Biphasic superoxide generation in potato tubers. A self-amplifying response to stress.

Potato (Solanum tuberosum) cultivars differ quantitatively in their responses to mechanical stress including the ability to synthesize melanin pigments in tuber tissues. Investigations into the cellular events induced by mechanical stress on tuber tissues have shown that an early cellular response is a significant and rapid synthesis of superoxide radicals. This burst of radical production distinctively displays a reproducible biphasic pattern over time with peaks of generation at 2 and 5 h. A concomitant consequence of the generation of these free radicals is elevated levels of oxidatively modified tuber proteins. Both radical generation and protein modification vary between cultivars but both are directly proportional to the amount of melanin pigments produced. Cell-free extracts of mechanically stressed tissues, pectic fragments, and scission products generated from cell walls are able to induce superoxide generation in non-stressed tissues, indicating the participation of a biologically active factor that induces a further a phase of radical synthesis.

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds.

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a 'hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.

Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions.

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6-8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene (PCP1) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea. Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F2 segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus.

SLR3: a modified receptor kinase gene that has been adapted to encode a putative secreted glycoprotein similar to the S locus glycoprotein.

A new member of the S gene family, SLR3 (S-Locus Related 3), was identified in Brassica oleracea. This gene had a novel pattern of expression compared with previously described members of the family, being expressed in petals, sepals and vegetative apices, in addition to stigmas and anthers. Moreover, use of SLR3-derived probes in RNA blot and RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) experiments has identified transcripts of genes closely related to SLR3 in leaves, cotyledons and, at high levels in developing anthers. SLR3 is not linked to the S locus but is linked to two or three closely related genes. Sequence analysis of the SLR3 gene indicates that it is derived from an ancestral receptor kinase gene that has been modified by a series of deletion events. As a result of these modifications, SLR3 is predicted to encode a secreted glycoprotein lacking both transmembrane and kinase domains. The putative SLR3 protein differs from the products of most other S gene family members in that several of the highly conserved cysteines have been lost. Within the S gene family, modification of receptor kinase genes by deletion may represent a general mechanism for the generation of genes encoding secreted glycoproteins.

The molecular basis for N-glycosylation in the 11S globulin (legumin) of lupin seed.

Ion exchange-HPLC under denaturing conditions was used to purify to homogeneity the major M(r) 44,000 alpha subunit of lupin seed (Lupinus albus, L.) 11S storage globulin (legumin). The carboxymethylated subunit was digested with trypsin and the peptide fragments separated by reverse phase HPLC. Only one glycosylated peptide reacting with concanavalin A was identified by dot-blotting. Its amino acid sequence allowed the location of this peptide within a highly conserved region in proximity to the N-terminus of the alpha subunits of the 11S globulins from other seeds. The unique presence of a serine residue in a sequence N-X-S of lupin 11S globulin, compared with all other 11S proteins, allows it to be the only protein of this class to bear covalently linked carbohydrate.

Characterisation of cDNA and genomic clones encoding homologues of the 65 kDa regulatory subunit of protein phosphatase 2A in Arabidopsis thaliana.

Two cDNA species encoding sequences homologous to the 65 kDa regulatory subunit (PR 65) of protein phosphatase 2A (PP2A) have been isolated from an Arabidopsis thaliana cDNA library. These were designated pDF1 and pDF2. pDF1 is 1795 bp long and by comparison with the human and porcine PP2A regulatory subunit sequences represents a full-length clone. It encodes a predicted polypeptide of 587 amino acid residues. pDF2 is truncated at the 5' end by 237 bp. The complete nucleotide sequences have been determined for both cDNA species. Comparison of the nucleotide and the deduced amino acid sequences showed that the two sequences were homologous but not identical and therefore must be derived from two different genes. Northern blot analysis was performed on total RNA and poly(A)+ RNA isolated from seed at various stages of development and from young leaf material of Brassica napus L. (oilseed rape). Both cDNA probes hybridised to a single major mRNA species of ca. 2.2 kb. The highest level of expression was observed in the total RNA from developing rape seed at about 33 days after flowering, and the transcript level in the poly(A)+ RNA of the seed was higher than in young leaf of oilseed rape. Southern blot analysis was performed on two varieties of A. thaliana and B. napus genomic DNA; this identified a small family of genes in A. thaliana consisting of at least 2 or 3 members and a larger multigene family in B. napus of at least 5 or 6 members. Two independent genomic clones were isolated from an A. thaliana genomic library. Sequencing of a fragment common to both revealed that the sequence was identical in both clones and, therefore, they were assumed to contain the same genomic sequence. The genomic sequence selected, designated regA, is 3639 bp long and the coding sequence contains eleven introns. The gene encodes a predicted polypeptide of 590 amino acid residues. The sequence comparison with both cDNA sequences showed that it is homologous but not identical to the two, confirming that at least three different genes exist in A. thaliana which encode PR65 of PP2A.

An S5 self-incompatibility allele-specific cDNA sequence from Brassica oleracea shows high homology to the SLR2 gene.

The sequence is reported of a cDNA molecule homologous to an mRNA from stigma tissue of Brassica oleracea plants homozygous for the S5 self-incompatibility allele. This cDNA is closely related to a previously published sequence designated SLR2, which was obtained from the same stigma cDNA library and is also related to the SLR1 gene, a cDNA for which has also been obtained from this library. Various B. oleracea lines differing in S alleles and of different varieties have been screened for the presence of particular S gene family sequences using a method involving hybridization of sequence-specific oligonucleotide probes to PCR products. The results indicate that a gene homologous to the sequence presented here is absent from a line lacking the S5 allele, though present in other lines containing the S5 allele, regardless of their genetic background. This finding suggests that the sequence represents a transcript of the SLG (S locus glycoprotein) gene. A similar approach has confirmed that a cDNA derived from a Brassica line containing the S29 allele is also S allele-specific. The predicted amino acid sequences derived from a number of S gene family sequences are compared using numerical methods and possible evolutionary relationships between them are discussed.

Expression of a Brassica napus extensin gene in the vascular system of transgenic tobacco and rape plants.

The organs and tissues where the Brassica napus extA extensin gene is expressed have been identified. The extA gene with 3.75 kb of 5' flanking sequence was transferred to tobacco via disarmed Agrobacterium tumefaciens vectors and transgenic plants regenerated. The gene was found to be inactive in transgenic tobacco leaf, but was active as measured by RNA transcript assays in both stem and root tissues. To determine the cell-specific expression pattern of the extA gene, a promoter-reporter gene fusion construct was made consisting of 1.0 kb of 5' extA sequence fused to the coding region of the glucuronidase (GUS) gene. This fusion construct was introduced into B. napus via Agrobacterium rhizogenes, and expression of GUS in transgenic rape hairy roots was examined. GUS activity was only seen in the vascular tissues of the rape root, and was found to be specifically localised in the phloem.

Characterisation of saporin genes: in vitro expression and ribosome inactivation.

A. Saponaria (soapwort) genomic library was screened with a PCR-derived saporin-specific gene probe. The nucleotide sequences of three saporin genomic clones were determined. One of the clones contained a full-length saporin coding sequence whilst the other two were truncated. A hybrid full-length saporin coding sequence was constructed using the two truncated clones. An SP6 promoter sequence and in-frame initiation codon was added to each of the coding sequences using PCR. In vitro translation of saporin coding sequence transcripts in rabbit reticulocyte lysates resulted in the specific depurination of 28S RNA. This indicated that the saporin sequences encoded functional polypeptides with RNA N-glycosidase activity.

The extensin gene family in oilseed rape (Brassica napus L.): characterisation of sequences of representative members of the family.

A family of cross-hybridising cDNA clones has been isolated from a cDNA library produced with poly(A)+ RNA from the roots of oilseed-rape (Brassica napus L.). The clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed. mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 times those in other organs, as shown by Northern blot analysis and RNase protection assays. Complete nucleotide sequence determination of the cDNA clones showed that they encoded proteins homologous to carrot extensin and were the products of at least three different genes. An extensin gene, designated extA, was obtained from an oilseed rape (B. napus L.) genomic library screened with a cDNA species encoding a protein expressed abundantly in roots. The gene is a member of a multigene family, consisting of about 3 members per haploid genome with strong homology to the probe, and a further 20 or so members with weaker homology. The isolated gene, although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species approximately 1,300 nucleotides in size. A single transcription start was identified by S1 mapping. The complete nucleotide sequence of the extA gene and its flanking regions has been determined and shown to encode a protein homologous to carrot and tomato extensins.

Synthesis of saporin gene probes from partial protein sequence data: use of inosine-oligonucleotides, genomic DNA and the polymerase chain reaction.

A strategy employing the polymerase chain reaction to synthesize gene-specific probes suitable for genomic Southern analyses and for screening genomic libraries is described. The method utilizes partial amino acid sequence data from the protein of interest, genomic DNA and inosine-containing oligonucleotide primers. An example of its application for the isolation of plant gene sequences encoding saporin, a ribosome inactivating protein, is described.

A gene from pea (Pisum sativum L.) with homology to metallothionein genes.

While searching for 'organ-specific' genes in pea (Pisum sativum L.) we have isolated a gene (designated PsMTA) which has an ORF encoding a predicted protein with some similarity to metallothioneins (MTs). The PsMTA transcript is abundant in roots which have not been exposed to elevated concentrations of trace metals.

A cDNA encoding an S-locus specific glycoprotein from Brassica oleracea plants containing the S5 self-incompatibility allele.

A cDNA sequence homologous to the Brassica self-incompatibility locus specific glycoprotein (SLSG) sequence was isolated from stigmas of B. oleracea plants homozygous for the S5 allele. The nucleotide sequence of this cDNA was obtained and compared with the S6 allelic form of the SLSG. Evidence is presented which indicates that this sequence does not specify the self-incompatibility response of pollen.

Transcriptional and posttranscriptional regulation of seed storage-protein gene expression in pea (Pisum sativum L.).

At least three classes of legumin, encoded by the gene families legA, legJ and legS, and a lectin, encoded by a single gene, accumulate in the developing cotyledons of Pisum sativum L. Transcription rates for the genes encoding these proteins were measured in nuclei isolated from cotyledons at 12 and 16 days after flowering (DAF). The steady-state levels of the corresponding mRNA species were also measured in absolute terms throughout cotyledon development, from 8-9 to 28 DAF. When transcription rates and steady-state mRNA levels of the different gene families are compared, there is little correlation. This indicates a posttranscriptional regulation of the level of expression of these storage proteins in the developing cotyledons. Expression of the legumin genes is known to be seed-specific, whereas expression of the lectin gene is found in both seed and root. When transcription rates were measured in leaf nuclei the levels of legumin and lectin transcripts detected approached background levels, indicating that these genes are either inactive or transcribed at very low levels in leaves; however, the rate of transcription of the chlorophyll a/b-binding protein gene was high. This points to transcriptional control as the major factor in the organ-specificity of legumin and lectin expression.

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.

Tissue-specific expression of a pea legumin gene in seeds of Nicotiana plumbaginifolia.

A 3.4-kilobase genomic DNA fragment from Pisum sativum L. containing the LegA gene, which encodes a major legumin storage protein, was transferred to Nicotiana plumbaginifolia using an Agrobacterium tumefaciens strain containing the Bin 19 binary vector system. Northern hybridisation analysis of legA-transformed plants demonstrated that legumin-specific RNA was present in developing seeds but not in developing leaves. Legumin protein was immunologically detected in the mature seeds of legA-transformed plants, and was present as the correct-size protein composed of disulphide-bonded polypeptides. It is concluded that the transferred pea genomic fragment contains all the information necessary for seed-specific expression of the legA gene, and for correct processing of the primary transcript and the precursor legumin protein.

The 5'-flanking regions of three pea legumin genes: comparison of the DNA sequences.

Approximately 1200 nucleotides of sequence data from the promoter and 5'-flanking regions of each of three pea (Pisum sativum L.) legumin genes (legA, legB and legC) are presented. The promoter regions of all three genes were found to be identical including the "TATA box", and "CAAT box', and sequences showing homology to the SV40 enhancers. The legA sequence begins to diverge from the others about 300bp from the start codon, whereas the other two genes remain identical for another 550bp. The regions of partial homology exhibit deletions or insertions and some short, comparatively well conserved sequences. The significance of these features is discussed in terms of evolutionary mechanisms and their possible functional roles. The legC gene contains a region that may potentially form either of two mutually exclusive stem-loop structures, one of which has a stem 42bp long, which suggests that it could be fairly stable. We suggest that a mechanism of switching between such alternative structures may play some role in gene control or may represent the insertion of a transposable element.

Sequence of a pseudogene in the legumin gene family of pea (Pisum sativum L.).

A second legumin gene, denoted psi Leg D, has been located on the pea genomic clone lambda Leg 1, approx. 1.3 Kbases 3' of Leg A, in the same orientation. The complete sequence of psi Leg D shows that it is a pseudogene, having two stop codons near the 5' end of its predicted coding sequence, as well as deletions and frame shift errors when compared to Leg A. No transcripts from this gene could be detected in developing pea seeds. Leg A and psi Leg D are homologous over their coding sequences, and partially homologous in the intron sequences and the immediate 5' flanking sequences. Other flanking sequences of the two genes show no significant homology, apart from the presence of polyadenylation signals 3' to both coding sequences. The introns in the two genes occur in corresponding positions in the sequences, but a deletion in psi Leg D affects the 3' boundary of IVS-2. Hybridisation of psi Leg D to pea genomic DNA suggests that it does not represent a hitherto undetected sub-family of legumin genes.