ComBio: teasing out the tasty tomato trichome

A truss of vine-ripened tomatoes advertises itself with an earthy perfume that says “fresh” better than any words. It emanates from tiny hair-like projections on the stems of the fruit, called trichomes.

Trichomes adorn many species in the tomato-potato family, the Solanaceae, and function as biochemical factories that synthesise and variously secrete or store a huge variety of defensive compounds to deter leaf-chewing insect pests and pathogens.

Around a third of the world’s vascular plants have trichomes – they have evolved independently in multiple families. Some of the best known are the aromatic mint family, the Lamiaceae, of rosemary and thyme fame; the Boraginaceae (comfrey, borage, echium), beloved of herbalists; and the Geraniaceae, whose leaves emit scents redolent of roses, apples, lemons, oranges, cinnamon and peppermint.

Trichomatous plants fascinate Professor Rob Last, a biochemist at Michigan State University and a lead researcher in the Solanum Trichome Project. Last will give a plenary lecture on the project at ComBio 2008 in Canberra from September 21 to 25.

The Solanum Trichome Project is focused around the tomato, Solanum lycopersicon, and four of its wild relatives from the same region of the Andes where the tomato was domesticated. Solanum habrochaites, S. pennellii, S. pimpinellifolium, and S. peruvianum have all contributed genes to the multi-hued cornucopia of tomatoes available to consumers today.

The project is employing a genetic approach to investigate trichome development and the biochemical pathways that operate in them.

Tomatoes were selected for their convenience as research subjects, rather than for potentially exciting biochemistry. “It’s not that tomatoes have the most interesting secondary or specialised chemistry – it that was our interest, we would have picked artemisins,” Last says.

[The genus Artemisia (Asteraceae) includes wormwood, source of the once popular but toxic liqueur absinthe. The traditional Chinese pharmacopoeia has long recognised wormwood’s anti-malarial properties; Western researchers are now interested in artemisin as a model for a new class of antimalarial drugs].

Tomatoes were the subject of choice because the Tomato Genome Project is making good progress, the emerging chromosomal map and gene catalogue simplifies the task of identifying the genes involved in synthesising the compounds in trichomes, and dissecting the metabolic pathways that generate them, he says.

The five Solanum species chosen for the trichome project are conveniently compatible, and their hybrids produce viable seeds. Researchers can study patterns of inheritance, and import genes of interest. Tomatoes are also amenable to experiments involving substituting chromosomal segments, importing transgenes, or silencing genes in situ.

---PB--- Tender tomatoes

Another attraction of tomatoes is that their trichomes are abundant and readily harvested from the surfaces of leaves and stems.

“We’re interested in the chemistry of the different types of trichome within each species,” Last says. “One species may have three or four types of trichomes, including both glandular and secretory forms.

“They are very different morphologically. Some are long and slender, others are shorter and broader, while others carry terminal four-celled rosettes. We know that the morphological differences correspond to biochemical differences, and we’re trying to determine the compounds synthesised by each.

“We’re also comparing the chemistry of equivalent trichome types across species – for example, how does the chemistry of secretory trichomes vary between S. lycopersicon, S. pimpinellifolium and S. peruvianum?”

Last says the analyses begin with identifying the various classes of compounds in each species, and in each trichome type in that species.

“We then go in and make libraries of expressed-sequence tags (ESTs) and cDNAs from the different trichomes in each species.

“We’re using 454 rapid sequencing, and we’re identifying the genes that are highly expressed at the messenger RNA level in, for example, type 6 glands and type 1 glands in the edible tomato versus the wild species. Are differences in chemistry reflected in differences in gene expression?

“If so, is a particular class of metabolites present, absent or more or less abundant in one species versus another?

“Then we put on our hats as biochemists and ask what sort of enzyme differences may contribute to the differences in chemistry. Do we see ESTs that are related to known examples of those enzymes, and does their expression correlate with the chemistry we see?

“We like to think of what we do as molecular biochemistry. The metabolonomic approach seeks to understand the specialised metabolites present in the different glands of each species.”

---PB--- Cool compounds

Last’s collaborators are coupling high tech genomics approaches with variations on traditional biochemical genetics in their search for new biochemical pathways.

Israeli geneticist Dr Dani Zamir, of the Hebrew University of Jerusalem, has used advanced plant breeding techniques to transfer chromosome segments carrying hundreds of genes from S. pennellii into cultivated tomatoes.

“Dani and colleagues have created 50 or 60 substitution lines, each carrying a different array of genes from a wild tomato. It means we can determine whether any genes on a particular piece of chromosome segment modify the chemistry of the trichomes.

“It’s a really fabulous resource that allows us to search for totally novel genes that cannot be discovered by analysing cDNA sequences.

“We can screen for any phenotype we’re interested in, such as fruit shape and colour, or changes in different classes of metabolites.

“We’re particularly interested in terpenes, acyl sugars and flavonides, as well as compounds whose structures haven’t been solved yet.

“We look for differences in compounds between the chromosome-substitution lines and parental lines. It’s very cool. We know where the altered traits map to, genetically, because the substituted chromosome segments have been well characterized by Zamir and others.

“If we see an altered phenotype, we can narrow the gene involved down to something as small as a tenth of a chromosome and treat it as a Mendelian trait.”

Where there is a change in the molecule accumulated in one trichome class, it is always to a phenotype expressed by one or the other parent. So far they have not found instances of intermediate or novel chemistry, but they’re on the lookout for new and potentially useful compounds.

“Where we see quantitative differences between parent and hybrid lines, it’s difficult to tell whether it’s due to real genetic interactions, or to environmental influences. Synthesis of these compounds tends to vary in response to environmental cues such as light levels and insect attack.”

The two major goals of the Solanum Trichome Project are to take advantage of the fact that trichomes are highly specialised factories for secondary metabolites, and ultimately, knowledge of the genes and synthesis pathways involved will enable them to be used to synthesise useful compounds.

“In a draft of a recent paper we talked about the glands being very single-minded – one reviewer didn’t like us anthropomorphising, but that’s pretty much how trichomes work: one compound per trichome.”

With the great diversity of plants with trichomes, the different classes of trichomes in each species and their different metabolite chemistry, Last says he has a fair idea what he will be doing for the remaining 15 years of his research career.