Overexpression of sweetpotato glutamylcysteine synthetase (IbGCS) in Arabidopsis confers tolerance to drought and salt stresses.

Various environmental stresses induce the production of reactive oxygen species (ROS), which have deleterious effects on plant cells. Glutathione (GSH) is an antioxidant used to counteract reactive oxygen species. Glutathione is produced by glutamylcysteine synthetase (GCS) and glutathione synthetase (GS). However, evidence for the GCS gene in sweetpotato remains scarce. In this study, the full-length cDNA sequence of IbGCS isolated from sweetpotato cultivar Xu18 was 1566 bp in length, which encodes 521 amino acids. The qRT-PCR analysis revealed a significantly higher expression of the IbGCS in sweetpotato flowers, and the gene was induced by salinity, abscisic acid (ABA), drought, extreme temperature and heavy metal stresses. The seed germination rate, root elongation and fresh weight were promoted in T3 Arabidopsis IbGCS-overexpressing lines (OEs) in contrast to wild type (WT) plants under mannitol and salt stresses. In addition, the soil drought and salt stress experiment results indicated that IbGCS overexpression in Arabidopsis reduced the malondialdehyde (MDA) content, enhanced the levels of GCS activity, GSH and AsA content, and antioxidant enzyme activity. In summary, overexpressing IbGCS in Arabidopsis showed improved salt and drought tolerance.

Genome-Wide Identification and Expression Analysis of the Starch Synthase Gene Family in Sweet Potato and Two of Its Closely Related Species.

The starch synthase (SS) plays important roles in regulating plant growth and development and responding to adversity stresses. Although the SS family has been studied in many crops, it has not been fully identified in sweet potato and its two related species. In the present study, eight SSs were identified from Ipomoea batatas (I. batata), Ipomoea trifida (I. trifida), and Ipomoea trlioba (I. trlioba), respectively. According to the phylogenetic relationships, they were divided into five subgroups. The protein properties, chromosomal location, phylogenetic relationships, gene structure, cis-elements in the promoter, and interaction network of these proteins were also analyzed; stress expression patterns were systematically analyzed; and real-time polymerase chain reaction (qRT-PCR) analysis was performed. Ipomoea batatas starch synthase (IbSSs) were highly expressed in tuber roots, especially Ipomoea batatas starch synthase 1 (IbSS1) and Ipomoea batatas starch synthase 6 (IbSS6), which may play an important role in root development and starch biosynthesis. At the same time, the SS genes respond to potassium deficiency, hormones, cold, heat, salt, and drought stress. This study offers fresh perspectives for enhancing knowledge about the roles of SSs and potential genes to enhance productivity, starch levels, and resistance to environmental stresses in sweet potatoes.

Caffeoylquinic acids: chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity.

Caffeoylquinic acids (CQAs) are specialized plant metabolites we encounter in our daily life. Humans consume CQAs in mg-to-gram quantities through dietary consumption of plant products. CQAs are considered beneficial for human health, mainly due to their anti-inflammatory and antioxidant properties. Recently, new biosynthetic pathways via a peroxidase-type p-coumaric acid 3-hydroxylase enzyme were discovered. More recently, a new GDSL lipase-like enzyme able to transform monoCQAs into diCQA was identified in Ipomoea batatas. CQAs were recently linked to memory improvement; they seem to be strong indirect antioxidants via Nrf2 activation. However, there is a prevalent confusion in the designation and nomenclature of different CQA isomers. Such inconsistencies are critical and complicate bioactivity assessment since different isomers differ in bioactivity and potency. A detailed explanation regarding the origin of such confusion is provided, and a recommendation to unify nomenclature is suggested. Furthermore, for studies on CQA bioactivity, plant-based laboratory animal diets contain CQAs, which makes it difficult to include proper control groups for comparison. Therefore, a synthetic diet free of CQAs is advised to avoid interferences since some CQAs may produce bioactivity even at nanomolar levels. Biotransformation of CQAs by gut microbiota, the discovery of new enzymatic biosynthetic and metabolic pathways, dietary assessment, and assessment of biological properties with potential for drug development are areas of active, ongoing research. This review is focused on the chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity recently reported for mono-, di-, tri-, and tetraCQAs.

Characterization of polyphenol oxidase from purple sweet potato (Ipomoea batatas L. Lam) and its affinity towards acylated anthocyanins and caffeoylquinic acid derivatives.

Biochemical characterization of polyphenol oxidase (PPO) present in purple sweet potato (PSP) is a key step in developing efficient methodologies to control oxidative damage caused by this enzyme to the valuable components of PSP, such as caffeoylquinic acid derivatives and acylated anthocyanins. Thus, this work focused on the assessment of the effects of pH, temperature, and chemical agents on the PPO activity as well as characterization of the PPO substrate specificity towards major phenolic compounds found in PSP. The optimum conditions of enzyme activity were pH 7 and a temperature range of 20-30 °C at which phenolic substrates were oxidized with 72.5-99.8% yield. Zn2+ ions remarkably reduced PPO activity while Cu2+ ions improved enzyme performance. The highest substrate preference was shown for 3,4,5-tri-caffeoylquinic and 3,5-di-caffeoylquinic acid, followed by 5-caffeoylquinic and caffeic acid, 3,4- and 4,5-di-caffeoylquinic acids, peonidin-3-caffeoyl-p-hydroxybenzoyl-sophoroside-5-glucoside. The highest Km values were found for 4,5-feruloyl-caffeoylquinic acid and catechol.

A novel sweetpotato RING-H2 type E3 ubiquitin ligase gene IbATL38 enhances salt tolerance in transgenic Arabidopsis.

Arabidopsis Toxicos en Levadura (ATL) proteins compose a subfamily of E3 ubiquitin ligases and play major roles in regulating plant growth, cold, drought, oxidative stresses response and pathogen defense in plants. However, the role in enhancing salt tolerance has not been reported to date. Here, we cloned a novel RING-H2 type E3 ubiquitin ligase gene, named IbATL38, from sweetpotato cultivar Lushu 3. This gene was highly expressed in the leaves of sweetpotato and strongly induced by NaCl and abscisic acid (ABA). This IbATL38 was localized to nucleus and plasm membrane and possessed E3 ubiquitin ligase activity. Overexpression of IbATL38 in Arabidopsis significantly enhanced salt tolerance, along with inducible expression of a series of stress-responsive genes and prominently decrease of H2O2 content. These results suggest that IbATL38 as a novel E3 ubiquitin ligase may play an important role in salt stress response.

A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale.

Changes in cell wall components and polysaccharide-degrading enzymes in relation to differences in texture during sweetpotato storage root growth.

Sweetpotato has special texture characteristics, which directly affect the eating quality and post-production processing quality of sweetpotato. To investigate the texture change mechanism of sweetpotato during the growth process, this study selected two varieties with significant differences in texture from 35 varieties. The storage roots were sampled at 50, 80, 110, and 140 days after planting. Measure the texture parameters, the cell wall composition content, cell wall-related enzyme activities and the expression of expansin genes of sweetpotato storage roots. The results show that the hardness, adhesiveness and chewiness parameters of 'Yushu No 10' were significantly lower than those of 'Mianfen No 1', they have significantly different texture properties. In terms of cell wall composition, the soluble pectin content of 'Yushu No 10' was more than twice that of 'Mianfen No 1', whereas the insoluble pectin content was lower than that of 'Mianfen No 1', with the cellulose content of 'Yushu No 10' being significantly higher than that of 'Mianfen No 1'. In terms of cell wall-related enzymes, 'Yushu No 10' hardness gumminess and chewiness had a significant correlation with hemicellulose activity, and 'Mianfen No 1' had insignificant correlation with four cell wall-related enzymes. Expansin genes were also expressed differently during the various stages of root tubers expansin. The expressions of IbEXP1, IbEXP2 and IbEXPL1 were significantly correlated with the changes in cell wall component content, and were related to the qualitative structure changes. The research conclusion shows that the texture changes during the growth of sweetpotato are related to cell wall composition, cell wall-related enzyme activity changes, and the expression of expansin genes. This study provides theoretical guidance for in-depth study of texture changes of sweetpotato, post-harvest processing and utilization and quality improvement of storage roots.

Characterization of a Novel Chitinase from Sweet Potato and Its Fungicidal Effect against Ceratocystis fimbriata.

Black rot, caused by Ceratocystis fimbriata, is a destructive disease of sweet potatoes (Ipomoea batatas). In this study, a novel chitinase (IbChiA) was screened from sweet potatoes, which showed a remarkably higher expression level in resistant varieties than in susceptible ones after inoculation with C. fimbriata. Sequence analysis indicated that IbChiA belongs to family 19 class II extracellular chitinase with a MW of 26.3 kDa and pI of 5.96. Recombinant IbChiA, produced by Pichia pastoris, displayed antifungal activity and stability. IbChiA could restrain the mycelium extension of C. fimbriata. FDA/PI double staining combined with transmission electron microscopy observation revealed the remarkable fungicidal effect of IbChiA on the conidia of C. fimbriata. The disease symptoms on the surface of slices and tuberous roots of sweet potatoes were significantly reduced after treatment with IbChiA. These results indicated that IbChiA could be used as a potential biofungicide to replace chemical fungicides.

Split application enhances sweetpotato starch production by regulating the conversion of sucrose to starch under reduced nitrogen supply.

Split application could improve nitrogen (N) uptake and increase sweetpotato yields under reduced N supply; however, little is known about how it affects the process of starch production in storage roots. An experiment was conducted to determine the effects of three N management strategies [conventional basal N management; 80% of the conventional N rate applied as a basal fertilizer; 80% of the conventional N rate equally split at transplanting and 35 days after transplanting] on starch accumulation, enzyme activity and genes expression in the conversion of sucrose to starch and the relationships among them. The results showed that, compared with conventional basal N management, split application decreased sucrose accumulation by 11.78%, but increased starch accumulation by 11.12% through improving the starch accumulation rate under reduced N supply. The ratio of sucrose synthetase to sucrose phosphate synthase, the enzymatic activity of ADP-glucose pyrophosphorylase (AGPP), starch synthase, and the expression of their corresponding genes were promoted by split application under reduced N supply and were positively correlated with starch accumulation rate. AGPP is the rate-limiting enzyme in starch synthesis in storage roots under different N management strategies. These results indicate that starch accumulation was enhanced by split application through regulating the activity and gene expression of key enzymes involved in the conversion of sucrose to starch under reduced N supply.

IbMPK3/IbMPK6-mediated IbSPF1 phosphorylation promotes tolerance to bacterial pathogen in sweetpotato.

KEY MESSAGE: IbSPF1, a novel target of IbMPK3/IbMPK6, regulates biotic stress response in sweetpotato. Environmental stresses due to biotic and abiotic factors negatively affect crop quality and productivity. To minimize the damage caused by these factors, numerous stress signaling pathways are activated in plants. Among these, the mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in diverse plant stress responses. MPK3 and MPK6 function in several cellular signaling pathways by phosphorylating downstream partner proteins in response to environmental stresses. However, little is known about the MPK3/MPK6 signaling pathway in sweetpotato [Ipomoea batatas (L.) Lam]. We recently confirmed that IbMPK3 and IbMPK6, two pathogen-responsive MAPKs, play essential roles in defense gene activation in sweetpotato. In this study, we show that sweetpotato SP8-binding factor (IbSPF1), a substrate of IbMPK3/IbMPK6, functions as a transcriptional regulator of biotic stress signaling in sweetpotato. IbSPF1 specifically interacts with IbMPK3 and IbMPK6, which phosphorylate Ser75 and Ser110 residues of IbSPF1. This increases the affinity of IbSPF1 for the W-box element in target gene promoters. Additionally, the expression of IbSPF1 was up-regulated under various stress conditions and different hormone treatments involved in plant defense responses. Interestingly, the phospho-mimicking mutant of IbSPF1 showed enhanced resistance to Pseudomonas syringae pv. tabaci, and transient expression of mutant IbSPF1 induced the expression of pathogenesis-related genes. These results indicate that the phosphorylation of IbSPF1 by IbMPK3/IbMPK6 plays a critical role in plant immunity by up-regulating the expression of downstream genes.

Acid Inhibition on Polyphenol Oxidase and Peroxidase in Processing of Anthocyanin-Rich Juice and Co-product Recovery from Purple-Fleshed Sweetpotatoes.

With high phytochemical and starch contents, purple-fleshed sweetpotatoes (PFSP) have been processed into various functional ingredients and food products including juices and natural colorants. For juice processing, PFSP are usually subjected to heat treatment for inactivation of pigment-degrading enzymes. However, heating of sweetpotatoes gelatinizes starch and produces thick slurry with cooked flavor, which are the drawbacks. Development of alternative processes to overcome the stated problems will be beneficial to sweetpotato processors. This study demonstrated that acidified water (≥3% w/v citric acid) was effective in inhibiting polyphenol oxidase and peroxidase in raw PFSP resulting in an attractive reddish juice. About 93% total phenolics (TP) and 83% total monomeric anthocyanins (TMA) in PFSP were extracted by two repeated extractions. The combined PFSP juice (3.2 L/kg PFSP) had high levels of TP (1,850 mg/L) and TMA (475 mg/L). With the developed process, 167 g dried starch, and 140 g dried high-fiber pomace were obtained for each kg raw PFSP, besides the highly pigmented juice. Pasteurization of the PFSP juice samples (pH 3.2) at 80 °C for 12 s resulted in 15% loss in TMA and had no effect on TP. The results indicated an efficient process to produce sweetpotato juice with high bioactive compounds and recovery of starch and high dietary fiber pomace as co-products. PRACTICAL APPLICATION: Purple-fleshed sweetpotatoes (PFSP) are rich in polyphenolics and antioxidant activities. In PFSP juice extraction, heat treatment to inactivate the pigment-degrading enzymes results in starch gelatinization and cooked flavor. A nonthermal process using acidified water was developed for producing anthocyanin-rich juice from PFSP and concurrently recovering native starch and dried pomace, which would increase the economic feasibility of the developed process. The results demonstrate an efficient process for the sweetpotato industry in producing PFSP pigmented juice and co-products for various food applications.

Changes in Nanoscale Chain Assembly in Sweet Potato Starch Lamellae by Downregulation of Biosynthesis Enzymes.

Granule-bound starch synthase I (GBSSI) and starch-branching enzymes I and II (SBEI and SBEII) are crucial enzymes that biosynthesize starches with varied apparent amylose contents and amylopectin branching structure. With a sweet potato ( Ipomoea batatas [L.] Lam. cv. Xushu22), this work shows that downregulating GBSSI (for waxy starch) or SBE (for high-amylose starch) activity allowed the formation of new semicrystalline lamellae (named Type II) in sweet potato starch in addition to the widely reported Type I lamellae. Small-angle X-ray scattering (SAXS) results show that, compared with Type I lamellae, Type II lamellae displayed increased average thickness and thickness-distribution width, with thickened amorphous and crystalline components. The size-exclusion-chromatography (SEC) data revealed mainly two enzyme sets, (i) and (ii), synthesizing amylopectin chains. Reducing the GBSSI or SBE activity increased the amounts of amylopectin long chains (degree of polymerization (DP) ≥ 33). Combined SAXS and SEC analyses indicate that parts of these long chains from enzyme set (i) could be confined to Type II lamellae, followed by DP ≤ 32 short chains in Type I lamellae and the rest of the long chains from enzyme sets (i) and (ii) spanning more than a single lamella.

The p38-like MAP kinase modulated H2O2 accumulation in wounding signaling pathways of sweet potato.

In sweet potato (Ipomoea batatas cv Tainung 57), MAPK cascades are involved in the regulation of Ipomoelin (IPO) expression upon wounding. p38 MAPK plays an important role in plant's responses to various environmental stresses. However, the role of p38-like MAPK in wounding response is still unknown. In this study, the levels of phosphorylated-p38-like MAPK (pp38-like MAPK) in sweet potato were noticeably reduced after wounding. In addition, SB203580 (SB), a specific inhibitor blocking p38 MAPK phosphorylation, considerably decreased the accumulation of pp38-like MAPK. Expression of a wound-inducible gene IPO was elevated by SB. Moreover, it stimulated hydrogen peroxide (H2O2) production rather than cytosolic Ca2+ elevation in sweet potato leaves. However, NADPH oxidase (NOX) inhibitor diphenyleneiodonium could not inhibit IPO induction stimulated by SB. These results indicated a p38-like MAPK mechanism was involved in the regulation of IPO expression through NOX-independent H2O2 generation. In addition, the presence of the protein phosphatase inhibitor okadaic acid or the MEK1/ERK inhibitor PD98059 repressed the H2O2- or SB-induced IPO expression, demonstrating phosphatase(s) and MEK1/ERK functioning in the downstream of H2O2 and pp38-like MAPK in the signal transduction pathway stimulating IPO. Conclusively, wounding decreased the amount of pp38-like MAPK, stimulated H2O2 production, and then induced IPO expression.

Antioxidative system in sweet potato root is activated by low-temperature storage.

BACKGROUND: Sweet potato is susceptible to chilling injury during low-temperature storage. To explore the correlation between chilling injury and reactive oxygen species (ROS) metabolism, the content of ROS and the activities and gene expression of antioxidant enzymes were analyzed in the typical storage-tolerant cultivar Xushu 32 and storage-sensitive cultivar Yanshu 25. RESULTS: The activities of antioxidant enzymes including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were enhanced rapidly in the early period of storage in response to chilling stress. Thereafter, the content of ROS metabolites increased consistently due to gradual decrease in ROS scavenging enzymes. Storage-tolerant cultivar Xushu 32 had higher antioxidant enzyme activities and gene expressions as well as higher content of antioxidant metabolites and lower content of ROS metabolites compared with storage-sensitive cultivar Yanshu 25, suggesting that the capacity of ROS scavenging by antioxidant enzymes and antioxidants is highly associated with the tolerance of sweet potato to chilling stress. CONCLUSION: These results indicated that the antioxidative system is activated in the storage root of sweet potato and the antioxidative capacity is positively associated with better storage performance in the storage-tolerant cultivar. © 2019 Society of Chemical Industry.

A novel glutathione S-transferase gene from sweetpotato, IbGSTF4, is involved in anthocyanin sequestration.

Anthocyanins are synthesized by multi-enzyme complexes localized at the cytoplasmic surface of the endoplasmic reticulum (synthesis site), and transported to the destination site, the vacuole. Three mechanisms for the vacuolar accumulation of anthocyanin in plant species have been proposed. Previous studies have indicated that glutathione S-transferase (GST) genes from model and ornamental plants are involved in anthocyanin transportation. In the present study, an anthocyanin-related GST, IbGSTF4, was identified and characterized based on transcriptome results. Phylogenetic analysis revealed that IbGSTF4 was most closely correlated to PhAN9 and CkmGST3, the anthocyanin-related GST of Petunia hybrida and Cyclamen. Furthermore, the expression analysis revealed that IbGSTF4 is strongly expressed in pigmented tissues, when compared to green organs, which is in agreement to the ability to correlate with anthocyanin accumulation. A GST activity assay uncovered that the IbGST4 protein owned similar activities with the GST family. Furthermore, the molecular functional complementation of Arabidopsis thaliana mutant tt19 demonstrated that IbGSTF4 might play a vital role in the vacuole sequestration of anthocyanin in sweetpotato. Moreover, the dual luciferase assay revealed that the LUC driven by the promoter of IbGSTF4 could not be directly activated by IbMYB1, suggesting that the regulatory mechanism of anthocyanin accumulation and sequestration in sweetpotato was intricate.

Biochemical characterization of isoprene synthase from Ipomoea batatas.

The bio-production process of isoprene, an essential chemical used in industry, is strongly limited by isoprene synthase. In our previous work, relatively high isoprene production was observed with isoprene synthase from Ipomoea batatas (IspSib). In this work the biochemical properties of IspSib were analyzed and compared with those of isoprene synthase from Populus alba (IspSpa) and other species. Firstly, IspSib and IspSpa were expressed, purified, and identified by SDS-PAGE and western blot analysis. Secondly, pH and temperature dependence of IspSib were performed and an optimum pH of 8.6 and an optimum temperature of 42 °C were resulted. Mg2+ with optimum concentration of 56 mM was proved to be needed for enzyme activation. In addition, in vivo and in vitro study of the thermostabilities of IspSib and IspSpa were performed. The enzyme activity of IspSib and IspSpa dropped very rapidly after incubation at 30 °C; almost 80% enzyme activity of IspSib was lost after 20 min of incubation. Moreover, the Michaelis-Menten constant was measured. IspSib showed a lower Km, 0.2 mM, and a higher kcat, 0.37 s-1, as compared with IspSpa. The high catalytic efficiency, which was reflected by the high kcat/Km ratio, indicates that IspSib is a good candidate for the bio-isoprene production, while its thermal instability remains as a challenge. Enzyme engineering efforts, such as direction evolution or semi-rational evolution, are planned for further research.

The effects of sequential enzyme modifications on structural and physicochemical properties of sweet potato starch granules.

Sweet potato starch products possess unacceptable hardness and poor transparency that in-turn reduces consumer acceptability. To expand the sweet potato starch utility with user acceptable and palatable food products herein enzyme modification has been carried out. Transglucosidase (TGAN) in combination with maltogenic α-amylase (MABS) and ß-amylase (BA) appears to be advantageous to modulate sweet potato starch properties. The MABS → BA → TGAN treatment increases the α-1, 6 glycosidic linkage ratio and short chain proportions (DP ≤ 24). Decrease in chain length, molecular weight and long chain proportions (DP > 24) is noticed. The initial C-type starch polymorphic structure transforms to B-type structure along with decreased crystallinity. Solubility increases substantially with concomitant decrease in viscosity, gelatinization temperature and melting enthalpy. The outcome is believed to open new pathways for regulating the physicochemical properties of sweet potato starch especially by enzyme modification to the design and development of novel sweet potato starch-based products.

Chemical Modification of Sweet Potato ß-amylase by Mal-mPEG to Improve Its Enzymatic Characteristics.

The sweet potato ß-amylase (SPA) was modified by 6 types of methoxy polyethylene glycol to enhance its specific activity and thermal stability. The aims of the study were to select the optimum modifier, optimize the modification parameters, and further investigate the characterization of the modified SPA. The results showed that methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) was the optimum modifier of SPA; Under the optimal modification conditions, the specific activity of Mal-mPEG5000-SPA was 24.06% higher than that of the untreated SPA. Mal-mPEG5000-SPA was monomeric with a molecular weight of about 67 kDa by SDS-PAGE. The characteristics of Mal-mPEG5000-SPA were significantly improved. The Km value, Vmax and Ea in Mal-mPEG5000-SPA for sweet potato starch showed that Mal-mPEG5000-SPA had greater affinity for sweet potato starch and higher speed of hydrolysis than SPA. There was no significant difference of the metal ions' effect on Mal-mPEG5000-SPA and SPA.

Histochemical observations and gene expression changes related to internal browning in tuberous roots of sweet potato (Ipomea batatas).

The mechanism underlying internal browning (IB), or brown discoloration, of the central region of tuberous roots of sweet potato (Ipomoea batatas) was examined. IB disorder begins in roots from approx. 90 days after transplanting, and the severity increases significantly with time. IB damage initially occurs in cells around the secondary vascular tissue, and the area per cell occupied by starch grains in this region was larger than in the unaffected region. High levels of reducing sugars, polyphenol oxidase (PPO) activities, chlorogenic acid, and hydrogen peroxide (H2O2) were detected in cells from the IB damaged regions. The content of sugar and polyphenols was higher in disks (transverse sections) with larger amounts of damaged tissues than in disks of sound root. The transcript levels of acid invertase (IbAIV) tended to be higher with greater IB severity, whereas fluctuation patterns of ADP-glucose pyrophosphorylase (IbAGPase), granule bound starch synthase (IbGBSS), and starch branching enzyme 1 (IbSBE1) were lower with higher IB severity. These observations suggest that the incidence of IB disorder in sweet potato is largely dependent on the excessive generation of reactive oxygen species (ROS) in cells around the secondary vascular tissues due to the abundant accumulation of sugar and/or starch grains during the root maturation period.

Structural insights on starch hydrolysis by plant ß-amylase and its evolutionary relationship with bacterial enzymes.

The conversion of starch to maltose is catalysed in plants by ß-amylase. The enzymatic mechanism has been well-characterized for the soybean and barley enzymes, which utilise a glutamic acid-glutamate pair. In the present study, we present a surprise observation of maltotetraose at the active site, the presence of which elucidates the clear role of Thr344 as a conformational "switch" between substrate binding and product release during hydrolysis. This observation is confirmed by the selection of maltotetraose by the crystallized enzyme although that carbohydrate was present in only trace amounts. The conformation of the residues in the substrate-binding site changed upon substrate binding, leading to the movement of threonine, glutamic acid, and the loop conformation, elucidating a missing link in the existing mechanism. By aligning our substrate-free and maltotetraose-bound structures with other existing structures, the sequence of events from substrate binding to hydrolysis can be visualized. Apart from this, the evolutionary relationship among ß-amylases of bacterial and amyloplastic origin could be established. The presence of a sugar-binding domain in the bacterial enzyme and its absence in the plant counterpart could be attributed to a carbohydrate-rich environment. Interestingly, cladogram analysis indicates the presence of N-terminal additions in some plant ß-amylases. Based on sequence similarity, we postulate that the role of such additions is important for the regulation of enzymatic activity, particularly under stress conditions.