Phytochemistry 70 (2009) 730–739

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Phytochemistry

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The leaf, inner bark and latex cyanide potential of Hevea brasiliensis:Evidence for involvement of cyanogenic glucosides in rubber yield

Panida Kongsawadworakul a,1, Unchera Viboonjun a,1, Phayao Romruensukharom b, Pisamai Chantuma b,Somjintana Ruderman b,c, Hervé Chrestin a,d,*

a Department of Plant Science, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailandb Chachoengsao Rubber Research Center, Sanam Chai Khet, Chachoengsao 24160, Thailandc Center for Agricultural Biotechnology (CAB), Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom 73140, Thailandd Institut de Recherche pour le Développement (IRD), UR060 CLIFA, CEFE-CNRS, 1919 route de Mende, F-34293 Montpellier, Cedex 5, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 June 2008Received in revised form 12 March 2009Available online 4 May 2009

Keywords:Hevea brasiliensisEuphorbiaceaeRubber treeLatexLeaf and bark cyanide potentialCyanide metabolismLinamarinRubber yield

0031-9422/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.phytochem.2009.03.020

* Corresponding author. Address: Department of PlaMahidol University, Rama VI Rd., Bangkok 10400, Thafax: +66 23 54 71 72.

E-mail address: chrestin@ird.fr (H. Chrestin).1 The first two authors equally contributed to the w

The latex of Hevea brasiliensis, expelled upon bark tapping, is the cytoplasm of anastomosed latex cells inthe inner bark of the rubber tree. Latex regeneration between two tappings is one of the major limitingfactors of rubber yield. Hevea species contain high amounts of cyanogenic glucosides from which cyanideis released when the plant is damaged providing an efficient defense mechanism against herbivores. In H.brasiliensis, the cyanogenic glucosides mainly consist of the monoglucoside linamarin (synthesized in theleaves), and its diglucoside transport-form, linustatin. Variations in leaf cyanide potential (CNp) werestudied using various parameters. Results showed that the younger the leaf, the higher the CNp. LeafCNp greatly decreased when leaves were directly exposed to sunlight. These results allowed us to deter-mine the best leaf sampling conditions for the comparison of leaf CNp. Under these conditions, leaf CNpwas found to vary from less than 25 mM to more than 60 mM. The rubber clones containing the highestleaf CNp were those with the highest yield potential. In mature virgin trees, the CNp of the trunk innerbark was shown to be proportional to leaf CNp and to decrease on tapping. However, the latex itselfexhibited very low (if any) CNp, while harboring all the enzymes (b-D-diglucosidase, linamarase and b-cyanoalanine synthase) necessary to metabolize cyanogenic glucosides to generate non-cyanogenic com-pounds, such as asparagine. This suggests that in the rubber tree bark, cyanogenic glucosides may be asource of buffering nitrogen and glucose, thereby contributing to latex regeneration/production.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction �20–40 cm laterally above the tapping cut) is called the ‘‘drainage

Hevea brasiliensis (rubber tree), a tree in the Euphorbiaceae fam-ily, is the only commercial source of natural rubber. Nowadays it isplanted as grafted selected clones in several humid tropical coun-tries. Rubber is synthesized in specialized ‘‘latex cells”. Uponmaturation, these cells fuse together to form a network of anasto-mosed latex vessels (the laticifers), described as a para-circulatorysystem in the inner bark of the rubber tree (Hébant and de Faÿ,1980; de Faÿ and Jacob, 1989). Upon bark wounding or tapping,the laticifers are severed and their cytoplasm (the latex) flowsout until coagulation processes are initiated, leading to the plug-ging of their extremity (Southorn, 1968; d’Auzac, 1989;Kongsawadworakul and Chrestin, 2003; Yeang, 2005). The barkarea from where the latex flows out (�40–70 cm below and

ll rights reserved.

nt Science, Faculty of Science,iland. Tel.: +66 22 01 52 93;

ork.

area” (Lustinec et al., 1968; d’Auzac, 1989).Ultracentrifugation of fresh latex results in the separation of

three major phases: supernatant rubber particles (�40% of the la-tex volume), an intermediate aqueous phase corresponding to thelatex cell cytosol, and a pellet (�15% of the latex) consisting of la-tex cell organelles, which are mostly micro-vacuoles (95%), calledlutoids (Moir, 1959; Ribaillier et al., 1971).

The rubber particles consist of clusters of long cis-polyisoprenechains surrounded by a phospho-lipoproteic membrane. The bio-synthesis of cis-polyisoprene occurs via glycolysis, followed bythe mevalonate pathway. Thus glucose is an ultimate precursorof rubber (Jacob et al., 1989; Keckwick, 1989).

After latex flow, the major limiting factor of latex yield is thecapability of laticifers to regenerate their lost cytoplasm betweentwo consecutive tappings (Jacob et al., 1989). Regeneration of thewhole latex cell cytoplasm, including rubber, mainly depends onthe availability and metabolism of sugar (Tupy, 1989) and of nitro-gen compounds (Pujade-Renaud et al., 1994; Tian and Hao, 1999).Several studies have focused on metabolism that affects latex yield,for instance studies on sucrose generated upon photosynthesis

Fig. 2. The most recent flush from a 7-year-old rubber tree. The most recent flushwas collected about 2 months after refoliation. The dark purple leaves correspondto stage B and the green leaves to stage D of leaf development. Yellow arrowsindicate the youngest and oldest mature leaves at stage D from the most recentflush whereas red crosses show the young mature leaves at stage D from the sameflush.

P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739 731

(Tupy, 1989), and glucose derived from starch (Chantuma et al.,2007; Silpi et al., 2007). Only a few studies have been conductedon the metabolism of nitrogen compounds in latex, except con-cerning its amino acid composition (Bzrozowska et al., 1974).

Like cassava (Manihot esculenta), another member of theEuphorbiaceae family, the rubber tree belongs to a large group ofplants that are highly cyanogenic (Lieberei, 2007). When the plantis damaged, they release cyanide, which is an efficient defensemechanism against herbivores (Ballhorn et al., 2005). Both cassava(McMahon et al., 1995; Siritunga and Sayre, 2004; Jørgensen et al.,2005) and rubber tree synthesize linamarin (1) (Fig. 1), a cyano-genic b-monoglucoside, especially in their young leaves. Linamarin(1) can be glycosylated into linustatin (2), the protected transportform of linamarin (1) that can be translocated into the whole plant(Selmar et al., 1988). By means of the possibly specific sequentialaction of b-diglucosidase, the rubber tree accumulates highamounts of linamarin (1) in its parenchyma cell vacuoles. Linamar-ase (b-D-glucosidase) (Selmar et al., 1987a) is apoplastic and is con-sequently separated from its linamarin (1) substrate (Selmar et al.,1988; Gruhnert et al., 1994). During tissue injury, both linamaraseand linamarin (1) are brought into contact, leading to cleavage ofglucose from its aglycone acetone cyanohydrin. The latter canspontaneously release hydrogen cyanide (HCN) and acetone atpH > 5. This reaction is accelerated by hydroxynitrile lyase (Selmaret al., 1989). Most living organisms harbor a cyanide detoxificationpathway using cyanide and cysteine to form asparagine via theactivities of either mitochondrial or cytosolic b-cyanoalanine syn-thase (Miller and Conn, 1980; Selmar et al., 1988).

All rubber tree tissues, including leaf, bark and seed tissues, arestrongly cyanogenic and accumulate both linamarin (1) and lina-marase (Moraes et al., 2002; Lieberei, 2007). The total amount ofcyanogenic precursor and free cyanide accumulated per gram offresh weight are used to calculate the cyanide potential (CNp) ofa given tissue (Loyd and Gray, 1970). The CNp corresponds to thetotal amount of free cyanide that would be locally released if allthe cyanogenic enzymes were brought into contact with theirrespective cyanogenic substrates.

Fig. 1. Hydrolysis of linustatin (2) into linamarin (1) and glucose by sequential b-D-diglucosidase.

Unlike most trees in humid tropical areas, mature rubber trees(�5–6 years old) shed their entire canopies at the beginning of themain dry season, and refoliate within a few weeks. The new leavesof the rubber tree develop in flushes, with one to three flushes ofnine to fifteen leaves a year, depending on the genotype and onenvironmental conditions. After bud burst, the very young leavesgrow exponentially. They become small, thin and pink, accumulateanthocyanin and then turn pale green (see Fig. 2 for an example).2

The leaves continue to harden and turn dark green when fully ma-ture. Maturation requires 10–21 days depending on the genotype.The developing leaves can be classified in four groups designatedA–D, according to morphological criteria (Djikman, 1951) and color(Hallé and Martin, 1968). Lieberei (2007) used physiological datato describe leaf developmental stages, in which stages A–C behaveas sink leaves, whereas stage D corresponds to a fully mature ligni-fied green source leaves capable of performing photosynthesis. TheCNp was reported to vary with the leaf stage with a maximum of37 lmol g�1 fresh weight (i.e. about 46 mM in the leaf tissue water)in the young leaves (stage C) of rubber trees (Lieberei, 2007).

In the seed of the rubber tree, linamarin (1) has been shown toaccumulate in the endosperm (Lieberei et al., 1986) and their CNplevels vary considerably (from 4 to 110 mmol kg�1) depending onage and rubber genotype (Selmar et al., 1991; Mallika et al.,1993). During germination of rubber tree seeds and early develop-ment of the organs, the CNp decreases to less than 15% of its initialvalue (Lieberei et al., 1986). According to Selmar et al. (1988), uponHevea seed germination, linustatin (2) is formed at the expense oflinamarin (1) and exudes from the endosperm. At the same time,the linustatin (2) cleaving b-diglucosidase and the cyanide assimi-lating b-cyanoalanine synthase in the young seedling display theirhighest enzyme activity. The cyanogenic glucosides have thus beensuggested to play a role in the primary metabolism of the develop-ing plant, and as nitrogen storage compounds.

2 For interpretation to color in Figs. 2 and 7, the reader is referred to the webversion of this article.

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a a a ab b c c c

Fig. 3. Variation in leaf CNp with leaf developmental stages and morning samplingtimes (PB260). Leaf cyanide potential (CNp) was measured at the youngest andoldest mature leaves (stage D) of the most recent flush (Fig. 2), as well as from theolder leaves from previous flush, at different times on a sunny morning from thehigh latex yielding PB260 clone fully exposed to sunshine (in April, southern Côted’Ivoire). CNp was expressed as the mean of three independent mixes of leaves. Thebars correspond to standard deviations and the letters correspond to the groupsexhibiting, or not, high significant differences determined by ANOVA (P < 0.05).

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Fig. 4. Variation in leaf CNp with leaf age and exposure to sunlight (RRIT251). Leafcyanide potential (CNp) was measured in leaves exposed or not to sunlight. Youngmature leaves (stage D) from the most recent flush and older leaves from theprevious flush collected from the high latex yield RRIT251 clone at 9:15 am on asunny day (May, eastern Thailand).

732 P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739

Cyanogenic metabolism has been extensively studied in rubbertree leaves for its putative involvement in the susceptibility of He-vea to the leaf fungus Microcyclus ulei, which is a major threat tothe species (Lieberei et al., 1996; Lieberei, 2007). Only a few datahave been published on the CNp of the inner bark of the rubbertree (Moraes et al., 2002) which harbors the laticifers, and noneon the CNp of Hevea latex.

In this paper, we confirm that leaf CNp varies with leaf stageand age. Other variables (diurnal vs. nocturnal, age, sunlight/shade) are reported herein which allowed us to define the bestconditions to sample leaves for comparative CNp studies. Weshowed that the rubber clones with the highest leaf CNp corre-sponded to clones with the highest latex yield potential, and viceversa. Further, the CNp of the inner bark at the bottom part ofthe trunk was proportional to the corresponding leaf CNp in ma-ture virgin trees (ready for exploitation) of different rubber clones.The CNp of the inner bark at the level of the drainage area de-creased in the tapped trees.

We report here for the first time that the fresh latex collectedfrom regularly tapped trees was characterized by a very low (orno) CNp. Yet the latex harbors all the enzymatic activities requiredeventually rapidly metabolizing the cyanogenic compounds andsubsequently freeing cyanide into non-cyanogenic compounds,such as amino acids. Finally, we propose that, in H. brasiliensis,the cyanogenic mono- and di-glucosides may represent sourcesof renewable buffering nitrogen and carbon for the latex regenera-tion process, and thus for rubber production.

2. Results

2.1. Variations in CNp and optimal conditions for leaf sampling

The cyanide potential (CNp) was determined among clones andunder different conditions to identify the best standard conditionsfor sampling leaves for further comparison studies of CNp.

An experiment was performed with a high latex yielding clone(PB260) to compare CNp between the youngest and oldest matureleaves at stage D (Fig. 3) of regularly tapped trees at three differentcollection times during a sunny morning. The results showed thatPB260 exhibited very high CNp (about 50 mM) in the most recentflushes between 06:00 am (dawn) and 08:00 am (early sunlight).There was significantly less CNp in older leaves from the previousflush at the same period of time (Fig. 3). The increase in sunlightbetween 08:00 and 10:00 am significantly reduced CNp in theclone tested. In another high latex yielding clone (RRIT251), be-tween 08:00 and 10:00 am on a sunny day, the CNp of young ma-ture leaves from the most recent flush was approximately two foldhigher in the shade than in direct sunlight (Fig. 4). A similar resultwas observed for old leaves, but with less increase in CNp than thatobserved in young leaves.

Further experiments at different time points from dawn tonightfall using trees of a medium latex yielding clone (RRIM600)(Fig. 5) demonstrated that the CNp of the mature leaf of the mostrecent flush decreased significantly during the period of maximumsunlight (10:45 am to 13:45 pm), but after sunset and during thenight again reached the maximum level recorded at dawn. Butthe leaf CNp of the medium latex yielding clone was about half thatof the high latex yielding clone during the same period (comparedwith Fig. 3). The CNp of the older leaves from the previous flush didnot follow the same kinetics as the younger leaves as can be seen inFig. 5, since their CNp remained high during the period of maxi-mum sunlight (10:45 am to 13:45 pm).

To determine whether developmental stages and ages affectCNp, different parameters were investigated. Variations in leafCNp were determined as a function of leaf developmental stages

and ages: i.e. young leaves at stages B–C (pink turning light green),C (entirely light green), D (hardened and dark green) from recentflushes and older leaves from previous flushes of trees of the highlatex yielding RRIT251 and the low latex yielding PR107 clones,collected early in the morning (Fig. 6). Maximum CNp was found

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Fig. 5. Variation in leaf CNp with leaf age and collection time (day and night) (RRIM600). Leaf cyanide potential (CNp) was measured in leaves of the medium latex yieldingRRIM600 clone, planted along a field border exposed to sunlight (May, eastern Thailand). Young mature leaves (stage D) from the most recent flush and older leaves from theprevious flush were collected at different times of the day and night on a sunny day.

P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739 733

in the youngest immature leaves (stage B–C and C) of both highand low latex yielding clones, but in all developmental stagesand ages tested, CNp was up to two fold higher in the high latexyielding clone than in low latex yielding clones. Young matureleaves (stage D) had lower levels of CNp than younger stages ofthe same flushes. The older leaves of the previous flush had lowerlevels of CNp than any leaves of the most recent flush. Finally, budand leaf CNp of growing scions was analyzed at different develop-mental stages of grafted trees a few weeks old and 2.5- and 10-

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Fig. 6. Variation in leaf CNp with leaf developmental stages and clonal yieldpotential (RRIT251 and PR107). Leaf cyanide potential (CNp) was measured inleaves from the most recent flush at different leaf developmental stages (B–C, C andD) as well as from older leaves from the previous flush. Each analysis wasperformed on mix of leaves collected in the shade from three trees of the high latexyielding RRIT251 and the low latex yielding PR107 clones, early in the morning(May, eastern Thailand).

year-old trees of another high latex yielding clone (PB217). TheCNp of the bursting bud shown in Fig. 7 was �20 mM, increasingto �45 mM in the leaf primordia and apex of 3–5 cm scions, andincreasing to �60 mM in young leaves (stage A–B) of the older(�15 cm) scions. The CNp of young mature leaves from 2.5- and10-year-old trees of the high latex yielding clone (PB217) was inthe same order of magnitude (as high as 50–55 mM).

For subsequent studies, we consequently retained as the sam-pling condition stage D leaves collected early in the morning fromthe most recent flushes and not directly exposed to sunlight.

2.2. Relationships between latex yield potential and the CNp of leavesand inner bark

Since linustatin (2) is synthesized in the leaves and subse-quently translocated to all parts of the tree (Moraes et al., 2002),we measured the CNp of leaves and trunk inner bark in thedrainage area (50 cm beneath the next tapping cut). Using the se-lected sampling condition described above, we measured the CNpof the leaves and the inner bark sampled from mature virgin un-tapped trees of clones with a known latex yield potential. In paral-lel, we measured the CNp of leaves and inner bark from maturetrees of the PB260 clone, which had been regularly tapped for15 months.

Table 1A shows that: (1) in virgin trees, there was a positiverelationship between the CNp of leaves and the inner bark at thebottom part of the trunk and the yield potential of the clones,and (2) an approximately equal percentage (about 40%) of CNp ofthe inner bark and of the leaves was observed in all mature virgintrees. Table 1A and B show that, while there was no significant dif-ference in leaf CNp between regularly tapped (Table 1B) and ma-ture virgin PB260 trees (Table 1A), the CNp of the inner bark washighly and significantly reduced (50%) in tapped trees, suggestingsome consumption of cyanogenic glucosides in the drainage areaof trees that had been regularly tapped for 15 months.

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Fig. 7. Leaf CNp of young grafted trees during growth of the scion as well as of young and mature trees of the same clone (PB217). Cyanide potential (CNp) was measured inthe bursting buds and at different leaf developmental stages (leaf primordia from �3 cm long scions, young immature leaves (stage A) from �15 cm long scions), as well as inmature leaves (stage D) from the most recent flush and in older leaves from the previous flush collected from 2.5- and 10-year-old trees of the same high latex yielding clone(PB217) on the same day (May, north Matto Grosso, Brazil). CNp was expressed as the mean of three independent mixes of plant material.

Table 1CNp of leaves and inner bark according to the yield potential of the rubber clones. (A): CNp was analyzed in young mature leaves (stage D) of the most recent flush and inner barkremoved 50 cm beneath the next tapping cut of mature virgin trees of clones with known latex yield potential. (B): CNp of leaves and inner bark was measured in 15-month-regularly-tapped trees of the PB260 clone. Means and standard deviations are shown. The letters correspond to groups exhibiting, or not, high significant differences determinedby ANOVA (P < 0.05). The yield index is expressed as % of the GT1 reference medium-yielding clone (April, southern Côte d’Ivoire).

Yield potential Clone (virgin) Yield index Leaf CNp (mM) Bark CNp (mM) Percentage bark/leaf CNp

A High PB260 146 48.5 ± 3.2 [a] 21.2 ± 3.2 [A] 43.8PB235 145 45.2 ± 5.9 [ab] 18.8 ± 3.7 [A] 41.7IRCA18 136 42.6 ± 3.6 [b] 17.6 ± 1.1 [A] 41.2PB217 125 46.6 ± 5.4 [ab] 19.6 ± 2.2 [A] 42.1

Medium and low GT1 100 27.2 ± 3.1 [c] 10.4 ± 2.1 [B] 38.2RRIM600 99 25.8 ± 1.9 [c] 11.4 ± 1.9 [B] 44.3PR107 61 24.9 ± 2.3 [c] 10.2 ± 1.0 [B] 40.9

Yield potential Clone (tapped) Yield index Leaf CNp (mM) Bark CNp (mM) Percentage bark/leaf CNp

B High PB260 146 54.3 ± 4.4 [a] 10.2 ± 3.2 18.8

734 P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739

2.3. Cyanogenic glucosides consumption in the drainage area of tappedtrees

Fig. 8 shows the kinetics of changes in the CNp of the leaves andinner bark in the bottom part of the trunk of virgin trees and in oneto three consecutive years of regularly tapped PB260 clone. Therewas a significant increase in the CNp of the inner bark of virgintrees sampled from the trunk from 50 cm above to 50 cm belowthe level of the tapping cut. The results indicated a slight but sig-nificantly higher accumulation of cyanogenic glycosides in thelower part of the trunk. Further, while leaf CNp did not vary signif-icantly over the course of the 3-year experiment, the CNp of the in-ner bark in the lower part of the drainage area (5–50 cm below the

tapping cut) decreased significantly and continuously after one tothree years of tapping. After one year of tapping, there was no sig-nificant change in the CNp of the inner bark 50 cm above the tap-ping cut, but there was a significant decrease after two and threeyears of tapping. Conversely, when compared with virgin treessampled three years earlier, the CNp of the inner bark of the treeswhich had not been tapped for three years, was slightly but signif-icantly higher at all levels of the trunk. In these untapped trees, theCNp at all levels was significantly higher than in trees that hadbeen tapped for one to three consecutive years, especially in thelower part of the trunk. These results suggested probable con-sumption of cyanogenic glucosides in the drainage area of thetapped trees.

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Fig. 8. CNp of leaf and inner bark measured at different levels of the trunk of virgin trees and after 1–3 years regular tapping. Young mature leaves (stage D) from the mostrecent flush and inner bark (50 and 25 cm above, and 5, 25 and 50 cm beneath the tapping cut), were collected as three independent mixes of three homogeneous virgin treesof the PB260 clone, before the first tapping, then after 1, 2 and 3 years of regular tapping. Three other sets of three trees were left untapped and their leaves and inner barkwere collected on the same day 3 years later (August, southern Côte d’Ivoire).

P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739 735

2.4. Possible link between leaf CNp and early estimation of yieldpotential of young progenies

The preliminary data shown in Fig. 9 suggest that there may bea positive relationship between leaf CNp and the early estimatedyield potential of the young (2.5 years old) seedling progenies ob-tained by hand pollination in the framework of a rubber treeimprovement and breeding program.

Fig. 9. Relationship between CNp of mature leaves and the early estimated latexyield potential of �3-year-old rubber tree progenies. Four individual young treeswith similar girth but different yield potential were selected from both progeniesproduced by hand pollination (open symbols: $PB260 � #RRIT251, and darksymbols: $RRIM600 � #RRII203). Yield potential was estimated at the age of2.5 years, by measuring their latex production during 3 � 10 micro-tappings. A fewmonths later, three young mature leaves (stage D) were individually sampled earlyin the morning from the most recent flush of each of these eight trees andimmediately processed for CNp analysis (May, eastern Thailand).

2.5. CNp, b-D-diglucosidase, b-D-glucosidase, linamarase andb-cyanoalanine synthase activities in the latex

Table 2 shows CNp and activities of enzymes related to cyanidemetabolism that were measured in the leaves, inner bark (in thedrainage area), and latex of PB260 trees tapped regularly for about15 months. Whereas the CNp levels of leaves (�54 mM) and of theinner bark (�11.5 mM) were at the expected level for tapped treesof this clone (Table 1B), the CNp of the latex was very low (or non-existent) in the cytosol and lutoid compartments. The b-D-glucosi-dase and the linamarase potential activities were in the same orderof magnitude in the inner bark extract and in the lutoid compart-ment of the latex. The linamarase activity in the lutoids was about40% of the total b-D-glucosidase in these organelles. The b-D-gluco-sidase and linamarase activities observed in the cytosol repre-sented only about 15% of the activities measured in latex pellet.Similar b-D-diglucosidase activities were detected, but at a farlower (nkat) level than linamarase (lkat level), in the bark extractand in the latex pellet, and even lower in the latex cytosol. Finally,

Table 2Estimation of CNp of the leaf, inner bark and latex compartments, and of enzymeactivities in cyanide metabolism of the inner bark and latex compartments. CNp andall enzyme activities were analyzed in the leaf, inner bark (50 cm beneath the tappingcut), and latex compartments of �1.5-year-regularly-tapped trees of the PB260 clone.

Samples CNp(mM)

b-D-Diglucosidase(nkat.mg�1

protein)

b-D-Glucosidase(lkat.mg�1

protein)

Linamarase(lkat.mg�1

protein)

b-CAS(pkat.mg�1

protein)

Leaf 54.1 ± 4.2 ND ND ND NDInner bark 11.5 ± 1.4 4.7 ± 0.6 3.2 ± 0.5 2.8 ± 0.4 8.7 ± 1.1Latex cytosol <0.001 1.2 ± 0.4 0.48 ± 0.12 0.2 ± 0.07 72.4 ± 4.2Latex pellet <0.003 3.3 ± 1.1 5.3 ± 0.8 2.1 ± 0.2 5.6 ± 0.7

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b-cyanoalanine synthase activity was the highest in the latex cyto-sol, while it was the lowest in the latex pellet and the bark extract.The undetectable level of CNp in latex observed in this experimentmight be due to uncontrolled degradation of cyanogenic glucosidesduring the long purification process of the cytosol and bottom frac-tion serum. Another experiment was then performed to measurethe CNp of the two main latex compartments after latex collectionand centrifugation. These results confirmed that the CNp of latexfrom tapped trees was very low (1.8 ± 0.4 lM in the cytosol and3.1 ± 0.8 lM in the pellet) and below the detection limit of theSpectroQuant kit.

3. Discussion

The methods used to measure the cyanide potential (CNp) ofHevea tissues possibly led to slight underestimation of the values,since almost all of the putative free cyanide might have been lostduring preparation of the samples with sulfuric acid. Also, the rel-atively low b-D-diglucosidase activity in the rubber tree tissuesmight not have been efficient enough to quantitatively hydrolyzethe diglucosides linustatin (2) and neolinustatin upon the enzy-matic hydrolysis of cyanogenic glucosides. However, free cyanideand these diglucosides have been reported to represent a verylow percentage (<1%) of the CNp of rubber tree tissues (Selmaret al., 1987b, 1988, 1991), consequently the CNp measured in ourconditions may be underestimated by only a few percent. Giventhis approximation, the leaf CNps reported here (25–60 mM) werein the same order of magnitude as, and even higher than, the CNpsreported by Moraes et al. (2002) (8–28 mM) or Lieberei (2007)(33–46 mM), depending on the rubber clone and leaf maturity,respectively.

Moraes et al. (2002) showed that the CNp of young and oldleaves from grafted compatible crown clones was high, and wasfar lower in the old mature leaves of incompatible clones fromother Hevea species. In contrast, we found that in H. brasiliensis,whatever the rubber clone or the age of the tree, the younger theleaves (from stage C to D), the higher the leaf CNp. Ballhorn et al.(2005) reported similar data on the obligate cyanogenic Phaseuslunatus species. This finding is quite significant since the youngestimmature leaves are more prone to herbivore attacks, suggestingthat high CNp confers high repellent and toxic efficiency againstpests.

Lieberei (2007) reported the highest CNp in the young greenimmature leaves (stage C) of rubber trees. However, in our studies,given the short leaf maturation time (including stage C) of H. bra-siliensis with respect to the long whole life span of stage D matureleaves, we chose to sample the latter and our experiments enabledus to define the best leaf sampling conditions to compare theirCNp. Young mature leaves (stage D) must be collected early inthe morning from the most recent flushes and from branches thatare not directly exposed to the sunlight. It is still unclear why theCNp of older leaves from previous flushes was systematically lowerthan that of stage D mature leaves from the most recent leaf flush,especially since these older leaves were not directly exposed tosunlight. It may be that in H. brasiliensis, the efficiency of cyano-genic glucoside biosynthesis continually decreases as proposedby Moraes et al. (2002), and/or that this compound is more effi-ciently drained from older leaves to the other tree organs.

This is the first report on diurnal variations in leaf CNp in H. bra-siliensis. The significant decrease in the CNp of young mature leavesdirectly exposed to sunlight, especially during the period of maxi-mum sunlight, could be explained by the fact that the biosynthesisof leaf cyanogenic glucosides may be inhibited during strong directsunlight, while their translocation from leaves to the whole treemay still be operational or even more efficient. The hypothesis of

the transport of cyanogenic glucosides from the Hevea canopy tothe trunk was supported by the results of Moraes et al. (2002) ina study of incompatibility phenomena observed when leavescrown clones with high CNp were grafted onto trunks of cloneswith high linamarase and b-D-diglucosidase activities in associa-tion with low b-cyanoalanine synthase activities.

The inhibition of cyanogenic glucoside synthesis in young ma-ture leaves exposed to intense sunlight may occur through genedown-regulation or protein degradation of the cytochromes P450(i.e. CYP79D isoform), which catalyze the first committed step inthe biosynthesis of cyanogenic compounds (Andersen et al.,2000; Forslund et al., 2004; Jørgensen et al., 2005). Various genesof the cytochrome P450 family have been reported to be differen-tially regulated (Mizutani et al., 1998; Schrøder et al., 1999) andthe corresponding proteins to be degraded (Hendry et al., 1981)by light.

The convergence of our numerous results indicates that in He-vea cyanogenic glucosides may be involved in latex regeneration/production: (1) the rubber clones with the highest latex yield po-tential had the highest leaf and inner bark (mature virgin trees)CNp; (2) there was a positive relationship between early estimatedyield potential and the leaf CNp of young rubber seedling proge-nies; (3) while the CNp of the leaves of a given clone remained rel-atively stable, in given environmental conditions and in givenseasonal periods, there was a marked decrease in the CNp of trunkinner bark (at least at the level of the latex drainage area) from thevirgin stage on, along with the number of years the trees had beenexploited.

Another striking result was that, in contrast to other surround-ing inner bark tissues, neither reliably detectable free cyanide norcyanogenic compounds were found in the latex. The presence oflinamarase activity has been reported in cassava latex (Nambisan,1999). The present study supports the presence of b-D-diglucosi-dase, unspecific b-D-glucosidase, linamarase, and b-cyanoalaninesynthase activities in the inner bark tissues of rubber tree, andthese activities were in the same order of magnitude as those pub-lished by Moraes et al. (2002). Interestingly this is the first reportof the presence of the four enzyme activities involved in cyaniderelease and detoxification in rubber tree latex. The non-specificb-D-glucosidase potential activity was found to be maximum, andrather high (a similar lkat level as that found in the inner bark)in the latex bottom fraction (Pujarniscle, 1968), together with thelinamarase potential activity. The latter accounted for about 40%of the total b-D-glucosidase. Compared with non-specific b-D-glu-cosidase, the lower potential activity of the linamarase pelletmay be due to the presence of different b-D-glucosidase isoformsin the lutoids, with more or less affinity for linamarin (1) and/orPNPP. The b-D-glucosidase and linamarase activities found in thecytosol represented only about 15% of the activities measured inthe latex pellet. They may correspond to contamination due toknown partial bursting of the lutoids, hence the release of thelutoidic hydrolytic enzymes into the cytosol during latex flow (Puj-arniscle, 1968). b-D-Diglucosidase activity was detected, but at a farlower (nkat) level than linamarase, in the two major latex com-partments that displayed 2.5 fold higher activity in the latex pellet,i.e. similar to the activity found in the inner bark. b-Cyanoalaninesynthase activity detected in the rubber tree latex cytosol was al-most 10 fold higher than in the bark, at a level comparable to thatfound in other tissues of cyanogenic plants such as cassava(Nambisan, 1996; Elias et al., 1997) with only traces in the bottomfraction. All our results showed that latex cells are able to consumelinustatin (2) and linamarin (1) and convert these cyanogenic glu-cosides, as well as free cyanide, into non-cyanogenic nitrogen com-pounds such as the amino acid asparagine, one of the mostimportant organic sources of nitrogen in plants (Blumenthalet al., 1990; Fischer et al., 1995; Wan et al., 2006).

P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739 737

4. Conclusion

It is well documented that during rubber (Lieberei et al., 1986;Selmar et al., 1988, 1991) and almond (Sánchez-Pérez et al., 2008)seed germination and/or development, as well as during cassavasprouting (Nambisan, 1999) or during the early stage of tuberiza-tion (Akonye and Osuagwu, 1998), cyanogenic glucosides can bemetabolized and used as a source of nitrogen. In the same way,we propose that in H. brasiliensis latex producing tissues, cyano-genic glucosides – typical secondary plant products – may serveas buffering nitrogen and glucose storage compounds favoring pri-mary metabolism, thus leading to better latex regeneration andhigher rubber yield.

Nevertheless, more detailed studies and statistical analyses areneeded with more rubber clones and with young progenies to con-firm whether leaf CNp is a possible early marker of rubber yield,and possibly of growth potential.

5. Experimental

5.1. Chemicals

All chemicals were of analytical grade and, unless otherwisespecified, were purchased from the Sigma–Aldrich Chemical Com-pany. Linamarin (1) and linustatin (2) were obtained from eitherICN Pharmaceutical or CHEMOS Gmbh. The SpectroQuant Kit pur-chased from Merck was used for cyanide measurements.

5.2. Plant material

Rubber tree leaf, bark and latex were collected from differentplantations. Samples from 10-year-old regularly tapped trees(PB260) and 6-year-old mature virgin trees (PB260, PB235, IRCA18,PB217, GT1, RRIM600 and PR107) were from the SOGB rubber es-tate and Bongo/SAPH industrial rubber estates in southern Côted’Ivoire (West Africa). The samples from young and mature graftedtrees of the PB217 clone were collected in the E. Michelin Planta-tion (PEM), North Mato Grosso, Brazil. Five-year-old regularlytapped trees of the clones RRIM600, RRIT251 and PR107 as wellas the 3-year-old seedling progenies from $PB260 � #RRIT251and $RRIM600 � #RRII203 were from the Chachoengsao RubberResearch Center (CRRC) in eastern Thailand.

5.3. Optimization of leaf sampling conditions

CNp variations as a function of leaf developmental stages andmorning sampling times: The youngest and oldest mature leaves(stage D, Fig. 1) from the most recent flush, as well as older leavesfrom the previous flush were compared. The samples consisting ofa total of nine leaves collected from three regularly tapped trees ofthe clone PB260 (homogeneous in girth and yield) were collectedat different times (06:00, 08:00 and 10:00 am) on a sunny morningof May 2002.

Day and nighttime variations in CNp: Young mature leaves (stageD) of the most recent flush and older leaves of the previous flushwere collected from the RRIM600 clone at different times duringthe day and night of a sunny day in May 2006.

Comparison of variations in CNp in leaves exposed or not to sun-shine: Young mature leaves (stage D) of the most recent flushand older leaves of the previous flush from trees of the RRIT251high latex yielding clone exposed or not to sunlight were collectedat 09:15 am on a same day of May 2006.

Comparison of variations in CNp in different developmental stagesfrom high/low yielding clones: Leaves from the B–C transition stageand from C and D stages of the most recent flush and older leaves

of the previous flush were collected from the RRIT251 high latexyielding clone and PR107 low latex yielding clone early in themorning of a same day of May 2006.

5.4. CNp of leaf and inner bark vs. clonal yield potential

Five 6-year-old mature virgin trees of different clones (PB260,PB235, IRCA18, PB217, GT1, RRIM600 and PR107) were selectedfor their growth homogeneity (trunk diameter 48–50 cm). Youngmature leaves (stage D) of the most recent flush and inner soft barklocated 50 cm below the next tapping cut were collected individu-ally and immediately deep-frozen in liq. N2, and then stored at�80 �C. The same sampling was performed on the regularly tappedtrees of the PB260 clone (homogeneous in girth). Leaf and barksamples were collected as three independent sets from three trees(mixed samples). All samplings were performed in April 2004.

5.5. CNp of leaf and inner bark vs. number of tapping years

Six sets of three homogeneous �6.5 year-old virgin trees of thePB217 clone were selected for their growth homogeneity (trunkdiameter 48–50 cm). Young mature leaves (stage D) and inner bark(50 and 25 cm above, 5, 25 and 50 cm below the tapping cut) werecollected as three independent mixes of three trees before the firsttapping and after about one, two, and three years regular tapping(half spiral, every 3–4 days: 1/2S d3d4). The other three sets ofthree virgin trees were left untapped for three more years. Theirleaves and inner bark were collected in the same way and on thesame day. All sampling was performed on mid-August 2002–2005.

5.6. Leaf CNp vs. yield potential of young seedling progenies

Eight individual �3-year-old seedling progenies produced byhand pollination (four from $PB260 � #RRIT251 and four from$RRIM600 � #RRII203) were selected for their relative girth homo-geneity (24.2 ± 2.1 cm measured 40 cm from the soil), and theirdifferent estimated yield potential (ranging from the lowest: 6 g/tree/10 tappings, to the highest: 41 g/tree/10 tappings). Each indi-vidual tree’s yield potential was estimated at the age of 2.5 yearsby measuring their latex production for 3 � 10 microtappings (atotal of 30, half spiral, every 3 days: 1/2S d/3), at a height of40 cm from the soil. The yield data were measured and correctedfor standardization to the same mean girth of this group of trees.Young mature leaves (stage D) were individually sampled fromthe most recent flush from eight individual trees and immediatelyprocessed for CNp analysis.

5.7. Latex collection and centrifugation

At SOGB (Côte d’Ivoire, West Africa), latex was sampled on April2004 from nine trees of the PB260 clone (homogeneous in girthand yield) that had been regularly tapped for �2.5 years. The latexwas collected in 50 ml centrifuge tubes, kept on ice, and immedi-ately transported to the laboratory. Three independent mixed latexsamples from three trees were centrifuged (Sigma 3 K, 1 h at20,000g) at 4 �C. A few ml of the clearest cytosol were recoveredusing a syringe and immediately stored at �80 �C. The bottom frac-tion was rinsed 2 times and centrifuged (30 min at 10,000g), with15 vol g�1 of a cold isotonic buffer (330 mM mannitol, 2.5 mMEDTA in 50 mM MOPS pH 7.0) according to a method adapted fromRibaillier et al. (1971), then stored at �80 �C. The samples weretransported in dry ice to the IRD laboratory where the cytosolicsamples were centrifuged again (1 h at 100,000g) at 4 �C to elimi-nate any remaining rubber particles. The pellet fractions werecompleted with 1% (v/w) of a plant protease inhibitor cocktail(Sigma P 9599), sonicated for 30 sec on ice, then centrifuged (1 h

738 P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739

at 40,000g) at 4 �C, before CNp, enzymatic activities and proteincontent were measured in the supernatants.

At CRRC (Thailand), latex was sampled on ice from three indi-vidual tapped trees of the RRIM600 clone on May 2006 and imme-diately centrifuged (20,000g for 1 h) at 4 �C. An aliquot (500 ll) ofthe cloudy cytosol or 500 mg of crude bottom fraction from eachindividual latex sample was immediately fixed in 4.5 ml of cold0.1 N H2SO4 and immediately processed for measurement of CNp.

5.8. Sampling of leaves and inner bark

In Africa, mature young leaves were collected from at leastthree different branches (with at least two leaf flushes) on eachtree, packed in aluminum foil and immediately submerged intoliq. N2. The leaves were then transported in dry ice to the labora-tory and stored at �80 �C before analysis. In Brazil and Thailand,three branch extremities (harboring two leaf flushes) per tree werecollected and immediately transferred to the laboratory, wherethree to six leaves per flush were immediately processed for anal-ysis of CNp.

For inner bark, in all cases a stainless steel cork borer (#2.5 cmin diameter) was used to recover bark samples up to the cambiumat different heights of the tree trunk. The first 2–3 mm of inner softbark including the cambium were quickly peeled off with a scalpeland the thin shavings were immediately deep frozen in liq. N2,then stored at �80 �C.

5.9. Protein extraction from inner bark

Deep-frozen soft bark shavings were ground to a fine powderunder liq. N2 using an IKA� A10 analytical grinder (IKA Laboratory,Staufen, Germany). Then aliquots (�300 mg) of powder were ex-tracted in 1 ml of extraction buffer (50 mM Ascorbic acid, 0.1% Tri-ton X-100, 1% b-Mercaptoethanol, in 50 mM Boric acid pH 9.0), in amortar (on ice). The homogenate was kept on ice for 30 min undershaking before spinning at 20,000g for 30 min at 4 �C to eliminatethe bark debris. The clear protein extracts were added with a plantprotease inhibitor cocktail (Sigma P 9599) to obtain a final concen-tration of 1% (v/v), then aliquoted for storage at �20 �C or forimmediate analysis.

5.10. Protein quantification

Latex fractions and bark protein contents were determined withthe Bio-Rad Protein Assay Kit based on Bradford’s method(Bradford, 1978).

5.11. Inner bark and latex enzyme activities

All analyses were performed in triplicate.Linamarase activity was determined as non-specific b-D-glucosi-

dase (EC 3.2.1.21) by adding either 10 to 50 ll of latex cytosol, or ofbottom fraction serum, or 5 to 20 ll of inner bark protein extract to3.5 ml of 50 mM phosphate buffer (pH 6.0) at 35 �C, then incubat-ing for 10 or 20 min with 10 mM p-nitrophenyl-glucopyranoside(PNPG) according to a method adapted from Conchie and Levy(1957). True linamarase specific activity was analyzed in the pres-ence of 10 mM linamarin (1) after 30 min incubation according tothe method of Selmar et al. (1987a), except that the released cya-nide was measured from the whole reaction medium, using theSpectroQuant Kit (Merck) after 10 min alkalinization with 0.2 NNaOH, to release cyanide from the acetone cyanohydrin, thenneutralization.

b-D-Diglucosidase activity was estimated in the same conditionsas for the true linamarase specific activity using 10 mM linustatin(2) as substrate.

b-Cyanoalanine synthase (EC 4.4.1.9) was measured according toa method derived from Blumenthal-Goldschmidt et al. (1963). Analiquot (50 ll) of each latex compartment was incubated in screwcap tubes at 35 �C for 30 and 60 min, in 4.5 ml of 50 mM Tris–HClbuffer (pH 8.9) containing 2.5 mM L-cysteine and 12.5 mM KCN.The released H2S was determined at OD 570 nm after 30 min incu-bation in the dark with N-N-dimethyl-P-phenylenediamine andFeCl3. Na2SO3 standard solutions were prepared immediately be-fore use to obtain a standard curve to evaluate the released sulfitecontent in the reaction mixture.

5.12. Measurement of total cyanogenic compounds (cyanide potential:CNp)

Deep-frozen plant material was ground into a fine powder inliq. N2 using an IKA� A10 analytical grinder (IKA Laboratory, Stau-fen, Germany). An aliquot (300 to 500 mg) of either fresh sliced orfrozen-ground plant material was weighed precisely, immediatelysuspended in 0.1 N H2SO4 (4.5 ml), and then finely ground in amortar with a pinch of silica sand. The crude acidic extract wastransferred into a screw cap centrifuge tube, incubated for20 min under shaking and then centrifuged (5000g for 20 min) toeliminate sand, cell debris and denatured proteins. An aliquot(400 ll) of the clear supernatant was added with 3.5 ml of 0.1 Mphosphate buffer and an appropriate volume of 0.2 N NaOH to ad-just the pH to 6 ± 0.3. This final extract of total cyanogenic com-pounds was quantified. To release acetone cyanohydrin fromlinustatin (2) and then linamarin (1), an aliquot of the final extractwas incubated for 30 min at 37 �C in the presence of a concentrateddialyzed crude protein extract from the Hevea inner bark (whichwas checked to make sure it contained high b-diglucosidase andlinamarase activities) added with 1% (v/v) of the plant proteaseinhibitor cocktail (Sigma P 9599). Alkalinization with 0.2 N NaOHenabled the immediate cleavage of acetone cyanohydrin into ace-tone and CN�. After neutralization with H2SO4, CNp was deter-mined at OD 585 nm using the SpectroQuant Kit (Merck)according to the manufacturer’s recommendations. A standardcurve was prepared with KCN in the same conditions as the plantextracts. All determinations were performed in triplicate. CNp wascalculated according to the percentage of dry matter of plant mate-rial and expressed in mM of CN� in internal tissue water.

5.13. Determination of leaf and inner bark dry matter and watercontents

An aliquot (300–500 mg) of either fresh or deep-frozen slicedleaves, inner bark pieces, or powder was weighed precisely beforeand after at least 48 h drying to constant dry weight in a ventilatedoven at 70 �C, after which the percentage of dry matter andinternal water contents were calculated.

5.14. Estimation of the average yield performance of clones

The average yield performance of each clone was calculated asthe average yield index from large scale rubber clones trialsperformed in southwestern Côte d’Ivoire, taking into account15–20 years of annual rubber production. The yield performanceof the medium yielding GT1 clone was used as a reference(index = 100). The original data were obtained from the plantationsand Dr A. Clément-Demange (personal communication).

Acknowledgments

We are grateful to the Institut Français du Caoutchouc (IFC), theMichelin Company, SOCFINCO and SIPH and the National Metaland Materials Technology Center (MTEC), Thailand (Grant No.

P. Kongsawadworakul et al. / Phytochemistry 70 (2009) 730–739 739

MT-B-49-POL-14-362-G) for funding this work. We are grateful tothe staffs of the SIPH (Bongo/Côte d’Ivoire and GREL/Ghana), SOCF-INCO (SOGB/Côte d’Ivoire) and Michelin (PEM/Brazil) industrialplantations, as well as of the Chachoengsao Rubber Research Cen-ter, Rubber Research Institute of Thailand/Department of Agricul-ture (CRRC/RRIT/DOA) for allowing us access to their plantmaterials and laboratories. We also acknowledge the help providedby B. Soumahoro and K. N’Guessan (SOGB), E. Serres, A. Attobra, M.Traore and M. Soumahoro (SAPH) as well as E. Cavaloc and M. Gas-sio (PEM) in the preparation and control of the experiments inplantation. We thank P. Trouslot and D. Morin for their technicalhelp in performing some of the biochemical analyses in IRD-Montpellier.

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