Journal of Experimental Botany, Vol. 52, No. 361, pp. 1593-1601,
August 1, 2001
© 2001 Oxford University Press
Review Article |
Using gene knockouts to investigate plant metabolism
Institute of Cell and Molecular Biology, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, UK
Received 5 February 2001; Accepted 23 March 2001
 |
Abstract |
|---|
 Top Abstract IntroductionThe reverse-genetics knockout...Discoveries in primary...Future perspectivesReferences |
|---|
Arabidopsis functional genomics resources now make the isolation of knockout mutants in any gene of choice both realistic and increasingly straightforward. Coupled with the completion of the genome sequence, this reverse genetics approach provides a platform facilitating dramatic progress in our understanding of fundamental aspects of plant metabolism. Recent experience shows that knockouts of genes encoding enzymes of primary metabolism can produce mutants with clear and sometimes unexpected phenotypes. They can provide new information about old pathways. Specific functions for individual members of multigene families can be revealed. Knockouts of enzymes of undefined function can lead to the discovery of those functions, and the analysis of enzymes which have previously never been studied at the biochemical level offers the potential to reveal new pathways of plant metabolism. Furthermore, the mutants isolated provide the starting point for genetic modification experiments to determine exactly how metabolism fuels growth and development, so providing a rational basis for the future modification of plant productivity.
Key words: Arabidopsis thaliana, insertional mutagenesis, gene knockouts, reverse genetics, plant metabolism, plant growth.
|
Introduction |
|---|
|
TopAbstractIntroductionThe reverse-genetics knockout...Discoveries in primary...Future perspectivesReferences |
|---|
Molecular genetic approaches to plant metabolism
Molecular genetic approaches to the study of plant metabolism can be traced back to the isolation of the first cDNA encoding a plant enzyme (Bedbrook et al., 1980
), the use of the Agrobacterium Ti plasmid to introduce foreign DNA into plant cells (Hernalsteens et al., 1980) and the establishment of routine plant transformation systems (Bevan, 1984; Horsch et al., 1985). It became possible to express foreign genes in plants and potentially to overexpress plant genes using cDNAs linked to strong promoters, with the aim of modifying metabolism. However, the discovery of the antisense phenomenon of plant gene silencing (van der Krol et al., 1988; Smith et al., 1988), and subsequently co-suppression (Napoli et al., 1990; van der Krol et al., 1990), provided the most powerful and widely-used methods for investigating the roles of specific enzymes in metabolism and plant growth. The antisense or co-supression of gene expression, collectively known as post-transcriptional gene silencing (PTGS), has been particularly versatile and powerful in studies of plant metabolism. With such molecular tools in place, plant metabolism became accessible to investigation and manipulation through genetic modification and dramatic progress was made in subsequent years (Stitt and Sonnewald, 1995; Herbers and Sonnewald, 1996), particularly in studies of solanaceous species (Frommer and Sonnewald, 1995).
Limitations of gene-silencing approaches
PTGS approaches suffer from a number of shortcomings (Bourque, 1995). Firstly, the effectiveness of gene silencing is variable and not controllable, leading to failure of many experiments. It is only recently that the structural features of transgenes required to achieve effective silencing have been discovered (Smith et al., 2000). Failure to obtain effective gene silencing may also be explained by a requirement for a particular enzyme during plant transformation and regeneration from culture, since the process of making such a transgenic plant would select against effective silencing. A further disadvantage of gene silencing is that its effectiveness may vary between cells or organs, depending upon the gene promoter employed. Even the commonly used CaMV 35S promoter is expressed at very different levels in different cell types (Jefferson et al., 1987; Tang et al., 1999; Wilkinson et al., 1997). PTGS can also be variable or unstable when comparing similar cells of the same tissue (van der Krol et al., 1988; Bourque, 1995) or when comparing different organs on the same plant (Palauqui et al., 1997; Wolters and Visser, 2000). With such potential variablity or instability, there is no opportunity to select mutants in which a phenotype caused by gene silencing is corrected by a mutation in another gene. A further limitation is that such gene silencing methods will potentially target all homologous genes, whether alleles, members of a family of closely-related genes or transgene introduced by re-transformation. The major disadvantage of PTGS is that it is never possible to eliminate an enzyme completely. There are several examples in which the amount or activity of an enzyme has been reduced to an almost undetectable level, without apparent phenotypic or metabolic effect (Gottlob-McHugh et al., 1992; Hajirezaei et al., 1994; Takaha et al., 1998). In such cases firm conclusions are difficult to reach, since it can always be argued that the small amount of residual enzyme is sufficient to fulfil the required role. Even with the newly-developed approach of intron-spliced hairpin formation which gives much more effective gene silencing (Smith et al., 2000), complete gene inactivation in all cells cannot be attained.
The power of gene knockouts generated by insertional mutagenesis
Transposon and T-DNA mutagenesis is now the prefered option for gene silencing in Arabidopsis (McKinney et al., 1995; Krysan et al., 1996, 1999) and increasingly for some other species (e.g. maize and Antirrhinum). With the creation of large populations of plants containing such mutagens (Table 1), and gene identification reaching an advanced stage through completion of the Arabidopsis genome sequence (The Arabidopsis Genome Initiative, 2000), a reverse-genetics screening approach is realistic and effective. Insertion of a transposon or T-DNA into a structural gene (whether into an exon or an intron) will usually disrupt gene expression completely and give a null mutation (see below). This is commonly referred to as a ‘knockout’. In these cases one can have complete confidence that a particular enzyme or enzyme isoform is totally absent from all cells throughout a homozygous plant. Unlike PTGS approaches, insertional mutagenesis allows the recovery of mutations in essential genes, as the plants can be maintained in the heterozygous state. A further advantage is that insertional mutagenesis will target individual genes within a family of closely-related genes, so that functions of individual members can be investigated (see below).
|
In the case of T-DNA and some transposon insertions there is no realistic possibility of reversion to wild type. Such mutants can be used to select second-site suppressors of specific phenotypes, and mutations can be combined through crossing of different mutants to obtain double or multiple mutations. Transgenic reconstruction of null mutants can also be used to generate a set of plants with varying levels of gene expression from zero to more than 100% that of wild type, so that metabolic control analysis (ap Rees and Hill, 1994
) can potentially be carried out. Null mutants can be used as the starting point for gene replacement such that an enzyme can be expressed under the control of a modified or novel promoter, thus giving expression which is inducible or with different spatial or temporal characteristics compared to the original wild-type gene. The subcellular location of an enzyme can effectively be changed by transforming a null mutant with a gene directing transport of the particular enzyme to a different compartment. Genes which encode an enzyme with amino acid sequence modifications can be introduced in order to study post-translational modification of specific enzymes, such as protein transport mechanisms, assembly, phosphorylation and allosteric regulation. At some point in the future such experiments will be carried out most effectively with homologous recombination so that the expression of the modified gene is as similar as possible to the original wild-type allele, but this technology is not yet sufficiently well developed in plants (Mengiste and Paszkowski, 1999).
Disadvantages of the knockout approach
The principal limitation is that it is only possible to investigate enzymes that are known about and for which the corresponding genes have been identified. Up to 40% of Arabidopsis genes of known or predicted function encode enzymes of metabolism or transport, amounting to about 7000 genes (The Arabidopsis Genome Initiative, 2000). However, there may be another 3000 such genes among those of unknown function. A reverse-genetics approach to discovering which of the 7500 genes with no predicted function are involved in metabolism, and the function of these genes, would require a massive systematic programme of mutant isolation and metabolite profiling, with only a limited probabilitiy of discovering gene function. Clearly then, forward genetic screening (also using metabolite profiling) will continue to be very important for the discovery of genes encoding enzymes of metabolism, as will traditional biochemical approaches. Even utilizing numerous different approaches, the function of many genes will remain unknown for many years to come. It is a sobering thought that of the 4300 Escherichia coli genes, about one-third are still of unknown function. A further consideration is that while there is currently a good probability of finding an insert in a target gene, there will be some genes for which insertions will not be found easily (Martinssen, 2000). In such cases, it may be necessary to target the locus by activation of a transposon from a neighbouring (closely-linked) launch pad, or to use the intron-spliced hairpin silencing method of PTGS (Smith et al., 2000). A further major disadvantage of current technology is that a knockout of any enzyme which is essential for gamete formation and function will not be recovered (also a problem for PTGS methods). There is no reliable estimate for the number of genes essential for gamete function, but it could be very significant. Presumably genes encoding enzymes of fundamental importance, such as those of respiratory metabolism, will be required for gamete function. A further complication common to all gene-silencing methods, is that pleiotropic effects such as altered source–sink relationships, may confound functional analysis of the phenotype. Such pleiotropic effects may subsequently be dissected by engineering organ-specific gene expression. At the other extreme, many knockout mutations may be without phenotype because of gene redundancy. It is apparent that Arabidopsis was once tetraploid, and while much of this redundant genetic information has subsequently been lost, duplications of much of the genome remain. Furthermore, there are numerous examples of other gene duplication events, with the result that 70% of Arabidopsis genes are present in more than one copy (The Arabidopsis Genome Initiative, 2000; Martinssen, 2000). However, divergence of gene sequence and function since gene or genome duplication will effectively increase the proportion of single copy genes.
|
The reverse-genetics knockout method |
|---|
|
TopAbstractIntroductionThe reverse-genetics knockout...Discoveries in primary...Future perspectivesReferences |
|---|
Principles
The principle of the reverse-genetics gene knockout approach relies on the general assumption that insertion of transposons and of T-DNA by Agrobacterium, occur at random sites within the plant genome, such that all structural genes are potential targets for insertional mutagenesis. Given a large enough population of transgenic plants, there is a measurable probability that an individual gene will be disrupted. There are two ways to find insertions in a target gene: firstly by analysis of DNA from pools of such plants, and secondly by asking if any of the currently sequenced insertion sites includes that target.
The method for PCR-based screening of DNA pools has been described previously (Krysan et al., 1996, 1999). Essentially it uses combinations of gene-specific and T-DNA or transposon-specific primers to detect an insertion in the target gene. In practice, many non-specific PCR products can be obtained, so Southern blotting and hybridization are necessary to detect the desired fragment. The use of ‘nested’ primers provides supporting evidence for the presence of an insertion in the target gene. DNA sequence analysis of putative junction fragments is then essential, because an appreciable number of ‘false-positives’ pass the first two selection criteria. Having found the desired product it is necessary to screen successively smaller pools until an individual plant is identified. Sometimes the trail ‘goes cold’ and no mutant is found. A variation on this approach is that in which transposon or T-DNA junction fragments are amplified by inverse PCR of pooled DNAs, and then loaded onto a membrane for hybridization with gene-specific probes. The main resources available for such screening methods are summarized in Table 1
. The service provided by the University of Wisconsin is particularly easy to use and has a high success rate. There are now increasing numbers of projects which aim systematically to sequence insertion sites, so that mutant isolation will simply involve interrogating databases and then requesting seed. Such databases currently exist (Table 1) but are still small.
Initial characterization of a mutant
Having isolated an individual knockout mutant plant, it is first necessary to establish if it is homozygous or heterozygous for the insertion. A pair of gene-specific primers spanning the insertion site will give a predictable PCR product from the single wild-type allele in a heterozygous plant, but give no product (or a larger than expected product) from a homozygous plant. If the plant genome contains only a single insert, marker gene inheritance in the subsequent generation could be used to confirm the genotype. However, many populations of transformed Arabidopsis plants contain, on average, more than one T-DNA insertion site per genome, while lines carrying transposons will probably contain multiple independent insertions (Table 1).
To establish if a particular T-DNA insertion mutant contains multiple insertion sites, it is initially helpful to carry out segregation analysis using the selectable phenotypic markers associated with the T-DNA (usually kanamycin, hygromycin or phosphinothricin resistance). It must be remembered, however, that deletions of T-DNA sequences can occur and may result in no, or a non-functional, marker being associated with a particular insert. Southern blotting using a T-DNA probe is also useful for gaining further insight into the number of insertions present. As the left border region of the T-DNA often remain intact, a probe derived from this region is particularly useful. Again, one must be aware that small T-DNA fragments may be missed with this approach. Furthermore, independent mutations not detectable by the methods described above, may have been introduced into the plant by one of several different mechanisms (for example, somaclonal variation or transposon footprinting). Thus, it is always essential to establish the link between target gene insert and phenotype (see below).
If it is clear that there is more than one insert present in the genome, attempts should be made to separate the desired mutation from all others by backcrossing the mutant to wild type. Assuming that the inserts are not linked, F2 individuals in which the additional T-DNA locus/loci has segregated away from that in the target gene can be identified by Southern blotting and checking the marker gene segregation ratio in the F3. However, if the insertion sites are tightly linked, it may not be possible to identify rare recombinants. In this case one would again proceed by establishing the link between genotype and phenotype.
Establishing the link between genotype and phenotype
One way to establish the link between genotype and phenotype is to isolate two or more independent mutants and show that they have the same phenotype (Eastmond et al., 2000a; Gottwald et al., 2000). The probability that both mutants have a second T-DNA or other mutation disrupting the same non-target gene, is so low as to be negligible. Similar arguments and requirements apply to mutants created by transposon mutagenesis. However, in this case, an alternative route to confirming the link between insert and phenotype can come from examples in which the transposon excises and the wild-type phenotype is re-established (Eastmond et al., 2000a). This approach cannot always be relied upon, however, as many excisions will leave a footprint which results in a stable mutation rather than a revertant. Even analysis of independent mutants or revertants does not provide conclusive proof that a particular mutant phenotype is the result of silencing the target gene because, potentially, such insertions could reproducibly disrupt expression of a neighbouring gene(s). This could be checked by Northern analysis of immediate neighbours, but would require transcriptome analysis for a complete picture. A further means of confirming the link between genotype and phenotype is to complement the mutant by transformation with a wild-type cDNA or gene (transgenic complementation). A genomic clone is preferable since it is more likely to reproduce the correct pattern and level of gene expression, and so restore wild-type phenotype, whereas use of the CaMV 35S promoter is likely to lead to ectopic or overexpression. As a general rule, one of the above three methods (multiple independent mutants, revertants or transgenic complementation) is the minimum necessary to establish the link between genotype and phenotype.
Once a mutant has been found, it is necessary to show that the target gene transcript is missing, which can be achieved using Northern blotting, or RT-PCR for increased sensitivity. A full-length cDNA is recommended for use as the probe to reveal any partial or aberrant transcripts that may be produced. The absence of protein product or enzyme activity should also be established. Finally, it is important to show whether knocking out one particular enzyme has any pleiotropic effects on the synthesis or activity of related enzymes, so that any phenotypes observed can be attributed to the mutation in the target gene. This may require assaying a range of enzymes (Eastmond et al., 2000a; Critchley et al., 2001), and will increasingly involve transcriptome analysis on DNA microarrays, or proteome analysis.
|
Discoveries in primary metabolism from the study of knockouts |
|---|
|
TopAbstractIntroductionThe reverse-genetics knockout...Discoveries in primary...Future perspectivesReferences |
|---|
Lipid metabolism and gluconeogenesis
Many seeds, including Arabidopsis, store triacylglycerol (TAG) in lipid bodies and this serves as a carbon source for heterotrophic growth of the seedling. During seed germination and seedling growth this TAG is hydrolysed by lipases and the resultant fatty acids subjected to ß-oxidation in the peroxiosme, producing acetyl CoA for conversion into succinate by the glyoxylate cycle (Fig. 1
). Succinate is transferred to the mitochondrion for conversion to malate and oxaloacetate, which provides the substrate for gluconeogenesis in the cytosol. 3-ketoacyl CoA thiolase is a key enzyme of fatty acid ß-oxidation, and is encoded by at least three genes in Arabidopsis. Remarkably, a knockout of one of these peroxisomal thiolases prevents seedling growth due to a block in ß-oxidation of TAG-derived fatty acids (Hayashi et al., 1998; Germain et al., 2001). This observation demonstrates quite clearly that such fatty acids cannot be metabolized via mitochondrial ß-oxidation and, furthermore, that TAG metabolism is essential to support Arabidopsis seedling growth. Seedling growth can be recovered by supplying exogenous sugar, showing that the TAG is simply providing a carbon and energy supply. Glyoxylate cycle enzymes and PEP carboxykinase are synthesized normally in the thiolase mutant (Germain et al., 2001) showing firstly that fatty acid metabolism is not required to generate a signal to activate expression of their genes, as previously hypothesized (Graham et al., 1992) and, secondly, that gluconeogenesis is apparently still functional. Therefore other gluconeogenic carbon sources are not in sufficient supply in the seed to support seedling growth.
|
Study of a promoter-trapped acyl CoA oxidase gene in Arabidopsis has revealed that this particular enzyme acts preferentially on medium-chain substrates, and is active in seedlings (Eastmond et al., 2000
b). Although the T-DNA insertion eliminates 95% of the medium-chain acyl CoA oxidase activity, TAG metabolism and seedling growth are not affected in the mutant. Acyl CoA oxidases are encoded by at least four genes in Arabidopsis, and although the other three act preferentially on long-chain and short-chain substrates, apparently one or more can substitute for the medium-chain enzyme in vivo. A mutant in which a T-DNA interupts a gene encoding a peroxisomal multifunctional protein has been isolated as a result of a forward screen for inflorescence development mutants (Richmond and Bleecker, 1999). This mutant clearly shows the importance of fatty acid ß-oxidation in reproductive growth. Consistent with this observation, a thiolase knockout also shows modified inflorescence development (S Footitt, JH Bryce, SM Smith, unpublished results). A reverse genetics approach should now lead to determination of the functions of all isoforms of ß-oxidation enzymes throughout growth and development.
In Arabidopsis, the key glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MS) are encoded by single-copy genes. Knockouts of both have been isolated in order to confirm the metabolic role of the glyoxylate cycle and also to determine how important this pathway is to the growth of seedlings (Eastmond et al., 2000a; Germain et al., 2000). The results establish that gluconeogenesis from acetate is drastically reduced in icl mutants, in keeping with our understanding of glyoxylate cycle function. Mutant seedling growth is compromised, but if sufficient light is available for photosynthesis, or if exogenous sugar is provided, seedlings readily grow and develop. Remarkably, even in the absence of the glyoxylate cycle, TAG is still metabolized, particularly if seedling growth is enhanced by exogenous sucrose. The lipid is apparently respired, showing that the carbon can be transferred to the mitochondrion even in the absence of the glyoxylate cycle, and therefore not as succinate. It could potentially be transferred from the peroxisome as either acetate or citrate (isocitrate is not synthesized in the peroxisome because aconitase is cytosolic: Hayashi et al., 1995). If citrate is synthesized in the peroxisome, a supply of oxalaoacetate must be maintained, which effectively means that the TCA cycle operates collectively between three cellular compartments (Fig. 2). It is not known if significant transfer of these substrates takes place in the presence of a functional glyoxylate cycle. A knockout of peroxisomal citrate synthase would establish if acetate can be transported to the mitochondrion. The results discussed here therefore provide very important new information about the metabolic capabilities of peroxisomes and mitochondria in lipid metabolism and raise new questions to be addressed.
|
Starch metabolism
Starch is of critical importance in plant metabolism and yet the details of its synthesis and breakdown are still to be deduced. This aspect of metabolism is directly amenable to analysis through knockout mutants since there is a limited number of enzymes, most of which have probably been identified. Insertions in 11 such genes were screened and knockouts were found for seven (D Thorneycroft, unpublished results). This illustrates the recent success rate in such screening, but also shows that knockouts of all enzymes of starch metabolism is a realistic goal for the near future.
D-enzyme (1,4-
-D-glucan: 1,4--D-glucan: 4--glucanotransferase) is found in a range of plants, but it has not been possible until now to examine its function in planta. Despite eliminating up to 98% of D-enzyme activity using an antisense approach in potato, no effect on starch metabolism was observed (Takaha et al., 1998). An Arabidopsis knockout mutant has now been isolated and shown to have impaired starch metabolism (Critchley et al., 2001). Detailed examination of the mutant revealed a very large accumulation of maltotriose in leaves in the dark, demonstrating that D-enzyme is required for malto-oligosaccharide metabolism. The conclusion from these studies is that leaf starch is degraded in the dark by amylases and starch phosphorylase to maltotriose but no further, and that the glucanotransferase activity of D-enzyme serves to convert maltotriose into larger malto-oligosaccharides upon which these enzymes can act (Fig. 3).
|
Not only can such studies provide information about mechanisms of starch metabolism, but they can also provide insight into metabolic pathways into which starch can feed. For example, it has been postulated that phosphorolytic starch breakdown at night is likely to lead to export of carbon from the chloroplast as triose phosphate, which can in turn fuel respiration, whereas amylolytic breakdown may lead to the export of glucose, which can preferentially fuel sucrose synthesis (Stitt et al., 1985
). A newly-isolated mutant of plastidial starch phosphorylase now provides the opportunity to test directly the function of the phosphorolytic pathway of starch breakdown in leaves (D Thorneycroft, SC Zeeman, AM Smith, SM Smith, unpublished results).
Some enzymes of -glucan metabolism occur in the cytosol of leaf cells, but it is not known if they participate in metabolism of starch or of other substrates (Duwenig et al., 1997; Tacke et al., 1991). There is a cytosolic form of D-enzyme (Okita et al., 1979), and a recently isolated knockout mutant (D Thorneycroft, unpublished results) shows a profound retardation of growth (Fig. 4). This mutant demonstrates the importance of cytosolic -glucan metabolism and provides the means to uncover potentially novel aspects of cytosolic carbohydrate metabolism in plants.
|
Sugar transport
Sugars provide the energy and carbon currencies of the plant, but also act as signalling molecules to regulate gene expression, growth and development (Jang and Sheen, 1997; Smeekens and Rook, 1997). Both sucrose transporters and hexose transporters play key roles in source–sink relationships, and are encoded by multigene families (Lalonde et al., 1999; The Arabidopsis Genome Initiative, 2000). The only realistic way to determine the function of each sugar transporter is through the isolation of knockouts of individual genes. A dramatic demonstration of the importance of the phloem-specific SUC2 sucrose transporter was provided earlier (Gottwald et al., 2000). Knockouts of this gene produce viable plants, but they show retarded growth and sterility. Sucrose is not transported efficiently from shoot to root or inflorescence, and source leaves contain a great excess of starch. These observations demonstrate unequivocally the importance of apoplastic loading of sucrose into sieve elements in Arabidopsis.
In another study, a mutant lacking hexose transporter AtSTP1 (Sherson et al., 2000) has no obvious morphological or growth phenotype, but has significantly impaired hexose uptake in germinating seeds and seedlings, and shows altered responses to exogenous sugars. This mutant provides the first opportunity to investigate the in planta characteristics of such a transporter. While phenotypes should not necessarily be expected in knockouts of individual members of sugar transporter gene families, these results show that they can potentially be revealed by appropriate experiments. Furthermore, creating lines with multiple knockouts by crossing individual mutants is expected to result in more profound phenotypes.
|
Future perspectives |
|---|
|
TopAbstractIntroductionThe reverse-genetics knockout...Discoveries in primary...Future perspectivesReferences |
|---|
The examples discussed here show that the knockout approach to plant metabolism is enormously powerful, and in its infancy. More mutants will rapidly become available and the means to identify knockouts of target genes will become easier. It is also possible to look forward to large collections of activation-tagged lines (Walden et al., 1994
) in which there is the possibility for overexpression of target enzymes with their correct spatial and temporal patterns of expression.
Some unexpected and exciting findings can be anticipated from the analysis of such mutants. For example, knockout of a plastidial phosphoenolpyruvate transporter results in compromised aromatic amino acid synthesis, underexpression of nuclear-encoded photosynthetic proteins, and mesophyll-specific cell defects causing reticulate leaves (Streatfield et al., 1999). Answers to questions that have occupied plant biochemists for many years are now within reach. For example, there has been a long-standing controversy concerning the role of mitochondria in ß-oxidation (Masterson and Wood, 2000), which can finally be resolved. The function of cytosolic enzymes of -glucan metabolism can now be determined. Other issues that can be addressed are the controls of glycolysis and gluconeogenic production of sucrose, the functional redundancy of cytosolic and plastidial pentose phosphate pathway enzymes, and the roles of sugar transporters, sucrose synthase and invertases in determining sink activity. The integration of carbon and nitrogen metabolism is amenable to detailed study. There are also novel areas of metabolism that have not been studied. For example, the discovery in Arabidopsis of citramalate (Fiehn et al., 2000), a potential precursor of pyruvate and acetate, suggests a novel aspect of carbon metabolism. If the net is spread wider to include secondary metabolism, the functions of the many cyctochrome P450 enzymes and glutathione transferases are obvious targets for study. Finally, a cautionary note: it must be remembered that Arabidopsis is not typical of all plants and that it will not help in the understanding of mycorrhizal interactions, nitrogen fixation, C4 and CAM photosynthesis, tuber metabolism, and so on. Equivalent studies in other species are therefore essential.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +44 131 650 5392. s.smith{at}ed.ac.uk
|
References |
|---|
|
TopAbstractIntroductionThe reverse-genetics knockout...Discoveries in primary...Future perspectivesReferences |
|---|
ap Rees T, Hill SA. 1994. Metabolic control analysis of plant metabolism. Plant, Cell and Environment 17, 587–600.
Bedbrook JR, Smith SM, Ellis RJ. 1980. Molecular cloning and sequencing of cDNA encoding the precursor to the small subunit of chloroplast ribulose-1,5-bisphosphate carboxylase. Nature 287, 692–697.
Bevan MW. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12, 8711–8722.
Bourque JE. 1995. Antisense strategies for genetic manipulations in plants. Plant Science 105, 125–149.
Critchley J, Zeeman S, Takaha T, Smith AM, Smith SM. 2001. A critical role for disproportionating enzyme in starch breakdown is revealed by a knockout mutation in Arabidopsis thaliana. The Plant Journal 26, 89–100.
Duwenig E, Steup M, Willmitzer L, Kossmann J. 1997. Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism. The Plant Journal 12, 323–333.[ISI][Medline]
Eastmond P, Germain V, Lange P, Bryce JH, Smith SM, Graham IA. 2000a. Post-germinative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle. Proceedings of the National Academy of Sciences, USA 97, 5669–5674.
Eastmond PJ, Hooks MA, Williams D, Lange P, Bechtold N, Sarrobert C, Nussaume L, Graham IA. 2000b. Promoter trapping of a novel medium chain acyl-CoA oxidase which is induced transcriptionally during Arabidopsis seed germination. Journal of Biological Chemistry 275, 34375–34381.
Fiehn O, Kopka J, Doermann P, Altmann T, Trethewey RN, Wilmitzer L. 2000. Metabolite profiling for plant functional genomics. Nature Biotechnology 18, 1157–1161.[ISI][Medline]
Frommer WB, Sonnewald U.1995. Molecular analysis of carbon partitioning in solanaceous species. Journal of Experimental Botany 46, 587–60.
Germain V, Footitt S, Dieuaide-Noubhani M, Raymond P, Renaudin J-P, Bryce JH, Smith SM. 2000. Role of malate synthase and the glyoxylate cycle in oilseed plants. Plant Molecular Biology Reporter 18, S20–26.
Germain V, Rylott E, Larson TR, Sherson SM, Bechtold N, Carde J-P, Bryce JH, Graham IA, Smith SM. 2001. Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid ß-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. The Plant Journal (in press).
Gottlob-McHugh SG, Sangwan RS, Blakeley SD, Vanlerberghe GC, Ko K, et al. 1992. Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase. Plant Physiology 100, 820–825.
Gottwald JR, Krysan PJ, Young, JC, Evert RF, Sussman MR. 2000. Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters. Proceedings of the National Academy of Sciences, USA 97, 13979–13984.
Graham IA, Leaver CJ, Smith SM. 1992. Induction of malate synthase gene expression in senescent and detached organs of cucumber. The Plant Cell 4, 349–357.
Hajirezaei M, Sonnewald U, Viola R, Carlisle S, Dennis DT, Stitt M. 1994. Transgenic potato plants with decreased expression of pyrophosphate : fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in tubers. Planta 192, 16–30.
Hayashi M, De Bellis L, Alpi A, Nishimura M. 1995. Cytosolic aconitase participates in the glyoxylate cycle in etiolated pumpkin cotyledons. Plant and Cell Physiology 36, 669–680.
Hayashi M, Toriyama K, Kondo M, Nishimura M. 1998. 2,4-dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid acid ß-oxidation. The Plant Cell 10, 183–195.
Herbers K, Sonnewald U. 1996. Manipulating metabolic partitioning in transgenic plants. Trends in Biotechnology 14, 198–205.[ISI][Medline]
Hernalsteens JP, Van Vliet F, De Beuckeleer M, Depicker A, Engler G. 1980. The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells. Nature 287, 754–656.
Horsch RB, Try JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG, Fraley RT. 1985. A simple and general method for transferring genes into plants. Science 227, 1229–1233.
Jang J-C, Sheen J. 1997. Sugar sensing in higher plants. Trends in Plant Science 2, 208–214.
Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions: ß-glucuronidase as a sensitive and versatile gene marker in higher plants. European Molecular Biology Organization Journal 6, 3901–3907.
Krysan PJ, Young JC, Tax F, Sussman MR. 1996. Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport. Proceedings of the National Academy of Sciences, USA 93, 8145–8150.
Krysan PJ, Young JC, Sussman MR. 1999. T-DNA as an insertional mutagen in Arabidopsis. The Plant Cell 11, 2283–2290.
Lalonde S, Boles E, Hellmann H, Barker L, Patrick JW, Frommer WB, Ward JM. 1999. The dual function of sugar carriers: transport and sugar sensing. The Plant Cell 11, 707–726.
Martinssen RA. 2000. Weeding out the genes: the Arabidopsis genome project. Functional and Integrated Genomics 1, 2–11.
Masterson C, Wood C. 2000. Mitochondrial ß-oxidation of fatty acids in higher plants. Physiologia Plantarum 109, 217–224.
McKinney EC, Nazeem A, Traut A, Feldmann KA, Belostotsky DA, McDowell JM, Meagher RB. 1995. Sequence-based identification of T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 and act4-1. The Plant Journal 8, 613–622.[ISI][Medline]
Mengiste T, Paszkowski J. 1999. Prospects for precise engineering of plant genomes by homologous recombination. Biological Chemistry 380, 749–758.[ISI][Medline]
Mettler IJ, Beevers H. 1980. Oxidation of NADH in glyoxysomes by a malate-aspartate shuttle. Plant Physiology 66, 555–560.
Napoli C, Lemieux C, Jorgensen R. 1990. Introduction of chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. The Plant Cell 2, 279–289.
Okita TW, Greenberg E, Kuhn DN, Preiss J. 1979. Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves. Plant Physiology 64, 187–192.
Palauqui JC, Elmayan T, Pollien JM, Vaucheret H. 1997. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. European Molecular Biology Organization Journal 16, 4738–4745.
Richmond TA, Bleecker AB. 1999. A defect in ß-oxidation causes abnormal inflorescence development in Arabidospsis. The Plant Cell 11, 1911–1923.
Sherson SM, Hemmann G, Wallace G, Forbes S, Germain V, Stadler R, Bechtold N, Sauer N, Smith SM. 2000. Monosaccharide/proton symporter AtSTP1 plays a major role in uptake and response of Arabidopsis seeds and seedlings to sugars. The Plant Journal 24, 849–857.[ISI][Medline]
Smeekens S, Rook F. 1997. Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiology 115, 7–13.[ISI][Medline]
Smith CJ, Watson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D. 1988. Antisense RNA inhibition of polygalacturonase genes in transgenic tomatos. Nature 334, 724–726.
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407, 319–320.[Medline]
Stitt M, Wirtz W, Gerhardt R, Heldt HW, Spencer C, Walker D, Foyer C. 1985. A comparative study of metabolit levels in plant leaf material in the dark. Planta 166, 354–364.
Stitt M, Sonnewald U. 1995. Regulation of metabolism in transgenic plants. Annual Review of Plant Physiology and Plant Molecular Biology 46, 341–368.[ISI]
Streatfield SJ, Weber A, Kinsman EA, Häusler RE, Li J, Post-Beittenmiller D, Kaiser WM, Pyke KA, Flügge U-I, Chory J. 1999. The phosphoenolpyruvate phosphate translocator is required for phenolic metabolism, palisade cell development and plastid-dependent nuclear gene expression. The Plant Cell 11, 1609–1621.
Tacke M, Yang Y, Steup M. 1991. Multiplicity of soluble glucan-synthase activity in spinach leaves: enzyme pattern and intracellular location. Planta 185, 220–226.
Takaha T, Critchley J, Okada S, Smith SM. 1998. Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4--glucanotransferase). Planta 205, 445–451.
Tang GQ, Luescher M, Sturm A. 1999. Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning. The Plant Cell 11, 177–189.
The Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.[Medline]
van der Krol A, Lenting PE, Veenstra J, van der Meer IM, Koes RE, Gerats AGM, Mol JNM, Stuitje AR. 1988. An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333, 866–869.
van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR. 1990. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expresssion. The Plant Cell 2, 291–299.
Walden R, Frike K, Hayaishi H, Miklasherichs E, Harling H, Schell J. 1994. Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development. Plant Molecular Biology 26, 1521–1528.[ISI][Medline]
Wilkinson JE, Twell D, Lindsey K. 1997. Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants. Journal of Experimental Botany 48, 265–275.
Wolters A-MA, Visser RGF. 2000. Gene silencing in potato: allelic differences and effect of ploidy. Plant Molecular Biology 43, 377–386.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. AGARWAL and A. GROVER Isolation and Transcription Profiling of Low-O2 Stress-Associated cDNA Clones from the Flooding-stress-tolerant FR13A Rice Genotype Ann. Bot., October 1, 2005; 96(5): 831 - 844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Weckwerth, M. E. Loureiro, K. Wenzel, and O. Fiehn Differential metabolic networks unravel the effects of silent plant phenotypes PNAS, May 18, 2004; 101(20): 7809 - 7814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Fong, J. Y. Marciniak, and B. O. Palsson Description and Interpretation of Adaptive Evolution of Escherichia coli K-12 MG1655 by Using a Genome-Scale In Silico Metabolic Model J. Bacteriol., November 1, 2003; 185(21): 6400 - 6408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Sherson, H. L. Alford, S. M. Forbes, G. Wallace, and S. M. Smith Roles of cell-wall invertases and monosaccharide transporters in the growth and development of Arabidopsis J. Exp. Bot., January 3, 2003; 54(382): 525 - 531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Smith, S. C. Zeeman, D. Thorneycroft, and S. M. Smith Starch mobilization in leaves J. Exp. Bot., January 3, 2003; 54(382): 577 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Zeeman, A. Tiessen, E. Pilling, K. L. Kato, A. M. Donald, and A. M. Smith Starch Synthesis in Arabidopsis. Granule Synthesis, Composition, and Structure Plant Physiology, June 1, 2002; 129(2): 516 - 529. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||