Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

Identification of laticifer-specific genes and their promoter regions from a natural rubber producing plant Hevea brasiliensis.

Latex, the milky cytoplasm of highly differentiated cells called laticifers, from Hevea brasiliensis is a key source of commercial natural rubber production. One way to enhance natural rubber production would be to express genes involved in natural rubber biosynthesis by a laticifer-specific overexpression system. As a first step to identify promoters which could regulate the laticifer-specific expression, we identified random clones from a cDNA library of H. brasiliensis latex, resulting in 4325 expressed sequence tags (ESTs) assembled into 1308 unigenes (692 contigs and 617 singletons). Quantitative analyses of the transcription levels of high redundancy clones in the ESTs revealed genes highly and predominantly expressed in laticifers, such as Rubber Elongation Factor (REF), Small Rubber Particle Protein and putative protease inhibitor proteins. HRT1 and HRT2, cis-prenyltransferases involved in rubber biosynthesis, was also expressed predominantly in laticifers, although these transcript levels were 80-fold lower than that of REF. The 5'-upstream regions of these laticifer-specific genes were cloned and analyzed in silico, revealing seven common motifs consisting of eight bases. Furthermore, transcription factors specifically expressed in laticifers were also identified. The common motifs in the laticifer-specific genes and the laticifer-specific transcription factors are potentially involved in the regulation of gene expression in laticifers.