Two Arabidopsis thaliana genes, KOR2 and KOR3, which encode membrane-anchored endo-1,4-beta-D-glucanases, are differentially expressed in developing leaf trichomes and their support cells.

The Arabidopsis KOR gene encodes a membrane-anchored endo-1,4-beta-D-glucanase involved in cell wall assembly. To obtain a more detailed knowledge of the small gene family encoding membrane-anchored endo-1,4-beta-D-glucanases in Arabidopsis thaliana, we have characterized two additional membrane-anchored endo-1,4-beta-D-glucanase genes. Sequence comparison indicates that KOR2 is distantly related to KOR and other plant membrane-anchored endo-1,4-beta-D-glucanases. The expression of KOR2 and KOR3 was followed by the beta-glucuronidase (gusA) reporter-gene method. While the KOR gene is most often expressed throughout the plant, KOR2::gusA and KOR3::gusA are active only in restricted cell types. We demonstrate that KOR2::gusA is expressed very early in the development of root hairs within the root differentiation zone (specialization zone) but not in the root-hair-bearing epidermal cells at the root/shoot junction (transition zone). Furthermore, KOR2::gusA is expressed in the proximal parts of leaves and floral organs (rosette and cauline leaves, sepals, petals and stamens), and in trichomes, as they develop at the tip of young leaves and later in more basal regions of the leaf blade. The KOR3::gusA construct is expressed in the trichome support cells that form a ring at the base of each trichome and in the bundle sheath cells which surround the vascular bundle within the leaf mesophyll tissue. Reverse transcription-polymerase chain reaction of Arabidopsis RNA confirmed the expression of KOR2::gusA and KOR3::gusA. In conclusion, although KOR2 and KOR3 have more restricted expression patterns than the previously characterized KOR gene, they are expressed in cell types at time points where cell wall assembly is likely to occur and, interestingly, differentially expressed in leaf trichomes and their support cells.

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.

Expression of a membrane-anchored endo-1,4-beta-glucanase from Brassica napus, orthologous to KOR from Arabidopsis thaliana, is inversely correlated to elongation in light-grown plants.

A PCR fragment derived from a membrane-anchored endo-1,4-beta-glucanase cDNA was amplified using degenerated oligonucleotides and mRNA from oilseed rape (Brassica napus L.) siliques. Sequence analysis of the corresponding gene, Cel16, showed that the predicted Cel16 protein has high identity with the Arabidopsis KOR protein (94%). High-stringency genomic Southern analysis further revealed that Cel16 and KOR are most likely orthologous genes performing a similar function in both species. Northern blot and GUS analysis of transgenic Arabidopsis containing a fusion between a 2.0 kb Cel16 promoter fragment and the GUS reporter gene showed that Cel16 was expressed at a low level in the primary raceme, the young lateral stems, the elongation zone of the primary root and the older root base. By contrast, a high level of Cel16 mRNA accumulation was found in the young root and in the main stem carrying flowers and young siliques. Cel16 transcripts were localized to the apical meristem, cambium, primary xylem and cortex of oilseed rape stem tissue by in situ RT-PCR. A similar pattern of activity was found in the GUS analysis of transgenic Arabidopsis. Cel16 mRNA accumulation in the main stem was lower in the zone of most rapid cell elongation than in the subjacent, fully elongated internodes. Similarly, Cel16 transcripts accumulated to a higher level in leaves as they reached full size than during early leaf expansion. Analysis of the expression pattern in elongating, light-grown seedlings showed that Cel16 mRNA accumulated at a lower level in the elongating upper third than elsewhere in the hypocotyl. This is contrary to etiolated hypocotyls, where we found a higher expression level in the rapidly elongating upper part. This difference in expression most probably reflects a difference in cell wall assembly between light- and dark-grown seedlings.

Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase.

Potato tuber pectin is rich in galactan (oligomer of beta-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.

Isolation and characterisation of a pod dehiscence zone-specific polygalacturonase from Brassica napus.

Seven distinct partial cDNAs, similar in sequence to previously described polygalacturonases (PGs), were amplified from cDNA derived from rape pod wall, dehiscence zone and leaves by the polymerase chain reaction. Northern analysis showed that one clone, PG35-8, was expressed at low levels in the dehiscence zone during the first five weeks after anthesis but was very abundantly expressed at week 6. In contrast, no PG35-8-related RNA was detected in the pod wall. Our data suggest that there are temporal and spatial correlations between the breakdown of the middle lamella, of the dehiscence zone cells and the pattern of synthesis of PG35-8 transcripts which may indicate a role for this particular PG in rape pod dehiscence. PG35-8 was used to isolate five cDNA clones from a rape dehiscence zone cDNA library. Restriction enzyme analysis and partial sequencing revealed that they were derived from four highly homologous transcripts which are probably allelic forms of a single gene. One full-length clone, RDPG1, was completely sequenced. The predicted protein of RDPG1 showed its highest identity with PG from apple fruit with an identity of 52%.

A surprising effect of extraction conditions on the mobility of some plant virus coat proteins in SDS-PAGE.

The coat protein from purified particles of pea seedborne mosaic potyvirus (PSbMV) moves in SDS-PAGE with an apparent molecular weight (M(r)) of 36 kDa. However, extracts of PSbMV infected plants prepared with SDS or urea contain PSbMV immunoreactive proteins with apparent M(r) 39 kDa as well as 36 kDa. The low mobility form may be generated from the apparent M(r) 36 kDa form by incubating purified PSbMV particles with healthy plant sap in the presence of denaturing agents. A similar effect is observed with bean yellow mosaic potyvirus, but not with three viruses outside the potyvirus group. Experiments suggest that a soluble plant enzyme is responsible for the conversion, which apparently takes place only in vitro under denaturing conditions. This phenomenon may lead to erroneous conclusions about the M(r) of some viral coat proteins. However, the conversion can be prevented by heat treatment of the plant tissue prior to extraction.

Detection of a 45 kD protein derived from the N terminus of the pea seedborne mosaic potyvirus polyprotein in vivo and in vitro.

A 45 kD protein (Pro1) derived from the N terminus of the pea seedborne mosaic potyvirus (PSbMV) polyprotein has been detected in extracts of infected pea plants and among in vitro translation products of PSbMV genomic RNA. The genomic region coding for the first 231 amino acids of the PSbMV polyprotein was cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase. A rabbit antiserum raised against the fusion protein recognized an approximately 45 kD protein in immunoblots of extracts of PSbMV-infected pea leaves that was not present in extracts of healthy leaves. The highest concentration of the 45 kD protein was found in extracts of young leaves, suggesting the protein may be rapidly degraded in vivo. After in vitro translation of PSbMV genomic RNA in a wheat germ extract, the antiserum immunoprecipitated a 45 kD polypeptide as well as some lower molecular weight translation products. On the other hand, an approximately 90 kD polypeptide was immunoprecipitated from in vitro translation products of genomic RNA in a rabbit reticulocyte lysate, corresponding to the combined molecular weights of Pro1 and the helper component predicted from genomic sequence data.

The 5' untranslated region from pea seedborne mosaic potyvirus RNA as a translational enhancer in pea and tobacco protoplasts.

We have exploited the transient expression of foreign genes introduced into plant protoplasts to investigate the effect of the pea seedborne mosaic potyvirus (PSbMV) 5' untranslated region (5'UTR) on the level of gene expression in pea and tobacco protoplasts. The plant viral 5'UTRs were found to increase translation significantly in comparison to a plasmid containing no 5'UTR of viral origin. The enhancement effect of the 5'UTRs of PSbMV and tobacco etch potyvirus (TEV) was found to be similar in pea and tobacco protoplasts, indicating a host-independent role of the potyviral 5'UTRs in enhancing gene expression. Translational enhancement of the two potyviral 5'UTRs was similar to that of the 5'UTR of tobacco mosaic virus (TMV). This observation makes it attractive to use potyviral 5'UTRs as general translational enhancers in future genetic transformations of plants.

The complete nucleotide sequence of pea seed-borne mosaic virus RNA.

The complete nucleotide sequence of the RNA genome of pea seed-borne mosaic virus (PSbMV) was determined from cloned cDNA and by direct sequencing of viral RNA. The PSbMV genomic sequence was determined to be 9924 nucleotides in length excluding the poly(A) tract. The RNA contained an open reading frame (ORF) of 9618 nucleotides with the potential to encode a polyprotein with a calculated Mr of 364000 (364K). The ORF was flanked by a 5' untranslated leader sequence of 143 nucleotides and a 3' untranslated region of 163 nucleotides. A comparison of the PSbMV polyprotein with the polyproteins of the potyviruses tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV) and potato virus Y (PVY) showed that PSbMV had a similar genome organization. The polyproteins had a high level of amino acid identity except in the N-terminal region, which varied in both sequence and length. Putative proteolytic cleavage sites were identified in the polyprotein of PSbMV by comparison with those identified for other potyviruses. The cleavage site between the 6K protein and the 49K proteinase is proposed to occur at the C-terminal side of glutamic acid and not at the C-terminal side of glutamine as in other potyviruses. In addition to the five proteolytic cleavage sites for the 49K proteinase identified previously, a sixth putative cleavage site was identified internally in the 49K proteinase of PSbMV, as well as in the 49K proteinases of TEV, TVMV, PPV, PVY and soybean mosaic virus. Cleavage at this site in the 49K proteinases of TEV, TVMV and PPV would result in an N-terminal protein of 22K to 24K, which is similar in size to the size determined for their VPgs.

Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene.

cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E. coli. Four overlapping clones that cover the complete PSbMV genome, except the extreme 5' terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing. Overlapping cDNA clones covering 1386 nucleotides of the 3' terminus were sequenced. The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract. The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein.

In vivo expression of the 29000 Mr protein from RNA-2 of pea early browning tobravirus.

The purpose of this work has been to investigate in vivo transcription and translation products from an open reading frame (ORF) located downstream of the coat protein (CP) cistron on RNA-2 of pea early browning virus (PEBV). This work was initiated as a step towards elucidation of the significance of this putative gene. Sequence data on RNA-2 suggest that a 29,600 Mr (29.6K) protein is translated from the ORF in question. Hybridization with ORF-specific probes on Northern blots with RNA from infected plants showed that PEBV synthesizes a subgenomic RNA encoding CP (RNA-2a) with a size of 3000 nucleotides (nt) and that a putative subgenomic RNA (RNA-2b) encoding the 29.6K protein appears to have a size of 1600 nt. This is 300 nt less than the size predicted from the sequencing data. For antibody production, a cDNA fragment harbouring 85% of the 29.6K ORF was cloned into the pUEX3 expression vector. The resulting plasmid expresses the 29.6K protein as a fusion protein with beta-galactosidase and this protein was used for raising antiserum containing specific anti-29.6K protein antibodies. By using these antibodies on immunoblots it was demonstrated that the 29.6K protein is expressed in infected plants.

Physical map of the mitochondrial DNA from the phycomycete Allomyces macrogynus including the position of the ribosomal RNA genes and of an intervening sequence in the large rRNA gene.

The mitochondrial (mt) DNA of the aquatic phycomycete Allomyces macrogynus is a circular molecule with a size of 56.1 kbp. The cleavage sites for the restriction enzymes SalI and PvuI were mapped by comparing the partial denaturation patterns of isolated restriction fragments with the pattern of the intact circle.The genes coding for the small and large ribosomal RNA (rRNA) were located on the restriction map by heteroduplex and R-loop analysis. The gene coding for the large rRNA contains an intervening sequence, app. 0.7 kbp in size, near the 3'-end of the gene. The two rRNA genes are encoded on the same strand of the mtDNA and separated by a region of 17-18 kbp.This rRNA gene organization is similar to that found with members of the Ascomycetes.

Paternal inheritance of the mitochondrial dna in interspecific crosses of the aquatic fungus allomyces.

We have demonstrated by gel analysis of restricted DNA paternal inheritance of mitochondrial DNA in interspecific crosses between A. macrogynus and A. arbuscula.

An electron microscopic analysis of transcription of nucleolar chromatin isolated from Tetrahymena pyriformis.

Transcriptionally active nucleoli and solubilized nucleolar chromatin were visualized by electron microscopy. The palindromic structure of the chromatin was demonstrated by spreading the chromatin on glow-discharged grids. In the presence of single-strand binding EcoHDP protein the preribosomal RNA transcripts are seen attached to the RNA-polymerase molecules in the electron micrographs. Each palindrome contains two preribosomal RNA genes. THe strict termination properties of the transcription are indicated by the absence of transcriptional complexes in the distal parts of the molecules. - Investigation of the process of transcription in nucleolar chromatin being depleted of a termination protein by ammonium sulfate-treatment showed in agreement with biochemical studies that only some of the RNA polymerases terminate properly while other transcribe into the distal spacer region. The elongation rate is estimated to be slightly lower than in the gene region. The results are discussed in relation to biochemical studies of the transcriptional properties of the chromatin.