Plant Molecular Biology

Johnson, Lowell; Palmer, Jeffrey

doi: 10.1007/BF00017442pmid: 24272712

Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.

Lubben, Thomas; Gatenby, Anthony; Ahlquist, Paul; Keegstra, Kenneth

doi: 10.1007/BF00017443pmid: 24272713

We have examined the import of a series of chimeric precursor proteins into chloroplasts. These fusion proteins contained the transit peptide, and various amounts of the amino-terminal region of the mature peptide, from the small subunit of ribulose 1,5-bisphosphate carboxylase, linked to the coat protein of brome mosaic virus. Chimeric genes were cloned into SP6 plasmids and in vitro transcription/translation was used to produce fusion proteins, which were examined in a quantitative in vitro import assay. A chimeric protein which contained only the transit peptide fused to the coat protein was imported into chloroplasts. A second chimeric precursor, which also contained a small portion of the mature peptide, was imported at nearly the same rate. A chimeric protein which contained the transit peptide and most of the mature peptide fused to the coat protein was not imported. These results suggest that secondary or tertiary structural features of precursor proteins are important for protein import, and that the presence of a transit peptide in a protein does not necessarily ensure import of that protein into chloroplasts.

Surin, B.; Downie, J.

doi: 10.1007/BF00017444pmid: 24272714

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.

Boulton, Margaret; Buchholz, Wallace; Marks, Melanie; Markham, Peter; Davies, Jeffrey

doi: 10.1007/BF00017445pmid: 24272715

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.

Wenzler, Herman; Mignery, Gregory; Fisher, Linda; Park, William

doi: 10.1007/BF00017446pmid: 24272716

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. To examine the regulation of the patatin genes, we constructed a chimeric gene containing 2.5 kb of 5′ flanking sequence from the class I patatin genomic clone PS20 transcriptionally fused to β-glucuronidase (GUS) and introduced it into potato plants using an Agrobacterium tumefaciens Tiplasmid vector. While the chimeric gene was expressed at high levels in tubers and in stolons attached to developing tubers, it was not normally expressed in leaves, stems, roots, or in stolons before tuberizatization. However, the expression of the class I patatin-GUS construct was not “tuber-specific” since leaf and stem explants cultured on medium containing 300 to 400 mM sucrose showed GUS activity equal or greater than that of tubers. The sucrose induction of GUS activity in leaf and stem explants was accompanied by the accumulation of patatin protein and large amounts of starch, but not by the morphological changes that normally are associated with tuberization. In contrast, the GUS reporter gene under the control of the 35S promoter of cauliflower mosaic virus showed an essentially uniform pattern of expression in transgenic potato plants and was not induced by sucrose.

Peyachoknagul, Surin; Matsui, Tamotsu; Shibata, Hiroshi; Hara, Saburo; Ikenaka, Tokuji; Okada, Yoshimi; Ohno, Takeshi

doi: 10.1007/BF00017447pmid: 24272717

The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a λgtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)+ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.

Spaink, Herman; Wijffelman, Carel; Okker, Robert; Lugtenberg, Ben

doi: 10.1007/BF00017448pmid: 24272718

The flavonoid-inducible nod promoters of Rhizobium are positively regulated by the nodD gene which is highly conserved in various Rhizobium species. The nodD gene are functionally different in (i) their response to various exogenously added flavonoid inducers, (ii) the extent to which they mediate the activation of the flavonoid-inducible promoters, and (iii) the extent to which they repress their own constitutive transcription. In order to localize the regions of the nodD product which determine these differences, two series of nodD hybrid genes have been constructed. In one series the 5′ moiety is derived from the R. meliloti nodD1 gene and the 3′ moiety from the R. trifolii nodD gene. In the other series, the origins of the nodD moieties are reversed. Two regions of the nodD product appeared to be involved in autoregulation and it was also shown that the nodD promoters differ in their susceptibility to autoregulation. Many regions, dispersed over the entire nodD product, are involved in the specificity of activation by flavonoids. Several hybrid nodD genes were characterized which activate transcription with novel inducers. Furthermore, two classes of hybrid nodD genes were found from which the activation characteristics differ completely from those of the parental nodD genes. The first class activates the nodABCIJ promoter to the maximum level in the absence of flavonoid inducer. This level can no longer be influenced, positively or negatively, by the presence of (iso-)flavonoids. With the second class of hybrids, activation of the nodABCIJ promoter, even in the presence of flavonoid inducers, is no longer possible.

Jouanin, Lise; Bouchez, David; Drong, Roger; Tepfer, David; Slightom, Jerry

doi: 10.1007/BF00017449pmid: 24272719

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the “core” overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome.

Thanh, Nguyen; Medgyesy, Peter

doi: 10.1007/BF00017450pmid: 24272720

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×104 colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This “potacco” plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.

doi: 10.1007/BF00017451pmid: N/A