Université Montpellier 2, France

Abstract

An important part of rubber research is and always will concern ways of opening up the creation and selection of new Hevea clones adapted to produce high yield with a low tapping frequency, adapted to marginal ecoclimatic conditions, selected for their resistance to foliar pathogens and insects but also selected for the quality of the latex, for the compatibility with technical processes and for some technological properties which are important to the final rubber product. Perhaps the ratio of sol and gel rubber, the length of the rubber chains and so on will be taken into account?

Besides these relatively classical, but essential, aspects of the research, it has become more and more necessary to develop other technical approaches to improve the productivity of the rubber-culture in order to maintain and develop its competitivity against synthetic rubber.

This paper reviews the state of basic rubber tree research and outlines areas for future investigation.

Introduction

More than two thousands plant species synthetize the 1-4 cis-polyisoprene molecule, in common with the natural rubber tree. Rubber belongs to the very important family of isoprenoids constituted of more than 200,000 different molecules which are present in the animal and plant kingdoms.

Everybody knows of the importance of cholesterol for mammals, and especially for humans, which is an isoprenoid compound. Consequently, much research has been carried out over many years on the isoprenoid family. These researches are of great interest in the knowledge of rubber biosynthesis and could be even more useful in the near future.

Some isoprenoids are equally needed for components of animal and plant cells membranes as is the case for cholesterol, phytosterol, polyprenols, dolichols, and also electron carriers such as ubiquinones and plastoquinones. The isoprenic chains of the haeminic nucleus of hemoglobin and chlorophyl are equally important and absolutely indispensable for life. Phytohormones, such as cytokinin, gibberellins and abscissic acid, control growth and development whilst numerous isoprenic phytoalexins protect plants against pathogen attacks (fungi, microbial).

Compared to the production of synthetic rubbers from petroleum, Hevea, the rubber-tree, has been called "The green rubber factory". On the one hand, Hevea uses the sun as a renewable energy source together with atmospheric CO₂, water and mineral elements of relatively poor soils for the building of its constituents, among them cis-polyisoprene.

On the other hand, the by-products of Hevea are to a high degree "biodegradable" and rubber itself is "recyclable". The rubber tree may be used to reconstitute new forests on poor or degradated soils. Rubber wood is increasingly used as timber. Hevea must be considered as a model for the "pure ecologists".

From the first quarter of this century, latex appears as a very original and interesting biological medium for numerous scientists working in different laboratories, particularly in the Rubber Research Institutes of the Far East, Europe, and America.

The principal characteristic of Hevea latex is that it contains up to 50% cis-polyisoprene (C₅H₈)n. Furthermore, monomerous isoprene can be produced by many plants and animals. Nobody knows the reasons for latex production in a lot of plant species, particularly in the rubber tree. According to some scientists1, the latex secretion is a defence against wounding and/or predators such as insects and microorganisms. According to another, rubber and other hydrocarbon compounds are carbon traps to maintain the balance between biomass and atmospheric CO₂. Another reason could be that isoprene emissions by numerous tropical plant species constitute an antidote against the toxicity of atmospheric ozone2.

From the rubber trees of the Amazonian basin, exploited by the seringueros to the rubber estates of the Far East, much knowledge has been accumulated by planters which has allowed increases in rubber yield from some hundred to more than two thousands kg/ha/year. This improvment has been obtained by agricultural techniques, by the selection of high yielding seedlings which, by grafting, have been vegetatively multiplied to obtain high yielding Hevea clones, by constant evolution of tapping systems and by the introduction of hormonal stimulation of latex yield.

How is the Hevea rubber factory constituted ?

All parts of the rubber tree produce latex, but only the bark of the trunk is exploited. A 3-D scheme shows the productive latex bark between the more external bark and the cambium. This cambium is of particular importance as a lateral meristem; it generates wood and xylem vessels to the inside and the soft bark containing the phloem and the latex vessels to the outside. Laticiferous vessels, organized in concentric layers around the cambium, are called latex mantles. In each mantle the different tubes are connected by anastomoses which constitute an advantage in the collection of latex.

Latex vessels are the factory where the rubber is synthetized. As in all factories, it must be supplied with raw material in different ways. Water and mineral elements absorbed by the roots go up into the trunk using xylem vessels located in the wood near the cambium. Sugar, amino acids, hormones and more generally elaborate molecules come from the source organs, ie. the leaves, and are carried by the sieve tubes located in the bark very near to the cambium. Cambium and sieve tubes are so fragile that they must not be wounded by the tapper's knife. Nevertheless, the latex vessels are not in direct contact with these two sources of nutriments and an horizontal circulatory system, constituted by the rays, links the wood and the fibre to the latex tubes.

Laticiferous vessels of tapped trees present an intensive metabolic activity. In situ they contain the usual organelles of plant cells: nucleus, mitochondria, plastes, vacuoles, ribosomes, golgi apparatus and endoplasmic reticulum.

The rubber particles are the more typical latex component. From 60nm to 6m in diameter and up to 50% of the latex by weight, these particles are wrapped in a monomembrane made of negatively-charged glycolipo-phosphoproteins. The negative charges allow stability of the latex colloidal suspension. A recent hypothesis by Na-Ranong et al³ implies that this membranFie protects the cis-polyisoprene molecules against oxidative degradation, responsible, after latex maturation, for decreases in some technological properties such as Plasticity Retention Index (PRI).

Among the proteins located in the membrane rubber particles, two enzymes implicated in the elongation of the cis-polyisoprene chain have been found: the cis-prenyl transferase (76Kd) and the Rubber Elongation Factor (REF, 14.6Kd), and also a glycoprotein (22Kd) which is implicated in latex coagulation by its glucidic moiety⁴.

The latex vacuoles, called lutoids, are monomembrane particles acting as polydispersed lysosomal vacuomes. Their membrane is negatively charged on its external face, the inside is acidic (pH 5) and positively charged (Mg++, Ca++, proteins (+ and -)).

Intralutoidic proteins and enzymes play an important role in latex physiology. Among the enzymes, all ranges of acidic hydrolases typical of animal and plant lysosomes, are present. These enzymes play a role in the recycling of worn molecules and by virtue of their lutoid contents like animal lysosomes, are the suicide bags of the latex cells, and their bursting leads to clamping of the cytoplasmic metabolism and to latex coagulation.

Some defence proteins (Pathogenesis Related Proteins) against pathogenic agents such as chitinase, b 1-3 glucanase, lysozymes, are accumulated in this compartment. Hevein is a very important protein for latex coagulation. Other proteins, which may be considered as a form of storage for reduced nitrogen (Vegetative Storage Protein), or stress proteins have also been found⁵.

During recent years, attention has turned to allergic phenomemons caused by some latex goods such as surgical gloves. Latex proteins are responsible for this allergy. Recent works have shown that some of the lutoid proteins are much more allergenic than cytosolic proteins^6,7,8.

Other kinds of particle were discovered in Bogor by Frey-Wyssling in the 1930s which possess a double membrane and show a complex internal structure which assimilate them to chromoplasts. They are characterized by a more or less important carotenoids content, which gives a slight yellow colour to the latex of some clones.

After the important discoveries on latex organelles at Bogor and after researches using optical and electron microscopy in England and Malaysia, the cytoplasmic nature of Hevea latex was clearly proved.

The laticiferous cells possess the genetic information (DNA) and the machinery needed for protein biosynthesis (RNA, ribosomes) which is indispensable to allow the restoration of the latex lost by the previous tapping.

Some years after the Second World War, biochemists, using new tools such as radioelements, discovered the biochemical pathway of isoprenoidbiosynthesis, simultaneously, in the animal and plant kingdom. Among numerous scientists, the Nobel Prizewinner Prof. F. Lynen using fresh latex coming from Brazil played a major role in advancing knowledge of the mevalonate (MVA) isoprenoid biosynthesis pathway.

Very recently, a new pathway, the ROHMER'S pathway⁹, producing the first isoprenic compound, isopentenyl pyrophosphate (IPP), has been discovered in some plants and particularly in organelles such as the chloroplasts. This could be implicated in the synthesis of some isoprenoids from Frey-Wyssling particles such as carotenoids, tocopherols, tocotrienols and plastoquinones.

The rubber biosynthesis pathway in the rubber tree

During the 1960s in Vietnam and Cambodia, it appeared that the latex sucrose content and its catabolism could be among the first limiting factors of the latex yield. More than 20 enzymatic reactions are involved in the transformation of sucrose into cis-polyisoprene. Numerous researchers have worked on these enzymes and their regulation. To quote some, Jacob et al¹⁰ for glycolysis and numerous major enzymes and Lynen¹¹ and Kekwick12 for rubber biosynthesis. Credits are also given to Tupy^13,14 since he had shown that the first reaction of this pathway, ie. transformation of sucrose into glucose and fructose, was a limiting factor in rubber biosynthesis. Such a phenomenon is typical in biochemistry in the beginning of a pathway. Nevertheless, other numerous enzymes of the latex are regulated.

Preparation of carbon skeleton of rubber molecules:

1. From sucrose to acetyl-COA

After the demonstration that latex sucrose content can often be positively correlated with latex yield (often but not always), a systematic study of the saccharose catabolism has been carried out in Vietnam and Cambodia and then in Paris, France and Côte d'Ivoire. Jacob et al¹⁰ and Tupy¹⁴ have practically studied all the enzymes linked to glycolysis and it is well demonstrated today that the invertase, the first enzyme able to use sucrose, is subject to strong regulation by variations of a few tenths of pH units. Downstream of the invertase step, other important enzymes are also tightly linked to small pH variations. This is the case for PEP-Case which drives PEP to organic acids of the Krebs cycle and plays a role in the regulation of latex pH by Davis pH-stat.

It must be underlined that the last step of this pathway, from pyruvate to acetylCoA, remains practically unknown even today.

2. From acetyl CoA to the first isoprenic molecule

According to the mevalonic acid (MVA) biosynthetic pathway of isoprenoids, acetyl-Coenzyme A, (acetyl COA) issued from glycolysis is transformed in isopentenyl pyrophosphate (IPP) and its dimethyl allyl pyrophosphate (DMAPP) isomer. The latter constitutes the foundation stone on which IPP molecules are added continously to produce C_lO, C₁₅, C₂₀, ...Cx isoprenic molecules.

Seven enzymes are implicated between acetyl-CoA and IPP-DMAPP. Acetyl-COA is generally considered as the first molecule of the MVA pathway. However, its formation in Hevea latex has been the object only of a few studies and it was observed that the rate of rubber biosynthesis in vitro is much more important with labelled acetate than with equivalent quantities of acetyl-COA and much more than with labelled pyruvate.

The conversion of acetyl-CoA to b-hydroxyl-b-methyl glutaryl Co-A (HMG-COA) is well documented for yeast and mammalian tissues¹⁵. Two enzymes are concerned: an acetyl-COA acetyl transferase (AACT) and a HMGsynthase able to acylate the acetoacetyl-COA. Little is known about HMG formation in plants, but a few authors, lastly Van der Heijden et al¹⁶, claimed that in some plants, at least one single protein catalyses HMG-CoA formation. Until today, nobody seems to have hypothesysed that HMG synthesis could limit isoprenoid biosynthesis. Presently, much research is focussed on the next step of the pathway: the reduction of HMG to MVA.

HMG-CoA reductase (HMGR): a limiting factor of isoprenoid biosynthesis ?

HMGR catalyses the synthesis of MVA which is considered as a major rate limiting factor of cholesterol biosynthesis in humans. Consequently, this enzyme has been extensively studied^17,18. Only one gene is encoded for HMGR in mammals: a 97 kDa transmembrane glycoprotein of the endoplasmic reticulum. This enzymatic activity is subject to numerous regulations at transcription, translation and post-translation levels.

In the plant kingdom, the greater diversity of isoprenoids is likely linked to the functioning of several HMGR genes. Only one gene has been found for the fungus Gibberella fujikuroi, two genes for Sacchammyces cerevisiae and Arabidopsis thaliana, three genes for tobacco and maybe four genes for potatoes^19,20. Three genes: hmg1, hmg2 and hmg3, have been discovered for the rubber tree²¹ and three m-RNAs encoding for HMGR are present in latex. The hmg1 gene is up to 100 times more expressed in latex than in leaves²²; c-DNA for hmg2 is far less expressed and the hmg3 gene appears equally present in leaves and latex. The hmg1 gene is considered responsible for rubber biosynthesis and ethylene induces its expression²¹. hmg3 will be less specialized and implicated in "house keeping". hmg2 could be linked to the defence reactions against wounding and pathogens.

3. From IPP to cis-polyisoprene

This important enzyme is also regulated by calmodulin, a calcium binding protein present in large quantities in latex cytosol as shown independantly in Thailand and in Côte d'Ivoire^23,24. Today, it appears that HMGR is submitted to regulation by calmodulin and very likely by a protein kinase cascade of reactions as recently suggested for higher plants²⁵.

A few experiments have been carried out in order to modify gene expression and HMGR activities for different plants such as tobacco^26,27.The transformed plants appear significantly richer in some phytosterols.

Since Lynen¹¹, it is admitted that HMGR activity of latex is particulary low. This lead Arokiaij et al²⁸ to insert the gene specific for HMGR-1 in Hevea anther-derived calli via particle bombardment. The HMGR-1 activities of transformed anther-derived calli ranged from 70 to 410% of the value of the wild type control and the activity of the transformed embryoids obtained ranged up to 250 - 300% but the enzymatic activities and the yield of future mature trees remains to be known.

The final steps of rubber biosynthesis have been clarified during recent years. Hence, the Rubber Transferase initially discovered by Archer and Cokbain²⁹ appears, on the one hand³⁰, as a cytosolic soluble trans-prenyl transferase allowing the transformation of C₅ monomerous into C₂₀ trans-geranyl-geranyl pyrophosphate (GGPP) and, on the other