Characterization of a phytochelatin synthase gene from Ipomoea pes-caprae involved in cadmium tolerance and accumulation in yeast and plants.

Phytochelatin synthases (PCSs) play a crucial part in heavy metal tolerance in plants via the synthesis of phytochelatins (PCs), which can chelate heavy metals (HMs) in the vacuole and decrease cell damage. Plant PCSs are commonly designated as key genes for phytoremediation. In this study, we identified a PCS gene (IpPCS1) from Ipomoea pes-caprae and investigated its role in regulating cadmium (Cd) tolerance and accumulation. The expression of a truncated IpPCS1t in yeast could complement the Cd-sensitive phenotype of the ycf1Δ mutant strain, as well as improve the Cd tolerance of the wild-type yeast strain, while promoting Cd accumulation in the yeast cells. The expression of IpPCS1 was induced in I. pes-caprae plants under Cd treatment. Compared with IpPCS1, the lack of a C-terminal in IpPCS1t did not affect its Cd tolerance, but might restrict the zinc (Zn) detoxification in yeast. The overexpression of IpPCS1t in Arabidopsis could improve the Cd tolerance slightly and had little impact on Cd accumulation in transgenic plant. Our results indicated that IpPCS1 has certain potential application value in Cd tolerance and detoxification, therefore provides a useful genetic resource for enhancing Cd tolerance and improving the Cd phytoremediation capacity of plants or organisms. In addition, our research is the first time to discover a new possible Cd activation site in the C-terminal of IpPCS1.

Genome-wide identification, characterisation and expression profile analysis of DEAD-box family genes in sweet potato wild ancestor Ipomoea trifida under abiotic stresses.

BACKGROUND: DEAD-box protein family is the largest subfamily of RNA helicases and plays an important role in RNA metabolism and plant growth, development, and stress responses. Although DEAD-box genes have been characterized in various major crop plants, their identification and characterization in Convolvulaceae is still in infancy. Sweet potato (Ipomoea batatas, in Convolvulaceae) is the seventh most important crop in the world. Ipomoea trifida is one of the ancestors of sweet potato and is an effective resource for sweet potato cross-breeding. OBJECTIVE: Identification and characterisation of DEAD-box transcription factors in sweet potato wild ancestor I. trifida genome. METHOD: A systematic genome-wide analysis was conducted to identify the DEAD-box transcription factors in the I. trifida genome. RESULTS: We identified 17 ItfDEAD-box genes which distributed unevenly on the nine chromosomes of I. trifida and encoded 29 DEAD transcripts. The phylogenetic analysis classified the DEAD-box proteins into nine groups named I-IX. Homology model prediction of ItfDEAD-box proteins obtained 14 models which lay a preliminary foundation for the further functional exploration of the ItfDEAD-box proteins. The tissue-specific and abiotic stress-responsive expression profiles of ItfDEAD-box genes were analyzed in different tissues and under abiotic stress responses by RNA-seq data and confirmed by quantitative PCR analysis. Some genes were significantly up- or down-regulated by different abiotic stress, suggesting that ItfDEAD-box plays a crucial role in stress responses in I. trifida. CONCLUSION: The identification and gene expression of the ItfDEAD-box gene family might shed light on the function exploration of DEAD-box gene in I. trifida and promote the molecular breeding of sweet potato.

CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B (DFR-B) locus in the Japanese morning glory Ipomoea (Pharbitis) nil.

CRISPR/Cas9 technology is a versatile tool for targeted mutagenesis in many organisms, including plants. However, this technique has not been applied to the Japanese morning glory (Ipomoea [Pharbitis] nil), a traditional garden plant chosen for the National BioResource Project in Japan. We selected dihydroflavonol-4-reductase-B (DFR-B) of I. nil, encoding an anthocyanin biosynthesis enzyme, as the target gene, and changes in the stem colour were observed during the early stages of plant tissue culture by Rhizobium [Agrobacterium]-mediated transformation. Twenty-four of the 32 (75%) transgenic plants bore anthocyanin-less white flowers with bi-allelic mutations at the Cas9 cleavage site in DFR-B, exhibiting a single base insertion or deletions of more than two bases. Thus, these results demonstrate that CRISPR/Cas9 technology enables the exploration of gene functions in this model horticultural plant. To our knowledge, this report is the first concerning flower colour changes in higher plants using CRISPR/Cas9 technology.

Plastidial α-glucan phosphorylase 1 complexes with disproportionating enzyme 1 in Ipomoea batatas storage roots for elevating malto-oligosaccharide metabolism.

It has been proposed that malto-oligosaccharides (MOSs) are possibly recycled back into amylopectin biosynthesis via the sequential reactions catalyzed by plastidial α-glucan phosphorylase 1 (Pho1) and disproportionating enzyme 1 (Dpe1). In the present study, the reciprocal co-immunoprecipitation experiments using specific antibodies against Pho1 and Dpe1 demonstrated that these two enzymes can form a complex (the PD complex) in Ipomoea batatas storage roots. The immunohistochemistry analyses also revealed the co-localization of Pho1 and Dpe1 in the amyloplasts, and the protein levels of Pho1 and Dpe1 increased gradually throughout sweet potato storage root development. A high molecular weight PD complex was co-purified from sweet potato storage root lysates by size exclusion chromatography. Enzyme kinetic analyses showed that the PD complex can catalyze maltotriose and maltotetraose to generate glucose-1-phosphate in the presence of inorganic phosphate, and it also performs greater Dpe1 activity toward MOSs than does free form Dpe1. These data suggest that Pho1 and Dpe1 may form a metabolon complex, which provides elevated metabolic fluxes for MOS metabolism via a direct transfer of sugar intermediates, resulting in recycling of glucosyl units back into amylopectin biosynthesis more efficiently.

S95C substitution in CuZn-SOD of Ipomoea carnea: impact on the structure, function and stability.

Superoxide dismutase (SOD) in general is a unique homo-dimeric enzyme that can scavenge toxic superoxide radicals by dismutation reaction. In IcSOD (Ipomoea carnea SOD), the presence of cysteine (Cys) plays an essential role in protein behaviour. This study analysed the role of Cys in modulating the stability and kinetic properties of IcSOD. To investigate the significance of the dimeric structure in modulating the structure/function relationship of CuZn-SODs, we have substituted a conserved serine by cysteine (Ser95Cys) in Ipomoea carnea CuZn-SOD. The results demonstrate that this mutation leads to an increase in dimeric strength, as reflected by size exclusion chromatography, differential scanning calorimetry, and high-temperature circular dichroism spectroscopy measurements. The mutant form, as compared to the native enzyme, shows a relatively low tendency to form aggregates but encountered a reduction in both dismutase and peroxidase activities. This study provides new mechanistic insight into the role of free cysteine in CuZn-SODs and such mutation may be used to increase dimeric strength. Protein docking and molecular dynamics simulations further demonstrate that Ser95Cys substitution in Ipomoea carnea CuZn-SOD leads to the creation of a new subunit interface resulting in increased dimeric strength of the protein.

Spontaneous mutations of the UDP-glucose:flavonoid 3-O-glucosyltransferase gene confers pale- and dull-colored flowers in the Japanese and common morning glories.

MAIN CONCLUSION: UDP-glucose:flavonoid 3- O -glucosyltransferase is essential for maintaining proper production quantity, acylation, and glucosylation of anthocyanin, and defects cause pale and dull flower pigmentation in morning glories. The Japanese (Ipomoea nil) and the common (I. purpurea) morning glory display bright blue and dark purple flowers, respectively. These flowers contain acylated and glucosylated anthocyanin pigments, and a number of flower color mutants have been isolated in I. nil. Of these, the duskish mutants of I. nil produce pale- and dull-colored flowers. We found that the Duskish gene encodes UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT). The duskish-1 mutation is a frameshift mutation caused by a 4-bp insertion, and duskish-2 is an insertion of a DNA transposon, Tpn10, at 1.3 kb upstream of the 3GT start codon. In the duskish-2 mutant, excision of Tpn10 is responsible for restoration of the expression of the 3GT gene. The recombinant 3GT protein displays expected 3GT enzymatic activities to catalyze 3-O-glucosylation of anthocyanidins in vitro. Anthocyanin analysis of a duskish-2 mutant and its germinal revertant showing pale and normal pigmented flowers, respectively, revealed that the mutation caused around 80 % reduction of anthocyanin accumulation. We further characterized two I. purpurea mutants showing pale brownish-red flowers, and found that they carry the same frameshift mutation in the 3GT gene. Most of the flower anthocyanins in the mutants were previously found to be anthocyanidin 3-O-glucosides lacking several caffeic acid and glucose moieties that are attached to the anthocyanins in the wild-type plants. These results indicated that 3GT is essential not only for production, but also for proper acylation and glucosylation, of anthocyanin in the morning glories.

Mechanistic study of CuZn-SOD from Ipomoea carnea mutated at dimer interface: enhancement of peroxidase activity upon monomerization.

The enzymatically active monomeric form of CuZn-superoxide dismutase has always been of interest to decipher the structure-function relationship in this class of enzymes. In the present study, spectroscopic and enzymatic characteristics of the dimeric and monomeric forms of recombinant Ipomoea carnea CuZn-superoxide dismutase were made to decipher their stability and altered catalytic properties. The monomeric form of protein was produced through site directed mutagenesis by replacing a conserved hydrophobic leucine with a polar lysine residue at the dimer-interface. Spectral characteristics of both the forms (monomer and dimer) showed the presence of novel electronic transitions. Superoxide scavenging activity of the mutated form was reduced to nearly half of the activity found in the native enzyme. Concomitantly, compared to native form the mutated enzyme showed an increase in peroxidase activity. High temperature dependent circular dichroism spectral analysis, differential scanning calorimetric profile, and the measurement of temperature dependent superoxide scavenging activity indicated an increased susceptibility of the mutated form to higher temperature as compared to the native form. The inhibitor studies like hydrogen peroxide, diethyldithiocarbamate and phenylglyoxal also indicate higher susceptibility, which might be due to, altered arrangement of active site residues as a consequence of the mutation. Molecular modeling and MD simulation studies further indicated that this specific mutation induces loss of hydrophobic interaction at dimer interface, resulting in the observed instability of the dimeric form. Increased peroxidative activity of the enzyme, upon monomerization may have physiological implication essentially in presence of high concentration of H2O2, as in case of plant cells specifically under stress conditions.

The molecular basis of incomplete dominance at the A locus of CHS-D in the common morning glory, Ipomoea purpurea.

The mutable a(flaked (a(f)) allele at the A locus of the common morning glory (Ipomoea purpurea) confers incomplete dominance in flower pigmentation and is caused by insertion of the DNA transposon Tip100 into CHS-D, which encodes chalcone synthase and is required for anthocyanin biosynthesis. Levels of CHS-D transcripts, CHS-D protein, and anthocyanin pigment in heterozygous flowers were about half that in homozygous flowers, indicating that dosage-dependent expression of CHS-D is the primary cause of the observed incomplete dominance. This contrasts with the Nivea locus in snapdragon (Antirrhinum majus) in which incomplete dominance is caused by semi-dominant CHS alleles.

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea.

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14-15%), has a molecular mass of 34.94 kDa (MALDI-TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0-9.0, 80 degrees C and the optimal activity is observed at pH 6.0 and 60 degrees C. Using p-nitrophenyl-N-acetyl-beta-D-glucosaminide, the kinetic parameters K(m), V(max), K(cat) and specificity constant of the enzyme were calculated as 0.5mM, 2.5 x 10(-8)mol min(-1)microg enzyme(-1), 29.0 s(-1) and 58.0mM(-1)s(-1) respectively. The extinction coefficient was estimated as 20.56 M(-1)cm(-1). The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G-E-I-A-I-Y-W-G-Q-N-G-G-E-G-S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.

Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carnea.

Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0 A resolution in-house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 126.9, c = 84.6 A, alpha = beta = 90, gamma = 120 degrees. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 A(3) Da(-1), corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.

Enhanced nitrogen and phosphorus removal from eutrophic lake water by Ipomoea aquatica with low-energy ion implantation.

Ipomoea aquatica with low-energy N+ ion implantation was used for the removal of both nitrogen and phosphorus from the eutrophic Chaohu Lake, China. The biomass growth, nitrate reductase and peroxidase activities of the implanted I. aquatica were found to be higher than those of I. aquatica without ion implantation. Higher NO3-N and PO4-P removal efficiencies were obtained for the I. aquatica irradiation at 25 keV, 3.9 x 10(16) N+ ions/cm(2) and 20 keV 5.2 x 10(16) N+ ions/cm(2), respectively (p < 0.05). Moreover, the nitrogen and phosphorus contents in the plant biomass with ion implantation were also greater than those of the controls. I. aquatica with ion implantation was directly responsible for 51-68% N removal and 54-71% P removal in the three experiments. The results further confirm that the ion implantation could enhance the growth potential of I. aquatica in real eutrophic water and increase its nutrient removal efficiency. Thus, the low-energy ion implantation for aquatic plants could be considered as an approach for in situ phytoremediation and bioremediation of eutrophic waters.

Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea.

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.

Escape from adaptive conflict after duplication in an anthocyanin pathway gene.

Gene duplications have been recognized as an important source of evolutionary innovation and adaptation since at least Haldane, and their varying fates may partly explain the vast disparity in observed genome sizes. The expected fates of most gene duplications involve primarily non-adaptive substitutions leading to either non-functionalization of one duplicate copy or subfunctionalization, neither of which yields novel function. A significant evolutionary problem is thus elucidating the mechanisms of adaptive evolutionary change leading to evolutionary novelty. Currently, the most widely recognized adaptive process involving gene duplication is neo-functionalization (NEO-F), in which one copy undergoes directional selection to perform a novel function after duplication. An alternative, but understudied, adaptive fate that has been proposed is escape from adaptive conflict (EAC), in which a single-copy gene is selected to perform a novel function while maintaining its ancestral function. This gene is constrained from improving either novel or ancestral function because of detrimental pleiotropic effects on the other function. After duplication, one copy is free to improve novel function, whereas the other is selected to improve ancestral function. Here we first present two criteria that can be used to distinguish NEO-F from EAC. Using both tests for positive selection and assays of enzyme function, we then demonstrate that adaptive evolutionary change in a duplicated gene of the anthocyanin biosynthetic pathway in morning glories (Ipomoea) is best interpreted as EAC. Finally, we argue that this phenomenon likely occurs more often than has been previously believed and may thus represent an important mechanism in generating evolutionary novelty.

Extraction and purification of Ipomoea peroxidase employing three-phase partitioning.

Three-phase partitioning (TPP) is a novel separation process used for the extraction and purification of biomolecules. The biomolecules are recovered in a purified form at the interface (precipitate), while the contaminants partition in t-butanol and aqueous phases. Peroxidase from the leaves of Ipomoea palmata was purified by using TPP. The ratio of the crude extract to t-butanol of 1:1 and 30% ammonium sulfate at 37 degrees C resulted in about 160% activity recovery and twofold purification in the aqueous phase of the first cycle of TPP. On subjecting the aqueous phase to the second cycle of TPP, a purification of 18-fold was achieved with about 81% activity recovery. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed substantial purification, and the molecular weight of peroxidase was found to be 20.1 KDa. The present study shows a higher degree of purification and activity yield as a primary purification process in comparison with existing literature values, thus demonstrating TPP as an attractive downstream process for the purification of peroxidase.

Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory).

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.

Allene oxide cyclase is essential for theobroxide-induced jasmonic acid biosynthesis in Pharbitis nil.

Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.

Distribution of gibberellins and expressional analysis of GA 20-oxidase genes of morning glory during fruit maturation.

Gibberellins A1/3 (GA1/3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1/3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1/3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible alpha-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.

Genomic organizations of two small subunit ADP-glucose pyrophosphorylase genes from sweetpotato.

The genomic features of the small subunit ADP-glucose pyrophosphorylase (AGPase) isoforms are different in barley and maize. The two isoforms found in barley originated from one single gene through alternative splicing, while two independent genes encode the two isoforms in maize. To ascertain the genomic organizations of two small subunit AGPase isoforms in sweetpotato (ibAGP1 and ibAGP2), we isolated genomic DNAs containing the entire coding regions of two genes. Complete genomic structures of ibAGP1 and ibAGP2 were ascertained by the sequencing of these genomic regions. The transcribed regions of ibAGP1 and ibAGP2, comprising nine exons and eight introns, were distributed over 3.9 and 4.0 kb, respectively. The eight introns differed in length, from 76 to 946 bp in ibAGP1, and from 76 to 811 bp in ibAGP2, while the locations of introns in ibAGP1 and ibAGP2 were identical. There was 46-58% sequence identity between the intron sequences of the two genes. Intron sequence analyses suggested that either duplication in each intron, or gene conversion between introns of two isoforms, might cause major intron size differences between the two genes. Altogether, these results indicate that two small subunit AGPase isoforms in sweetpotato are encoded by two independent genes, in a fashion similar to that of maize small subunit AGPase genes.

Multiple transcripts of a gene for a leucine-rich repeat receptor kinase from morning glory (Ipomoea nil) originate from different TATA boxes in a tissue-specific manner.

TATA boxes are the most common regulatory elements found in the promoters of eukaryotic genes because they are associated with basal transcription initiation by RNA polymerase II. Often only a single TATA element is found in a given promoter, and tissue-, stage- and/or stimulus-specific expression occurs because the TATA box is associated with other cis -acting elements that enhance or repress transcription. We used software tools for gene analysis to assist in locating potential TATA box(es) in an AT-rich region of the promoter of a gene, inrpk1, which codes for a leucine-rich receptor protein kinase in morning glory (Ipomoea nil). Through the use of RT-PCR and various combinations of forward primers bracketing most of the promoter region we were able to define the 5'-ends of transcripts in this region. The region was then targeted for analysis by RNA Ligase-Mediated-5' Rapid Amplification of cDNA Ends (RLM-5' RACE) to identify the transcript initiation site(s). Positioning of initiation sites with respect to TATA boxes identified by gene analysis tools allowed us to identify three operational TATA elements which regulate basal transcription from this gene. Two TATA boxes were responsible for all of the inrpk1 transcripts found in leaves and cotyledons, and about 25-30% of the transcripts in roots. A third TATA box was involved only in expression in roots and accounted for the remaining 50-70% of root transcripts. RNAs expressed from this element lack two potentially functional upstream AUG codons, and may be translated more efficiently than transcripts originating from the other TATA boxes.

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds.

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.