Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis. We have isolated and characterized a cDNA clone that encodes a protein tightly bound on a small rubber particle. We named this protein small rubber particle protein (SRPP). Prior to this study, this protein was known as a latex allergen, and only its partial amino acid sequence was reported. Sequence analysis revealed that this protein is highly homologous to the rubber elongation factor and the Phaseolus vulgaris stress-related protein. Southern and Northern analyses indicate that the protein is encoded by a single gene and highly expressed in latex. An allergenicity test using the recombinant protein confirmed that the cloned cDNA encodes the known 24-kDa latex allergen. Neither ethylene stimulation nor wounding changed the transcript level of the SRPP gene in H. brasiliensis. An in vitro rubber assay showed that the protein plays a positive role in rubber biosynthesis. Therefore, it is likely that SRPP is a part of the rubber biosynthesis machinery, if not the rubber polymerase, along with the rubber elongation factor.Rubber (cis-1,4-polyisoprene), an isoprenoid polymer with no known physiological function to the plant, is produced in about 2000 plant species with varying degrees of quality and quantity (1). Rubber is the raw material of choice for heavy duty tires and other industrial uses requiring elasticity, flexibility, and resilience. Hevea brasiliensis has been the only commercial source of natural rubber mainly because of its abundance in the tree, its quality, and the ease of harvesting. The diminishing acreage of rubber plantations and life-threatening latex allergy to Hevea rubber, coupled with an increasing demand, have prompted research interests in the study of rubber biosynthesis and the development of alternative rubber sources.In H. brasiliensis, rubber synthesis takes place on the surface of rubber particles suspended in the latex (the cytoplasm of laticifers). The laticifers are specialized vessels that are located adjacent to the phloem of the rubber tree. When severed during tapping, the high turgor pressure inside the laticifers expels latex containing 30 -50% (w/w) cis-1,4-polyisoprene. The latex can be fractionated by centrifugation into three phases: the top fraction containing mostly rubber particles, the metabolically active middle fraction (called C-serum), and the bottom fraction of mainly vacuole-like organelles called lutoids. More than 240 expressed sequence tags (ESTs) 1 have been identified from the latex of H. brasiliensis 2 Kush et al. (2) have shown differential expression of several rubber biosynthesis-related genes in latex. The rubber elongation factor (REF), an enzyme involved in rubber biosynthesis (3), is highly expressed in laticifers (4). Laticiferous cells actively translate the transcribed genes into proteins. About 200 distinct polypeptides are present in the latex of H. brasiliensis (5). Arokiaraj et al...

cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Despite potential significance of cis-prenyltransferase in plant growth and development, no gene of the enzyme has been cloned from higher plants. Using sequence information of the conserved region of cis-prenyltransferase cloned recently from Escherichia coli, Micrococcus luteus, and yeast, we have isolated and characterized the first plant cis-prenyltransferase from Arabidopsis thaliana. Sequence analysis revealed that the protein is highly homologous in several conserved regions to cis-prenyltransferases from M. luteus, E. coli, and yeast. In vitro analyses using the recombinant protein overexpressed in E. coli revealed that the enzyme catalyzed the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130 with a predominance of C 120 . The enzyme exhibited a higher affinity for farnesyl diphosphate than for geranylgeranyl diphosphate, with the K m values being 0.13 and 3.62 M, respectively, but a lower affinity for isopentenyl diphosphate, with a K m value of 23 M. In vitro rubber biosynthesis analysis indicated that the Arabidopsis cis-prenyltransferase itself could not catalyze the formation of higher molecular weight polyprenyl diphosphates similar to natural rubber. A reverse transcriptase-polymerase chain reaction analysis showed that the gene was expressed at low levels in Arabidopsis plant, in which expression of the cis-prenyltransferase in leaf and root was higher than that in stem, flower, and silique. These results indicate the tissue-specific expression of cis-prenyltransferase and suggest a potential role and significance of the enzyme in the polyisoprenoid biosynthesis in plants.Prenyltransferase is an enzyme that catalyzes the synthesis of linear prenyl diphosphates involved in the biosynthesis of various isoprenoid compounds, including sterols, carotenoids, terpenes, quinones, glycosyl carrier lipids, prenyl proteins, and natural rubber. Based on the configuration of isoprene units in the final reaction products, prenyltansferases are classified into two classes: trans-and cis-prenyltransferase. In both prokaryotes and eukaryotes, trans-prenyltransferases catalyze the formation of isoprenoid compounds, such as geranyl diphosphate (C 10 ), farnesyl diphosphate (FPP 1 ; C 15 ), and geranylgeranyl diphosphate (GGPP; C 20 ), which serve as initiating molecules to produce many other longer chain length isoprenoid compounds necessary for cellular growth and survival. The structural genes for FPP synthase (1-6) and GGPP synthase (7-13) have been cloned and characterized from various organisms. In addition, the genes for hexaprenyl diphosphate synthase (14), heptaprenyl diphosphate synthase (15), octaprenyl diphosphate synthase (16), solanesyl diphosphate synthase (17), and decaprenyl diphosphate synthase (18) have been cloned. Mutational analyses and x-ray crystallographic investigations o...

Rubber (cis-1,4-polyisoprene), an important raw material for many industrial uses, is synthesized in the latex of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. We postulated that the genes uniquely or preferentially expressed in the latex may be important for rubber biosynthesis. We constructed cDNA libraries from the latex of H. brasiliensis to investigate the genes expressed in the latex by single-run partial sequencing of the cDNA clones. Sequence analyses identified 245 expressed sequence tags (ESTs), of which 57% showed homology to previously described sequences in public databases. About 16% of the database-matched ESTs encode rubber biosynthesis-related proteins such as rubber elongation factor (REF) and small rubber particle protein (SRPP). The second most frequent transcripts next to rubber biosynthesis-related genes were defense genes and protein metabolism-related genes (12.6% each). About 27% of the database-matched ESTs had sequence homology with genes of unknown function. Among the redundantly expressed genes, REF was the most frequently expressed (6.1%), followed by SRPP (3.7%) and HbLAR (2.9%). Northern blot analyses showed that ten (71%) of the 14 ESTs studied were expressed at a higher level in latex than in leaves.

We describe a protocol, and several experiments that helped lead to its development, for sunflower regeneration. Important factors for sunflower regeneration were explant age, cytokinin type and concentration, basal medium, and explant source. We could not induce shoot regeneration from the explants derived from mature tissues including leaf, petiole, and stem. However, use of juvenile explants such as embryo meristem and primordial leaf tissues allowed routine regeneration of 17 different sunflower genotypes. High frequency of shoot regeneration was achieved with these explants taken from seedlings up to 5 d after germination. Explant age was less critical for embryo meristem explants than for primordial leaf tissues. Of the four basal media tested, MS and B5 media produced higher shoot-regeneration frequencies than did Anderson and woody plant media. The highest shoot-regeneration frequency was obtained with MS medium supplemented with 2 mM BA and without auxin. Addition of 1 mM naphthalene-acetic acid to the medium significantly reduced both the percentage of explants producing shoots and average number of shoots per explant. Regenerated shoots were grown to maturity in a greenhouse.

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