Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton.

Sophorae Radix (Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.

Down-regulation of GIGANTEA-like genes increases plant growth and salt stress tolerance in poplar.

The flowering time regulator GIGANTEA (GI) connects networks involved in developmental stage transitions and environmental stress responses in Arabidopsis. However, little is known about the role of GI in growth, development and responses to environmental challenges in the perennial plant poplar. Here, we identified and functionally characterized three GI-like genes (PagGIa, PagGIb and PagGIc) from poplar (Populus alba × Populus glandulosa). PagGIs are predominantly nuclear localized and their transcripts are rhythmically expressed, with a peak around zeitgeber time 12 under long-day conditions. Overexpressing PagGIs in wild-type (WT) Arabidopsis induced early flowering and salt sensitivity, while overexpressing PagGIs in the gi-2 mutant completely or partially rescued its delayed flowering and enhanced salt tolerance phenotypes. Furthermore, the PagGIs-PagSOS2 complexes inhibited PagSOS2-regulated phosphorylation of PagSOS1 in the absence of stress, whereas these inhibitions were eliminated due to the degradation of PagGIs under salt stress. Down-regulation of PagGIs by RNA interference led to vigorous growth, higher biomass and enhanced salt stress tolerance in transgenic poplar plants. Taken together, these results indicate that several functions of Arabidopsis GI are conserved in its poplar orthologues, and they lay the foundation for developing new approaches to producing salt-tolerant trees for sustainable development on marginal lands worldwide.

Metabolic engineering of carotenoids in transgenic sweetpotato.

Sweetpotato [Ipomoea batatas (L.) Lam], which contains high levels of antioxidants such as ascorbate and carotenoids in its storage root, is one of the healthiest foods, as well as one of the best starch crops for growth on marginal lands. In plants, carotenoid pigments are involved in light harvesting for photosynthesis and are also essential for photo-protection against excess light. As dietary antioxidants in humans, these compounds benefit health by alleviating aging-related diseases. The storage root of sweetpotato is a good source of both carotenoids and carbohydrates for human consumption. Therefore, metabolic engineering of sweetpotato to increase the content of useful carotenoids represents an important agricultural goal. This effort has been facilitated by cloning of most of the carotenoid biosynthetic genes, as well as the Orange gene involved in carotenoid accumulation. In this review, we describe our current understanding of the regulation of biosynthesis, accumulation and catabolism of carotenoids in sweetpotato. A deeper understanding of these topics should contribute to development of new sweetpotato cultivars with higher levels of nutritional carotenoids and abiotic stress tolerance.

Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity.

Differential responses of three sweetpotato metallothionein genes to abiotic stress and heavy metals.

Metallothioneins (MTs) are cysteine-rich, low molecular weight, metal-binding proteins that are widely distributed in living organisms. Plants produce metal-chelating proteins such as MTs to overcome the toxic effects of heavy metals. We cloned three MT genes from sweetpotato leaves [Ipomoea batatas (L.) Lam.]. The three IbMT genes were classified according to their cysteine residue alignment into type 1 (IbMT1), type 2 (IbMT2), and type 3 (IbMT3). IbMT1 was the most abundantly transcribed MT. It was predominantly expressed in leaves, roots, and callus. IbMT2 transcript was detected only in stems and fibrous roots, whereas IbMT3 was strongly expressed in leaves and stems. The IbMT expression profiles were investigated in plants exposed to heavy metals and abiotic stresses. The levels of IbMT1 expression were strongly elevated in response to Cd and Fe, and moderately higher in response to Cu. The IbMT3 expression pattern in response to heavy metals was similar to that of IbMT1. Exposure to abiotic stresses such as methyl viologen (MV; paraquat), NaCl, polyethylene glycol (PEG), and H2O2 up-regulated IbMT expression; IbMT1 responded strongly to MV and NaCl, whereas IbMT3 was induced by low temperature and PEG. Transgenic Escherichia coli overexpressing IbMT1 protein exhibited results suggest that IbMT could be a useful tool for engineering plants with enhanced tolerance to environmental stresses and heavy metals.

Down-regulation of sweetpotato lycopene ß-cyclase gene enhances tolerance to abiotic stress in transgenic calli.

Lycopene ß-cyclase (LCY-ß) is a key enzyme involved in the synthesis of α- and ß-branch carotenoids such as α-carotene and ß-carotene through the cyclization of lycopene. IbLCY-ß had a length of 1,506 bp and approximately 80 % nucleotide sequence identity with that of tomato LCY-ß. IbLCY-ß was strongly expressed in leaves, and expression was enhanced by salt-stress and osmotic-stress conditions. To characterize the LCY-ß gene (IbLCY-ß) of sweetpotato (Ipomoea batatas), it was isolated and transformed into calli of white-fleshed sweetpotato using an IbLCY-ß-RNAi vector. Transgenic IbLCY-ß-RNAi calli had yellow to orange color and higher antioxidant activity compared to that of white, nontransgenic (NT) calli. Transgenic cells had significantly higher contents of total carotenoids, although lycopene was not detected in transgenic or NT cells. All transgenic calli had strongly activated expression of carotenoid biosynthetic genes such as ß-carotene hydroxylases (CHY-ß), cytochrome P450 monooxygenases (P450), and carotenoid cleavage dioxigenase 1 (CCD1). Transgenic cells exhibited less salt-induced oxidative-stress damage compared to that of NT cells, and also had greater tolerance for polyethylene glycol (PEG)-mediated drought compared to that of NT cells, due to the higher water content and reduced malondialdehyde (MDA) content. The abscisic acid content was also higher in transgenic cells. These results show that a study of IbLCY-ß can facilitate understanding of the carotenoid biosynthetic pathway in sweetpotato. IbLCY-ß could be useful for developing transgenic sweetpotato enriched with nutritional carotenoids and with greater tolerance to abiotic stresses.

Down-regulation of the IbEXP1 gene enhanced storage root development in sweetpotato.

The role of an expansin gene (IbEXP1) in the formation of the storage root (SR) was investigated by expression pattern analysis and characterization of IbEXP1-antisense sweetpotato (Ipomoea batatas cv. Yulmi) plants in an attempt to elucidate the molecular mechanism underlying SR development in sweetpotato. The transcript level of IbEXP1 was high in the fibrous root (FR) and petiole at the FR stage, but decreased significantly at the young storage root (YSR) stage. IbEXP1-antisense plants cultured in vitro produced FRs which were both thicker and shorter than those of wild-type (WT) plants. Elongation growth of the epidermal cells was significantly reduced, and metaxylem and cambium cell proliferation was markedly enhanced in the FRs of IbEXP1-antisense plants, resulting in an earlier thickening growth in these plants relative to WT plants. There was a marked reduction in the lignification of the central stele of the FRs of the IbEXP1-antisense plants, suggesting that the FRs of the mutant plants possessed a higher potential than those of WT plants to develop into SRs. IbEXP1-antisense plants cultured in soil produced a larger number of SRs and, consequently, total SR weight per IbEXP1-antisense plant was greater than that per WT plant. These results demonstrate that SR development was accelerated in IbEXP1-antisense plants and suggest that IbEXP1 plays a negative role in the formation of SR by suppressing the proliferation of metaxylem and cambium cells to inhibit the initial thickening growth of SRs. IbEXP1 is the first sweetpotato gene whose role in SR development has been directly identified in soil-grown transgenic sweetpotato plants.

Downregulation of the lycopene ε-cyclase gene increases carotenoid synthesis via the ß-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli.

Lycopene ε-cyclase (LCY-ε) is involved in the first step of the α-branch synthesis pathway of carotenoids from lycopene in plants. In this study, to enhance carotenoid synthesis via the ß-branch-specific pathway [which yields ß-carotene and abscisic acid (ABA)] in sweet potato, the expression of IbLCY-ε was downregulated by RNAi (RNA interference) technology. The RNAi-IbLCY-ε vector was constructed using a partial cDNA of sweet potato LCY-ε isolated from the storage root and introduced into cultured sweet potato cells by Agrobacterium-mediated transformation. Both semi-quantitative Reverse transcription polymerase chain reaction (RT-PCR) of carotenoid biosynthesis genes and high-performance liquid chromatography (HPLC) analysis of the metabolites in transgenic calli, in which the LCY- εgene was silenced, showed the activation of ß-branch carotenoids and its related genes. In the transgenic calli, the ß-carotene content was approximately 21-fold higher than in control calli, whereas the lutein content of the transgenic calli was reduced to levels undetectable by HPLC. Similarly, expression of the RNAi-IbLCY-ε transgene resulted in a twofold increase in ABA content compared to control calli. The transgenic calli showed significant tolerance of 200 mM NaCl. Furthermore, both the ß-branch carotenoids content and the expression levels of various branch-specific genes were higher under salt stress than in control calli. These results suggest that, in sweet potato, downregulation of the ε-cyclization of lycopene increases carotenoid synthesis via the ß-branch-specific pathway and may positively regulate cellular defenses against salt-mediated oxidative stress.

Expression of the sweetpotato R2R3-type IbMYB1a gene induces anthocyanin accumulation in Arabidopsis.

R2R3-type MYB transcription factors (TFs) play important roles in transcriptional regulation of anthocyanins. The R2R3-type IbMYB1 is known to be a key regulator of anthocyanin biosynthesis in the storage roots of sweetpotato. We previously showed that transient expression of IbMYB1a led to anthocyanin pigmentation in tobacco leaves. In this article, we generated transgenic Arabidopsis plants expressing the IbMYB1a gene under the control of CaMV 35S promoter, and the sweetpotato SPO and SWPA2 promoters. Overexpression of IbMYBa in transgenic Arabidopsis produced strong anthocyanin pigmentation in seedlings and generated a deep purple color in leaves, stems and seeds. Reverse transcription-polymerase chain reaction analysis showed that IbMYB1a expression induced upregulation of several structural genes in the anthocyanin biosynthetic pathway, including 4CL, CHI, F3'H, DFR, AGT, AAT and GST. Furthermore, overexpression of IbMYB1a led to enhanced expression of the AtTT8 (bHLH) and PAP1/AtMYB75 genes. high-performance liquid chromatography analysis revealed that IbMYB1a expression led to the production of cyanidin as a major core molecule of anthocyanidins in Arabidopsis, as occurs in the purple leaves of sweetpotato (cv. Sinzami). This result shows that the IbMYB1a TF is sufficient to induce anthocyanin accumulation in seedlings, leaves, stems and seeds of Arabidopsis plants.

Comparative characterization of sweetpotato antioxidant genes from expressed sequence tags of dehydration-treated fibrous roots under different abiotic stress conditions.

Drought stress is one of the most adverse conditions for plant growth and productivity. The plant antioxidant system is an important defense mechanism and includes antioxidant enzymes and low-molecular weight antioxidants. Understanding the biochemical and molecular responses to drought is essential for improving plant resistance to water-limited conditions. Previously, we isolated and characterized expressed sequence tags (ESTs) from a full-length enriched cDNA library prepared from fibrous roots of sweetpotato subjected to dehydration stress (Kim et al. in BMB Rep 42:271-276, [5]). In this study, we isolated and characterized 11 sweetpotato antioxidant genes from sweetpotato EST library under various abiotic stress conditions, which included six intracellular CuZn superoxide dismutases (CuZnSOD), ascorbate peroxidase, catalase, glutathione peroxidase (GPX), glutathione-S-transferase, thioredoxin (TRX), and five extracellular peroxidase genes. The expression of almost all the antioxidant genes induced under dehydration treatments occurred in leaves, with the exception of extracellular swPB6, whereas some antioxidant genes showed increased expression levels in the fibrous roots, such as intracellular GPX, TRX, extracellular swPA4, and swPB7 genes. During various abiotic stress treatments in leaves, such as exposure to NaCl, cold, and abscisic acid, several intracellular antioxidant genes were strongly expressed compared with the expression of extracellular antioxidant genes. These results indicated that some intracellular antioxidant genes, especially swAPX1 and CuZnSOD, might be specifically involved in important defense mechanisms against oxidative stress induced by various abiotic stresses including dehydration in sweetpotato plants.

A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato.

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. 'White Star') and characterized its activity in transgenic Arabidopsis, carrot, and potato using the ß-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 µM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5' deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.

Three Brassica rapa metallothionein genes are differentially regulated under various stress conditions.

The expression profiles of three Brassica rapa metallothionein genes (BrMT 1-3) were determined in 7-day-old seedlings exposed to various exogenous factors including plant hormones, heavy metals and abiotic stresses. BrMT1, BrMT2, and BrMT3 were representatives of MT gene type 1, type 2, and type 3, respectively, according to their cysteine alignment. BrMT2 showed a relatively higher basal expression level compared to BrMT1 and BrMT3 under normal conditions. The BrMT1 transcript was markedly increased by various factors including ethephon, polyethylene glycol and hydrogen peroxide, with no down-regulation evident. On the contrary, BrMT2 expression was down-regulated by abscisic acid, salicylic acid, and methyl jasmonate. Heavy metals did not increase BrMT2 expression. BrMT3 expression was only marginally and non-significantly up- and down-regulated by the stress conditions tested. Promoter regions of BrMT1 and BrMT2 display different cis-acting elements supporting the different responses of both genes against various stresses. The results demonstrate the differential regulation of BrMT1-3 by various plant exogenous factors, and indicate the utility of the BrMT1 promoter as a multiple stress inducible promoter.

Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli.

Late embryogenesis abundant 14 (LEA14) cDNA was isolated from an EST library prepared from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Quantitative RT-PCR revealed a variety of different IbLEA14 expression patterns under various abiotic stress conditions. IbLEA14 expression was strongly induced by dehydration, NaCl and abscisic acid treatments in sweetpotato plants. Transgenic sweetpotato non-embryogenic calli harboring IbLEA14 overexpression or RNAi vectors under the control of CaMV 35S promoter were generated. Transgenic calli overexpressing IbLEA14 showed enhanced tolerance to drought and salt stress, whereas RNAi calli exhibited increased stress sensitivity. Under normal culture conditions, lignin contents increased in IbLEA14-overexpressing calli because of the increased expression of a variety of monolignol biosynthesis-related genes. Stress treatments elicited higher expression levels of the gene encoding cinnamyl alcohol dehydrogenase in IbLEA14-overexpressing lines than in control or RNAi lines. These results suggest that IbLEA14 might positively regulate the response to various stresses by enhancing lignification.

Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance.

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (Populus alba × Poplus glandulosa) expressing the AtNDPK2 gene under the control of a SWPA2 promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant's tolerance to MV-mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV-treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin-response genes IAA2 and IAA5. These results suggest that enhanced AtNDPK2 expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar.

Tobacco seeds simultaneously over-expressing Cu/Zn-superoxide dismutase and ascorbate peroxidase display enhanced seed longevity and germination rates under stress conditions.

Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal growth and seed development. Furthermore, the transgenic seeds displayed increased CuZnSOD and APX enzymatic activities during seed development and maintained antioxidant enzymatic activity after two years of dried storage at room temperature. The two-year stored non-transgenic seeds (aged NT seeds) had higher levels of ion leakage than the two-year stored transgenic seeds (aged CA seeds), indicating membrane damage caused by ROS was more severe in the aged NT seeds than the aged CA seeds. The aged CA seeds decreased germination rates as compared to newly harvested transgenic and non-transgenic seeds. The aged CA seeds, however, significantly increased germination rates under various abiotic stress conditions as compared to aged NT seeds. These data strongly suggest that simultaneous over-expression of the CuZnSOD and APX genes in plastids improves seed longevity and germination under various environmental stress conditions by attenuating the effects of oxidative stress produced by elongated storage conditions and harsh environmental stresses.

SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweetpotato (Ipomoea batatas).

A sweetpotato (Ipomoea batatas cv. 'Jinhongmi') MADS-box protein cDNA (SRD1) has been isolated from an early stage storage root cDNA library. The role of the SRD1 gene in the formation of the storage root in sweetpotato was investigated by an expression pattern analysis and characterization of SRD1-overexpressing (ox) transgenic sweetpotato plants. Transcripts of SRD1 were detected only in root tissues, with the fibrous root having low levels of the transcript and the young storage root showing relatively higher transcript levels. SRD1 mRNA was mainly found in the actively dividing cells, including the vascular and cambium cells of the young storage root. The transcript level of SRD1 in the fibrous roots increased in response to 1000 muM indole-3-acetic acid (IAA) applied exogenously. During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root. SRD1-ox sweetpotato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants. The metaxylem and cambium cells of the fibrous roots of SRD1-ox plants showed markedly enhanced proliferation, resulting in the fibrous roots of these plants showing an earlier thickening growth than those of wild-type plants. Taken together, these results demonstrate that SRD1 plays a role in the formation of storage roots by activating the proliferation of cambium and metaxylem cells to induce the initial thickening growth of storage roots in an auxin-dependent manner.

Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes.

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)-induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non-transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV-treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.

Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses.

Plants synthesize compatible solutes such as glycinebetaine (GB) in response to abiotic stresses. To evaluate the synergistic and protective effect of GB, transgenic potato plants expressing superoxide dismutase (SOD) and ascorbate peroxidase (APX) targeting to chloroplasts (referred to as SSA plants) were retransformed with a bacterial choline oxidase (codA) gene to synthesize GB in chloroplast in naturally occurring non-accumulator potato plants (including SSA) under the control of the stress-inducible SWPA2 promoter (referred to as SSAC plants). GB accumulation resulted in enhanced protection of these SSAC plants and lower levels of H(2)O(2) compared with SSA and non-transgenic (NT) plants after methyl viologen (MV)-mediated oxidative stress. Additionally, SSAC plants demonstrated synergistically enhanced tolerance to salt and drought stresses at the whole-plant level. GB accumulation in SSAC plants helped to maintain higher activities of SOD, APX and catalase following oxidative, salt and drought stress treatments than is observed in SSA and NT plants. Conclusively, GB accumulation in SSAC plants along with overexpression of antioxidant genes rendered the plants tolerant to multiple environmental stresses in a synergistic fashion.

The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system.

MYB transcription factors play important roles in transcriptional regulation of many secondary metabolites including anthocyanins. We cloned the R2R3-MYB type IbMYB1 complementary DNAs from the purple-fleshed sweet potato (Ipomoea batatas L. cv Sinzami) and investigated the expression patterns of IbMYB1 gene with IbMYB1a and IbMYB1b splice variants in leaf and root tissues of various sweet potato cultivars by reverse transcription-polymerase chain reaction. The transcripts of IbMYB1 were predominantly expressed in the purple-fleshed storage roots and they were also detectable in the leaf tissues accumulating anthocyanin pigments. In addition, transcript levels of IbMYB1 gene were up-regulated by treatment with methyl jasmonate or salicylic acid in leaf and root tissues of cv. White Star. To set up the intragenic vector system in sweet potato, we first evaluated the utilization of the IbMYB1 gene as a visible selectable marker. The IbMYB1a was transiently expressed in tobacco leaves under the control of a constitutive cauliflower mosaic virus 35S promoter, a root-specific and sucrose-inducible sporamin promoter, and an oxidative stress-inducible sweet potato anionic peroxidase2 promoter. We also showed that overexpression of IbMYB1a induced massive anthocyanin pigmentation in tobacco leaves and up-regulated the transcript levels of the structural genes in anthocyanin biosynthetic pathway. Furthermore, high-performance liquid chromatography analysis revealed that the expression of IbMYB1a led to production of cyanidin as a major core molecule of anthocyanidins in tobacco leaves. These results suggest that the IbMYB1 gene can be applicable to a visible marker for sweet potato transformation with intragenic vectors, as well as the production of anthocyanin as important nutritive value in other plant species.

Molecular characterization of the sweet potato peroxidase SWPA4 promoter which responds to abiotic stresses and pathogen infection.

Previously, the swpa4 peroxidase gene has been shown to be inducible by a variety of abiotic stresses and pathogenic infections in sweet potato (Ipomoea batatas). To elucidate its regulatory mechanism at the transcriptional level under various stress conditions, we isolated and characterized the promoter region (2374 bp) of swpa4 (referred to as SWPA4). We performed a transient expression assay in tobacco protoplasts with deletions from the 5'-end of SWPA4 promoter fused to the beta-glucuronidase (GUS) reporter gene. The -1408 and -374 bp deletions relative to the transcription start site (+1) showed 8 and 4.5 times higher GUS expression than the cauliflower mosaic virus 35S promoter, respectively. In addition, transgenic tobacco plants expressing GUS under the control of -2374, -1408 or -374 bp region of SWPA4 promoter were generated and studied in various tissues under abiotic stresses and pathogen infection. Gel mobility shift assays revealed that nuclear proteins from sweet potato cultured cells specifically interacted with 60-bp fragment (-178/-118) in -374 bp promoter region. In silico analysis indicated that four kinds of cis-acting regulatory sequences, reactive oxygen species-related element activator protein 1 (AP1), CCAAT/enhancer-binding protein alpha element, ethylene-responsive element (ERE) and heat-shock element, are present in the -60 bp region (-178/-118), suggesting that the -60 bp region might be associated with stress inducibility of the SWPA4 promoter.