No abstract

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.The directed engineering of plant metabolism has been a major focus for both academic and applied plant research in recent years. An enormous amount of work describes the use of transgenic technologies to manipulate genes on important biochemical pathways. In the vast majority of cases, single genes have been manipulated, often by down-regulating their activity using antisense RNA or cosuppression. Although this work has been extremely successful and illuminating, full exploitation of the potential for plant metabolic engineering will likely necessitate the manipulation of multiple genes.We have been researching the lignin biosynthetic pathway for many years and have encountered a number of situations where it would be desirable to manipulate the expression of multiple genes in a coordinate fashion. A major aim of our research is to understand how the lignin biosynthetic pathway operates in vivo so as to elucidate fundamental questions such as the exact sequence and identity of enzymes involved. Although the basic pathway was outlined many years ago, recent data have prompted some revisions and multiple alternative routes have been suggested for the synthesis of certain intermediates. Lignin has significant commercial importance and modified-lignin transgenics can also provide improved raw materials for industrial and agricultural uses. Manipulating combinations of genes involved in monolignol biosynthesis offers the best potential for gaining additional evidence on the organization of the pathway in planta and may give rise to novel, commercially valuable lignins.A small number of reports recently have described the combined suppression of t...

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants.

Phosphoenolpyruvate carboxylases (PEPCs) are encoded by a small multigenic family. In order to characterise this gene family in sugarcane, seven DNA fragments displaying a high homology with grass PEPC genes were isolated using polymerase chain reaction-based cloning. A phylogenetic study revealed the existence of four main PEPC gene lineages in grasses and particularly in sugarcane. Moreover, this analysis suggests that grass C4 PEPC has likely derived from a root pre-existing isoform in an ancestral species. Using the Northern-dot-blot method, we studied the expression of the four PEPC gene classes in sugarcane cv. R570. We confirmed that transcript accumulation of the C4 PEPC gene (ppc-C4) mainly occurs in the green leaves and is light-induced. We also showed that another member of this gene family (ppc-aR) is more highly transcribed in the roots. The constitutive expression for a previously characterised gene (ppc-aL2) was confirmed. Lastly, the transcript accumulation of the fourth PEPC gene class (ppc-aL1) was not revealed. Length polymorphism in non-coding regions for three PEPC gene lineages enabled us to develop sequence-tagged site PEPC markers in sugarcane. We analysed the segregation of PEPC fragments in self-pollinated progenies of cv. R570 and found co-segregating fragments for two PEPC gene lineages. This supports the hypothesis that diversification of the PEPC genes involved duplications, probably in tandem.

Four caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) cDNA clones were isolated from RNA extracted from TMV-infected tobacco leaves using an heterologous DNA probe. The cDNAs were 84-93% identical in their nucleotide sequences, indicating that they are the products of four closely related genes. A comparison of the CCoAOMT cDNAs with database sequences and Southern blot analysis indicated that they are encoded by a new CCoAOMT family of tobacco. Overall expression of this gene family in tobacco tissues was investigated by RNA blot analysis. The expression of each individual gene was studied by RT-PCR coupled with RFLP analysis of PCR products, taking advantage of the presence of specific restriction sites in each cloned cDNA. Two members of the CCoAOMT gene family appeared to be constitutively expressed in various plant organs and tissues whereas the two others were preferentially expressed in flower organs, after tobacco mosaic virus (TMV) infection or elicitor treatment of leaves. The CCoAOMT enzymatic protein expressed in bacteria was purified and shown to be specific for the caffeoyl-CoA and 5-hydroxyferuloyl-CoA esters and to have no activity against free caffeic acid and 5-hydroxyferulic acid. The pattern of CCoAOMT transcript accumulation during development of tobacco stem was found closely related to that of COMT I genes which have been shown to be specifically involved in lignin biosynthesis. Moreover, the inhibition of COMT I gene expression in transgenic tobacco was also shown to decrease CCoAOMT gene expression, particularly in the most lignified tissues. Thus, the expression pattern and the substrate specificity of tobacco CCoAOMT sustain a preferential role in lignin biosynthesis.