Evidence for specific variation of protein pattern during tapping panel dryness condition development in Hevea brasiliensis

CiENCE Plant Science 105 (1995) 207-216

ELSEVIER

Evidence for specific variation of protein pattern during tapping panel dryness condition development in Hevea brasiliensis Kouadio Diana, Abdourahamane

Sangare* b, Jacques Kore Diopohb

aIDEFOR-DPL, 01 BP 1536, Abidjan 01, Ivory Coast, Africa bUniversi14 Nationale de C&e d’lvoire, F.A.S.T., 22 BP 582. Abidjan 22, Ivory Coast, Africa

Received 5 August 1994; revision received I8 November 1994; accepted 28 November 1994

Abstract Tapping panel dryness (TPD) condition affects the production of latex in Heveu brasiliensis plantations. Latex production is severely decreased in diseased plants and can be completely shut down in the ultimate stage of the phenomenon. In search of the molecular basis of the disease, we have analysed the changes in latex protein pattern during the development of this condition, Five proteins specific to the cytosolic compartment of latex were found to be related to the disease. Major changes consisted of a dramatic increase of a 26-kDa and a 145kDa protein in diseased plants and minor changes affected a SkDa, a 34kDa and a 21-kDa protein. The 26-kDa protein was found to be linked to the coagulation process. Its accumulation is specifically correlated to the disease development and is inhibited by ethylene. The 14.5kDa protein accumulates preferentially in the severe stages of the condition. A disfunctioning of the coagulation process is proposed to be the major cause of the syndrome. Keywork:

Tapping

panel dryness

related proteins;

Hevea brusiliensis

1. Introduction The latex of Hevea brasiliensis is the primary industrial source of natural rubber. It is obtained by

collecting and processing exuded latex from notches made on the bark of 5-30-year-old plants. In this process called tapping, incisions are made diagonally and cover half of the tree’s circumference. Latex drains from the notches for a cer* Corresponding author. Elsevier Science Ireland Ltd. SSDI 0168-9452(94)04034-E

tain period of time until the flow slowly decreases as rubber in the latex begins to polymerize. Occasionally, in apparently healthy trees, cuts will produce only drops of latex and if the phenomenon progresses it can ultimately lead to the complete shut down of latex production with subsequent browning of the bark. The phenomenon is known as irreversible tapping panel dryness (TPD). In some cases, production can revert to normal if the condition is detected early enough and latex harvesting is stopped: the disease is then qualified

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as reversible. However, reversible TPD is thought to be different from the irreversible disease [l]. TPD was first reported in Brasilia on wild rubber trees by Croos in 1885, cited by Evers in 1974 [2]. In Malaysia, the importance of the disease in rubber tree plantations was recently reported, particularly in precocious high yielding clones [3]. In CBte d’Ivoire (The Ivory Coast), the syndrome was reported only as recently as 1962 with the first Zf. brusiliensis plantations. Currently, the disease is estimated to result in a 15-20% decrease of yield per year [4]. A number of approaches have been used to describe and study the development of the disease. Cytological disorders associated with disease development were reported [5,6], which might appear to agree with the hypothesis that a fungal infection was the cause of TPD. Studies on viruses and viroids were inconclusive [7-lo]. The syndrome was also hypothesized to be associated with physiological disorders [ 1 l- 151and several studies yielded different hypotheses about the origin and the development of the disease. Important in situ coagulation in laticifers of TPD affected plants was reported by de Fay [5] suggesting a correlation between the two phenomenons. Whether coagulation is a cause or a consequence of the disease has not been demonstrated. In this study, we examined the changes in protein patterns of different fractions of H. brasiliensis latex during the development of the disease and correlated quantitative variations of certain cytosolic proteins to TPD. We also present evidence that a coagulation associated protein strongly accumulates in plants showing typical symptoms as well as in healthy plants suspected to be in early stages of the syndrome. The involvement of latex coagulation pathway in disease induction is also discussed. 2. Material and methods 2.1. Plant material Two clones of H. brasiliensis were used in this study, namely clone PB 235, which is known to be sensitive to TPD, and PB 217, less sensitive to the syndrome. Both clones were grafted onto GTl clone rootstocks. They have been exploited since 1986 and are tapped twice a week without ethylene

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stimulation. PB 235, PB 217 and GTl clones are originally from Malaysia (Prang Besar and Gondang Tapen). The work presented here was carried out with latex collected from trees which are still being harvested. 2.2. Methoa!s Fractionation of latex and protein purification.

Total proteins from cytosolic and lutoidic compartments of latex were used in this study. Lutoids are inclusions described by Pujarniscle [ 151 and Gomez [16] found in latex cytosol that display both vacuole-like and lysosome-like features. Latex fractions were obtained after differential centrifugation and total proteins were purified from each compartment as follows. Fresh latex was collected in pre-cooled plastic tubes and centrifuged at 15 000 rev./min for 15 min in a Beckman 30ti rotor. The three fractions obtained are composed of aggregated rubber particles floating at the surface of the supernatant (which is mainly cytosolic but still contains small particles of polyisoprene), and a pellet formed by lutoid, Frey-Wissling particles and mitochondria. The obstructing rubber aggregate was discarded and the cytosolic fraction was purified by a l-h centrifugation at 40 000 rev./mm in a Beckman 50ti rotor. The pellet was washed three times with an isotonic washing buffer (pH 7.5; 50 mM Tris-HCl, 400 mM mannitol, 0.05 mM ammonium molybdate, 5 mM magnesium chloride, 0.5 mM digitonine and 1.5 mM fi-mercaptoethanol). Digitonine destroys double membrane particles and the final pellet was thereby composed of only lutoidic particles. Lutoids were broken by heat shock (75”C, 5 min), centrifuged at 12 000 rev./min for 15 min and the supematant containing lutoidic proteins was recovered. The pellet containing lutoidic membranes was washed three times with washing buffer without mannitol. Purified membranes were then resuspended in 10% SDS (sodium dodecyl sulfate) and boiled for 2 min before centrifugation at 12 000 rev./min for 15 min. Protein concentration of latex fractions is determined by the method of Sedmak and Grosberg [17]. SDS-PAGE (SDS polyacrylamide gel electrophoresis) was performed follow-

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ing conditions described by Laemmli [ 181. Protein samples were not heat denatured prior to electrophoresis (with the exception of lutoid membrane proteins). Protein bands were visualized after silver nitrate staining [ 191. For long-term storage, protein extracts were kept in 15% glycerol at -80°C. No obvious degradation was observed after 1 year using this method. 2D-IEF (two-dimensional iso-electric focusing) was conducted according to O’Farrel [20]. Ampholyte (Bio-lyte) mixtures used for gel preparation were in a l/9 ratio of 10 ampholytes (pH 3) to 7 ampholytes (pH 5), respectively. Symptom severity records. Symptom severity was scored from 0 to 5: 0, normal flowing of latex; 1, l-20% of dry notch; 2,21-40% of dry notch; 3, 41-60% of dry notch; 4,61-80% of dry notch; and 5, 81-100% of dry notch. Healthy plants used in our experiments were selected based on the absence of symptoms and scored 0 for 5 years. Ethylene stimulation of latex production. Ethrel(Z chlorothylphosphonic acid), which produces ethylene, was applied to the incisions as 2.5% of active component of a mixture containing palm oil. 3. Results 3.1. SDS-PAGE protein pattern of different compartments of latex Fig. 1 shows the electrophoresis patterns of cytosolic and lutoidic compartments purified from healthy plants of clone PB 235. Cytosolic proteins display a homogeneous pattern amongst the extracts in our experimental conditions while a certain variability was detected in both membrane and intra-lutoidic protein profiles. The two lutoidic fractions were often very similar, indicating that the fractionation system used in this study was not efficient enough to clearly separate them. Therefore, the results shown below represent intra-lutoidic fractions which were found to be less variable in healthy plants and also less likely to contain components from other cytosolic organellar membranes. The patterns observed were repeatedly confirmed in all clones used in this work. Typical variations observed are indicated in Fig. 1 where a group of proteins of approximately 28-30

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Fig. 1. Analysis by SDS-PAGE (12.5%) of latex proteins recovered from cytosolic fraction (lanes l-4), intra-lutoidic fraction (lanes 5-8) and lutoid membranes (lanes 9-12) in clone PB 235. Lanes 1,5 and 9 are extracts from the same individual, lanes 2, 6 and IO from another individual and so forth. Total proteins from each fraction were loaded on SDS-PAGE immediately after extraction, regardless of protein concentration. Thus, the pattern actually shows the relative amount of proteins in each fraction compared to each other. Lane 13: molecular weight marker.

kDa shows a variation from one healthy sample to another. 3.2. Protein synthesis modification in latex of diseased plants The heavy lutoidic fraction is not present in latex when the disease reaches an advanced stage. This phenomenon is due to the de stabilization of lutoids and makes it impossible to obtain lutoidic proteins from highly afflicted plants. Therefore, lutoidic proteins could be analysed only for plants scored ‘1’ (l-200/o of dry notch). Such a handicap had indeed no consequence in our study as the electrophoresis pattern instability observed in lutoidic compartment would have rendered doubtful any conclusion about disease impact on protein synthesis in latex. On the contrary, the relative stability of the cytosolic protein pattern made this compartment the ideal candidate for the search of disease related proteins. Comparison of electrophoretic patterns of lutoids and total cytosolic proteins from latex of healthy and affected plants are shown in Fig. 2. There are no noticeable differences between healthy and diseased plant lutoidic protein patterns (Fig. 2a) even though a 36-kDa protein seems

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a

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b 123456789 -

66 kDa 66 kDs 55 kDa

36 kh

b

36 kDa 29 kDa

26 kDa L>

36 kDa 34 kDe 20 kDa

29 kDa 26 kDs

14.2 kDa 21 kDa 20 kDa

14.5 kDa

D

14.2 kDa

Fig. 2. SDS-PAGE protein patterns from lutoidic (Fig. 2a) and cytosolic (Fig. 2b) fractions of healthy and brown bast affected PB 235. In Fig. 2a, four randomly selected healthy plants (lanes 1-4) and four diseased plants with a score of ‘I’ (lanes 6-9) were compared. Cytosolic fraction of a typical healthy plant (lane 1) and three diseased plants scored as ‘5’ (lane 2), ‘4’ (lane 3) and ‘3’ (lane 4) are shown in Fig. 2b. Opened triangles indicate the position of the lutoidic 36-kDa and 26-kDa (Fig. 2a) and the cytosolic 55-kDa. 34-kDa, 26-kDa, 21-kDa and 145kDa (Fig. 2b) proteins discussed in the text. Each lane contains approximately I5 pg total protein. Lanes 5: molecular weight marker.

to accumulate in the lutoid of some but not all samples taken from sick plants. In the case of cytosolic proteins (Fig. 2b), there are two different major changes between healthy and diseased plants: a 26-kDa and a 14.5-kDa protein are dramatically increased in sick plants, although the latter seems to be inconstant. A closer analysis of the protein patterns also reveal some minor changes consisting essentially in the decreased amount of a 55-kDa, a 34-kDa and a 21-kDa proteins in the cytosol of highly affected plants. These patterns were observed in both sensitive PB235 and less susceptible PB217 clones, indicating that the variations are solely related to the physiological state of the plants and not to clonal variation. 3.3. Changes in the accumulation of the disease related 26-kDa protein during symptom

development

In order to correlate the disease appearance with the accumulation of the 26-kDa protein,

which is the most obvious and constant change in affected plants, electrophoresis patterns of cytosolit proteins from 30 individuals selected from apparently healthy plants of the PB235 clone were monitored during a period of 10 months. During this period four individuals (namely Al, A6, E21 and H2) showed a remarkable increase in intensity of the 26-kDa protein at different times, although no apparent disease symptom could be detected. Two weeks after Al displayed a relatively high level of the 26-kDa protein, the normal cytosolic profile was regained and that individual never showed external symptoms. Plant A6 did show symptoms corresponding to level 1 in our scoring system but regained the healthy phenotype 1 week later, and never showed symptoms again. The normal cytosolic profile was also regained in that case. Plants E21 and H2 developed the disease and reached the highest score after 7 months. However, fluctuations were observed in the evolution of the disease severity. Fig. 3 shows the example of

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a_B

*, I *I

-

66 kDa

_

36 kDa

-

29 kDa *

26 kDa

-

20 kDa

-

* 14.6 kDa 14.2 kDa

Fig. 3. Evolution of brown bast disease syndrome and accumulation of the 26-kDa protein in a healthy plant chat became diseased (E21). The severity of the disease (upper) is put in parallel with the protein pattern from latex collected at the same time as symptoms were recorded (lower). In that particular example, the first signs of the 26-kDa accumulation were detected in July 1991. EZl was definitively affected by the Tapping panel dryness condition in January 1992.

clone E21 in which accumulation of the 26-kDa protein in cytosol is correlated to symptom severity. None of the 26 remaining healthy plants that express neither the 26-kDa nor the 14.SkDa protein at a noticeable level showed disease symptoms during the study period. A population of PB 217 observed for the same period of time remained 100% healthy and we never detected an increased level of the 26-kDa protein. These observations strongly suggest that the 26-kDa protein accumulation is correlated to disease development. 3.4. Accumulation of the 26-kDa protein during latex flow

The early symptom observed during the development of TPD is a very rapid coagulation of the first drops of exuding latex. Therefore, one may assume that the variations in cytosolic protein pattern and particularly the changes in the amount of the 26-kDa protein are related to latex coagula-

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tion process. To further analyse this question, latex was collected at intervals of 10 min starting from the first drop of latex flowing after notching, to the end of draining. Healthy and symptomatic plants were used for the experiments. Cytosolic proteins extracted from latex at different time points showed three types of responses depending on the physiological state of the plant. In plants showing increasing symptoms of the disease such as H2 and E21, the heavy fraction is destabilized in the very first latex samples and it continues to diminish until its total disappearance in the last collected fraction. Fig. 4a shows the changes in amount of the 26-kDa protein in these plants. At a relatively low amount in the first fraction, the 26kDa protein accumulates very rapidly and reaches a maximal level in 20 min. Latex coagulates within 40 min. Plants in which the symptoms of the disease are attenuated have their heavy fraction destabilized in the first time point sample. Then, after a period of recovering, lutoids are destroyed again and the last collected fraction has a very low quantity of heavy fraction. The electrophoretic pattern of such a plant (A6 in this case) is shown in Fig. 4b. Again, the instability of the heavy fraction is correlated to the accumulation of the 26kDa protein and the time necessary to achieve total coagulation of the latex is delayed in these plants with respect the highly affected plants. Interestingly, in the last cytosolic fraction taken before total coagulation new proteins (23-25 kDa) that have not been seen elsewhere sometimes appear. However, this phenomenon has been noticed only in A6 clones and needs to be further analysed. Healthy plants did not show a dramatic variation in the consistency of the lutoids, although the last fraction contained less pellet. In this case, the 26kDa protein accumulates only in the very last fraction which was collected 2 h after the incision (Fig. 4c). These observations indicate that the 26-kDa protein synthesis is induced during latex flowing although plants in the acquisition phase of the disease have already a 3-5-fold higher level of the protein compared with normal plants at the first time point. Only very diseased plants showed accumulation of the 14.5kDa protein in some cases which typically occurred around 30 min after incision (Fig. 4a).

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a 10’

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b 20’

30’

40’ kDs

kD a

*r -

28 k.Da

26 kDa

26 kDa

14.5 kDe

-

14.5 kDa

14.5 kDa

Fig. 4. Changes in cytosolic proteins during latex flow. In all cases fractions were collected every IO min until latex stopped flowing. Fig. 4a shows the profiles obtained with latex of plants that are developing the disease (in the example shown, latex flow in plant E2I discussed in the text was analysed in July and December 1991). Fig. 4b shows the changes in the cytosolic protein pattern of plants that are showing signals of recovering from the disease (for plant A6 in the case shown. two different samples were analysed during July 1991). Fig. 4c shows the profile of healthy plants latex. Because latex flows for a longer period of time, cytosolic patterns are shown for IO, 30, 50 and 120 min in the case of normal plants. Different healthy trees from different clones constantly showed the same pattern of accumulation of the 26-kDa during latex flow. The putative degradation products of the 26-kDa protein are shown by stars.

3.5. Effect of ethrel on the 26kDa cumulation

protein ac-

Ethrel is usually used as ethylene source to increase latex production and is believed to act by I

2

34567.5

26 kD -

inhibiting latex coagulation. In search of more evidence of a linkage between the 26-kDa protein accumulation and the coagulation process, plants showing different degrees of disease severity were treated with ethrel and latex was collected 24 and 48 h later. The cytosolic protein pattern of each fraction was then analysed. Fig. 5 summarizes the reaction of plants after ethrel treatment. The 26kDa protein is strongly inhibited in ethrel treated plants but this effect does not last 48 h. Also, in very sick plants, the inhibition is brief in time and does not persist for even 24 h. 4. Discussion

Fig. 5. Effect of ethylene on the accumulation of the 26-kDa proteins of tapping panel dryness affected plants. Cytosolic proteins from sick plants scored 4 (lanes 1 and 2). 3 (lanes 3 and 4), 2 (lanes 5 and 6). and I (lanes 7 and 8) collected 24 h (lanes I, 3, 5 and 7) and 48 h (lanes 2. 4, 6 and 8) after ethrel stimulation

SDS-PAGE analysis of different compartments of the latex of H. brasiliensis revealed the existence of disease related proteins in the cytosolic fraction. Two major proteins - 26 kDa and 12.5 kDa accumulate specifically in latex of affected plants

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213

b IEF

IEF

12.5% SDS

26 kDa

26 kDa

14.5 kDc

14.6 kDa

PH

10

3

PH

)

b

10

3

Fig. 6. Two dimensional gel electrophoresis of total soluble proteins extracted from cytosolic compartment of (a) normal and (b) TPD affected plants. The molecular size of the 26-kDa and the 145kDa discussed in the text is indicated.

but many other minor changes can be observed. Among these minor changes, one can notice a slight decrease in the expression of a 55-kDa, a 34kDa and a 21-kDa protein (Fig. 2b). Observing these minor changes requires a very close analysis of electrophoretic patterns. They are therefore very difftcult to point out as typical disease related proteins. Another minor change is observed in the lutoidic fraction which consists of a slight increase of a 36-kDa protein in sick plants. However, this protein is a lutoid membrane associated protein (Fig. 1) which could contaminate the supernatant during intra-lutoidic proteins purification because of the increasing de stabilization of lutoids membranes in the course of disease development. Even though its accumulation on intra-lutoidic protein pattern might give an indication of lutoid destabilization, the 36-kDa protein cannot be considered as a disease related protein. The most constant and obvious disease related protein is a 26-kDa cytosolic protein which accumulates very early in disease development and actually before any external symptoms are visible. Its accumulation is coupled to the lutoid destabilization that is believed to be the cause of latex co-

agulation [21,22]. One may therefore speculate that the 26-kDa protein is a lutoidic protein that is released in the cytosolic compartment during disease development as a result of lutoid destruction. A very weak band of 26 kDa can be observed in lutoids (Fig. 2a) but no significant difference is noticeable between healthy and affected plants. Nevertheless, we cannot rule out the possibility that the 26-kDa could be a lutoidic protein as it is practically impossible to obtain lutoids from highly diseased plants in which one could find an equivalent accumulation of the 26-kDa protein. However, lutoid destabilization can also be observed in latex after a heavy rain, independently of TPD. This phenomenon is not accompanied by 26kDa protein accumulation (unpublished data). Three observations made here suggest the involvement of the 26-kDa protein in the coagulation phenomenon: (1)

(2)

the protein accumulates in the last fractions of coagulating latex in normal individuals even though it never reaches the level observed in TPD affected plants; plants that are developing the disease coagu-

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(3)

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late very rapidly and these plants show an early accumulation of the 26-kDa protein: and ethylene, which is known to increase the pH in laticifer medium [22-251 and delay coagulation, acts as a strong inhibitor of the 26kDa protein accumulation.

ZD-IEF (Fig. 6) indicates that the 26-kDa protein has a low pH. Its accumulation in the latex could therefore influence the pH of the medium and provoke the destabilization of lutoids that are known to be stable only at a pH close to neutrality [ 15,161. Such an event could then lead to a massive concentration increase of coagulants such as bivalent cations (Mg2+ and Ca2+) and lutoidic components reported earlier [26-281. As observed by de Fay [5], in situ coagulation characterizes TPD affected plants. Lutoids destabilization was therefore suggested as a possible cause of early activation of coagulation with TPD as consequence [29]. Previously, Chrestin [29] showed an abnormal production of toxic oxygen molecules (02-)in over stimulated diseased plants. Then, he hypothesized that an excess of ethylene could activate the lutoidic NADH-oxidase which then produces toxic oxygen that in turn destabilizes lutoid membranes. The acidic content of lutoids would consequently be in contact with the cytosol and provokes latex coagulation. However, production of 02- molecules has not been observed by Chresti [29] in the case of naturally occurring TPD. With our data shown here, this hypothesis tends to prove a very complex regulation of the coagulation process, involving ethylene. Anyhow, it appears that the same condition (TPD) with probably the same molecular causes (disorder in coagulation process) can be triggered by different factors. The 26-kDa protein which accumulates only in the last drops of latex of normal plants seems to be directly related to coagulation. The fact that this protein appears in the very first drops of latex of TPD affected plants suggest a precocious induction of coagulation of latex in those plants. If the 26-kDa protein is assumed to be involved in the coagulation of a normal plant’s latex, its accumulation in sick plants suggests that factors other than the stress evoked by the cutting are in-

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volved in its induction. This would therefore be a case of a defense mechanism developed by the plant against aggressions yet to be characterized. Latex is thought to be a defense substance [30] and our data support this hypothesis. However, the induction of the coagulation mechanism observed in this study is different from other cases of defense strategies. Martin’s hypothesis is based on the presence of high chitinase and chitinase-lysozyme activities in latex [30] which recalls a PR-protein defense mechanism [31,32]. In our study, we did not investigate proteins induced in leaves and we are therefore unable to put in parallel the induction of the 26-kDa in latex and possible PR proteins in leaves. The cause of the naturally occurring TPD is still unknown, but evidence shown in this study support a physiological disorder rather than a pathogenic origin for the syndrome. Deduction from previous works [33] suggest that an unbalanced endogenous ethylene concentration could be involved in the phenomenon. The fact that ethylene used in ‘non-stressing’ conditions inhibits coagulation and ameliorates disease symptoms, supports its direct or indirect involvement in latex coagulation pathway. Consequently, the inhibition of the accumulation of the 26-kDa protein by ethylene in diseased and healthy plants also allows us to establish a relation between the 26-kDa and the coagulation process. In some plants recovering from the disease, the last fraction contain novel types of proteins between 25 and 26 kDa that are not present in other categories of plants. We believe that these proteins are degradation products of the 26-kDa protein although we do not have direct evidence for that assumption. More investigation is needed in that direction as this could be an indication of a protease activity destroying the 26-kDa protein. Such an occurrence could explain why plants recover from the disease and would give more certainty to the direct involvement of the 26-kDa protein in the coagulation mechanism and its importance for the disease development. Beside the 26-kDa protein accumulation, highly affected plants often accumulate a 14.5-kDa protein in the last collected latex fraction. This protein is also suspected to be involved in the coagulation process. Its molecular size suggest that it could be the ‘rubber elongation factor’ [34-361 which

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loosely binds to the rubber particle and can therefore be extracted with the cytosolic serum. The 26-kDa protein could also be the 24-kDa protein described by Hasma [37] which was found to be an ammonia extractable protein from rubber particles. In that case, the accumulation of both 14.5-kDa and 26-kDa proteins in diseased plant also gives a solid support to the hypothesis of an early activation of rubber particle synthesis subsequently leading to a rapid coagulation of exuding latex from notches. This study demonstrates unequivocally that specific proteins accumulate during tapping panel dryness development. We suggest to designate these proteins as ‘tapping panel dryness related proteins’. The disease is proposed to be related to a disorder in natural latex coagulation mechanism. We intentionally focused on major changes, although many minor differences have been noted. Some of the minor changes are reported here and Prematillaka et al. [38] also reported disappearing proteins in latex of tapping panel dryness affected plants. However, more analysis is needed particularly using 2D electrophoresis to better characterize protein synthesis variation induced by tapping panel dryness condition. As an example, the 14.5-kDa protein band appears to be more complex in 2D electrophoresis (Fig. 6) than it could be suspected with monodimensional electrophoresis. Such studies should also take into account naturally and frequentIy occurring fluctuations of latex protein content in relation to other physiological changes. A better characterization of these changes could lead to the development of tools for early detection of physiological abnormality that could help to prevent abandoning diseased plants and therefore improve plantation productivity. ELISA test systems can be developed for example to detect abnormal accumulation of the 26-kDa and 14.5-kDa proteins. Naturally, our efforts are continuing to further characterize biochemically these protein. Acknowledgments

This study was supported by the Ministry of Scientific Research of CBte d’rvoire and IDEFORDPL. The authors would like to thank

21s

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