THE RESPIRATORY PROCESSES IN PLANT CELLS IN RELATION
TO THE FORMATION OF COACERVATES
J. DUFRENOY AND H. S. REED
(WITH TWO FIGURES)
Life depends on the existence of a water phase within the cytoplasm.
Generally the water phase, as represented by the vacuolar solution is optically empty: in whatever condition the solutes are, they are hydrated and
randomly distributed'so that they appear to be in solution. The vacuolar
solution, however, may itself become the site of a separation of phases
through coacervation, as we intend to show by the data to be presented.
Coacervation may occur in the vacuolar solution of plant cells under
the influence of various agencies apt to unbalance cell respiration, such as
zinc deficiency, virus or fungal infection. Auto complex coacervates, made
up of a central core of polymerized phenolic compounds, surrounded by
phosphatides, once formed, appear as permanent structures in the cell and
have frequently been misinterpreted by observers. Recent advances in
cytochemistry emphasize that the vacuole is the reaction chamber of the cell.
The process of coacervation in it affords an unusual opportunity to visualize
one of the delicately balanced equilibria so essential for life (13).
The polyphenols, chiefly catechol, which are so widely and almost universally distributed in the vacu6lar solution of plant cells, have during
these recent years been recognized as the most important mediators between
the metabolites furnishing the hydrogen and the atmospheric oxygen accepting it in the process of aerobic respiration. In the normal "compensated" respiration these compounds are reduced back to the original condition of polyphenols as fast as they are dehydrogenated to quinones.
Therefore they retain their original physical as well as chemical properties.
When the respiration is "non-compensated" the phenolic compounds,
having been dehydrogenated to quinones, no more have made available to
them the activated hydrogen that can reduce them to their original condition (9). Instead of being reversibly reduced, they become subject to
further oxidations resulting in concomitant changes of their physical conditions. Some of the linkages which should normally have been C-OH
linkages become C-C linkages, meaning linkages bet.ween the C of different
molecules, building large molecular weight units, or polymers, which assume
the consistency of a gum, and the color of a pigment-either the brownishred phlobaphenes, or the vivid red anthocyanols.
The phenomenon of molecular aggregation, or polymerization, was correlated with changes in chemical affinity by BERTHELOT (2), GULDBERG and
WAAGE (8) and PASTEUR (11). BERTRAND (3) applied his newly acquired
knowledge of oxidases to explain the concomitant oxidation and polymerization of Boletol and other phenolic derivatives. COMBES (4) recognized that
416
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DUFRENOY AND REED: COACERVATES
417
when anthocyanic pigments are formed, oxygen is fixed by the organs which
become red; then those organs are the site of enhanced oxidative phenomena.
Conversely, when anthocyanic pigments disappear, the organs whence they
disappear release oxygen. It is possible to demonstrate an aggregation of
oxidizable molecules into more or less soluble gummy masses in cells which
are the sites of unbalanced or non-compensated respiration. The phenomenon may be correlated with a process of dehydrogenation not only in vivo
but in vitro.
Relation between coacervates and the non-compensated
respiration
Diseased tissues which evidence discoloration, absorb more 02 than
normal, and release less CO2; oxygen acts as a hydrogen acceptor whereby
the phenolic compounds become dehydrogenated into pigments. Oxygen,
however, is not fixed by the pigment. As phenolic compounds, such as
catechol, normally dispersed in solution in the vacuolar sap, are being
dehydrogenated, due to non-compensated respiration, both the rH and the
pH of the vacuolar solution would shift; correlatively the behavior of the
compounds toward water will be altered. Molecules of polyphenols, originally dispersed at random in the vacuolar solution, become grouped into
larger units of polymers and those units may not remain distributed at
random, but may become more densely congregated at certain loci.
This unequal distribution of phenolic compounds within the vacuolar
sap may be achieved through one of two ways:
1. The vacuolar sap in the cell becomes apportioned to different vacuoles,
some of which may contain phenolic compounds, others not. In other cases
the initially single vacuole containing the whole of the vacuolar solution
in the cell may bud out, at its periphery, a number of drops which will
become individualized into many small vacuoles. These drops may contain a solution richer in phenolic compounds than the original solution, in
which case the central vacuole may be described as secreting or excreting
phenolic compounds, or, on the contrary the exudate may be devoid of
phenolic compounds, in which case the original vacuole excretes water, and
concent.rates the phenolic solution.
2. Instead of being achieved through the fragmentation of the vacuole,
the concentration of the phenolic compounds can occur in the vacuole itself
which retains its full volume, while the phenolic compounds contract spontaneously and become condensed as a mass floating in the vacuolar liquid,
now impoverished in colloids.
Many investigators actually stained coacervates with dyes like neutral
red, but failed to realize that they were floating in the vacuolar solution.
MAST (10), studying the relations between the kinds of food, growth, and
structure, reported that Amoeba proteus, which had fed for several days
exclusively on Chilomonads, contained an extraordinarily large number
of spherical bodies which showed a definitely differentiated structure;
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41848PLANT PHYSIOLOGY
namely, a central rnass surrounded by a distinct, though fragile, shell which
was covered with a thin layer of oily substance. In solutions containing
neutral red the outer layer became crimson in color, but the central portion
and the shell were not stained by the dye. Coacervates thus express the
ultimate effect of a gradient in the distribution of the vacuolar materials.
The phenolic compounds as they lose hydrogen develop ability for C linkages; hydrogen ties up into H20 or H202; less and less hydrogen is available to redisperse the polymerized phenolic compounds, or rather the dehydrogenase systems (whereby hydrogen should have been made available)
fail to perform, either because the active phosphorus linkages have been
broken or because the active SH groups have been oxidized to disulfides.
The loosening of phosphorus linkages concomitant with the dispersion of
the dehydrogenase systems, provides for a supply of ionic P04 which goes
into the vacuolar solution, there to become incorporated into the amphoteric
lipid complexes at the boundary of the auto complex coacervates.
The non-compensation of respiration, following dispersion of dehydrogenases, lack of active hydrogen for rehydrogenation of coacervated phenols,
immigration of P04 ions into the vacuoles onto the boundary of the coacervates, can be experimentally induced by treatment with agents such as
salicylaldoxime interfering with the normal activity of dehydrogenases.
Coacervates in plant cells
The effect of disrupting the dehydrogenase system was convincingly
shown in the experiment where sugar cane was treated with 100 p.p.m. sodium
xanthogenate. Within a few hours living cells were in a condition represented by figure 1. Following treatment with the molybdenum reagent, the
nuclear mass gave a diffuse reaction for phosphorus, mitochondria were
swollen, phenolic material was coacervated within the network of the phospholipid and was floating in the vacuolar sap. This condition can be interpreted as the result of the disequilibrium between the dehydrogenase
system and the oxidase system. The former being more sensitive, most
reagents are likely to induce a non-compensated respiration as expressed
by coacervation. This will explain the common occurrence of coacervates
in pathological tissues as will be shown later.
About 1940, in some of the sugar cane fields of Louisiana were first
detected a few stalks affected by an infectious disease, which was niamed
"chlorotic streak." ABBOTT and INGRAM (1) obtained evidence t.hat it is a
virus disease and showed that it could be transmitted by a leaf-hopper. A
red discoloration of vascular bundles at the nodes was revealed when the affected stalks were split lengthwise. Freehand longitudinal sections of the
nodes showed in most perivascular cells a large coacervate, the contents of
which gave positive reactions for phenolic compounds, and most specifically
gave the positive Scudi reaction for pyridoxin (6). The coating of phospholipids separating the inner solution (rich in pyridoxin) from the depleted
outer vacuolar solution could be easily demonstrated by the molybdenic
reagent.
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DUFRENOY AND REED: COACERVATES
419
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420
PLANT PHYSIOLOGY
These coacervates could also be demonstrated in permanent mounts of
sections of tissues fixed in the HELLY's killing fluid, and properly stained
with haematoxylin or with acid fuschsin. Ultimate stages of cell disintegration in sugar cane, either due to physiological senescence, or pathological
breakdown are expressed by the prevalence of coacervates which may
eventually be the only recognizable features. In fact the intermediate phase
of the coacervate is so permanent a structure that the inner core of phenolic
I_
FIG. 2. A cell from stalk of sugar cane penetrated by hyphae, F, of C. falcatum
fixed in HELLY fluid and stained with haematoxylin. The nucleus, N, at the center of the
cell shows numerous prochromosomes; mitochondria, M, are lined up between a number
of vacuoles. The segregated vacuole in the upper right corner contains a large coacervate,
C, outlined by the deeply stained phospholipoid envelope.
compounds can be dissolved out by proper solvents (for instance tetra
ethylene glycol or methyl ethers), leaving the envelope intact as an empty
shell.
Whereas coacervates are constantly present in nodes of young canes
affected by chlorotic streak, they generally do not occur in homologous tissues
of young, actively growing, healthy canes, but they make their appearance
later, toward the end of the growth cycle, about the time for harvest. They
also appear in cells affected by various diseases besides chlorotic streak, and
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DUFRENOY AND REED: COACERVATES
421
they can be made to appear at will, following injection of the stalks by dilute
solutions of agents acting as inhibitors of some respiratory systems, for instance salicylaldoxime (10 to 100 p.p.m.) or sodium xanthogenate (100
p.p.m.) .
Coacervates similar to those observed in chlorotic streak also occur in
cells of cane infected by Colletotrichum falcatur,m the pathogenic fungus
responsible for the Red Rot disease (fig. 2). The disease is characterized
by a discoloration due to anthocyanol formation in the vacuolar solution of
cells adjoining the area of infection. Conidia of the fungus travel upwards
and downwards along the pitted vessels from the point of inoculation, having a tendency to lodge and germinate a short distance below the nodes.
The germ tubes which grow through pits into the adjoining slender, elongated parenchyma cells cause the vacuolar solutions to be dispersed into a
number of vacuoles possessing high oxidase activity, and soon producing the
above-mentioned red pigment. Some of this oxidized, polymerized material
oozes from the vacuoles through pits in the wall into tyloses. That reaction
efficiently blocks any further progress of the germ tubes in resistant canes.
In susceptible canes, however, the germ tubes grow as hyphae from one
parenchyma cell to the next, or in the intercellular spaces, inducing noncompensated respiration in cells as much as five or six rows distant, as evidenced by the oxidation of paraphenylene diamine hydrochloride to the
red quinoid derivative (Wuirster red) in the vacuolar solutions. The red
anthocyanol pigment formed in the parenchyma cells of susceptible cane
oozes from the vacuoles into the intercellular spaces and coacervates may
appear in some vacuoles. In Red Rot tissues we therefore witness both the
external secretion of vacuolar material, even to the intercellular spaces, and
also that peculiar type of "internal secretion" which results in coacervate
formation.
Cell inclusions in the vine disease, now called "Pierce's disease," were
observed by VIALA and SAUVAGEAU in 1892 (15) and then misinterpreted as
plasmodia of a Myxomycete. DUCOMET (5) abandoned the erroneous idea
of plasmodial parasites and gave a cytochemically correct interpretation of
the small and large spherical bodies in the vacuolar sap, opening the way
to the recognition of the prevalence in other plants of those phenolic or
tannic compounds which may become conspicuously coacervated or otherwise
polymerized (12). Pierce's disease is characterized physiologically by the
failure of internodes to elongate and cytochemically by coacervation of
phenolic compounds. A similar correlation between dwarfed shoots and
coacervation was demonstrated as the result of zinc deficiency in apricots
and walnuts (REED and DUFRENOY) (13).
Cytochemical examination of grapes experimentally infected in the
greenhouses of the University of California by Dr. J. H. FREITAG demonstrates the prevalence of coacervates in the perivascular cells of the stems.
A longitudinal section after proper fixation in the HELLY 's killing fluid and
staining with haematoxylin, showed distorted nuclei (some with amyloplasts
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422
PLANT PHYSIOLOGY
closely appressed) and one or several coacervates sharply outlined. In contiguous cells the phenolic material was often flocculated in the vacuole, and
many times could be distinguished as a mass of small spherical drops. In
other cases the vacuolar space was almost filled with a diffuse material giving
the chromaffinic reaction.
CYTOCHEMICAL REACTION
Coacervates in the vacuolar solution appear as features in a three phase
system. The inner phase may be defined as a "gelatinous hydrate" of
phenolic compounds and the outer phase as the depleted vacuolar solution.
The intermediate phase is made of amphoteric phospholipids. The identification of coacervates therefore calls for the cytochemical localization of the
phenolic compounds and of the phospholipids.
PHENOLIC COMPOUNDS
The cytochemical reactions may range from those indicative of some
active C-OH group or dienol HO-C-C-OH or paraphenol, to those of
highest specificity for some definite configuration of the ring structure or
active groups.
The reactions resulting in the formation of bright azo-dyes have been
shown by LISON to grade from the general localization of phenolic compounds to the specification of definite compounds, according to the cytochemical technique applied. The diazo-reaction, which SWAMINATHAN (14)
used for the quantitative estimation of vitamin B6 may also be used for the
detection of pyridoxin in coacervates, or in sections of fresh plant material.
A convergent line of evidence as to the presence of pyridoxin in coacervates
may be obtained by simply immersing freehand sections of tissues in a
suspension of 2.6 dichloroquinone chloroimide buffered at pH 8.8 with
sodium borate (6).
DEPLETED VACUOLAR SOLUTIONS
The vacuolar solution in which coacervates are floating is evidently
depleted in dispersed materials which would respond to vital staining, and
concomitantly depleted in P04 ions, since the phenolic compounds apt to
absorb the vital dyes have become coacervated in the inner phase of the
coacervate, and the ionic P04 has entered into combination with lipids to
form the third or intermediate phase of the auto-complex coacervate.
Sections of tissues at the site of non-compensated respiration may show
cells where coacervates are sharply outlined from the otherwise empty
vacuole, in contrast to the neighboring cells, where phenols have not been
coacervated as yet, and whose vacuolar solution stains as a whole with vital
dyes, or responds to the chromaffin reaction.
THIRD (INTERMEDIATE) PHASE
This phase may be identified as to either of its constituents: The phosphorus being responsible for the formation of molybdenum blue with
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DUFRENOY AND REED: COACERVATES
423
molybdenum reagent, and the fatty materials staining solid black a f ew
minutes after the section has been immersed in the hydrated methylal solution of Sudan Black (7). The intermediate phase represents a solid physical structure, which persists as an empty shell after the phenolic contents
have been dissolved out by proper solvents such as polyethylene glycol or
by methyl ethers. In healthy cells the P04 ions from the vacuoiar solution
are used almost as fast as they become available for the building up of
nucleic acids and other energy-rich phosphorus compounds through esterification. In hypoplastic cells, coacervation ties up phosphorus in the solid
auto-complex structure, thus making it unavailable for phosphorylation of
carbohydrates in cell metabolism, which may account for the dwarfed shoots
characteristic of rosette of fruit trees or the "Court Noue'" of vines.
It remains for future investigations to show how the activity of the
enzymic systems may be influenced by the alterations in equilibria due to
the formation of auto-complex coacervates in cell vacuoles.
Summary
1. The study of coacervation in hypoplastic cells demonstrates some important correlations with the respiratory activities in the vacuoles.
2. Auto-complex coacervates appear as refringent spherical bodies in the
vacuoles of hypoplastic cells affected by virus, or parasitized by certain
fungi, or deficiency of some essential microelement. They have also been
induced in tissues treated with compounds tending to block some component
of the cell respiratory system.
3. The data presented show that these refringent spherical bodies result
from the coacervation of phenolic compounds from the vacuolar solution
concomitant with the adsorption of phosphatides at the interphase between
the coacervated compounds and the water phase of the vacuole. Both
physico-chemical phenomena are related to a shift of the respiratory systems toward a higher rH.
4. Coacervation is one of the several ways whereby a phase originally
homogeneous may become differentiated into several. Coacervation of phenolic materials within the vacuole does not imply any change of cytoplasmic
permeability (although the same disorders in cell respiration which induce
coacervation may enhance permeability).
A portion of the work herein reported was accomplished while the senior
author was an exchange professor at the University of Louisiana.
UNIVERSITY OF CALIFORNIA
BERKELEY 4, CALIFORNIA
LITERATURE CITED
1. ABBOTT, E. V., and INGRAM, J. W. Transmission of chlorotic streak of
sugar cane by the leaf-hopper Draeculacephala portola. Phytopathology 32: 99. 1942.
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424
PLANT PHYSIOLOGY
2. BERTHELOT, M., et SAINT-GILEs, L. P. DE Recherches sur les affinites.
De la formation et de la decomposition des ethers. Compt. Rend.
Acad. Sci. 53: 474-478. 1861.
3. BERTRAND, G. Sur 1'extraction du boletol. Compt. Rend. Acad. Sci.
134: 124-126. 1902.
4. COMBES, R. Du role de l'oxygene, dans la formation et la destruction
des pigments rouges anthocyaniques chez les vegetaux. Compt.
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5. DUCOMET, V. Recherches sur la brunisure des vegetaux. Ann. Ecole
Nat. Agric. Mont. 11: 170-282. 1900.
6. DUFRENOY, J. The occurrence of pyridoxin in sugar cane tissues.
Proc. Louisiana Acad. Sci. 7: 15-16. 1943.
, and REED, H. S. A technic for staining cells with Sudan
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Ostwald's Klassiker. 104, Leipzig. 1899.
9. HUMPHREY, H. B., and DUFRENOY, J. Host-parasite relationship between the oat plant (Avena spp.) and crown rust (Puccin'ia coronata). Phytopath. 34: 21-40. 1944.
10. MAST, S. 0. The relation between kind of food, growth, and structure
in Amoeba. Biol. Bull. 77: 391-398. 1939.
11. PASTEUR, Louis. iAtudes sur la biere. Gauthier-Villars. Paris. 1876.
12. REED, H. S. Cytology of leaves affected with little-leaf. Amer. Jour.
Bot. 25: 174-186. 1938.
, and DUFRENOY, J. Catechol aggregates in the vacuoles
13.
of zinc-deficient plants. Amer. Jour. Bot. 29: 544-551. 1942.
14. SWAMINATHAN, M. Chemical estimation of vitamin B, in foods by
means of the diazo reaction and the phenol reagent. Nature 145:
780. 1940.
15. VIALA, P., et SAUVAGEAU, C. La brunisure et la maladie de Californie.
Ann. Ecole Nat. Agric. Mont. 7: 87-108. 1892.
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