Herbivore-induced, indirect plant defences.

Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D4-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.

/pdf/nicotine-s-defensive-function-in-nature-2uxrz9yhis.pdf

Nicotine's defensive function in nature.

The Solanaceae is a plant family of great economic importance. Despite a wealth of phylogenetic work on individual clades and a deep knowledge of particular cultivated species such as tomato and potato, a robust evolutionary framework with a dated molecular phylogeny for the family is still lacking. Here we investigate molecular divergence times for Solanaceae using a densely-sampled species-level phylogeny. We also review the fossil record of the family to derive robust calibration points, and estimate a chronogram using an uncorrelated relaxed molecular clock. Our densely-sampled phylogeny shows strong support for all previously identified clades of Solanaceae and strongly supported relationships between the major clades, particularly within Solanum. The Tomato clade is shown to be sister to section Petota, and the Regmandra clade is the first branching member of the Potato clade. The minimum age estimates for major splits within the family provided here correspond well with results from previous studies, indicating splits between tomato & potato around 8 Million years ago (Ma) with a 95% highest posterior density (HPD) 7–10 Ma, Solanum & Capsicum c. 19 Ma (95% HPD 17–21), and Solanum & Nicotiana c. 24 Ma (95% HPD 23–26). Our large time-calibrated phylogeny provides a significant step towards completing a fully sampled species-level phylogeny for Solanaceae, and provides age estimates for the whole family. The chronogram now includes 40% of known species and all but two monotypic genera, and is one of the best sampled angiosperm family phylogenies both in terms of taxon sampling and resolution published thus far. The increased resolution in the chronogram combined with the large increase in species sampling will provide much needed data for the examination of many biological questions using Solanaceae as a model system.

/pdf/a-phylogenetic-framework-for-evolutionary-study-of-the-3ynaz5v92j.pdf

A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree

http://dspace.library.uu.nl/bitstream/1874/27851/1/TiPS-Pieterse-2007.pdf

Plant interactions with microbes and insects: from molecular mechanisms to ecology

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2β-hydroxylation: a larger class of C19 GA2oxs and a smaller class of C20 GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C20 GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C20 GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C20 GA2oxs were found to cause less severe GA-defective phenotypes than C19 GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C20 GA2oxs.

A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice.

Research into the genetic basis of the ecological sophistication of plants is hampered by the availability of transformable systems with a wealth of well-described ecological interactions. We present an Agrobacterium-mediated transformation system for the model ecological expression system, Nicotiana attenuata, a native tobacco that occupies the post-fire niche in the Great Basin Desert of North America. We describe a transformation vector and a transformation procedure that differs from the standard cultivated tobacco transformation protocols in its use of selectable markers, explants, media and cultivation conditions. We illustrate its utility in the transformations with genes coding for key enzymes in the oxylipin cascade (lipoxygenase, allene oxide synthase, hydroperoxide lyase) in antisense orientations and present high-throughput screens useful for the detection of altered phenotypes for the oxylipin cascade (green leaf volatiles and jasmonic acid after wounding).

Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: (i) an Agrobacterium-based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

Michelle Lim

Papers

Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system

Solanum nigrum: A model ecological expression system and its tools