Evidence of neutral transcriptome evolution in plants.

* The transcriptome of an organism is its set of gene transcripts (mRNAs) at a defined spatial and temporal locus. Because gene expression is affected markedly by environmental and developmental perturbations, it is widely assumed that transcriptome divergence among taxa represents adaptive phenotypic selection. This assumption has been challenged by neutral theories which propose that stochastic processes drive transcriptome evolution. * To test for evidence of neutral transcriptome evolution in plants, we quantified 18 494 gene transcripts in nonsenescent leaves of 14 taxa of Brassicaceae using robust cross-species transcriptomics which includes a two-step physical and in silico-based normalization procedure based on DNA similarity among taxa. * Transcriptome divergence correlates positively with evolutionary distance between taxa and with variation in gene expression among samples. Results are similar for pseudogenes and chloroplast genes evolving at different rates. Remarkably, variation in transcript abundance among root-cell samples correlates positively with transcriptome divergence among root tissues and among taxa. * Because neutral processes affect transcriptome evolution in plants, many differences in gene expression among or within taxa may be nonfunctional, reflecting ancestral plasticity and founder effects. Appropriate null models are required when comparing transcriptomes in space and time.

Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae.

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

Integration of environmental and host-derived signals with quorum sensing during plant-microbe interactions.

Many plant-associated microbes use secreted autoinducer molecules, including N-acylhomoserine lactones (AHLs), to regulate diverse behaviours in association with their population density (quorum sensing). Often, these responses are affected by environmental conditions, including the presence of other AHL-producing bacterial species. In addition, plant-derived metabolites, including products that arise as a direct result of the bacterial infection, may profoundly influence AHL-regulated behaviours. These plant products can interact directly and indirectly with the quorum-sensing network and can profoundly affect the quorum-sensing behaviour. Local conditions on a microscopic scale may affect signal molecule longevity, stability and accumulation, and this could be used to give information in addition to cell density. Furthermore, in many Gram-negative bacteria, AHL signalling is subservient to an additional two-component signalling system dependent upon homologues of GacS and GacA. The signal(s) to which GacS responds are not known, but recent research suggests that a self-produced ligand may be being detected. This review will focus on two well-studied examples of AHL-regulated plant-associated behaviour, Erwinia carotovora and Agrobacterium tumefaciens, to illustrate the complexity of such signalling networks.

Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model.

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission.

Using the Arabidopsis ethylene receptor ETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical to ETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomato Never ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.

Nucleotide sequence and expression of a ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins.

The nucleotide sequence and derived amino acid sequence were determined for a full-length version of the tomato cDNA clone, pTOM75, the mRNA for which has previously been shown to accumulate in roots, ripening fruit and senescing leaves. Computer analysis of the predicted protein product, which we have named tomato ripening-associated membrane protein (TRAMP) indicates strong homology to known transmembrane channel proteins from other organisms. Northern analysis showed that this gene was induced by waterstress and that this induction was unaffected in an ABA-deficient genetic background.

Molecular genetics of tomato fruit ripening.

Tomato ripening is an excellent system for studying control of gene expression in plants. A multiplicity of well-defined biochemical and genetic changes occur in a precise sequence, regulated by a gaseous hormone. The generation of targeted mutations using sense and antisense genes provides a means of manipulating endogenous gene expression, both for answering fundamental questions and for crop improvement.

Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct.

Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5' end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectin-esterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit. The paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.

Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression.

A tomato phytoene synthase gene, Psy1, has recently been isolated as the clone GTOM5 and shown by sequence identity to be the gene from which the major fruit-ripening cDNA clone TOM5 was derived. Sequence analysis of transcripts from two allelic yellow-fruited tomato mutants, mapped to chromosome 3, has shown the lack of carotenoids in fruit of these mutants to be due to the production of aberrant TOM5 transcripts which are unlikely to encode a functional phytoene synthase enzyme. In one mutant (yellow flesh) the aberrant transcript contained a sequence that, by its strong hybridization to a wide size range of genomic fragments, appeared to be repeated many times within the genome. Southern and PCR analysis of the phytoene synthase genes in the mutant revealed restriction fragment length polymorphisms, suggesting that the production of altered mRNAs was associated with specific genomic rearrangements. Constitutive over-expression of a TOM5 cDNA clone in transgenic mutant plants restored synthesis of the carotenoid lycopene in ripening fruit and also led to unscheduled pigment production in other cell types. In some mutant plants transformed with the TOM5 cDNA construct, inhibition of carotenoid production in immature green fruit, leaves and flowers was observed, due to the phenomenon of co-suppression, indicating that different insertion events with the same gene construct can lead to overexpression or co-suppression in transgenic plants. Green organs of these plants were susceptible to photobleaching, due to the lack of carotenoids. These results suggest the existence of separate Psy genes for carotenoid synthesis in green organs.