. Magnification X60. C, Accumulation of nitroso-positive compoundsthat correspond to phenols (cherry red color) in the ovules of a 2-mm-diameter fruit. Magnification X20. D, Accumulationof PPO preferentially in inner and outer epidermis of ovule integument in a 5-mm-diameter (7 d postanthesis) fruit.Magnification XI20. E and F, Localization of PPO D and E/F transcripts on ovule of a 5-mm-diameter fruit, respectively.Magnification X70. C, Immunogold localization of PPO in carpellar phloem. Magnification XI20. H, PPO in carpellaridioblasts. Magnification X50. I, PPO accumulation in embryo and in endosperm of mature seed. Magnification X50. ] andK, Localization of PPO A/C and E/F transcripts on mature seed, respectively. I, No detectable expression of PPO A/C.Apparent signal is comparable to that of sense control probe. Magnification X75. K, PPO E/F expression in both embryo andendosperm. Magnification X75. Ed, Endosperm; Em, embryo; Ep, epidermis; Es, embryo sac; Ib, idioblast; lt, innerintegument epidermis; Ov, ovule; PC, placenta; Ph, phloem.

The expression patterns of PPO during flower develop-ment are complex. During early anther development pollenmother cells accumulate PPO; at later stages PPO accumu-lates primarily in the tapetum; and by maturation very little

PPO can be detected in pollen. However, pollen germinationand pollen tube growth in the style are accompanied byexpression of PPO B and by accumulation of PPO in thepollen tube. PPO is present at high levels in the style itself




only at early stages of flower development, as previously described by Shahar et al. (1992). However, this accumulation is derived primarily from expression of PPO B, in which mRNA specifically accumulates in the connective cells surrounding transmitting tissue of the developing style.

The function(s) of PPOs in pollen tubes and flower tissues remain unclear. In Brassica species tapetum-specific mRNAs that resemble protease inhibitors were suggested to have a role in protecting the anther and developing microspores from microbial or insect attack (Scott et al., 1991). PPO B may have such a defensive role in tapetum cells. In addition, the polymerized phenylpropanoid con- stituents of pollen exine (sporopollenin [Herminghaus et al., 1988; Gubatz and Wiermann, 19931) may originate with the release of PPO during the breakdown of the tapetal layer. In this connection it is interesting to note the associ- ation of PPO expression with programmed cell death. In the tapetal layer accumulation of quinones during deteri- oration of these cells would not only hasten cell death, but would contribute to formation of pollen exine. Similarly, expression of PPO during senescence or wounding could be expected to promote cell death and to generate an environment that would discourage adventitious coloniza- tion by pathogens. In support of such a role, transformed plants containing a PPO B promoter:GUS fusion exhibit strong GUS expression in abscission zones and also show ethylene-responsive regulation of this gene (S.M. Newman, P. Thipyapong, J.C. Steffens, unpublished data).

The relatively high abundance of PPO and nitroso- positive phenolics in developing fruits may support a role for PPO in discouraging herbivory of the immature fruits (Mayer and Harel, 1981). Nitroso-positive phenolics were also found to be abundant in type VI trichomes, in very young leaf mesophyll, in the connective tissue of styles, and in root tips (P. Thipyapong, J.C. Steffens, unpublished data), a11 of which also accumulate high levels of PPO. The co-localization of PPO and phenolics at high levels may be taken as further evidence that the primary role for PPO is a protective one achieved through the generation of quinones upon cell disruption.

In addition, idioblasts scattered throughout mature leaves (node 5) , stem cortex, fruit pericarp, and placenta1 tissues also accumulate high amounts of phenolics and PPO. Similar idioblasts containing high amounts of phenolic compounds and PPO have been observed previously in water hyacinth leaves and were suggested to have a defensive role against insects or pathogens (Martyn, 1977). Distortion of funga1 hyphae upon idioblast penetration was reported, which may result from quinones generated by PPO upon cell disruption (Martyn, 1977). Therefore, these idioblasts may function in direct defense against pathogens or herbivores.

In other plant species such as potato and broad bean, PPOs are encoded by a multigene family (Cary et al., 1992; Hunt et al., 1993; Newman et al., 1993; Thygesen et al., 1995). In both tomato and potato, PPO is encoded by similarly clustered gene families organized on chromosome 8 (Hunt et al., 1993; Newman et al., 1993). In the case of potato, although the gene family is considerably larger than in tomato, several PPO

cDNAs are direct homologs of tomato PPO genes (Hunt et al., 1993; Thygesen et al., 1995). Therefore, it would not be unex- pected to find that homologous PPOs are regulated similarly in potato and tomato. During the preparation of this work Thygesen et al. (1995) reported RNA-blot-analysis evidence for differential expression patterns of several PPO cDNAs isolated from developing tubers. Transcripts of a potato gene homologous to tomato PPO E / F are similarly abundant in young flowers and leaves and share the tomato pattern of expression in that the potato PPO E/F homolog is also present at high levels in anthers and ovary, which may correspond to the pollen mother cells and ovules disclosed in our study by in situ hybridization. In contrast, only tomato petal primordia contain detectable PPO E/F transcripts, not the unopened flower, as reported for the potato homolog of PPO E/F (Thygesen et al., 1995). In tomato flowers at anthesis or postanthesis, PPO E/F transcripts are detectable only in ovules, which might account for the low potato PPO E/F homolog transcript level observed in RNA blots from ex- plants at this stage of flower development (Thygesen et al., 1995). The potato homolog of PPO D is also expressed in roots, consistent with our observation of PPO D in lateral root primordia and root phloem cells. However, PPO D mRNA also accumulates in young tomato leaves, stems, flowers, and fruits, whereas the potato PPO D homolog does not. High levels of PPO B transcripts are observed in young tomato leaves and flowers, in contrast to potato PPO B homolog, which is only highly expressed in tuber tissue (Thygesen et al., 1995). Although the similarities in PPO expression are striking, the above comparisons clearly indicate the limita- tions to which tomato can be considered a model system for the regulation of PPO, particularly with regard to the regulation of postharvest PPO physiology (Bachem et al., 1994).

PPO expression in tomato is a complex temporal and spa- tia1 mosaic of overlapping and sequential patterns of accumulation and turnover. In most cases PPO accumulation patterns reflect those of PPO transcripts, suggesting that PPO accumulation is primarily controlled at the mRNA level. In some tissues PPOs appear to be quite stable. For example, PPO persists in petal parenchyma long after PPO B and E / F transcripts become undetectable. In contrast, PPOs are rap- idly degraded in other tissues, e.g. PPO B accumulation in connective tissues of styles is highly transient. Similarly, in pollen, PPO accumulates during differentiation but is lost upon maturation, and PPO B is resynthesized upon germina- tion. Leaves also present a complex regulatory pattern. Al- though PPOs B and E / F are expressed in epidermis of leaves, sepals, and petals early in development, turnover of PPO appears to be more rapid in the epidermal leucoplasts of leaves and sepals than in the chromoplasts of petal epidermis. Similarly, the persistence of PPO in anther abaxial epidermal chromoplasts, while PPO is lost in connective cells and endothecium at anthesis, may suggest a differential stability of PPO in these cell types. Plastid differentiation is an active developmental process that involves posttranslational as well as mRNA-leve1 mechanisms of regulation (Lincoln and Fi- scher, 1988; DellaPenna et al., 1989; Kobayashi, 1991; Law- rence et al., 1993). Although the state of plastid differentiation affects the processing of PPO (Sommer et al., 1994), its poten-



71 8 Thipyapong et al. Plant Physiol. Vol. 11 3 , 1997

tia1 importance in governing PPO turnover remains to be determined.

ACKNOWLEDCMENTS

We are grateful to Dr. M.V. Parthasarathy, N. Rizzo, and C. Dougherty for the use of microtome facilities, and Dr. S.D. Tank- sley for the use of the microscope. Dr. D.M. Joel was on sabbatic leave at Cornell University.

Received August 29, 1996; accepted November 24, 1996. Copyright Clearance Center: 0032-0889/97/113/0707/ 12.

LITERATURE ClTED

Bachem CWB, Speckmann GJ, van der Linde PCG, Verheggen FTM, Hunt MD, Steffens JC, Zabeau M (1994) Antisense expression of polyphenol oxidase genes inhibits enzymatic brown- ing in potato tubers. Bio/Technology 12: 1101-1105

Beckman CH, Mueller WC, McHardy WE (1972) The localization of stored phenols in plant hairs. Physiol Plant Pathol 2: 69-74

Cary JW, Lax AR, Flurkey WH (1992) Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase. Plant Mo1 Biol 20: 245-253

Constabel CP, Bergey DR, Ryan CA (1995) Systemin activates synthesis of wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway. Proc Natl Acad Sci USA 92: 407-411

DellaPenna . D, - . .. Lincoln JE, Fischer RI, Bennett AB (1989) Tran- scriptional analysis of polygalacturonase and other ripening associated genes in Rutgers, rin, nor, and Nr tomato fruit. Plant Physiol 90: 1372-1377

Duffey S, Felton G (1991) Enzymatic antinutritive defenses of the tomato plant against insects. Zn P Hedin, ed, Naturally Occur- ring Pest Bioregulators. American Chemical Society, Washing- ton, DC, pp 166-197

Ellis JG, Lawrence GJ, Finnegan EJ, Anderson PA (1995) Con- trasting complexity of two rust resistance loci in flax. Proc Natl Acad Sci USA 92: 41854188

Fang G, Hammar S, Grumet R (1992) A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques 13: 52-54

Gubatz S, Wiermann R (1993) Studies on sporopollenin biosyn- thesis in Cucurbita maxima. I. The substantial labeling of s oro ollenin from Cucurbita maxima after application of

Herminghaus S, Gubatz S, Arendt S, Wiermann R (1988) The occurrence of phenols as degradation products of natural sporopollenin-a comparison with “synthetic sporopollenin.” Z Naturforsch 43c: 491-500

Hind G, Marshak D, Coughlan S (1995) Spinach thylakoid polyphenol oxidase: cloning, characterization, and relation to a pu- tative protein kinase. Biochemistry 34: 8157-8164

Hunt MD (1995) Cloning, expression and functional analyses of polyphenol oxidase. PhD thesis, Cornell University, Ithaca, NY

Hunt MD, Eannetta NT, Yu H, Newman SM, Steffens JC (1993) cDNA cloning and expression of potato polyphenol oxidase. Plant Mo1 Biol 21: 59-68

Kobayashi H (1991) Differentiation of amyloplasts and chromoplasts. In L Bogorad, IK Vasil, eds, The Photosynthetic Appara- tus: Molecular Biology and Operation. Academic Press, San Diego, CA, pp 395415

Kowalski SP (1989) Insect resistance in potato: purification and characterization of a polyphenol oxidase from type A glandular trichomes of Solanum berthaultii Hawkes. PhD thesis, Cornell University, Ithaca, NY

Kowalski SP, Eannetta NT, Hirzel AT, Steffens JC (1992) Purifi- cation and characterization of polyphenol oxidase from glandular trichomes of Solanum berthaultii. Plant Physiol 100: 677-684

Lawrence SD, Cline K, Moore GA (1993) Chromoplast-targeted proteins in tomato (Lycopersicon esculentum Mill.) fruit. Plant Physiol 102: 789-794

[ P 4 p Clphenylalanine. Z Naturforsch 48c: 10-15

Lax AR, Vaughn KC (1991) Colocalization of polyphenol oxidase and photosystem I1 proteins. Plant Physiol 96: 26-31

Lincoln JE, Fischer RL (1988) Diverse mechanisms for the regulation of ethylene-inducible gene expression. Mo1 Gen Genet 212: 71-75

Luckwill LC (1943) The Genus Lycopersicon: An Historical, Biolog- ical, and Taxonomic Survey of the Wild and Cultivated Toma- toes. The Aberdeen University Press, Aberdeen, Scotland

Maeda N, Smithies O (1986) The evolution of multigene families: human haptoglobin genes. Annu Rev Genet 20: 81-108

Martin GB, Brommonschenkel SH, Chunwonges J, Frary A, Ga- na1 MW, Spivey R, Wu T, Earle ED, Tanksley SD (1993) Map- based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262 1432-1436

Martyn RD (1977) Disease resistance mechanisms in water hya- cinths and their significance in biocontrol programs with phy- topathogens. PhD thesis, University of Florida, Gainesville

Mayer AM (1987) Polyphenol oxidases in plants-recent progress. Phytochemistry 26: 11-20

Mayer AM, Harel E (1979) Polyphenol oxidases in plants. Phyto- chemistry 18: 193-215

Mayer AM, Harel E (1981) Polyphenol oxidases in fruits-changes during ripening. In J Friend, MJC Rhodes, eds, Recent Advances in the Biochemistry of Fruit and Vegetables. Academic Press, London, pp 159-180

Newman SM, Eannetta NT, Yu H, Prince JP, de Vicente MC, Tanksley SD, Steffens JC (1993) Organisation of the tomato polyphenol oxidase gene family. Plant Mo1 Biol 21: 1035-1051

Rathjen AH, Robinson SP (1992) Aberrant processing of polyphe- no1 oxidase in a variegated grapevine mutant. Plant Physiol 99: 1619-1625

Reeve RM (1951) Histochemical tests for polyphenols in plant tissues. Stain Technol 26: 91-96

Scott R, Hodge R, Paul W, Draper J (1991) The molecular biology of anther differentiation. Plant Sci 80: 167-191

Shahar T, Hennig N, Gutfinger T, Hareven D, Lifschitz E (1992) The tomato 66.3-kD polyphenoloxidase gene: molecular identi- fication and developmental expression. Plant Cell 4: 135-147

Sherman TD, Vaughn KC, Duke SO (1991) A limited survey of the phylogenetic distribution of polyphenol oxidase. Phyto- chemistry 30: 2499-2506

Smith AG, Hinchee M, Horsch R (1987) Cell and tissue specific expression localized by in situ RNA hybridization in floral tissues. Plant Mo1 Biol Rep 5: 237-241

Sommer A, Néeman E, Steffens JC, Mayer AM, Harel E (1994) Import, targeting and processing of a plant polyphenol oxidase. Plant Physiol 105: 1301-1311

Steffens JC, Harel E, Hunt MD (1994) Polyphenol oxidase. ln BE Ellis, GW Kuroki, HA Stafford, eds, Genetic Engineering of Plant Secondary Metabolism. Plenum Press, New York, pp 275-312

Sudupak MA, Bennetzen JL, Hulbert SH (1993) Unequal ex- change and meiotic instability of disease-resistance genes in the RpI region of maize. Genetics 133: 119-125

Thipyapong P, Hunt MD, Steffens JC (1995) Systemic wound induction of potato (Solanum tuberosum) polyphenol oxidase. Phytochemistry 40: 673-676

Thygesen PW, Dry IB, Robinson SP (1995) Polyphenol oxidase in potato. A multigene family that exhibits differential expression patterns. Plant Physiol 109: 525-531

Trebst A, Depka B (1995) Polyphenol oxidase and photosynthesis research. Photosynth Res 46: 41-44

Vámos-Vigyázó L (1981) Polyphenol oxidase and peroxidase in fruits and vegetables. CRC Crit Rev Food Sci Nutr 15: 49-127

Vaughn KC, Duke SO (1981) Tissue localization of polyphenol oxidase in sorghum. Protoplasma 108: 319-327

Vaughn KC, Duke SO (1984) Function of polyphenol oxidase in higher plants. Physiol Plant 60: 106-112

Verwoerd TC, Dekker BMM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17: 2362

Yu H, Kowalski SP, Steffens JC (1992) Comparison of polyphenol oxidase expression in glandular trichomes of Solanum and Lyco- persicon species. Plant Physiol 100: 1885-1890



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