Homeobox genes contain sequences coding for DNA-binding motifs. These sequences are highly conserved across both the animal and plant kingdoms. Members of this gene family code for transcription factors that are key regulators of developmental organization. In an attempt to further elucidate the developmental process of tuberization in the potato plant, a full-length homeobox cDNA has been isolated via sequence homology from an early tuberization stage cDNA library constructed from 4-day axillary bud tubers. This cDNA, POTH1, has been sequenced and characterized by Southern blotting, northern analysis, sequence comparison, and in situ hybridization. POTH1 is shown to be a class I homeobox gene with 45% overall similarity to Kn-1 of maize and 73% match in the homeobox region. Messenger RNA accumulation studies indicate that POTH1 mRNA, unlike most homeobox transcripts, is not limited to a particular organ or developmental stage. Instead, POTH1 mRNA accumulates in rapidly growing cells of the potato plant: the apical meristems, the vascular cambium, the edges of young leaves, axillary buds, and root tips. In situ studies indicate accumulation of POTH1 mRNA in the tunica and corpus layers of the apical dome of the shoot apex and the stolon apex. In the stolon, growth and proliferation of the parenchymal cells associated with the vascular cambium contribute to swelling during early stages of tuberization, and this tissue accumulates POTH1 mRNA. It is possible that POTH1 may be posttranscriptionally regulated in a particular organ or stage of growth, or that it is involved in a wider range of growth processes than most plant homeobox genes.
Jennifer K. Hart and David J. Hannapel
Gary F. Polking, David J. Hannapel, and Richard J. Gladon
Our recent research has focused on the control of genes and enzymes involved with the synthesis of chlorophyll, especially as it relates to tomato fruit development and ripening. Glutamate-1-semialdehyde-2,1-aminomutase (GSAAM) is one of the first committed enzymes in the chlorophyll biosynthetic pathway, and it is one of three enzymes that catalyze the conversion of glutamate into 5-aminolevulinic acid. We have isolated a full-length cDNA clone of GSAAM from a tomato fruit library. The tomato primary sequence shows extensive homology to GSAAM sequences found in other plant species. The primary structure also predicts a 46.7-kDa, 437-amino acid, mature protein and a transit peptide of 44 amino acids. Southern analysis indicated that GSAAM was present as a single copy. Northern blot analysis showed that GSAAM was expressed differentially in various tomato organs and that GSAAM transcripts decreased with increased fruit age. Immunoblot analysis also indicated that GSAAM protein decreased dramatically with increased fruit age. These results show that there is developmental regulation of the expression of GSAAM in tomato fruits.
Marios C. Kyriacou, David J. Hannapel, and Richard J. Gladon
Tomato fruit ripening is characterized by a decrease in chlorophyll content and an increase in lycopene synthesis. 5-Aminolevulinic acid (ALA) dehydratase (ALAD) is the fruit committed enzyme in the chlorophyll and heme biosynthetic pathways, and it catalyzes the dimerization of two ALA molecules into porphobilinogen We have focused our attention on the potential pivotal role of ALAD in the developmental regulation of chlorophyll biosynthesis during tomato fruit growth, development, and ripening. We have standardized an assay procedure for measuring the enzymatic activity of ALAD in tomato fruit tissues. The activity of ALAD was assayed from ten days past anthesis to day 60, when fruits where void of chlorophyll. We observed a several-fold decline in ALAD activity to residual levels during fruit ontogeny. Our data also show greater ALAD activity in chlorophyllous organs (leaves, stems, immature fruits) than in nonchlorophyllous organs (roots, ripe fruits), where heme production is predominant.
Gary F. Polkinz, David J. Hannapel, and Richard J. Gladon
Tomato fruit ripening is characterized by a decrease in chlorophyll content and an increase in lycopene synthesis. We are interested in the role of chlorophyll metabolism as it relates to tomato fruit ripening. 5-Aminolevulinic acid dehydratase (ALAD) is the first committed enzyme in the chlorophyll biosynthetic pathway, and it catalyzes the conversion of two 5-aminolevulinic acid molecules into porphobilinogen. We have isolated a full-length tomato ALAD cDNA clone from a tomato fruit library. Sequence analysis showed that this tomato ALAD was highly homologous to ALAD found in spinach and pea, and the analysis predicted a protein of 46.8 kDa. Southern analysis indicated that 1 to 3 copies of the ALAD gene are present in the tomato genome. Northern analysis suggested that the gene is expressed constitutively throughout tomato fruit development. Currently, we are subcloning the fragment into an E. coli expression vector in order to obtain protein for antibody production for Western analysis.
Mikhailo V. Kolomiets, Richard J. Gladon, and David J. Hannapel
Due to apparent participation of plant lipoxygenases (LOXs) in the biosynthetic pathways for jasmonic acid, methyl jasmonate, traumatin, and several C-6 volatile compounds, LOXs are believed to have a role in senescence, plant growth and development, and wound- and pathogen-induced defense responses. Multiple functions that are ascribed to this enzyme family are in accordance with the heterogeneity of LOX isozyme forms. It is possible that different LOX isoforms may be involved in different physiological processes. In our search for a gene that encodes a LOX isozyme form specifically involved in potato defense responses against pests and pathogens, we have screened an abscisic acid-induced potato leaf cDNA library, and we have isolated, sequenced, and characterized a cDNA clone that we have designated POTLX-3. The high sequence homology of our cDNA clone to other reported plant LOX genes provided evidence that POTLX-3 is a lipoxygenase. This cDNA clone represents a novel potato LOX gene in that it shares the least nucleotide and amino acid sequence homology to other isolated potato LOX genes. Northern analysis indicated that POTLX-3 transcripts did not accumulate in untreated potato leaves, but it was highly induced by treatment with physiological levels of ethylene. Northern analysis also was performed to study whether the POTLX-3 mRNA accumulation could be induced by other plant hormones that affect expression of the other plant LOX and defense-related genes. Treatment of potato leaves with methyl jasmonate, abscisic acid, gibberellic acid, auxin (NAA), and cytokinin (BA) did not induce POTLX-3 gene expression. Because the pattern of POTLX-3 gene expression is similar to that of pathogenesis-related (PR) proteins, especially the PR-1 and PR-5 groups, we suspect that POTLX-3 may be involved specifically in ethylene-induced defense responses against pathogens.
Mikhailo V. Kolomiets, David J. Hannapel, and Richard J. Gladon
Plant lipoxygenases (LOXs) (linoleate:oxygen oxidoreductase, EC 22.214.171.124) catalyze the oxygenation of polyunsaturated fatty acids such as linolenic and linoleic acids. Some of the final products of LOX-catalyzed reactions are traumatin, jasmonic acid (JA), methyl jasmonate (MJ), and C-6 volatile compounds, and they serve hormone-like regulatory and defense-related roles in plants. Recently, it has been proposed that LOXs play a role in potato tuberization processes because JA, MJ, and structurally similar tuberonic acid and tuberonic acid glycoside have been shown to be tuber-inducing substances. In order to study possible lipoxygenase involvement in potato tuberization, we have isolated, sequenced, and characterized the expression pattern of two cDNA clones, designated POTLX-1 and POTLX-2, that represent similar, but distinct, LOX genes. Within the scope of our experiments, northern hybridization studies with mRNA extracted from various organs of `Superior' potato plants indicated that the expression of these two genes is restricted to developing tubers and roots only. Moreover, there is a positive correlation between POTLX-1 and POTLX-2 mRNA accumulation and the stage of potato tuber development, and this implicates LOX in tuberization processes. Accumulation of their transcripts was not detected in leaves, flowers, stems, shoot tips, or axillary buds. These results indicate that the isozyme forms encoded by these two genes are tuber-specific, and they are good candidates to study LOX involvement in potato tuberization processes. Treatment of potato leaves with abscisic acid, MJ, gibberellic acid, auxin (NAA), and cytokinin (BA) did not trigger transcriptional activation of either of these genes.
Sang-Gon Suh, Yong-Sun Moon, and David J. Hannapel
The 22-kDa potato proteinase inhibitors (22-kDa PPI) are synthesized as a preprotein with a hydrophobic signal sequence of 40-residue amino acids. The amino-terminal amino acid sequence (10-mer amino acids: 18-Ala-Phe-Ala-Arg-Ser-Phe-Thr-Ser-Glu-Asn-27) of signal peptide of 22-kDa PPI was synthesized. The 22-kDa PPI signal peptide specific anti-peptide antibodies were raised in New Zealand white rabbits against the 22-kDa PPI synthetic signal peptide. Immunoblot and Northern blot analysis were performed by using 22-kDa PPI anti-peptide antibody and cDNA probe, p34021, which codes for the 22-kDa PPI, respectively. In this paper, we determined the process of the 22-kDa potato proteinase inhibitor in tuber and wounded leaves.
Fredy R. Romero, David J. Hannapel, and Kathleen Delate
Echinacea is one of the best-selling medicinal plants in the United States. It was historically harvested from wild populations, but its demand has increased so significantly that commercial production has become a necessity to supply the increasing demand and to protect wild populations. The medicinal properties of echinacea are associated with secondary metabolites that are produced mainly in the roots. Hairy roots, induced by the Ri plasmid of Agrobacterium rhizogenes, have been produced in other crops as alternative sources of secondary metabolites that commonly are produced and synthesized in the roots of mature plants. This method of production offers some advantages over traditional agricultural systems, such as the possibility of producing novel compounds year-round. The overall goal of this project is to explore the utility of hairy root cultures (mediated by A. rhizogenes) as an efficient, alternative, and enriched source of secondary compounds with medicinal properties, such as alkamides, flavonoids, and caffeic acid derivatives. We have been successful in transforming roots from E. angustifolia, E. pallida, and E. purpurea plants, and confirming the presence of rol ABC genes in hairy roots using molecular techniques. Roots from control plants show no active growth under dark conditions, whereas transformed roots from E. pallida and E. purpurea show a low degree of branching with a slow growth rate on solid media under darkness. However, transformed E. angustifolia show a faster growth rate and higher degree of branching under the same conditions. Currently, we are working on the optimization of the growing conditions of the transformed roots and will proceed to the biochemical analysis phase of the project.
Fredy R. Romero, Kathleen Delate, and David J. Hannapel
With the increase in popularity of echinacea as a botanical supplement, organic production of this herb continues to grow. Echinacea seeds typically show a high percentage of dormancy that can be broken by ethephon or gibberellic acid, but these methods are not accepted in organic production. We examined in three experiments the effects of varying seed source and germination conditions on echinacea growth. To determine the efficacy of nonchemical treatments, we evaluated the effect of light with and without cold-moist stratification on seed germination of the three most important medicinal species of echinacea, E. angustifolia, E. purpurea, and E. pallida. We used cold-moist stratification under 24 h light, 24 h dark, and 16/8 h (light/dark) to break seed dormancy. We found that germination was enhanced in seeds from a commercial organic seed source, compared to a public germoplasm source. When seeds were not cold-moist stratified, light increased germination in E. angustifolia only, suggesting differential dormancy among the three species. We found that when seeds were cold-moist stratified under 16–24 h of light for 4 weeks, the percentage and rate of germination increased 10% over the control, suggesting this method as an alternative to chemical seed treatments.
Fredy R. Romero, Kathleen Delate, and David J. Hannapel
Organic production of one of the most popular botanical supplements, Echinacea, continues to expand in the U.S. Echinacea seeds typically show a high degree of dormancy that can be broken by ethephon or gibberellic acid (GA), but these methods are currently disallowed in organic production. In order to determine the efficacy of nonchemical seed treatments, we evaluated the effect of varying seed source and supplying light, with and without cold-moist stratification, on seed germination of the three most important medicinal species of Echinacea, E. angustifolia DC, E. purpurea (L) Moench, and E. pallida (Nutt.) Nutt. Treatments included cold-moist stratification under 24 hours of light, 24 hours of dark, and 16/8 hours of light/dark to break seed dormancy. We found that germination was greater in the E. purpurea and E. pallida seeds from a commercial organic seed source compared to a public germplasm source. When seeds were not cold-moist stratified, 16 to 24 hours light increased germination in E. angustifolia only. Echinacea angustifolia, E. purpurea, and E. pallida seeds that were cold-moist stratified under 16 to 24 hours of light for 4 weeks had a significantly greater percentage and rate of germination compared to seeds germinated in the dark. Therefore, cold-moist stratification under light conditions is recommended as a method to break seed dormancy and increase germination rates in organic production of Echinacea.