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Comparative Study
, 123 (3), 1133-42

Identification of Natural Rubber and Characterization of Rubber Biosynthetic Activity in Fig Tree

Affiliations
Comparative Study

Identification of Natural Rubber and Characterization of Rubber Biosynthetic Activity in Fig Tree

H Kang et al. Plant Physiol.

Abstract

Natural rubber was extracted from the fig tree (Ficus carica) cultivated in Korea as part of a survey of rubber producing plants. Fourier transform infrared and (13)C nuclear magnetic resonance analysis of samples prepared by successive extraction with acetone and benzene confirmed that the benzene-soluble residues are natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of fig tree was about 4%, whereas the rubber content in the bark, leaf, and fruit was 0.3%, 0.1%, and 0.1%, respectively. Gel-permeation chromatography revealed that the molecular size of the natural rubber from fig tree is about 190 kD. Similar to rubber tree (Hevea brasiliensis) and guayule (Parthenium argentatum Gray), rubber biosynthesis in fig tree is tightly associated with rubber particles. The rubber transferase in rubber particles exhibited a higher affinity for farnesyl pyrophosphate than for isopentenyl pyrophosphate, with apparent K(m) values of 2.8 and 228 microM, respectively. Examination of latex serum from fig tree by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed major proteins of 25 and 48 kD in size, and several proteins with molecular mass below 20 and above 100 kD. Partial N-terminal amino acid sequencing and immunochemical analyses revealed that the 25- and 48-kD proteins were novel and not related to any other suggested rubber transferases. The effect of EDTA and Mg(2+) ion on in vitro rubber biosynthesis in fig tree and rubber tree suggested that divalent metal ion present in the latex serum is an important factor in determining the different rubber biosynthetic activities in fig tree and rubber tree.

Figures

Figure 1
Figure 1
FTIR spectra of the rubber extracted from fig tree (A) and rubber tree (B) in potassium bromide disc. Four scans were co-added in the spectral range of 4,000 to 370 cm−1 at a resolution of 2 cm−1. Characteristic bands for cis-1,4-polyisoprene are indicated with their wave numbers.
Figure 2
Figure 2
13C NMR spectra of the rubber extracted from fig tree (A), F. elastica (B), and rubber tree (C) in C6D6. About 5,000 scans were collected at a spectral width of 20,000 Hz. Representative peaks for cis-1,4-polyisoprene are indicated as α through ε.
Figure 3
Figure 3
Time course- (A) and WRP- (B) dependent incorporation of IPP into rubber. Reactions were carried out in 50 μL of 100 mm Tris-HCl, pH 7.5, containing 1 mm MgSO4, 1 mm DTT, 20 μm FPP, 0.1 mm [14C]IPP (55 mCi mmol−1), and 5 mg WRP in A and the indicated amount of WRP in B. Reactions in B were performed at 25°C for 5 h, the rubbers were extracted with benzene as described in text, and the resulting radioactivities of the 14C-labeled rubber were measured by a liquid scintillation counter. ♦, Rubber tree; ▪, fig tree.
Figure 4
Figure 4
Rubber transferase activity of the WRP of fig tree (white bars) and rubber tree (black bars). Reactions were carried out in 50 μL of reaction mixture containing 5 mg of WRP as described in Figure 3. −FPP, Without FPP; +EDTA, with 25 mm EDTA; Boil, WRP-boiled for 5 min; 0, reaction at 0°C.
Figure 5
Figure 5
Substrate dependence of rubber transferase activity in fig tree WRP. Five milligrams of WRP was incubated with increasing amounts of FPP at saturating IPP concentration (1 mm; A), and increasing amounts of IPP at saturating FPP concentration (20 μm; B) at 25°C for 5 h. Product formation was plotted against substrate concentration, and the apparent Km values were calculated by a Lineweaver-Burk analysis.
Figure 6
Figure 6
Effect of Mg2+ ion on rubber transferase activity of fig tree and rubber tree WRPs (A), and dependence of IPP incorporation by the latex of fig tree and rubber tree on EDTA additions (B). Reactions were carried out as described in Figure 3 with 50-μL reaction mixtures containing 5 mg of WRP (black symbol) to determine the effects of Mg2+ (A), or 5-μL aliquots of latex serum (white symbol) were evaluated for effects of EDTA (B). ♦ and ⋄, Rubber tree; ▪, fig tree WRP; □ and ○, fig tree latex collected at different time of the year; ▵, fig tree latex filtered to 3-k membrane centricon (Amicon, Beverly, MA) to remove smaller molecules, including Mg2+ ions.
Figure 7
Figure 7
Analysis of the proteins in the latex and WRP of fig tree and rubber tree by 12% (a) and 6% (b) to 17% gradient SDS-PAGE. Rubber particle proteins were solubilized by incubating fig tree WRP in a detergent solution containing 0.1% (w/v) Triton X-100 and 1% (w/v) SDS. After electrophoresis, proteins were detected by Coomassie Blue staining. L, Latex; RP-1, first WRP; RP-2, second WRP; H, rubber tree latex; M, marker.
Figure 8
Figure 8
A, Inhibition of rubber biosynthesis of rubber tree WRP, but not fig tree WRP by an antibody to the SRPP from rubber tree. Reactions were carried out as described in Figure 3 in 50 μL of reaction mixture containing 5 mg WRP and indicated amount of antibody raised against the SRPP from rubber tree. ▪, Fig tree WRP; ●, control serum; ♦, rubber tree WRP. B, Western analysis of rubber particle proteins. F, Fig tree; H, rubber tree; 24, purified SRPP.
Figure 9
Figure 9
Mr distribution of endogenous rubber extracted from the latex of fig tree (□) and distribution of 14C-labeled rubber synthesized in vitro (●). Reaction was carried out in 1 mL of reaction mixture containing 200 mg WRP as described in Figure 3. The rubbers synthesized in vitro were extracted with benzene and subjected to a GPC. The eluent was monitored by ELSD and assayed for radioactivity.

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