Peroxidase as a tool in the study of the flowering process

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Reference

Peroxidase as a tool in the study of the flowering process

GREPPIN, Hubert

GREPPIN, Hubert. Peroxidase as a tool in the study of the flowering process. In: Hubert Greppin, Claude Penel & Thomas Gaspar. Molecular and physiological aspects of plant peroxidases . Genève : Université de Genève, Centre de botanique, 1986. p. 333-339

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H.GREPPIN

Plant Physiology Laboratory, 3, place de l'Universite, Universite de Geneve, CH 1211 GENEVE 4

Introduction

The flowering process is a very important part of plant development (6, 7, 27). But at present no compounds or biochemical reactions are known that occur exclusively in plants induced to flower. However, with the numerous publications in this field, much is known about the various steps in a descriptive sense, but unfortunately, we are neither able to express it in terms of molecular physiology to uncover the underlying mechanism, nor have we found relevant markers in terms of biochemistry. Only indirect means are available to measure the propensity to flower.

The mechanism

Since the works made in the past by Sachs and Klebs, we not clearly know how plant flower. Many hypotheses have been made to explain flowering; the most accepted is the concept of hormonal floral stimulus. Despite some limited success with extraction, this stimulus remains a physiological concept rather than a chemical reality. Recently Tran Thanh Van et al. (26) have made conspicuous the role of some oligosaccharins (cell wall compounds) in the induction of flowers in tissue culture.

In a plant, what are the genes and proteins specifically implicated in flowering induction? What is the number of

334 H GREPPIN

obligatory, sequential and/or concomitant biochemical events possibly imposed in the course of ontogenesis? What is the part of positive inductions or alternatively of sequential release from inhibitions? Further progress in methodology must be made to probably bring us closer to fundamental problems in flowering.

The overall flowering process involves many steps, usually starting with perception of environmental factors (leaves, roots), as light and temperature that provoke the induction of leaves, the production of floral circulating stimulus, and terminating with the differentiation of the apex to produce floral morphogenesis.

Since between reactions effect.

Strategy

a certain degree many cells is cannot be studied

In our laboratory (13), flowering and especially remains vegetative in short during 6 to 8 weeks. We transfer of 4 weeks old light.

of organization and communication required for flowering, individual in one cell. We have a network

since many years, we are working on on spinach, a long day plant that days (8 hrs light, 16 hrs night) induce the start of flowering by a plants from short day to continuous Our goal was to detect the first events of the floral induction in shoot apex, where flowers are made, and to have markers, sooner as possible, of the initiation and acquisition of floral state by the apex, and accordingly by leaves that have produced the signals for flowering, a few hours before; evocation is the period of cellular differentiation in apex that consecutively permits floral embryogenesis.

The markers must be always present when we induce flowering by different ways: light, temperature, chemical products; they must be absent when flowering is inhibited and in vegetative state. We can propose for spinach such type of markers.

The correlation between markers and floral induction will not necessarily mean they are the dynamic factors that initiate flowering. But if we always observe upstream in the induction process, some specific markers, we could infer, with a great probability that, at least, certain could play a key-role in flowering or are closely associated to the ground of this mechanism. A new type of experiments can be looked on this base.

Results Apex

Shoot meristems are the target of leaves signals (differentiation, mitosis). As indicated by Bernier evocation does not seem to be a single sequence of events, but a number of

interacting sequences. Each one may be triggered by one or several components of floral signals: ions as secondary messengers, autocatalytic change in symplastic membranes, carbohydrates flux, oligosaccharins, hormones, etc ... Evocation should be regarded as the essential part of the process of flowering initiation because it is probably one part which is common to all plants(7).

The first changes appearing in apex of spinach are in ultrastructure and in nucleic acid metabolism (3,4). We observe structural alterations and membranes proliferation, biosynthesis of nucleic acid.

The pentosephosphate pathway is important for this type of modifications. Although its products may return to the glycolysis pathway, intermediary metabolites are of consequence for other metabolic processes (ribose and deoxyribose phosphate, erythrose for shikimic acid and aromatic compounds, NADPH for lipids synthesis).

The rise of Glucose-6 phosphate dehydrogenase activity and carbohydrates level in axial zone of apex can be employed as a quick marker of evocation in spinach. We have tested G6PD in different conditions of induction or inhibition of flowering to propose it as specific marker (1, 2, 8, 9) 3-4 hours after the onset of induction in leaves.

Leaf

Peroxidases have been implicated in and associated directly or indirectly with various physiological processes (12); but there are many problems in trying to assign a particular role for isoperoxidases in these events (10, 11).

Total peroxidasic activity is depending on numerous factors, however the period of onset of flowering corresponds in spinach to the time where the lowest le�el of the peroxidase activity is observed (16). At this moment, we can see a quick change of the ratio between anodic and cathodic peroxidases activity, a ratio which increases. This is the consequence of a fast decrease of cathodic peroxidases activity and a progressive increase of anodic peroxidases, at the moment of the critical photoperiod (11-12 hrs light). Moreover, we have a rapid change in compartmentation, inside and outside the cell of the different peroxidases activities. All these effects are integrated in all the leaves with a few delay (11, 19, 20, 23)

The regulation of peroxidase by phytochrome was described by several authors like Mohr and Schopfer. In spinach, we have extracted at high pH, a peroxidase with a basic isoelectric point that was shown to rapidly react after short irradiations of leaves with red or far-red light.

This enzymatic activity underwent a fluctuation after an irradiation of 1 or 2 minutes and the shape of the fluctuation

336 H GREPPIN

was dependent on the light quality. When we compare vegetative to induced spinach leaves, we observe an inversion of the type of response to light quality. This is probably bound to modification of the interaction between phytochrome, membranes and basic peroxidases (17-25).

irradiated leaf

PLANT non - irradiated leaf

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Fig. 1. Tentative model to explain the interaction between phytochrome, plasma membrane (ions pumps) and peroxidases : direct modulation by red/far red light, indirect modulation in non-irradiated leaves after the transfer of electro-chemical signals by the symplast (after Penel C, F Karege, R Greppin 1980 Correlations rapides au niveau des feuilles chez l'epinard. XIeme Rencontre de Meribel,281-283).

Several physical or chemical factors which promote or delay floral induction can be studied by using the photomodulation of basic peroxidase activity as an indicator. This can be employed as a marker of floral induction in leaves of spinach (18, 19).

An interesting fact concerning this effect was that the photoconversion of phytochrome in some leaves of the plant immediately modified the peroxidase activity in other leaves(20, 23). This implied the fast transmission of some signals from leaves to leaves which was triggered by phytochrome photoconversion. This transmission was inhibited by several substances including lithium chloride (inhibitor of flowering), EGTA and the Ca-ionophore A23187.

The model we propose is the following: the signal generated by phytochrome and membranes would result from changes in K distribution; K changes are correlated with modification of cellular Ca distribution which itself controls peroxidase activity distribution and/or synthesis within the cells (Fig. I).

During flowering the properties of membranes are modified (5, 14, 1 5).

We have seen that many properties of leaves peroxidases are changed during the induction of flowering. The fast variation in the ratio between acid to basic peroxidases activities or the compartmentation inside and outside the cells, or the immediate photomodulation of basic peroxidases activity can be employed as quick indicators of the start of floral induction. New proteins characterize the floral state (Fig.2). All this information must be checked by the G6PD test in apex during the following hours after the start of induction in the leaf.

Spinacia oleracea, cv Nobel (4weeks S.D.)

S.D.

8 16 24 hrs: local time

PETIOLE

• EVOCATION • fo:u,�fer toQ_

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(ll-12hrs)C.P.: Induction _{, time} Floral state(specific proteins) -- senescence

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Fig. 2. Schematic view of events occurring in shoot apex of spinach, after transfer from 4 weeks short days (vegetative state) to continuous light (induction of flowering : evocation, then floral organogenesis). G6PDH, rise of glucose-6- phosphate dehydrogenase activity, pentose phosphate pathwayindicator. DNA biosynthesis in plastids and mitochondria, then division; DNA biosynthesis in nuclei (48 hrs), (see, 1 to 4). Leaf, petiole list of some markers to detect flowering induction. C.P.: critical photoperiod (onset of induction).

338 H GREPPIN

Flowering initiation is a an important role likewise plant.

process where fast regulation plays the membranes network of all the

References

l AUDERSET G, PB GAHAN, AL DAWSON, H GREPPIN 1980 Glucose-6- phosphate dehydrogenase as an early marker of floral induction in shoot apices of Spinacia oleracea, c. v.

Nobel. Plant Sci Lett 20: 109-113

2 AUDERSET G, PB GAHAN, GOC ONYIA, H GREPPIN 1985 Increased pentose phosphate pathway activity linked to floral induction in apices of Spinacia oleracea during short days. Ann Bot 55: 61-64

3 AUDERSET G, H GREPPIN 1976 Etude de l'apex caulinaire d'epinard avant et apres l'induction florale. Saussurea 7: 73-103

4 AUDERSET G, H GREPPIN 1977 Effet de l'induction florale sur l'evolution ultrastructurale de l'apex caulinaire de Spinacia oleracea, c.v. Nobel. Protoplasma 91: 281-301 5 AUDERSET G, AS SANDELIUS, C PENEL, A BRIGHTMAN, H GREPPIN,

DJ MORRE 1986 Isolation of highly purified fractions of plasma membrane and tonoplast from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction. Physiol Plant (in press)

6 BERNIER G, JM KINET, RM SACHS 1981 The physiology of flowering. CRC Press, Boca Raton, Florida

7 CHAMPAGNAT P, R JACQUES 1979 La physiologie de la floraison.

Coll. Intern CNRS n° 285, CNRS, Paris

8 ELTOUKHI M, G AUDERSET, H GREPPIN 1985 Glucose-6-phosphate dehydrogenase activity in the shoot apices of Spinacia oleracea after the floral evocation in continuous light.

Saussurea 16: 1-5

9 GAHAN PB, G AUDERSET, H GREPPIN 1979 Pentose phosphate pathway activity during floral induction in Spinacia oleracea, c. v. Nobel. Ann Bot 44: 121-124

10 GASPAR Th ,C PENEL, FJ CASTILLO, H GREPPIN 1985 A two-step control of basic and acidic peroxidases and its

significance for growth and development. Physiol Plant 64: 418-423

11 GASPAR Th, C PENEL, H GREPPIN 1975 Peroxidases and isoperoxi­

dases in relation to root and flower formation. Plant Biochem J 2: 33-47

12 GASPAR Th, C PENEL, T THORPE, H GREPPIN 1982 Peroxidases 1970-1980. A survey of their biochemical and physiolo­

gical r6les in higher plants. Universite de Geneve, Centre de Botanique, Geneve

13 GREPPIN H 1975 La floraison: ebauche d'une nouvelle strategie. Saussurea 6: 245-252

14 GREPPIN H, G AUDERSET, M BONZON, C PENEL 1978 Changement d'etat membranaire et mecanisme de la floraison.

Saussurea 9: 83-101

15 GREPPIN H, R GAGLIARDI 1980 Quelques aspects de la

regulation chez les plantes superieures. Saussurea 11:

43-48

16 KAREGE F, C PENEL, H GREPPIN 1977 Evolution de l'activite peroxydasique dans les feuilles de l'epinard lors de l'ontogenese en photoperiode courte ou continue.

Saussurea 8: 75-83

17 KAREGE F, C PENEL, H GREPPIN 1979 Reaction of a peroxidase activity to red and far red light in relation to the floral induction of spinach. Plant Sci Lett 17: 37-42 18 KAREGE F, C PENEL, H GREPPIN 1982 Floral induction in

spinach leaves by light, temperature and gibberellic acid: use of the photocontrol of basic peroxidase activity as biochemical marker. Z Pflanzenphysiol 107:

357-365

19 KAREGE F, C PENEL, H GREPPIN 1982 Detection de l'etat vegetatif et floral de la feuille de l'epinard: emploi d'un indicateur biochimique. Arch Sci Geneve 35: 331-340 20 KAREGE F, C PENEL, H GREPPIN 1982 Rapid correlation between

the leaves of spinach and the photocontrol of a peroxidase activity. Plant Physiol 69: 437-441 21 PENEL C, H GREPPIN 1973 Action des lumieres rouge et

infrarouge sur l'activite peroxydasique des feuilles d'epinard avant et apres l'induction florale. Ber Schweiz Bot Ges 83: 253-261

22 PENEL C, H GREPPIN 1974 Variation de la photostimulation de l'activite des peroxydases basiques chez l'epinard.

Plant Sci Lett 3: 75-80

23 PENEL c, H GREPPIN 1975 Photocontrole immediat de l'activite peroxydasique et correlation entre les feuilles

d'epinard. Saussurea 6: 287-291

24 PENEL C, H GREPPIN 1975 The balance between acid and basic peroxidases and its photoperiodic control in spinach leaves. Plant Sci Lett 5: 41-48

25 PENEL C, H GREPPIN 1979 Effects of calcium on subcellular distribution of peroxidases. Phytochemistry 18: 29-33 26 TRAN TANH VAN K, P TOUBART, A COUSSON, AG DARVILL,

DJ GOLLIN, P CHELF, P ALBERSHEIM 1985 Manipulation of the morphogenetic pathways of tobacco explants by

oligosaccharins. Nature 314: 615

27 VINCE-PRUE D, B THOMAS, KE COCKSHULL 1984 Light and the flowering process. Academic Press, London. Coll Intern CNRS n° 285