Hevein is a chitin-binding protein that is present in laticifers of the rubber tree (Hevea brasilensis

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H202 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a AoxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the AoxyR mutant to grow in the presence of H202. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H202 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H202 stress.

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Hevea brasiliensis is the major producer of natural rubber which is cis-1,4-polyisoprene. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is involved in the biosynthesis of rubber and other plant products. We have used a hamster HMGR cDNA clone as a heterologous hybridization probe to isolate and characterize cDNA and genomic clones of HMGR from H. brasiliensis. Sequence analysis revealed that these clones fall into two different classes, HMGR1 and HMGR2. Comparison of the two classes shows 86% nucleotide sequence homology and 95% amino acid homology. The carboxy-termini of Hevea HMGRs are highly homologous to those of hamster, yeast and Arabidopsis HMGR. The amino-terminus of Hevea HMGR contains two potential membrane-spanning domains as in Arabidopsis HMGR while seven such domains are found in the HMGRs of other organisms. The apparent molecular mass of Hevea HMGR was estimated in western blot analysis to be 59 kDa. Northern blot analysis indicated that the HMGR1 transcript of 2.4 kb is more highly-expressed in laticifer than in leaf. Genomic Southern analysis using 3'-end cDNA probes indicates the presence of at least two HMGR genes in Hevea.

Natural rubber, cis-1,4-polyisoprene, is obtained from a colloidal fluid called latex, which represents the cytoplasmic content of the laticifers of the rubber tree (Hevea brasiliensis). We have developed a method of extracting translatable mRNA from freshly tapped latex. Analysis of in vitro translation products of latex mRNA showed that the encoded polypeptides are very different from those of leaf mRNA and these differences are visible in the protein profiles of latex and leaf as well. Northern blot analysis demonstrated that laticifer RNA is 20-to 100-fold enriched in transcripts encoding enzymes involved in rubber biosynthesis. Plant defense genes encoding chitinases, pathogenesis-related protein, phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, cinnamyl alcohol dehydrogenase, and 5-enolpyruvylshikimate-3-phosphate synthase show a 10-to 50-fold higher expression in laticifers than in leaves, indicating the probable response of rubber trees to tapping and ethylene treatment. Photosynthetic genes encoding ribulose-bisphosphate carboxylase small subunit and chlorophyll a/b-binding protein are not expressed at a detectable level in laticifers. In contrast, genes encoding two hydrolytic enzymes, cellulase and polygalacturonase, are more highly expressed in laticifers than in leaves. Transcripts for the cytoplasmic form of glutamine synthase are preferentially expressed in laticifers, whereas those for the chloroplastic form of the same enzyme are present mainly in leaves. Control experiments demonstrated that ,/-ATPase, actin, and ubiquitin are equally expressed in laticifers and leaves. Therefore, the differences in specific transcript abundance between laticifers and leaves are due to differential expression of the genes for these transcripts in the laticifers.

Seed germination is an important developmental switch when quiescent seed cells initiate oxidative phosphorylation for further development and differentiation. During early imbibition of soybean seeds (Glycine m a L. cv. Weber), a superoxide dismutase (SOD) activity peak was observed, in embryonic axes, after 6 h imbibition. Peroxidase activities, including catalase, were significantly increased after 12 h imbibition and during germination phase 111. Catalase was the most efficient enzyme in catabolizing H,O, in embryonic axes. When stored at 42°C and 100% relative humidity, seeds were stressed and lost their viability in a time-dependent manner. A significant increase in the Cu, Zn-superoxide-dismutase activity, and to a lesser extent, Mn superoxide dismutase activity was observed during germination in low-viability (stressed) seeds as compared to high-viability (unstressed) seeds. Northern blot analysis confirmed that superoxide dismutase induction resulted from an accumulation of its transcripts in response to the production of 0;. The induction of catalase did not occur in low-viability seeds, resulting in dramatic accumulation of H,O,. Using capillary electrophoresis, HPLC and NMR we found that the endogenous cytokinin, zeatin riboside, was present in large quantities in the high-viability seeds, but it was oxidized into adenine in the low-viability seeds. In vitro superoxide anion could also oxidize the cytokinin. Zeatin riboside, but not adenine, was found to act as a scavenger of superoxide anions and may help to maintain seed viability by detoxifying reactive oxygen species. Germination of stressed seeds was partially restored by the addition of exogenous cytokinin (zeatin riboside). Protection against oxidative stress by cytokinin seemed to be a general phenomenon, as Escherichia coli cells were also protected against superoxide stress in the presence of cytokinin.Seed germination can be separated into three distinct phases (Bewley and Black, 1985). In phase I, both oxygen uptake and seed-mass increase; phase I1 is characterized by a metabolic plateau with a stable oxygen uptake and seed mass increase; in phase I11 the germination process is completed with a marked increase in oxygen uptake associated with radicle protrusion. Reactive oxygen species (ROS) arise as by-products of oxidative phosphorylation. ROS are key compounds in the etiology of numerous diseases in human beings (Halliwell and Gutteridge, 1990) and in some stress situations in plants ( Thompson et al., 1987). To limit the damage caused by deleterious oxygen species, aerobic organisms maintain a battery of repair and protective enzymes. These include superoxide dismutase (SOD), which scavenges the toxic superoxide radical, and catalase, ascorbate peroxidase and glutathione peroxidase, which catalyze the decomposition of H,O,. The production of ROS increases under Correspondence to A. Kush,

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.

Severa1 homeotic genes that are involved in floral differentiation have recently been isolated and characterized from Antirrhinum majus and Arabidopsis thaliana (Sommer et al., 1990; Yanofsky et al., 1990). Based on genetic and molecular studies, a model has been proposed to explain the roles of different homeotic genes in the specification of floral organ identity (Bowman et al., 1991). To see whether this model might also apply to Petunia, we initiated a project to isolate Petunia genes that show sequence homology to known floral homeotic genes in Antirrhinum and Arabidopsis.A11 of the floral homeotic genes isolated to date contain a putative DNA-binding domain that shows homology with that of other so-called MADS box proteins (Schwarz-Sommer et al., 1990; Angenent et al., 1992). We used a polymerase chain reaction technique to synthesize a DNA fragment encoding the MADS box region (amino acids 2-93) of Antirrhinum DEF A (Sommer et'al., 1990). This 280-bp polymerase chain reaction fragment was used as a probe to screen a Petunia hybrida W115 petal cDNA library. Twelve independent clones were purified and excised from X Zap I1 according to the manufacturer's instructions (Stratagene). Nine clones were found to differ in their restriction pattems. The nucleotide sequences of two clones (Petunia MADS 1 and 2, Tables I and 11) were detennined using the United States Biochemical Sequenase kit.The nucleotide sequence of the PMADS 1 cDNA is 881 bp in length and encodes a protein with 231 amino acid residues and a predicted molecular mass of 27,022 D. Sequence comparison shows that PMADS 1 is highly homologous to the DEF A gene of Antirrhinum majus (Sommer et al., 1990). The amino acid homology is 93% within the MADS box region (amino acids 3-60) and 77% in the remainder of the protein.Northem blot analysis showed that PMADS 1 is preferentially expressed in the second and third whorl of Petunia flowers (van der Krol et al., 1993). No expression of PMADS 1 was observed in leaves. Consistent with this expression pattern, we found that the PMADS 1 gene is deleted from a green petal mutant that shows a homeotic conversion of petal '