Plant Molecular Biology

Loopstra, Carol; Stomp, Anne-Marie; Sederoff, Ronald

doi: 10.1007/BF00017719pmid: 1966486

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.

Guerrero, Felix; Jones, Jennifer; Mullet, John

doi: 10.1007/BF00017720pmid: 1715781

Reduction of turgor in pea shoots caused the accumulation of several poly(A) RNAs. cDNA clones derived from three different poly(A) RNAs which accumulate in wilted pea shoots were isolated, sequenced and expression of the corresponding genes examined. Clone 7a encoded a 289 amino acid protein. The C-terminal 180 amino acids of this protein were homologous to soybean nodulin-26. RNA hybridizing to cDNA 7a was abundant in roots, and induced in shoots by dehydration, heat shock and to a small extent by ABA. Hydropathic plots indicate that the protein encoded by cDNA 7a contains six potential membrane spanning domains similar to proteins which form ion channels. Clone 15a encoded a 363 amino acid protein with high homology to cysteine proteases. RNA hybridizing to cDNA 15a was more abundant in roots than shoots of control plants. Dehydration of pea shoots induced cDNA 15a mRNA levels whereas heat shock or ABA treatment did not. Clone 26g encoded a 508 amino acid protein with 30% residue identity to several aldehyde dehydrogenases. RNA hybridizing to cDNA 26g was induced by dehydration of shoots but not roots and heat shock and ABA did not modulate RNA levels. Levels of the three poly(A) RNAs increased 4–6-fold by 4 h after wilting and this increase was not altered by pretreatment of shoots with cycloheximide. When wilted shoots were rehydrated, RNA hybridizing to cDNA 26g declined to pre-stress levels within 2 h. Run-on transcription experiments using nuclei from pea shoots showed that transcription of the genes which encode the three poly(A) RNAs was induced within 30 min following reduction of shoot turgor. One of the genes showed a further increase in transcription by 4 h after dehydration whereas transcription of the other 2 genes declined. These results indicate that plant cells respond to changes in cell turgor by rapidly increasing transcription of several genes. Furthermore, the expression of the turgor-responsive genes varies with respect to the time course of induction and reversibility of the wilting-induced changes.

Hamill, John; Robins, Richard; Parr, Adrian; Evans, David; Furze, Judith; Rhodes, Michael

doi: 10.1007/BF00017721pmid: 2103440

Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.

Cannon, Maura; Platz, Jerry; O'Leary, Maureen; Sookdeo, Cathleen; Cannon, Frank

doi: 10.1007/BF00017722pmid: 2103441

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS ‘gene’ dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially ‘down mutations’ in essential genes where only a short 5′ sequence of the mRNA is required. They also suggest that the ‘position effect’ on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.

Silverthorne, Jane; Wimpee, Charles; Yamada, Takashi; Rolfe, Stephen; Tobin, Elaine

doi: 10.1007/BF00017723pmid: 2103442

The gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase in the monocot Lemna gibba contains approximately twelve members. We have isolated six of these genes from a genomic library, and sequenced five of the coding regions. The transit peptide nucleotide sequences are conserved, but less highly than the mature polypeptide coding sequence. The mature polypeptide amino acid sequences are identical to each other and to the sequence deduced from a cDNA clone derived from a seventh gene. Each of the five fully characterized genomic sequences contains a single intron in precisely the same position as the second intron of several dicots. The intron sequences differ in length and are less conserved than the coding sequences.

Ishibashi, Nobuhiro; Yamauchi, Daisuke; Minamikawa, Takao

doi: 10.1007/BF00017724pmid: 2103443

By differential hybridization screening, we previously selected a class of cDNA clones from a λgt10 cDNA library that was constructed from the total poly(A)+ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39–44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)+ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at development stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at development stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of ‘stored’ mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.

Skuzeski, James; Nichols, Lindy; Gesteland, Raymond

doi: 10.1007/BF00017725pmid: 2103444

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of β-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

Quattrocchio, Francesca; Tolk, Marianne; Coraggio, Immacolata; Mol, Joseph; Viotti, Angelo; Koes, Ronald

doi: 10.1007/BF00017726pmid: 2103445

The activity, tissue specificity and temporal expression of the tandem promoter region preceding a maize zein gene (zE19, encoding a 19 kDa zein protein) were tested in transgenic Petunia plants. To simplify the analysis, the tandem promoter as well as each of the two separate promoter regions were fused to the β-glucuronidase (GUS) reporter gene. All of the three constructs directed the synthesis of GUS in the endosperm of transformed seeds indicating that both separate promoters are independently activated and show the same tissue and cell type specificity observed for zein genes in maize. The kinetics of accumulation and the localization of GUS activity are not coordinated with those of Petunia endogenous seed storage proteins during the development of transformed seeds.

Meer, Ingrid; Spelt, Cornelis; Mol, Joseph; Stuitje, Antoine

doi: 10.1007/BF00017727pmid: 2103446

In order to scan the 5′ flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5′ end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from −67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from −142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (−59 and −52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.

Bennett, D.; Rogers, Sharon; Chen, Liang-Jwu; Orozco, Emil

doi: 10.1007/BF00017728pmid: 1715782

A fifth, previously undetected transcript for the plastid gene encoding the β subunit of spinach chloroplast ATPase (atpB) has recently been identified in vitro. In this report we show that this transcript is present in vivo and its 5′ end is at the translation start codon. When synthesized in vitro using spinach chloroplast RNA polymerase, the 5′ end of this novel transcript is located at the beginning of the second codon of the atpB coding sequence. Although this atpB transcript lacks an untranslated leader region, it is an abundant RNA in vivo and is associated with crude polysomal fractions, as are the four larger atpB transcripts. In vitro synthesis of the leaderless transcript does not occur for a plasmid DNA in which the ‘−35’ region of the promoter is deleted.