Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.) Lam) senescent leaves

Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxyl side of asparagine residues, and is possibly involved in the post-translational processing of proproteins. In this report one full-length cDNA, SPAE, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPAE contained 1479 nucleotides (492 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (c. 61-68%) with asparaginyl endopeptidases of Vicia sativa, Phaseolus vulgaris, Canavalia ensiformis, and Vigna mungo. SPAE probably encoded a putative precursor protein. Via cleavage of the N- and C-termini, it produced a mature protein containing 325 amino acids (from the 51st to the 375th amino acid residues), the conserved catalytic residues (the 173rd His and 215th Cys amino acid residues), and the putative N-glycosylation site (the 332nd Asn amino acid residue). Semi-quantitative RT-PCR and western blot hybridization showed that SPAE gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was much less in mature green leaves, stems, and roots. Phylogenic analysis showed that SPAE displayed close association with vacuolar processing enzymes (legumains/asparaginyl endopeptidases), which function via cleavage for proprotein maturation in the protein bodies during seed maturation and germination. In conclusion, sweet potato SPAE is probably a functional, senescence-associated gene and its mRNA and protein levels were significantly enhanced in natural and induced senescent leaves. The possible role and function of SPAE associated with bulk protein degradation and mobilization during leaf senescence were also discussed.     

Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas) leaves

Metallothionein (MT) is a group of proteins with low molecular masses and high cysteine contents, and is classified into different types, which in general contains two domains (domain 1 and domain 2) with typical amino acid sequences (Rauser 1999). In this report two full-length cDNAs (Y459 and G14) encoding MT-like proteins were isolated from leaves of sweet potato (Ipomoea batatas). Their open reading frames contained 249 and 195 nucleotides (82 and 64 amino acids) for Y459 and G14, respectively, and exhibited a relatively low amino acid sequence similarity (ca. 25.8%). Gene structure studies showed that Y459 had the conserved domain 1 region of type 2 MT; however, the domain 2 region was not conserved and contained additional amino acids between the CxC and CxC spacing. G14 had conserved domains 1 and 2 of type 4 MT except that the last CxC of domain 2 was changed to RxC. Semi-quantitative RT-PCR showed that Y459 was expressed in significant quantity in roots and stems, but was much less in green leaves. During natural and induced (with dark and ethephon, an ethylene-releasing compound, treatments) leaf senescence, Y459 gene expression was significantly enhanced. In contrast, relatively constant gene expression levels were found for G14 in all tissues or treatments analyzed. In conclusion, the two MT-like protein genes of sweet potato display differential gene structures and gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues.     

Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.     

Isolation and characterization of some proteolytic enzyme inhibitors in sweet potato (Ipomoea batatas)

Ten trypsin (EC 3.4.21.4) inhibitors have been isolated and purified by gel filtration and ion-exchange chromatography from the tubers of sweet potato (Ipomoea batatas). The molecular weights of the three most active inhibitors were estimated by molecular sieve chromatography and found to be 12 000, 10 000 and 9300, respectively. They showed maximum activity at pH 7.5–8.5 as well as maximum K_i within this pH range. They displayed different trypsin inhibitory activity, and this activity was completely lost on boiling for 40 min.     

Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomoea batatas)

Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.)

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1^–1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1^–1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.Abbreviations MS Murashige and Skoog basal medium - IAA Indol-3-acetic acid - NAA naphthaleneacetic acid - 2,4-D dichlorophenoxyacetic acid - Mes 2-(N-morpholino)-ethanesulfonic acid - Cpw cell and protoplast washing solution     

Isolation of antidiabetic components from white-skinned sweet potato (Ipomoea batatas L.)

We have already reported that white-skinned sweet potato (Ipomoea batatas L.) (WSSP) shows antidiabetic activity in streptozotocin (STZ) induced diabetic rats and genetically diabetic models (yellow KK, db/db mice and Zucker fatty rats). In this study, isolation and purification of the antidiabetic component of WSSP were attempted. Almost all antidiabetic activity was found in the cortex of WSSP. The fractionation of the antidiabetic component in the WSSP cortex was done by the following methods: dialysis of the water extract, 85% ethanol precipitation, 15% trichloroacetic acid (TCA) treatment, butyl-, phenyl-hydrophobic column chromatography, and ultrafiltration treatment. The antidiabetic component was not eliminated during dialysis and was soluble in 85% ethanol and 15% TCA, but it passed through a filter that allows the passage of substances of a molescular weight of 30,000. The uniformity of this isolated active component was analyzed using HPLC. A single peak was seen with three different columns (C8 reverse-phase column, anion exchange QA column, and gel filtration column (GFC)), indicating that the component is a uniform substance. The molecular weight of this antidiabetic component was estimated to be 22,000 by GFC analysis. This active component was presumed to be an acidic glycoprotein because it contained protein and sugar and was adsorbed onto the QA column at pH 7.0.     

Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas)

An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H(2)O(2) at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 10(3)-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72).     

Touch-induced gene (IbTCH1) from sweet potato [Ipomoea batatas (L.) Lam.]: molecular cloning and functional analysis

The cDNA of the touch-induced genes (TCH) of the sweet potato [Ipomoea batatas (L.) Lam.] has been cloned and analyzed. IbTCH1, which exists as at least two-copy genes in the genome of the sweet potato, encodes for 148-amino acid polypeptides, and harbors four conversed Ca²⁺-binding motif EF-hands. IbTCH1 was shown to be expressed in the flower, leaf, thick pigmented root, and particularly in the white fibrous root, but expressed only weakly in the petiole. IbTCH1 is upregulated upon exposure to environmental stresses, dehydration, and jasmonic acid. Furthermore, IbTCH1 is developmentally regulated in the leaf and root. These results strongly indicate that the gene performs functions in both plant development and in defense/stress-signaling pathways.     

Starch granule size and the domestication of manioc (Manihot esculenta) and sweet potato (Ipomoea batatas)

Archaeological studies of plant remains have indicated that an increase in seed size is frequently correlated with both intensive cultivation and domestication of seed crop plants. To test if starch granules of domesticated root crops are significantly larger than those of wild or less intensively cultivated plants, archaeological and modern specimens of manioc and sweet potato were sampled for starch granules, and granule size was compared across a temporal sequence. The results indicate that a gross generalization can be made that modern specimens of both manioc and sweet potato yield larger starch granules than some archaeological specimens. It does appear, however, that modern domesticated manioc roots produce significantly larger-sized starch granules than those of its purported wild ancestor. Additionally, there exist two lines of evidence that the coastal Peruvian and lowland Neotropical regional types of manioc differ from one another and have been separate for several millennia. These findings indicate that manioc may have been domesticated more than once.     

Molecular,morphological and agronomic characterization of the sweet potato (Ipomoea batatas L.) germplasm collection from Mozambique: Genotype selection for drought prone regions

Sweet potato (Ipomoea batatas (L.) Lam) is one of the most important root crops in Mozambique, ranking in the 3rd position, after cassava and maize. Within the scope of the national and regional strategies/initiatives, we have used a multi-analysis approach to characterize the national sweet potato germplasm collection at two different levels: i) genetic, morphological and agronomic diversity; and ii) agronomic potential (storage root yield, vine weight, biomass, harvest index and dry matter content) toward drought tolerance. This collection, composed by 44 accessions, comprises 28 genotypes cultivated in three different provinces of Mozambique (Gaza, Inhambane and Zambezia), nine from other African countries (Kenya, South Africa, Uganda and Zimbabwe), one from the United States of America, and six from CGIAR research centers (IITA and CIP). According to our results, the Mozambican germplasm bank presents a high level of diversity, comparable to those from the collections of the primary centers of origin and South Africa, therefore constituting of a good source of agronomic traits for breeding. Regarding drought tolerance, six Mozambican genotypes (Admarc, Chingova, Nhacoongo-1, Xihetamakote, Nwanatuyo, and Chissicuana-2), one from Uganda (NASPOT-5), one from Zimbabwe (Moz_white), one from Kenya (SPK 004), and one from the USA (Resisto) seem to have the highest potential to be used in regions with frequent drought seasons and in future breeding programs. The results showed that such integrated analysis can be used to successfully characterize the genetic material in terms of suitability to drought-prone regions, therefore helping sweet potato crop management, with economic and food security impacts.     

Abscisic acid-induced growth inhibition of sweet potato (Ipomoea batatas L.) in vitro

The inhibitory effects of abscisic acid (ABA) on in vitro growth and development of axillary buds from nodal segments of sweet potato (Ipomoea batatas L.) was investigated. ABA at concentrations of 0.01, 0.1, 1.0 or 10.0 mg 1^-1 inhibited axillary bud and root development and subsequent plantlet growth. ABA at 10 mg 1^-1 completely inhibited axillary shoot development but did not affect the viability of cv. Jewel explants over a culture period of 365 days. Transfer of nodal segments cultured for 90, 180 or 365 days from basal medium containing 10 mg 1^-1 ABA to growth regulator-free media resulted in rapid and normal plantlet development. Gibberellic acid at 0.1, 1.0 or 10.0 mg 1^-1 in the presence of ABA at 0.1, 1.0 or 10.0 mg 1^-1 did not counteract the ABA-induced growth inhibition. Although ABA totally inhibited the growth of 6 sweet potato plant introductions at a concentration of 10.0 mg 1^-1, the efficacy of ABA as a suppressant of shoot growth varied with genotype.Abbreviations ABA abscisic acid - GA gibberellic acid - cDNA complementary DNA - PI plant introduction - SE standard error     

Occurrence and cytological mechanism of 2n pollen formation in a tetraploid accession of Ipomoea batatas (sweet potato)

We evaluated a 4x accession of Ipomoea batatas (L.) Lam. and four 2x accessions of Ipomoea triloba for 2n pollen production. Approximately 90% of the genotypes of accession 81.2 (I. batatas, 4x) produced 2n pollen with different frequencies. In contrast, none of the genotypes of I. triloba produced 2n pollen. The diameter of the 2n pollen was approximately 30% ((3) sqrt 2) larger than that of the n pollen, making it easy to identify, measure, and quantify. The correlation (r = 0.93**) between the frequency of giant pollen and the frequency of dyads and triads was highly significant, strongly suggesting that the giant pollen grains were 2n pollen. The 2n pollen producers presented either a parallel or tripolar spindle arrangement (Y shaped) at anaphase II instead of the normal 60 degrees crossed spindle orientation. These two abnormal spindle configurations produced dyads and triads, with different frequencies (13-67%), instead of tetrads. Occasionally a metaphase II spindle variation was found with a single fused spindle, which also forms a dyad. The correlation (r = 0.89**) between the frequency of 2n pollen and the frequency of parallel, fused, and tripolar spindle arrangements was also highly significant, suggesting that these abnormal spindle configurations are involved in the production of 2n pollen in I. batatas. When we evaluated the efficiency of 2n pollen in polyploidization using 4x x 4x (2n) crosses, all progenies were 4x, suggesting the existence of barriers to crossability between 4x genotypes and their 2n pollen-producer counterparts.     

Growth and morphogenesis of immature embryos of sweet potato (Ipomoea batatas L.) in vitro

Sweet potato (Ipomoea batatas L.) embryos excised from the fertilized ovules of 6- to 12-day-old capsules were cultured on MS medium supplemented with NAA, BA, GA separately and in combinations. GA was found essential for initial morphogenesis of globular and heart stages. Seedlings were recovered from late globular stage onwards but recovery was best from advanced embryo stages. Differentiated embryos produced multiple shoots on MS medium +1M NAA÷2M BA +0.5M GA.     

A dithiol glutaredoxin cDNA from sweet potato (Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies

Glutaredoxins (Grx) play an important role in reduction of protein glutathione mixed disulphides. An IbGrx cDNA (561 bp, EF362614 ) encoding a putative dithiol Grx was cloned from sweet potato (Ipomoea batatas [L.] Lam). The deduced amino acid sequence is conserved among the reported dithiol Grx, having a CGYC dithiol motif at the active site. A 3‐D structural model was created based on the known crystal structure of a poplar Grx (GrxC1). To characterise the IbGrx protein, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His₆‐tagged IbGrx was expressed and purified by metal affinity chromatography. The purified enzyme showed a monomeric band, as demonstrated with 15% SDS‐PAGE. The Michaelis constant (K_M) for ß‐hydroxyethyl disulphide (HED) was 0.50 ± 0.08 Mm . The enzyme retained 60% activity at 80 °C for 16 min. The enzyme was active over a broad pH range from 6.0 to 11.0, and in the presence of imidazole up to 0.4 M . The enzyme was susceptible to protease.     

Morphohistobiochemical characteristics of embryogenic and nonembryogenic callus cultures of sweet potato (Ipomoea batatas L.)

Ipomoea batatas callus culture raised in a medium supplemented with 2,4-D (2,4-dichlorophenoxy acetic acid) alone or 2,4-D in combination with benzyl adenine, were found to be embryogenic. Supplementation of exogenous chemicals, such as 5 g/l NaCI or 0.7 g/l proline together with a mild dose of 0.2 mg/l 2,4-D, enhanced somatic embryogenesis significantly in all the genotypes tested. Morphological, growth, physiological, histological, and biochemical characteristics of the embryogenic callus were different from the nonembryogenic callus. The former was compact, slow growing, and nodular compared with the fast growing, fragile, nonembryogenic callus. The embryogenic callus tissue had more dry matter, protein and reducing sugar contents compared with the less embryogenic callus. The somatic embryogenic response remained steady in the cultures for up to 96 weeks.     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Cryopreservation of embryogenic tissue of sweet potato (Ipomoea batatas): use of sucrose and dehydration for cryoprotection

Embryogenic tissue of two sweet potato (Ipomoea batatas (L) LAM) genotypes, TIB 10 and Nemanete (Nem), was established from in vitro axillary meristems on Murashige and Skoog (1962) media supplemented with 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid respectively. Embryogenic aggregates of approximately 1.5–2.0 mm in diameter were subjected to a rapid or a two-step freezing protocol in liquid nitrogen following alginate encapsulation, sucrose preculture and varying degrees of dehydration. Up to 28% of encapsulated embryogenic aggregates of TIB 10 survived rapid freezing without dehydration. This was not enhanced by dehydration prior to freezing. However, survival after dehydration was enhanced up to 74% by incorporating an initial slow cooling step prior to plunging the tissue into liquid nitrogen. Following freezing, embryogenic tissue appeared to develop normally and retained its competence to produce mature embryos and plantlets. Similar results were obtained with Nem, although the survival percentages were much lower.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium