Enzymatic Browning and Its Prevention - American Chemical Society

Chapter 8

Implications of the Phylogenetic Distribution of Polyphenol Oxidase in Plants 1

1

Timothy D. Sherman , Theaux Le Gardeur , and Alan R. Lax

2

Downloaded by UNIV OF ROCHESTER on August 15, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch008

1

2

Department of Biological Sciences, University of South Alabama, Mobile, AL 36688-0002 Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70179

A limited phylogenetic survey of the distribution of polyphenol oxidase (PPO: EC 1.10.3.2) was conducted in order to determine if this enigmatous enzyme is present in all plants groups or in only certain phylogenetic lines. Evidence for presence of the enzyme was gathered via enzyme assays using various recognized substrates and through the use a heterologous DNA sequence from the PPO of Vicia faba in DNA hybridization experiments and in polymerase chain reaction amplification of conserved sequence within the gene. Enzyme assays showed PPO activity in members of all eukaryotic groups tested, albeit at low levels in some groups, including green algae, mosses, and gymnosperms. DNA hybridization experiments showed varying degrees of signal strength obtained on the dot blots. This could mean limited homology between Vicia probe and target DNA or different copy number in the target DNA. Amplification experiments using primers for a 111 base pair region of the Vicia copper A binding region yielded 111 base pair products in all members of all phylogenetic groups tested. These data strongly suggest the presence of active PPO in all photosynthetic organisms more advanced than cyanophytes. This, along with data from studies with PPO-less mutants, implies some essential function of the enzyme in plants.

Polyphenol oxidase (PPO; EC 1.10.3.2), also known as catechol oxidase, phenolase, and o-diphenol oxygen oxidoreductase, has no clearly established function (1-3). The enzyme is widely distributed in higher plants (4-7), and is found in the plastids of all tissues types in species possessing PPO activity (2). A number of nuclearly inherited isozymes have been shown to exist in tobacco (8,9). Recent investigations at the DNA-level have shown that PPO is represented as a multigene family in broadbean (70), tomato (77) and potato (72). In the Solanaceae, different members 0097-6156/95/0600-0103$12.00Α) © 1995 American Chemical Society

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


104

ENZYMATIC BROWNING AND ITS PREVENTION

of the family are expressed in various tissue types, with certain forms that are involved in pathogen defense being expressed exclusively in epidermis and type A glandular trichomes on the surface of the leaf (13-15). There is certainly evidence that the enzyme plays a part in deterrence of insect and fungal pathogens in many, but not all, species and cell types. This role has been reviewed elsewhere (16) and will be addressed by others in this book. There has long been speculation that PPO's have other roles in plants as well. The multigenic nature of the enzyme and differential tissue-specific gene expression implies that there is more than a single function for PPO. PPO activity has been monitored previously in a number of individual plant species (7, 17-22). Also, there have been two more broadly based surveys of PPO published (4, 5). That conducted by Cambie and Bocks (5) was limited to the gymnosperms, and focused primarily on the family, Podocarpaceae. The other covered a wide spectrum of the plant kingdom, including representatives from the green algae, mosses, liverworts, homworts, ferns, gymnosperms, and angiosperms. In that work, three assay protocols were utilized (spectrophotometric assays, PAGE enzymic activity stains, and cytochemistry) to determine the presence of PPO activity in a given species (4). Although PPO was found almost ubiquitously in the angiosperms, a number of groups had no detectable activity. These including most of green algae, the mosses, and essentially all of the gymnosperms tested. The question remained, was the enzyme present but in an inactive form, unable to utilize the substrates used for assay, or simply absent? With the cloning of PPO in a number of species (10, 11, 13, 23-26), it is now possible to look for the presence of the PPO gene in these previously examined phylogenetic groups and to address the question as to the presence or absence in groups that previously show no enzyme activity. The survey conducted here, builds upon this previous work (4), covering a wider spectrum of the higher plant kingdom, and examines a number of groups that have not been previously assayed. Here, spectrophotometric assays utilizing a group of different PPO substrates have been utilized to verify the presence of PPO enzyme activity in newly assayed and previously recalcitrant species, and genomic DNA has been isolated from many representatives within each group and examined with a heterologous probe for a highly conserved region of the PPO genefromVicia faba. From this, we hoped to gain a more comprehensive and accurate view of the distribution of PPO in the algae, the bryophytes, and higher plants, and to make inferences concerning essentiality of this enigmatous protein. Experimental Plant Material. Plant materials were collected locally (Mobile, AL) when possible or obtained from commercial sources. Monotropa uniflora was collected in Perry Co., AR. Axenic Trebouxia erici was obtained from Ward Biologicals. Chlorella vulgaris was a gift from E. Funkhouser (Texas A & M Univ., College Station, TX). Chlamydomonas reinhardtii strain cw 92 mt was a gift from J. Salisbury (Mayo Clinic, Rochester, MN). Chorafibrosaand Phaeoceros laevis were giftsfromK. +


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SHERMAN ET AL.

Phylogenetic Distribution of PPO in Plants

105

Renzaglia (Ε. Tenn. St. Univ., Johnson City, TN). Anthoceros fusiformis and Megoceros aenigmaticus were gifts from K. Vaughn (USDA, Stoneville, MS). Notothylas breutelii was a gift from R. Brown (Univ. of S. LA, Lafayette, LA). Artemisia vulgaris was a giftfromS. Weller (Purdue Univ., West Lafayette, IN). PPO Extraction and Assay. Tissue employed for assay varied with the size of the organism. Algae and bryophytes were homogenized as whole plants or large pieces of plants. For vascular plants, frond or leaf tissue was used, with woody midribs removed. Cells or tissues were homogenized at a ratio of 200 mg tissue/1 mL buffer in cold (0-4°C) 0.1 M sodium cacodylate buffer, pH 7.2, containing 0.1% ( /v) sodium dodecyl sulfate using glass, hand-held homogenizers or with a Brinkmann Polytron. The homogenate was then clarified by centrifugation at 12,000 χ g for 2 min. In the standard PPO assay, the conversion of DL-dihydroxyphenylalanine (DOPA) to quinone polymers in the supernatant was monitored spectrophotometrically at 490 nm. The assay solution consisted of 1 mL of 5 mg/mL DOPA in 0.1 M sodium cacodylate, pH 7.2, which had been aerated for 5 min prior to assay. To prevent oxidation of the substrate by peroxidase, 280 units of bovine liver catalase in 0.1 mL of water was added. The assay was initiated by addition of 0.05 mL of the plant extract and monitored for 2 minutes at 30°C. Some of the species with very low or no activity in the standard assay were also examined in a modified assay in which samples were incubated in the presence of each of four different commonly used PPO substrates, along with appropriate controls in 96-well plates for 10 h at 22°C with intermittent shaking and hourly absorbance readings taken at 490 nm. These assays were conducted in a volume of 0.2 mL and contained either 5 mg/mL DOPA, 10 mM catechol, 10 mM caffeic acid, or 10 mM chlorogenic acid in 0.1 M sodium cacodylate, pH 7.2. All assays also contained 40 units/mL catalase to prevent peroxidation of the substrate. Assays were started with the addition of 0.01 mL of plant extract (prepared as outlined above). One set of control wells contained additional 1 mM diethyldithiocarbamate as an inhibitor of PPO. A second control set contained plant extract but no additional substrate. Absorbance readings from these two controls were used to correct for non-PPO induced browning and oxidation of substrate that was introduced into the assay along with the plant extract, respectively.


w

DNA Isolation. A modification of the method of Lodhi et al (27) was used for DNA isolationfromall plant species. Briefly, this was a follows. Approximately 500 mgfreshweight plant material, similar to that used for PPO activity assays, was frozen and ground to afinepowder in liquid nitrogen. While stillfrozen,the slurry was transferred to preheated (60°C) extraction buffer containing 5 mL of 100 mM Tris (pH 8 at 20°C), 20 mM N a E D T A , 1.4 M NaCl, 2% ( /v) cetyltrimethylammonium bromide, 0.2% ( /v) beta-mercaptoethanol and 50 mg insoluble polyvinylpyrrolidone polymer. The slurry was mixed thoroughly and allowed to incubate for 30 min at 60°C. The sample was then allowed to cool and extracted once with one half volume of chloroform: isoamyl alcohol (24:1). After w

2

v


106


centrifugation at 1,600 χ g for 10 min., the upper phase was transferred to clean tube and the nucleic acids were ethanol precipitated through the addition of one half volume of 5 M NaCl and two volumes of 95% ethanol and allowed to stand overnight at -20°C. Nucleic acids were collected by centrifugation at 1,600 χ g for 10 min., washed with 76% ethanol, and resuspended in a small volume of 10 mM Tris (pH 8 at 20 °C), 1 mM Na EDTA. RNA was degraded by treatment with 100 μg/mL RNAse A and 10 U/mL RNAse T l for 1 h at 37°C. The remaining DNA was quantified using the method of Cesarone et al (28) and checked for integrity by agarose gel electrophoresis (29). Downloaded by UNIV OF ROCHESTER on August 15, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch008

2

DNA Hybridizations. One microgram samples of genomic DNA isolated as described above were spotted onto nitrocellulose, allowed to air-dry, and baked at 80°C for 1 hr in a vacuum oven prior to hybridizations. A 798 base pair segment of the cDNA clone for Vicia faba PPO covering the area between amino acids 175 to 441 and including the copper A and Β regions (10) was random primed labelled with oc P-dCTP using a Prime-a-Gene kit (Promega) according to manufacturers instructions. Hybridization were conducted in the presence of 50% formamide, 5x Denhardt's solution, 1% sodium dodecyl sulfate, 2x SSPE (29) at 42°C. Blots were washed at low stringency (2x SSPE, 0.1% sodium dodecyl sulfate), and exposed to X-Omat AR film (Kodak) overnight or longer. Hybridization signals were empirically determined and scaled from 0 (no hybridization signal) to 4 (strong hybridization signal). Because it was not know how many copies of the PPO gene would be present in the genomic DNA of each species, it was decided that more quantitative determination of hybridization signal would have little meaning and was not attempted. 32

Amplification of Conserved Regions of the Copper A Region of PPO. DNA samples from representatives of various phylogenetic groups were chosen from samples isolated as described above. Oligonucleotide primers were synthesized that correspond to the left and right borders of the conserved copper-binding domain (copper A) of the PPO cDNA clone from Vicia Faba (10). DNA samples (100 ng) from the various species were combined with 100 pmol of eachrightand left primer and amplified using a Perkin-Elmer AmpliTaq polymerase chain reaction (PCR) kit according to the manufacturers instructions. Thermal cycling conditions were: 94°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min for 30 cycles followed by 72°C for 5 min. PCR reaction products were analyzed by electrophoresis on a 5% acrylamide gel using a TBE buffer system (29), stained with ethidium bromide, and photographed under ultraviolet radiation. Plasmid pBR322 DNA that had been digested with the restriction enzyme Mspl was used as molecular mass standards. Vicia faba DNA used under these conditions is expected to generate DNA of 111 base pairs.


8.

SHERMAN ET AL.


107


Results and Discussion Ubiquitously found in healthy green plants, there is a form of PPO that appears to be localized exclusively in the chloroplast and is associated with the thylakoid membrane. This is evidenced by immunogold labelling and the fact that cytochemical reaction product accumulates in the lumen of the thylakoid (4, 30-32). It has also been demonstrated that the protein precursor of the enzyme contains the targeting sequence necessary for import into the chloroplast and insertion in the thylakoid membrane (33). The data that we will report here indicates that PPO may be present in all plant ranging in complexity from the green algae through the angiosperms and gymnosperms (Table I). In light of this distribution, PPO may well serve some indispensible function to the chloroplast. A model for a possibility of this function will be outlined first, providing a conceptual framework to interpret the data. Although the enzyme takes its name from its ability to catalyze the oxidation of o-diphenols, and is most commonly assayed by this activity, this may in fact be a secondary function or have no physiological relevance in many plastid types. One possible function that has been speculated is in the modulation of photosystem I (PSI) reduction of molecular oxygen (the Mehler reaction or pseudocyclic photophosphorylation) (2, 3, 34, 35). Normally, photosynthetic energy is utilized in the reduction of intermediates necessary for biosynthetic processes (NADP , nitrite, etc.), but it has been suggested that under circumstances that limit carbon dioxide availability, destructive, high oxygen concentrations can occur temporarily due to the continued operation of the photosythetic apparatus. A model of a possible scenario where energy can be diverted to dissipation of molecular oxygen via a Mehler-like reaction involving PPO is shown in Figure 1. The mechanism depicted in this figure is different slightly from that outlined for the Mehler reaction as it has been previously presented (2, 35), because the electrondonating end of the PSI complex, believed to be involved in Mehler activity, is on the stromal side of the thylakoid membrane, whereas PPO appears to be associated with PSII on the lumenal side of this membrane (36, 37). In the scheme shown here, PPO acts to oxidize the reduced quinones (internal to the membrane) generated by PSI activity, thereby preventing molecular oxygen accumulation and regenerating substrate for continued operation of the cycle. It is well known that there are bound quinones associated with PSII (Q and Qt>) and are involved in electron transport so the possiblity of other membrane localized quinones can not be ruled out. Additionally, this model requires quinones to be present in only catalytic quantities within the plastid, and thus wolild be possible even in species that are not known to possess high levels of tissue-quinones. Furthermore, a manganese-containing superoxide dismutase has long been thought to be associated with the thylakoids of cyanobacteria and green algae (38, 39). The presence of a similar enzyme in higher plants was reported earlier (40), but was later disputed (47). If such an enzyme exists, it would allow for hydrogen peroxided that is not particularly reactive in itself (42) to cross the membrane for further processing in the stroma. In this model, a role for PPO in an oxygen mediating reaction is possible that is consistent with +

a



Group

1

1 (Charlean algae)

(other algae)

1 I

I Chlorophytes 1 (Green algae)

Γ

none none none none

Microspora sp.

Stigeoclonium tenue

Trebouxia erici (axenic)

Ulva faciata

+

low none none

Chondrus sp.

Monostroma sp.

Chora fibrosa

low

+

none

Laminaria sp.

Nitella sp.

+

trace

+

+

+ +

trace

Fucus sp.

Rhodymenia sp.

-

+

trace none

Spirogyra grevileana

+

+

DNA collected

Chlamydomonas reinhardtiistrain: cw92 mt (+) Choleochaete scutata

trace

PPO Activity ι none

Common name or discriptor

Chlorella vulgaris

Genus/species

Table I. Summary of PPO data concerning enzyme activity and DNA hybridization


0

1

1

4

0

1

1

(0-4)2

Hyb. Signal


D Polypodiophytes (ferns)

(mosses)

(anthocerotes)

1 (hepatics)

1 Bryophytes

H Table 1. (continued)

-

none none

Sphagnum sp.

Thuidium delicatulum

+

low low

Salvinia sp.

-

Polystichum acrostichoides Christmas fern

low

+

low

Mnium sp.

Water clover

+

trace

Marsilea quadrifolia

+ +

none

Dicranum sp.

Hypnum sp.

+

-

low

Phaeoeros laevis

low

-

low

Megoceros anaegmaticus

Ebony spleenwort

+

low

Anthoceros fusiformis

Asplenium platyneuron

+

none none

Riccia fluitans

Riccia sp.

+

+

0.5

4

3

1

1

0

Hyb. Signal (0-4)2

Continued on next page

DNA collected

high

1

Marchantia pofymorpha

PPO Activity low

Common name or descriptor

Conocephalum conicum

Genus/species



(gymnosperms)

Pinophytes

(fern allies)

Lycopodiophytes

Table 1. (continued)

+ none none

Ginkgo Northern Redcedar Longleaf pine White pine

Ginkgo biloba

Juniperus virginiana

Pinus palustris

Pinus strobus

Pinus virginiana

Coontie

trace

none

+

-

+

none

Podocarpus macrophyllus Buddhist-pine

Taxodium distichum

+

none

Virginia pine

Zamiamîegnfolia

-

none

Baldcypress

+

none

+

-

none none

+

+

Atlantic White-cedar

none

mod

+

Chamaecyparis thyoides

Spikemoss

SelagineUa apoda

high

+

Lebanon Cedar

Wisk fern

Ρsiloturn nudum

n.a.

-

Cedrus lebani

Club moss

Lycopodium sp.

low

+

DNA collected

Norfolk Island Pine

Quillwort

Isoetes sp.

low

PPO Activity!

Araucaria excelsa

Horsetail


Equisetum sp.

Genus/species


1

1

1

0

4

3

4

Hyb. Signal (0-4)2


(angiosperms)

Magnoliophytes

Table 1. (continued)

-

trace low n.a. low

Duckweed False loosestrife Parrot feather Eel grass

Lemna minor

Luawigia arcuata Walt

Myriophyllum sp.

Vallisneria sp.

+ + +

n.a. v.high n.a. n.a. n.a. n.a.

Ragweed Mugwort Sea Oxeye Mockernut hickory Southern Catalpa Spiderplant

Ambrosia artemesifolia

Artemisia vulgaris

Borrichia frutescens

Carya tomentosa

Catalpa bignonioides

Chlorophytum comosium

+

+

n.a.

Senstive plant

+

+ +

Albizia julibrissin

(terrestrial)

+

trace

Burhead

Echinodorus sp.

-

-

low

3

0.5

1

1

4

0.5

Hyb. Signal (0-4)2

Continued on next page

DNA collected

Cryptocoryne affinis

1

PPO Activity

+

Brazilian waterweed

Common name or descriptor

v.high

Elodea densa (Anacharis densa)

(aquatic)

Genus/species



angiosperms (cont)

Il Table 1. (continued)

+ +

n.a. n.a.

Common Persimmon Saltgrass Rush Pennywort

Diospyros virginiana

Distichlis spicata

J uncus sp.

Hydrocotyle bonariensis

Lactuca sauva

+

+ +

n.a. n.a. n.a. n.a. low

Orontium aquaticum (L.) Golden Club E. Sycamore S. Red Oak Virginia Live Oak Black Locust

Plantanus occidentalis

Quercusfalcata

Quercus virginiana.

Robinia pseudoacacia

+

+

trace

Indian Pipes

Monotropa virginiana

v.high

Sweetbay Magnolia

Magnolia virginiana

+

n.a.

Lycospersicon esculentum Tomato

n.a.

+

v.high

Lettuce Southern Magnolia

+

low

Magnolia grandiflora

+ +

n.a.

+

n.a.

Cyrilla

Cyrilla racemiflora

-

trace

Cucumber

Cucurbita maxima

+

DNA collected

n.a.

PPO Activity ι

Flowering Dogwood


Cornus florida

Genus/species


4

3

3

4

0

0

4

3

(0-4)2

Hyb. Signal


Genus/species

2

1

+ + +

low n.a. n.a. none

Summer grape Muscadine grape Christmas cactus

Vitus aestivulus

Vitus rotundifolia

Zygocactus sp.

Vigna unguiculata

Cowpea

Mungbean

Vignaradiata

+

high

-

Broadbean

Vicia faba

+ +

low n.a. v.high

Spinach Turtle grass

-

n.a.

SaltmarshCordgrass

Spatina alternaflora

Thalassia testudinum

0

+

n.a.

Sea purslane

Sesuvium sp.

Spinacia oleracea

0

+

n.a.

3

0.5

3

1

Hyb. Signal (0-4)2

Sapium sebiferum (L.)Roxb.Tallowtree

Salicornia biglovti

trace: < 1 unit; low: 2-33; moderate: 34-66; high: 67-100; very high > 100 (1 unit = A490 min^g^fr.wt.) n.a. (not assayed) relative hybridization signal one xray film. 0 (no signal) - 4 (dark signal)

1 I

+ +

n.a. n.a.

+

DNA collected

Arrowhead

n.a.

1

PPO Activity

Glasswort

Marsh Pink


Sagittaria sp.

1 angiosperms (cont) Sabatia sp.

Il Table 1. (continued)


114



photosystem and membrane topology. In support of this theory, it has been reported that cells that generate little or no molecular oxygen, such as agranal bundle sheath cells of C4 plants (57) and guard cells (43, 44), generally have no PPO. There is an additional benefit to the plant of having an oxygen consuming system localized within the thylakoid. Oxygen, which diffuses easily across biological membranes, becomes an additional problem when it arrives in the stroma because of its competitive inhibition of ribulose-l,5-bisphosphate carboxylase-oxygenase (RuBisCO).

Figure 1. Model for PPO's possible involvement in a Mehler-like reaction for detoxifying oxygen species. This is only one possible role for PPO and it is entirely possible that it has a number of unrelated functions in normal plant growth and development that utilize its ability to interact in redox reactions with quinones. Whatever its role, some form of PPO seems to be essential for some aspect of plant function. This is evidenced by the fact that there are no reported naturally-occurring or experimentally-generated viable mutants that completely lack all PPOs. In mutants of grape that have sections of the leaves that lack of PPO activity, these sections are lacking in competent photosynthesis and appear pale yellow (25). Also, in plants that have been treated with tentoxin, a cyclic peptide of fungal origin, PPO import into the chloroplast is blocked (33, 45). These plants are photosynthetically incompetent and have this same yellow pigmentation as that seen in the grape mutant. Recently, Steffens'



8.

SHERMAN ET AL.

Phylogenetic Distribution of PPO in Plants 115

group (72) has expressed PPO-anti-sense mRNA in potato so as to decrease PPO activity to less than 5% of that found in the untransformed variety, and yet the transformed plants seem to be morphologically normal. These data indicate that either the essential form of PPO remained uneffected by the treatment or that very little PPO is necessary for normal plant growth. If it is true that PPO plays some important role in defense against pathogens and/or oxidative damage within the plastid, it is stands to reason that this enzyme should have wide distribution within the plant kingdom. If, in fact, it is physiologically necessary, it should be present in all green plants. In this case, data presented here is strongly supportive of an important physiological role. DNA sequence corresponding to the copper-binding region (copper A) is highly conserved among those PPOs that have been cloned and sequenced (10, 11, 13, 24, 26) and the sequence in this region is shared by no other proteins reported to the genetic databases to date. It is apparent, based upon the results of the PCR experiments, that this 111 base pair sequence is present in all of the phylogenetically dissimilar genera that were examined here (Figure 2). Homology of this region between Vicia faba and a phylogenetically distant member of the group, Chlamydomonas, was determined by subcloning and sequencing the amplified algal DNA. Sequence analysis of this DNA showed only a single amino acid substitution of an isoleucine for a histidine at position number 211 in the 37 amino acids coded for in the 111 base pair section. This region of DNA could exist as a vestigial remnant of some ancestral gene, but there is a great likelihood that mutations would create changes in the sequence over the hundreds of millions of years since the divergence of these two groups. In fact, the sequence homology reported for the chloroplastically-encoded large subunit of RuBisCO varies by 21% between Chlamydomonas and Lycopersicon, and the rate of silent substitution in chloroplastic genes is in the order of two to three times lower than that of nuclear encoded genes (of which PPO is a member) (46). Based upon the highly conserved nature of this copper-binding region that has been reported here and elsewhere, it would seem that the sequence and length of this region is necessary for function and that the gene encoded in these various species is likely to be functional. The DNA evidence is also supported by the enzyme activity assays reported here. Oxidative activity was found to be present at low levels and with different substrate affinitiesin groups that showed no activity in the previous survey (the green algae, mosses and gymnosperms). Representatives of these assays are shown in Figure 3. Here catechol was a preferred substrate to DOPA in all cases, but measured activity was very low in each case, relative to the activity monitored in species such as Vicia faba or Lactuca sativa that reached off-scale absorbance readings in minutes rather than hours (data not shown). The substrates chlorogenic acid and caffeic acid, that are preferred by the trichome form of PPO in potato (14) showed essentially no activity with these species that showed very low activity levels. It is tempting to speculate that this low level of activity found in these species is in keeping with the "catalytic" quantities described above that would be involved in a PPO-based "Mehler'Mike scenario. Also, this data emphasizes the point that traditional enzyme



116


Figure 2. PCR amplification products of genomic DNA corresponding to the 111 bp fragment of the copper-binding region (copper A) of Vicia faba PPO. (Mr standards: Mspl digested pBR322 DNA) assays used to monitor PPO may be monitoring a secondary and possibly physiologically irrelevant activity, at least in this ubiquitous form of PPO reported here. In conclusion, the data presented reinforce the idea that some form of PPO has an essential role in plant growth and/or metabolism. That function is still open to speculation. One possibilty is presented here that encompasses a number of ideas and observations from other published work. Further analysis of the gene encoding the Chlamydomonas PPO, its use in anti-sense expression studies, and search for the presence of PPO in the more primitive photosynthetic cyanophytes, will be examined in the future and will hopefully add pieces to the rapidly assembling puzzle of PPO function. Acknowledgments The work in the early stages of DNA isolation by Dr. Matilde Tellaetxe-Isusi was essential in finding a technique appropriate for use with the diverse collection of plant material surveyed here. The cytochemistry performed by Dr. Kevin Vaughn on plant samples, although not presented here, was enormously helpful in interpretation of the enzyme data. The efforts of both are acknowledged and greatly appreciated.


SHERMAN ET AL.

0.5


J

Chlamydomonas

0.4 ο

ν

Sphagnum V — ν y V • 0 •

Os

Ζ 0.3

CATECHOL DOPA CHLOROGENIC CAFFEIC

I 0.2


1

< 0.1

- /

—

0.0 - i - — ο — ο — ο — ; — τ — t — • Mnium I 0.4 ο

ο Ζ 0.3 ce υ §ο 0.2 -Ρ < 0.1 0.0 0.7 i 0.6

-•^8=8=8=8=81

1

1

1

1

1

Hypnum

ν

Ρ^ •1/ Pinus | » o O—o—ο—ο , ιpalustris Τ — t — · — · - Podocarpus ι Τ 1 1 * · V—ν V V

* 0.5 03

8 0.4

V — ν — ν — ν - — ν

·£ 0.3 2

3 °· 0.1 0.0

V %

% % Φ

2

Φ%

4 6 8 10 Time (h)

I

• I

2

τ

I ,1 J ι_ 4 6 8 10 Time (h)

Figure 3. Long-term assays for PPO in species showing little activity in standard assay.


:

118


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1. 2. 3. 4.

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