Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Genetic distance detected with RAPD markers among selected Australian commercial varieties and boron-tolerant exotic germplasm of pea (Pisum sativum L.)

The optimisation of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in pea was investigated and the results were applied to an analysis of five representative Australian varieties and five selected boron-tolerant accessions derived from different geographical regions. Genotypes were compared using 34 random primers (Operon Technologies, Alameda, CA) which generated 180 polymorphic bands. Genetic similarity among genotypes was estimated on the basis of the percentage of common bands between genotypes and a dendrogram was constructed by the unweighted pair grouping method. A pattern of RAPD reaction corresponding to two main groups was discerned. The genetic divergence between Australian varieties and the boron-tolerant accessions suggests an intensive back-crossing programme would be required to transfer boron tolerance to a locally adapted genetic background. Our results show RAPD to be useful for clarifying phylogenic relationships within a species and also to provide useful genetic markers for varietal identification in pea.     

Fate of the nucleolar vacuole during resumption of cell cycle in pea cotyledonary buds

Summary Meristematic cells of pea cotyledonary buds blocked in G_0–1 state contain a small nucleolus with a large central clear area surrounded by a fibrillar rim. The nucleolar structure varies according to the cell cycle from the G_0–1-blocked state until the first mitoses occurring between 24 and 27h after removal of the main stem. In order to better identify and understand the role of the central area in the nucleolar function, its content was investigated by cytochemical and terminal deoxynucleotidyl transferase-immunogold methods. The central area showed the characteristics of a vacuole commonly constituted of the condensed chromatin, ribonucleoprotein granules, and lack of argyrophilic proteins. 3 h after decapitation, a thickening of the fibrillar rim occurred, accompanied by an increase of granules in the vacuole. After 6h, the unique vacuole broke up into two to four small vacuoles in which the granules are more abundant. After 12 h the nucleolus acquired compact structure with few minute vacuoles dispersed over the fibrillar component. During the whole cell cycle, the condensed chromatin is always observed in the vacuole. Our findings suggest that the appearance of the vacuoles is subsequent to the output of preribosomes from nucleolus. These vacuoles might play a role in condensation and decondensation of the chromatin.     

The pea lectin gene family contains only one functional gene

Molecular hybridization experiments have shown that the pea genome contains four regions which hybridize with pea lectin cDNA (Kaminski, Buffard, and Strosberg, 1986. Plant Science 46, 111–116). The complete organization of the pea lectin gene family was investigated. Four partial EcoRI genomic libraries were screened with a lectin cDNA (pPS 15–50) covering the entire coding region. Four positive recombinant phages, I 101, I 52, III 51 and IV 22, were isolated and the DNA sequences of the subclones, designated respectively PSL1, PSL2, PSL3 and PSL4, were determined. PSL2, PSL3 and PSL4 are incomplete genes; the presence of several stop codons in the correct reading frames indicate that these genes cannot code for a functional lectin protein. The sequences of PSL1 and pPS 15–50 have identical coding regions. The pea lectin gene has no intervening sequences and is flanked at its 5 region by a sequence containing an exceptionally high A+T content (73%). Eucaryotic consensus sequences such as a TATA box and a polyadenylation signal are also found in the flanking regions of the PSL1 clone.     

Survival and growth of pea protoplasts after transformation by electroporation

Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.     

Excessive aluminium accumulation in the pea mutant E107 (brz)

E107 is a pleiotropic mutant of peaPisum sativum cv. ‘Sparkle’, characterized by forming few nodules and developing bronze necrotic spots on older leaves. The mutant accumulates Al and has symptoms typical of Al toxicity. The lateral roots of E107 are fewer (40%) and shorter (50%) than those of its parent. High concentrations of Al accumulate in E107 shoots (1000 mg kg^-1) and roots (3000 mg kg^-1), and three-week old E107 plants extrude 2.5 times more protons than ‘Sparkle’ plants of similar age. Al concentrations of the roots of the mutant and of its parent ‘Sparkle’ are similar for the first two weeks of growth. Thereafter they differ. In 2 week old plants Al continues to accumulate in excessive amounts in E107 primary and lateral roots whereas in ‘Sparkle’ roots, it reaches a plateau. In E107, Al is erratically distributed in the walls of root hairs and epidermal cells in both primary and lateral roots. Some of these cells have also Al in their nucleus.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

Location of the replication origin in the 9-kb repeat size class of rDNA in pea (Pisum sativum)

The replication origin of the 9-kb rDNA repeat size class of pea (Pisum sativum cv. Alaska) was identified by benzoylated naphthoylated DEAE-cellulose column chromatography and Southern blotting procedures. The origin is located at or near a 0.19-kb EcoR I fragment in the non-transcribed spacer region between the 25S and 18S rRNA genes. Identification of the origin was based on three criteria: (i) an enrichment of the 0.19-kb fragment in replicating rDNA from asynchronously dividing root meristematic cells, (ii) the scarcity of the 0.19-kb fragment in rDNA from non-dividing carbohydrate starved cells, and (iii) a 60-min periodic enrichment of the 0.19-kb fragment in replicating rDNA that temporally coincides with the sequential initiation of replication of replicon families in synchronized pea root cells.     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M_r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M_r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Studies on the nodulation of strain-specific resistant pea lines using single,mixed and delayed inoculation by Rhizobium leguminosarum

Symbiotic interactions between peas and Rhizobium leguminosarum were investigated by inoculating four pea lines, three of which are strain-specific resistant to the European strain 311d, with various combinations of two strains of Rhizobium, 311d and Tom⁺⁺. The strains were almost equally good to infect the susceptible European cultivar Hero when added singly inoculated. After mixed inoculation (1:1 proportion) strain analysis by ELISA revealed that the nodules were preferentially formed by 311d, although some Tom⁺⁺ nodules were also found mainly on the upper part of the root. Our conclusion is that Tom⁺⁺ is less compatible in comparison with 311d. In addition, we found that as the Hero plants emerged, they were becoming more resistant towards infection with not adapted bacteria. The strain-specific resistant lines from Afghanistan belong to two different systems: Afgh. I, completely resistant to 311d and highly nodulating with Tom⁺⁺, and Afgh. III, incompletely resistant to 311d and poorly nodulating with Tom⁺⁺. Mixed inoculations resulted in nodule depressions, as compared to single inoculations with Tom⁺⁺ ranging from 87% to 14%. The ability of 311d to block infection sites on the roots were found to depend on the degree of symbiotic adaptation between Afgh. I and Tom⁺⁺, respectively Afgh. III and Tom⁺⁺. Strain analysis after double strain inoculation of Afgh. I plants revealed that some nodules were induced by strain 311d. Thus, the presence of Tom⁺⁺ in this case influences the degree of host resistance. However, in Afgh. III plants the resistance towards nodulation were unaffected by the presence of Tom⁺⁺. We suggest that the degree of symbiotic adaptation may change the barrier of resistance towards infection.     

Culture of pea embryo axes for studies on the reactivation of the cell cycle at germination

Summary A method for culturing excised pea embryo axes, which allows growth of large batches of embryo axes in a reduced volume of medium and is suitable for performing biochemical analyses and early treatment or labeling of embryos with exogenous substances, has been devised. Pea embryo axes were excised from the seeds after 3 h of imbibition and cultured in a mineral medium containing sucrose, vitamins, and casamino acids. Under these conditions, the reactivation of the cell cycle occurs much earlier than in intact seeds: with flow cytometry and bromodeoxyuridine labeling, cells in the S phase were found in the root meristems after only 12 h from the beginning of imbibition. The transition of cells from a quiescent to a proliferative state is accompanied by changes in the electrophoretic pattern of nuclear proteins, particularly with regard to two proteins of apparent molecular weight of 90 and 46 kDa, as shown previously in normally germinating seeds.     

The effect of surface soil consolidation on the early root development of pea (Pisum sativum L.)

Cores of repacked soil were consolidated with a compressive strength testing machine, after peas had been planted in the centre of the core. The number that emerged were counted and root and shoot lengths and diameters were measured. Consolidation had no effect on emergence, root length or root diameter of the peas grown in a loamy sand, whereas emergence, root length and root diameter were affected by a small increase in load in a clay loam.     

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.