Lipoxygenase is involved in the control of potato tuber development.

Plant lipoxygenases (LOXs) are a functionally diverse class of dioxygenases implicated in physiological processes such as growth, senescence, and stress-related responses. LOXs incorporate oxygen into their fatty acid substrates and produce hydroperoxide fatty acids that are precursors of jasmonic acid and related compounds. Here, we report the involvement of the tuber-associated LOXs, designated the Lox1 class, in the control of tuber growth. RNA hybridization analysis showed that the accumulation of Lox1 class transcripts was restricted to developing tubers, stolons, and roots and that mRNA accumulation correlated positively with tuber initiation and growth. In situ hybridization showed that Lox1 class transcripts accumulated in the apical and subapical regions of the newly formed tuber, specifically in the vascular tissue of the perimedullary region, the site of the most active cell growth during tuber enlargement. Suppression mutants produced by expressing antisense coding sequence of a specific tuber LOX, designated POTLX-1, exhibited a significant reduction in LOX activity in stolons and tubers. The suppression of LOX activity correlated with reduced tuber yield, decreased average tuber size, and a disruption of tuber formation. Our results indicate that the pathway initiated by the expression of the Lox1 class genes of potato is involved in the regulation of tuber enlargement.

A leaf lipoxygenase of potato induced specifically by pathogen infection.

Lipoxygenase (LOX) activity has been identified consistently during pathogen-induced defense responses. Here we report the involvement of a specific leaf LOX gene of potato (Solanum tuberosum), designated POTLX-3 (GenBank/EMBL accession no. U60202), in defense responses against pathogens. The sequence of POTLX-3 does not match any other LOX genes of potato and has the greatest match to a tobacco LOX gene that contributes to a resistance mechanism against Phytophthora parasitica var nicotianae. POTLX-3 transcript accumulation was not detected in untreated, healthy potato organs or in wounded mature leaves. POTLX-3 mRNA accumulation was induced in potato leaves treated with ethylene or methyl jasmonate or infected with either virulent or avirulent strains of Phytophthora infestans, the causal agent of late blight. During the resistance response, POTLX-3 was induced within 6 hours, increased steadily through 24 hours, and its mRNA continued to accumulate for a week after inoculation. In contrast, when a plant was susceptible to P. infestans, induction of mRNA accumulation in response to inoculation was inconsistent and delayed. LOX activity assayed during an incompatible interaction in leaves peaked 3 days earlier than during a compatible interaction. POTLX-3 mRNA accumulation also was induced during hypersensitive response development caused by the incompatible pathogen Pseudomonas syringae pv phaseolicola. Our results show that POTLX-3 may be involved specifically in defense responses against pathogen infection.

A petunia MADS box gene involved in the transition from vegetative to reproductive development.

We have identified a novel petunia MADS box gene, PETUNIA FLOWERING GENE (PFG), which is involved in the transition from vegetative to reproductive development. PFG is expressed in the entire plant except stamens, roots and seedlings. Highest expression levels of PFG are found in vegetative and inflorescence meristems. Inhibition of PFG expression in transgenic plants, using a cosuppression strategy, resulted in a unique nonflowering phenotype. Homozygous pfg cosuppression plants are blocked in the formation of inflorescences and maintain vegetative growth. In these mutants, the expression of both PFG and the MADS box gene FLORAL BINDING PROTEIN26 (FBP26), the putative petunia homolog of SQUAMOSA from Antirrhinum, are down-regulated. In hemizygous pfg cosuppression plants initially a few flowers are formed, after which the meristem reverts to the vegetative phase. This reverted phenotype suggests that PFG, besides being required for floral transition, is also required to maintain the reproductive identity after this transition. The position of PFG in the hierarchy of genes controlling floral meristem development was investigated using a double mutant of the floral meristem identity mutant aberrant leaf and flower (alf) and the pfg cosuppression mutant. This analysis revealed that the pfg cosuppression phenotype is epistatic to the alf mutant phenotype, indicating that PFG acts early in the transition to flowering. These results suggest that the petunia MADS box gene, PFG, functions as an inflorescence meristem identity gene required for the transition of the vegetative shoot apex to the reproductive phase and the maintenance of reproductive identity.

A novel MADS-box gene of potato (Solanum tuberosum L.) expressed during the early stages of tuberization.

A potato MADS-box gene cDNA (POTM1-1) from an early tuber cDNA library has been isolated and characterized. The deduced amino acid sequence of POTM1-1 cDNA encodes 250 amino acids of a putative transcription factor containing a MADS-box domain and a K-box domain. These conserved domains share high homologies to those of flower-specific homeotic proteins, TM4 of tomato and AP1 of Arabidopsis, indicating that POTM1-1 gene is a homologue of the AP1 gene family. The levels of POTM1-1 transcripts were high in axillary buds, underground stolen tips, and newly formed tubers, but relatively low in mature tubers. During axillary bud development in a model petiole-leaf cutting system, the levels of POTM1-1 transcripts were abundant in actively growing shoots and during the early stages of microtuber development. It is possible that POTM1-1 functions as a transcription factor that regulates plant developmental processes in a number of tissue types.

Agrobacterium T-DNA mutation causes the loss of GUS expression in transgenic tobacco.

A new freezer stock of an Agrobacterium tumefaciens clone of LBA4404/pBI121, designated 8999, was found to contain a mutation in the T-DNA region. GUS activity in Agrobacterium 8999 was reduced to levels in negative controls of LBA4404. Additionally, GUS activity in T1 seedlings from tobacco plants transformed with 8999 was reduced to that of untransformed plants. Southern and northern blotting showed that Agrobacterium clone 8999 transferred its T-DNA into the plant, that the correct sizes of 35S promoter and GUS coding region were integrated into the plant's genome in the correct orientation, but that no transcript was detectable after 24 h. Genomic DNA from a T1 seedling from 8999 transformation, digested with HpaII and MspI, indicated no methylation in the promoter region. We conclude from this data that Agrobacterium 8999 has a stable mutation that reduces expression at the mRNA level and is responsible for the lack of GUS expression in plants transformed with this Agrobacterium clone. Therefore, unselected genes within the T-DNA region may suffer mutations in Agrobacterium.

Nucleotide sequences of novel potato (Solanum tuberosum L.) MADS-box cDNAs and their expression in vegetative organs.

Two similar, apparently full-length potato cDNAs, POTM1-1 and POTM1-2, from an early tuber cDNA library have been identified and sequenced. These two cDNAs encode 250 identical amino acids (aa) including 56 aa of the MADS-box domain and 53 aa of the K-box domain, indicating that they may function as transcription factors. The transcripts of these cDNAs accumulate abundantly in vegetative organs, suggesting that these novel MADS genes may be involved in vegetative organ development of potato.

Expression of a cDNA for chloroplast ribosomal protein S16 of potato (Solanum tuberosum L.).

A cDNA encoding the potato (Solanum tuberosum L.) chloroplast ribosomal protein S16 (cp rps16) was isolated and characterized. The deduced amino acid sequence of the chloroplast rps16 coding sequence from potato shows 33% homology to the E.coli ribosomal protein S16 and 95% homology to the tobacco chloroplast ribosomal protein S16. The 13 amino acids encoded by exon-1 are highly conserved (100% identity) among higher plants. Two sizes of rps16 transcripts have been identified from northern blot analysis. The 1.5 kb transcript contains the 855 bp intron and represents an unprocessed RNA molecule, whereas the 0.7 kb transcript represents the mature form. The expression of rps16 is developmentally regulated, with transcript levels decreasing during tuber development and increasing during shoot development. The potato cp rps16 gene appears to be constitutively expressed in unswollen stolons and axillary buds, and its expression is enhanced by light.

Characterization of a cDNA encoding 5-aminolevulinic acid dehydratase in tomato (Lycopersicon esculentum Mill.).

A full-length cDNA clone encoding tomato (Lycopersicon esculentum Mill.) 5-aminolevulinic acid dehydratase (ALAD) was isolated and characterized. The primary structure predicts a 430-amino acid precursor which comprises a 41.7 kDa, 388-amino acid mature protein and a 47-amino acid transit sequence. The tomato primary sequence shows extensive homology to those of pea and spinach. Southern analysis indicated that 1 to 2 copies of the ALAD gene are present in the tomato genome. Northern blot analysis shows differential expression in various tomato organs, and constitutive developmental expression in tomato fruits.

Transformation of Solanum brevidens using Agrobacterium tumefaciens.

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l(-1) kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.

A wound-inducible potato proteinase inhibitor gene expressed in non-tuber-bearing species is not sucrose inducible.

Sequences homologous to a potato cathepsin D inhibitor cDNA, p749, were identified in the genomic DNA of tomato (Lycopersicon esculentum) and of two non-tuber-bearing potato species (Solanum etuberosum and S. brevidens) by means of Southern blot analysis. The expression of these p749 genes in leaves was induced at the RNA level in response to wounding. High levels of p749 transcripts were detected in polyadenylated RNA extracted from locally wounded leaves 12 h after wounding. Systemic induction of the cathepsin D inhibitor gene also occurred in nonwounded leaves of wounded plants. Both potato and tomato leaves treated with the oligosaccharide chitosan showed an induced accumulation of p749 transcripts. Even though the cathepsin D inhibitor genes from tomato and from non-tuber-bearing potato species are wound inducible, they could not be induced in leaf explants cultured on medium containing very high concentrations of sucrose. Only leaf explants from the tuber-bearing potato (S. tuberosum) accumulated p749 transcripts when cultured on high sucrose medium. A sequence related to the 22-kD potato proteinase inhibitor cDNA, p34021, was identified in tomato by means of genomic Southern blot analysis. Northern blot hybridization showed that p34021 transcripts accumulated in potato (S. tuberosum) leaf explants, but not in tomato explants, when cultured on high sucrose medium. This study demonstrates that the expression of a potato cathepsin D inhibitor gene in tomato and in non-tuber-bearing potato species is wound inducible, but not sucrose inducible.

Proteinase-inhibitor activity and wound-inducible gene expression of the 22-kDa potato-tuber proteins.

Using a proteinase-inhibition assay, we have demonstrated that the 22-kilodalton (kDa) potato (Solanum tuberosum L.) tuber proteins are strong inhibitors of serine proteinases. Two out of three purified proteins from the 22-kDa family of potato-tuber proteins were effective inhibitors of both trypsin and chymotrypsin, while the third, with a molecular mass (Mr) of approx. 24 kDa, inhibited only trypsin activity. Comparison of the amino-acid sequence of the putative reactive sites of several proteinase inhibitors with the deduced sequence of the 22-kDa protein showed that the 22-kDa protein contained sequences potentially possessing "doubleheaded" sites of inhibition, one against trypsin and another against chymotrypsin. The genes coding for the 22-kDa proteins were developmentally regulated in tubers and environmentally regulated in leaves. Wound induction of the genes coding for the 22-kDa potatotuber proteins was detected at the RNA level. In leaves, transcripts of the 22-kDa protein family were detected 6 h after wounding and were highest after 12 h in locally wounded leaves. The strongest induction occurred systemically in response to mechanical wounding in non-wounded leaves. Cross-hybridization of a cDNA, p34021, which codes for the 22-kDa tuber protein, with both proteinase-inhibitor I and II cDNAs and with a second family of 20-kDa potato-tuber cDNAs showed no cross-homology. Members of this second group of 20-kDa potato-tuber proteins also exhibited wound-induction in leaves at the RNA level.

Differential expression of potato tuber protein genes.

Patatin and the 22-kilodalton protein complex make up more than 50% of the soluble protein present in potato (Solanum tuberosum) tubers and these two proteins are coordinately regulated during tuber development. Although genomic sequences related to these tuber genes exist in the genome of potato species that do not bear tubers, they cannot be induced into expression under the tested conditions. These genes are not expressed during substantial starch accumulation in petioles from a model petiole-leaf cutting system in nontuber-bearing plants, indicating that starch accumulation and synthesis of the major tuber proteins occur independently. Tuber protein gene expression also has been examined in hybrid potato plants that contain genomes from both tuberizing and nontuberizing species. One such triploid hybrid produced only stolons, whereas a pentaploid hybrid with an increased number of tuber genomes produced tubers. It was shown, using immunoblotting and Northern blot hybridization, that these two hybrids actively expressed both patatin and the 22-kilodalton tuber protein in induced petioles from the leaf-cutting system. The induced accumulation of patatin transcripts was consistent in all genotypes containing some tuberizing genome. The induced accumulation of the 22-kilodalton protein transcripts, however, was lower in genotypes containing some nontuberizing genome. Sucrose induction of these genes in leaves corroborates the induction patterns in petioles. A correlation exists between 22-kilodalton protein gene expression and a potato plant's ability to produce stolons or tubers.

Purification and characterization of the 22-kilodalton potato tuber proteins.

Three abundant proteins of approximate molecular masses of 22, 23, and 24 kilodaltons were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange column chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel-purified 22-kilodalton protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24-kilodalton proteins are immunologically related and that these proteins are present in tubers and as higher molecular mass forms in leaves, but not in stems, roots, and stolons. The ratios of amino acid composition were compared among the three purified proteins, and the aminoterminal amino acid sequences were determined for these three proteins. All three proteins have identical amino-terminal sequences that match the deduced amino acid sequence of an abundant tuber protein cDNA.

Comparison of axillary bud growth and patatin accumulation in potato leaf cuttings as assays for tuber induction.

Single-node leaf cuttings from potatoes (Solanum tuberosum L.) cvs. Norland, Superior, Norchip, and Kennebec, were used to assess tuber induction in plants grown under 12, 16, and 20 h daily irradiation (400 micromol s-1 m-2 PPF). Leaf cuttings were taken from plants at four, six and 15 weeks after planting and cultured for 14 d in sand trays in humid environments. Tuber induction was determined by visually rating the type of growth at the attached axillary bud, and by measuring the accumulation of the major tuber protein, patatin, in the base of the petioles. Axillary buds from leaf cuttings of plants grown under the 12 h photoperiod consistently formed round, sessile tubers at the axils for all four cultivars at all harvests. Buds from cuttings of plants grown under the 16 and 20 h photoperiods exhibited mixed tuber, stolon, and leafy shoot growth. Patatin accumulation was highest in petioles of cuttings taken from 12 h plants for all cultivars at all harvests, with levels in 16 and 20 h cuttings approx. one-half that of the 12 h cuttings. Trends, both in visual ratings of axillary buds and in petiole patatin accumulation, followed the harvest index (ratio of tuber to total plant dry matter), suggesting that either method is an acceptable assay for tuber induction in the potato.

Regulation of Acyl Carrier Protein Messenger RNA Levels during Seed and Leaf Development.

We have examined the expression of acyl carrier protein (ACP) mRNA levels and ACP activity in leaves, where fatty acids function primarily in membrane synthesis, and in developing soybean seeds, where fatty acids are primarily used for oil storage. An RNA probe transcribed from a synthetic spinach ACP-I gene hybridized on Northern blots to ACP mRNA from both seed and leaf tissue from soybean, spinach, and rapeseed. In each species, the ACP transcript from leaf was slightly larger than that from seed. Both the amounts of ACP protein and the levels of ACP mRNA were substantially higher in young leaf tissue of spinach and soybean when compared to mature leaf tissue. Light-grown spinach leaves also contained higher ACP activity and accumulated more ACP mRNA than dark-grown leaves. ACP mRNA levels measured in developing soybean seeds peaked at 20 days after flowering then decreased 10-fold by 70 days after flowering. In each tissue, the developmental changes in ACP protein levels can be accounted for by changes in ACP mRNA abundance. Comparison of the relative prevalence of mRNA and protein for ACP and lectin in soybean seeds suggests a major difference in mRNA translational efficiency and/or protein stability for these two proteins.

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene.

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.

The two classes of genes for the major potato tuber protein, patatin, are differentially expressed in tubers and roots.

The major potato tuber protein, patatin, is a family of 40kd glycoproteins that constitutes forty per cent of the soluble protein in tubers but is generally undetectable in other tissues. Fused rocket immunoelectro-phoresis was used to detect in roots patatin that is immunologically different from tuber patatin. Western blots of SDS-polyacrylamide gels show root patatin to have a different molecular weight distribution than tuber patatin isoforms, but immunoprecipitation of in vitro translation products shows the patatin precursors to be of similar molecular weight in both tissues. This suggests that post-translational processing may differ in tubers and roots. Northern blots show that tuber and root patatin mRNAs are of similar size, but tuber transcripts are about 100-fold more abundant. 5' S1 nuclease and primer extension mapping suggests the class of patatin transcripts expressed in roots (class II transcripts) to be a subset of patatin transcripts expressed in tubers (classes I and II). Class II patatin mRNAs differ from class I transcripts by the presence of a 22 nucleotide insertion just upstream of the initiation codon. These data demonstrate that expression of the patatin multigene family is differentially regulated in tubers and roots.