Charles Ainsworth

Leaf senescence in Brassica napus: cloning of senescence related genes by subtractive hybridisation

A subtractive hybridisation technique was developed to clone cDNAs representing genes that showed enhanced expression during leaf senescence in Brassica napus. A number of different genes were identified that, when analysed by northern hybridisation, showed different patterns of expression during leaf development but were all expressed at increased levels during senescence. Sequence analysis of these cDNAs showed that several types of genes were found including two different proteases, glutamine synthetase, ATP sulphurylase, catalase, metallothionein, ferritin and an antifungal protein. The possible roles of these gene products in the senescence process are discussed.

Male and female flowers of the dioecious plant sorrel show different patterns of MADS box gene expression.

Male and female flowers of the dioecious plant sorrel (Rumex acetosa) each produce three whorls of developed floral organs: two similar whorls of three perianth segments and either six stamens (in the male) or a gynoecium consisting of a fertile carpel and two sterile carpels (in the female). In the developing male flower, there is no significant proliferation of cells in the center of the flower, in the position normally occupied by the carpels of a hermaphrodite plant. In the female flower, small stamen primordia are formed. To determine whether the organ differences are associated with differences in the expression of organ identity genes, cDNA clones representing the putative homologs of B and C function MADS box genes were isolated and used in an in situ hybridization analysis. The expression of RAD1 and RAD2 (two different DEFICIENS homologs) in males and females was confined to the stamen whorl; the lack of expression in the second, inner perianth whorl correlated with the sepaloid nature of the inner whorl of perianth segments. Expression of RAP1 (a PLENA homolog) occurred in the carpel and stamen whorls in very young flower primordia from both males and females. However, as soon as the inappropriate set of organs ceased to develop, RAP1 expression became undetectable in those organs. The absence of expression of RAP1 may be the cause of the arrest in organ development or may be a consequence.

Expression, organisation and structure of the genes encoding the waxy protein (granule-bound starch synthase) in wheat

A full-length cDNA clone representing the waxy protein (GBSSI) isolated from a hexaploid wheat developing grain cDNA library has been used to characterise the organisation and expression of the waxy genes in wheat. The genes are organised as a triplicate set of single copy homeoloci on chromosome arms 4AL, 7AS and 7DS. The genes are active throughout grain filling where the main 2.3 kb transcript accumulates to high levels. The 2.3 kb transcript is not expressed in leaves where the presence of a related, but less homologous, transcript of 1.6 kb suggests that a different set of genes operates. Gel analysis and purification of the waxy protein isolated from starch granules, followed by N-terminal amino acid sequencing in conjunction with data from hybrid select translation experiments and sequence analysis of the cDNA, shows that the mature protein has a molecular weight of 60kDa (615 amino acids) and that the preprotein includes a chloroplast/amylopast transit peptide of 7kDa (75 amino acids). Analysis of the derived amino acid sequence and alignment with five other plant waxy proteins shows that they exhibit substantial homology. The wheat protein differs from all others in that it contains an 11 amino acid insertion towards the N-terminus. The protein contains the conserved motif KTGGL found in other waxy proteins and which has been implicated as the active site in glycogen synthase.

Nucleotide sequence of a wheat (Triticum aestivum L.) cDNA clone encoding the waxy protein

The waxy mutation in maize is characterised by the complete loss of amylase in the starch fraction of the kernel [1, 2, 3]. In addition, there is a significant reduction in the amount of granule-bound starch synthase activity in the mutant [2] which correlates with the absence of a protein of 60 kDa, which in wild-type kernels is the most abundant protein associated with starch granules [4, 5]. For this reason, it has been assumed that the 60 kDa waxy protein is the granule-bound starch synthase. Unequivocal evidence for this is lacking and in pea, evidence suggests that another protein, only distantly related to the waxy protein, is the granule-bound starch synthase [6]. It remains to be seen whether this is the case in cereals. The waxy locus has been cloned from a number of plants including maize [ 3 ], rice [ 7 ], barley [ 8 ] and potato [9]. We report here the isolation of a fulllength cDNA clone for the waxy protein of hexaploid wheat and present the complete nucleotide sequence. A cDNA library of was constructed in 2gtl0 from poly(A) + RNA isolated from wheat grains harvested at 20 days post anthesis from the hexaploid variety Chinese Spring. Isolation of RNA, selection of poly(A) + RNA and cDNA synthesis were carried out according to manufacturers protocols (Amersham). After addition of linkers the cDNAs were ligated into the Eco RI site of 2gtl0 and packaged to produce 1.5 x 10 5 recombinants per/~g poly(A) + RNA. The library was screened for waxy protein clones by hybridising plaque lifts with a barley waxy protein cDNA (pcwx27 [4]) and 9 clones with inserts that ranged in size from 0.5 to 4.4 kb were isolated and plaque-purified. The insert from a single cDNA clone of 2.2 kb was subcloned into pUC18 for further analysis since northern blots of wheat RNA hybridised with pcwx27 allowed the waxy protein mRNA size to be estimated at 2.2-2.3 kb (data not shown). The plasmid clone containing the 2.2 kb insert was designated pcSS22. The complete nucleotide sequence of the cDNA insert of pcS

Isolation of RNA from floral tissue ofRumex acetosa (Sorrel)

22 was determined by dideoxy chain termination sequencing [ 10] of Bal 31 deletions and restriction fragments cloned into pUC18. Synthetic oligonucleotide primers to the waxy cDNA were also used. Staden programs were used for sequence assembly [11] and the UWGCG programs for sequence analysis. The

Adenosine diphosphate glucose pyrophosphorylase genes in wheat: differential expression and gene mapping

Flower tissue ofRumex acetosa was previously intractable for the isolation of RNA using standard methods, due probably to its high level of polysaccharides. Extraction at low pH, precipitation of polysaccharides with potassium acetate followed by precipitation of RNA with lithium chloride yielded high-quality RNA that was suitable for northern hybridisation,in-vitro translation, poly(A)+ RNA selection, and subsequent cDNA synthesis.

Isolation and analysis of a cDNA clone encoding the small subunit of ADP-glucose pyrophosphorylase from wheat

A full-length cDNA clone representing the large (shrunken-2) subunit of ADP-glucose pyrophosphorylase (AGP; EC 2.7.7.27) has been isolated from a cDNA library prepared from developing grain of hexaploid wheat (Triticum aestivum L., cv. Chinese Spring). The 2084-bp cDNA insert contains an open reading frame of 1566 nucleotides and primer-extension analysis indicated that the 5′ end is 10 nucleotides shorter than the mRNA. The deduced protein contains 522 amino acids (57.8 kDa) and includes a putative transit peptide of 62 amino acids (6.5 kDa). The similarity of the deduced protein to the small subunit of AGP and to other AGP genes from plants and microorganisms is discussed. Northern hybridisation shows that the Agp1 genes (encoding the small subunit in the wheat endosperm) and the Agp2 genes (encoding the large subunit in the wheat endosperm) are differentially expressed in the wheat grain. Transcripts from both gene sets accumulate to high levels in the endosperm during grain development with the majority of the expression in the endopsperm rather than the embryo and pericarp layers. Although enzyme activity is detected in developing grains prior to 10 d post anthesis, only the Agp1 genes are active at this time (the Agp2 genes are not expressed until 10 d post anthesis). The possibility that the enzyme expressed during early grain development is a homotetramer of small subunits is discussed. The Agp1 and Agp2 genes are arranged as triplicate sets of single-copy homoeoloci in wheat. The Agp2 genes are located on the long arms of chromosomes 1A, 1B and 1D, about 80 cM from the centromere. The Agp1 genes have been mapped to a position just distal to the centromere on the long arms of chromosomes 7A, 7B and 7D.

ISOLATION AND CHARACTERISATION OF CDNA CLONES REPRESENTING THE GENES ENCODING THE MAJOR TUBER STORAGE PROTEIN (DIOSCORIN) OF YAM (DIOSCOREA CAYENENSIS LAM.)

A full-length cDNA clone from hexaploid bread wheat, encoding the small subunit of ADP-glucose pyrophosphorylase, has been isolated from an endosperm cDNA library. The cDNA insert has an open reading frame which encodes a protein of 473 amino acids (52.1 kDa). The presence of a chloroplast/amyloplast transit peptide of 22 amino acids is proposed. The deduced amino acid sequence exhibits a high degree of homology with the small subunit ADP-glucose pyrophosphorylase proteins from rice (with 90% of identical amino acids) and potato (with 86% of identical amino acids) and contains conserved sequence elements which are thought to represent the substrate binding and allosteric activator sites. The genes are organised as single-copy loci on chromosomes 7A, 7B and 7D in the wheat genome and are highly expressed during grain development. Homologous transcripts are expressed in leaves and roots.

Endophytic bacteria expressing β-glucuronidase cause false positives in transformation of Dioscorea species

AbstractcDNA clones encoding dioscorins, the major tuber storage proteins (Mr 32000) of yam (Dioscorea cayenensis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of Mr 30015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

SummaryFalse positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express β-glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.