Impact of changes in root biomass on the occurrence of internal browning in radish root.

The purpose of this study was to investigate the effects of root biomass during the later stage of growth on fatty acid composition and lipid peroxidation, and to clarify the physiological mechanisms by which these differences affect internal browning (IB) development in radish roots. Therefore, we controlled the enlargement of roots by changing the thinning period and generated plots composed of roots with different biomass in the latter half of growth. The earlier the radish seedlings were thinned, the more vigorous the root growth from an earlier stage was achieved. Earlier thinning caused IB from the early stage of root maturation, and IB severity progressed with subsequent age progression; however, IB damage did not occur when root size during the later growth stage was kept small by later thinning. Higher levels of hydrogen peroxide, peroxidase activity, NADPH-dependent reactive oxygen species (ROS) burst-related genes, and carbonyl compounds were detected in earlier-thinned large-sized roots compared to later-thinned small-sized ones. Compared with the latter small-sized roots, the former large-sized roots had a lower ratio of linoleic acid (18:2) and a higher ratio of α-linolenic acid (α-18:3). Furthermore, in earlier-thinned large-sized roots, higher levels of phospholipase- and/or lipoxygenase-related genes were detected compared to later-thinned small-sized ones. These facts suggest the possibility that root biomass in the later stage of growth affects the desaturation of membrane fatty acids, ROS concentration, and activity of fatty acid degrading enzymes, and controls the occurrence of IB injury through membrane oxidative degradation.

Effects of heat stress on the biological Maillard reaction, oxidative stress, and occurrence of internal browning in Japanese radish (Raphanus sativus L.).

Internal browning (IB) of Japanese radish, in which the entire central part of the root changes from light tan to dark brown, is a physiological disorder caused by heat stress at the root maturation stage and represents significant economic losses to farmers. In this study, we cultivated three cultivars with different tolerance to IB disorder under high-temperature conditions, and examined the physiological factors involved in IB disorder differences among cultivars. There was a close correlation between the severity of IB disorder among cultivars and the size of root parenchymal cells. Significantly smaller cells were observed in the IB-resistant cultivars compared to the IB-susceptible one. Glucose concentration and invertase gene expression level tended to be higher in the normal roots of the IB-susceptible cultivar than in those of the resistant cultivars. Results of immunohistochemical analysis revealed that hexanoyl-lysine and acrolein, which are indicators of oxidative stress, in parenchymal cells were significantly elevated in the normal roots of the IB-susceptible cultivar than in those of the resistant ones. Similarly, the normal roots of the IB-susceptible cultivar showed elevated levels of Nε-(carboxymethyl)lysine, which is an indicator of the Maillard reaction, compared with those of the resistant cultivars. In addition, the immunohistochemistry of these indicators of oxidative stress and the Maillard reaction were more strongly observed in the damaged roots of the IB-susceptible cultivar. These results indicate that the occurrence of IB in Japanese radish is closely related to the increase in cell size, enhancement of the Maillard reaction by elevation in reducing sugar concentration, and increase in intracellular oxidative stress.

Oxidative stress via the Maillard reaction is associated with the occurrence of internal browning in roots of sweetpotato (Ipomoea batatas).

Internal browning (IB) of sweetpotato is a physiological disorder that causes great losses not only for growers but also for consumers. Differences in sugar metabolism in tissues were compared for normal and disorderly regions of roots of an IB-susceptible cultivar. Compared to the normal region, the disorderly region had higher content of reducing sugar. Likewise, higher levels of genes related to sugar biosynthesis were detected in the disorderly region than in the normal region. Through immunohistochemical screening of IB-susceptible and -resistant cultivars, we identified markers of oxidative stress, including due to the Maillard reaction, in the disorderly region. 8-Nitroguanosine, hexanoyl-lysin, acrolein, and dityrosine were markedly accumulated, especially in parenchyma cells around secondary vascular tissue (SVT) in the disorderly region of the IB-susceptible cultivar. Likewise, in this region, marked accumulation of methyglyoxal and 3-DG imidazolone was observed in parenchymal cells around SVT. These results show that reactive oxygen species in roots of growing sweetpotato may be caused by non-enzymatic reactions via the biological Maillard reaction due to sugar accumulation within the cells and that this phenomenon is related to IB occurrence.

Enhancement of Iron Acquisition in Rice by the Mugineic Acid Synthase Gene With Ferric Iron Reductase Gene and OsIRO2 Confers Tolerance in Submerged and Nonsubmerged Calcareous Soils.

Iron (Fe) is an essential micronutrient for plants. Plants encounter Fe deficiency when grown in calcareous soil with low Fe availability, leading to reduced crop yield and agricultural problem. Rice acquires Fe from the soil via Strategy I-related system (ferrous ion uptake by OsIRT1) and Strategy II system (ferric ion uptake by chelation). However, rice plants have a weak ability in Fe(III) reduction and phytosiderophore secretion. We previously produced an Fe deficiency-tolerant rice harboring OsIRT1 promoter-refre1/372 (for higher Fe(III) reductase ability) and a 35S promoter-OsIRO2 (for higher phytosiderophore secretion). In this study, we produced a new Fe deficiency-tolerant rice by the additional introduction of a barley IDS3 genome fragment with refre1/372 and OsIRO2 (named as IRI lines) for further enhancement in Strategy II phytosiderophore productivity and better growth performance in various environments. Our results show that an enhanced tolerance was observed in OsIRO2 introduced line at the early growth stage, refre1/372 introduced line in the late stage, and RI line in all stages among five types of cultivation method. Moreover, we demonstrated that new IRI rice lines exhibited enhanced tolerance to Fe deficiency compared to nontransgenic (NT) rice and rice lines harboring the overexpressing OsIRO2 or the IDS3 fragment under submerged calcareous soil. The yields of IRI lines were ninefold higher than the NT line. Furthermore, under Fe-limited nonsubmerged calcareous soil condition (a new cultivation condition), IRI lines also conferred enhanced tolerance than NT, lines introducing only the OsIRT1 promoter-refre1/372 or overexpressing OsIRO2, and lines harboring both. Our results demonstrate that further enhancement of the Strategy II Fe uptake system by the mugineic acid synthase gene in addition to Fe uptake by enhanced ferric Fe reduction and phytosiderophore production in rice contributes Fe deficiency tolerance and broaden its utility in calcareous soil cultivation under paddy or nonpaddy field conditions.

Occurrence of internal browning in tuberous roots of sweetpotato and its related starch biosynthesis.

Although sweetpotato is an important crop worldwide, there has been almost no research on the occurrence of internal browning (IB) to date. In this study, we clarified the mechanism of occurrence of the disorder by using two types of cultivars with different IB susceptibility. In cells around the secondary vascular tissue, large size of starch grains accumulated in IB-susceptible cultivar compared with resistant one. Histochemical observation performed on cells around the secondary vascular tissues showed the presence of high levels of polyphenol oxidase activity, chlorogenic acid, and hydrogen peroxide in cells from the IB-affected regions in IB-susceptible cultivar. Likewise, high levels of starch content, hydrogen peroxide concentration, and polyphenol content were detected in the affected regions of IB-susceptible cultivar. In IB-susceptible cultivar, both the transcript levels of gens related starch and polyphenol biosynthesis were higher at an early stage of root maturation, while the levels in resistant cultivar were low at this stage and thereafter increased relatively more moderately. These observations suggest that the occurrence of IB disorder in sweetpotato largely depends on the morphology and timing of accumulated starch grain in cells around the secondary vascular tissues.

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.

Carbohydrate components in sweetpotato storage roots: their diversities and genetic improvement.

Carbohydrates are important components in sweetpotatoes in terms of both their industrial use and eating quality. Although there has been a narrow range of diversity in the properties of sweetpotato starch, unique varieties and experimental lines with different starch traits have been produced recently both by conventional breeding and genetic engineering. The diversity in maltose content, free sugar composition and textural properties in sweetpotato cultivars is also important for their eating quality and processing of storage roots. In this review, we summarize the current status of research on and breeding for these important traits and discuss the future prospects for research in this area.

A new transgenic rice line exhibiting enhanced ferric iron reduction and phytosiderophore production confers tolerance to low iron availability in calcareous soil.

Iron (Fe) deficiency is a critical agricultural problem, especially in calcareous soil, which is distributed worldwide. Rice plants take up Fe(II) from soil through a OsIRT1 transporter (Strategy I-related system) and also take up Fe(III) via a phytosiderophore-based system (Strategy II system). However, rice plants are susceptible to low-Fe conditions because they have low Fe(III) reduction activity and low-level phytosiderophore secretion. Previously, we produced transgenic rice plants expressing a mutationally reconstructed yeast ferric chelate reductase, refre1/372, under the control of the OsIRT1 promoter. This transgenic rice line exhibited higher Fe(III) chelate reductase activity and tolerance to Fe deficiency. In addition, we produced transgenic rice overexpressing the Fe deficiency-inducible transcription factor, OsIRO2, which regulates the expression of various genes involved in the strategy II Fe(III) uptake system, including OsNAS1, OsNAAT1, OsDMAS1, OsYSL15, and TOM1. This transgenic rice exhibited improved phytosiderophore secretion ability and tolerance to Fe deficiency. In the present research, transgenic rice plants that possess both the OsIRT1 promoter-refre1/372 and the 35S promoter-OsIRO2 (RI lines) were produced to enhance both Strategy I Fe(II) reductase ability and Strategy II phytosiderophore productivity. RI lines exhibited enhanced tolerance to Fe-deficient conditions at the early and middle-late stages of growth in calcareous soil, compared to both the non-transgenic line and lines harboring either OsIRT1 promoter-refre1/372 or 35S promoter-OsIRO2 alone. RI lines also exhibited a 9-fold higher yield than the non-transgenic line. Moreover, we successfully produced Fe-deficiency-tolerant Tachisugata rice, which is a high-biomass variety used as fodder. Collectively, our results demonstrate that combined enhancement of two Fe uptake systems in rice is highly effective in conferring tolerance to low Fe availability in calcareous soil.

Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata.

A class III chitinase belonging to the GH18 family from Nepenthes alata (NaCHIT3) was expressed in Escherichia coli. The enzyme exhibited hydrolytic activity toward colloidal chitin, ethylene glycol chitin, and (GlcNAc)(n) (n=5 and 6). The enzyme hydrolyzed the fourth glycosidic linkage from the non-reducing end of (GlcNAc)(6). The anomeric form of the products indicated it was a retaining enzyme. The colloidal chitin hydrolytic reaction displayed high activity between pH 3.9 and 6.9, but the pH optimum of the (GlcNAc)(6) hydrolytic reaction was 3.9 at 37 °C. The optimal temperature for activity was 65 °C in 50 mM sodium acetate buffer (pH 3.9). The pH optima of NaCHIT3 and NaCHIT1 might be related to their roles in chitin degradation in the pitcher fluid.

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata.

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (ß-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), ß-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.

Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L.

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata.

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [ß-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata.

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants.

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear alpha(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.

Reduction of trienoic fatty acid content by expression of a double-stranded RNA of a plastid omega-3 fatty acid desaturase gene in transgenic tobacco.

Plastid omega-3 fatty acid desaturase catalyzes the conversion of dienoic fatty acids (16:2 and 18:2) to trienoic fatty acids (16:3 and alpha-18:3) in glycerolipids which are the main constituents of chloroplast membranes. We produced transgenic tobacco plants that express the transcript of a double-stranded RNA (dsRNA) of tobacco plastid omega-3 fatty acid desaturase gene, NtFAD7. In these transgenic plants, 16:3 and alpha-18:3 content in leaves decreased to less than 2.7% and 7.5-10.4%, respectively, when compared with the control plant. The steady-state NtFAD7 mRNA was not detected in the transgenic plants. These results indicate that down-regulation of the transcript level in the NtFAD7 by introduction of NtFAD7 dsRNA constructs is useful to decrease the trienoic fatty acid contents of the vegetative tissues in higher plants.

Starch-branching enzyme I gene (IbSBEI) from sweet potato (Ipomoea batatas); molecular cloning and expression analysis.

The cDNA of the starch-branching enzyme I gene (IbSBEI) in the sweet potato (Ipomoea batatas) has been cloned and sequenced. The IbSBEI amino acid sequence was 81% identical to that of potato StSBEI. DNA gel-blot analyses demonstrated that at least two copies of IbSBEI are present in the sweet potato genome. IbSBEI was strongly expressed in tuberous roots. Transcript levels in the roots of single leaf cuttings were extremely low during the first 15-40 d after planting and continuously increased up to 50 d, by which time the tuberous roots had almost completely developed. This indicates that IbSBEI may work in concert with the AGPase large subunit during the primary phase of starch granule formation.

Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam.

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.