Improved tolerance to various abiotic stresses in transgenic sweet potato (Ipomoea batatas) expressing spinach betaine aldehyde dehydrogenase

Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea batatas), a root crop with worldwide importance. The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavorable conditions. The gene encoding betaine aldehyde dehydrogenase (BADH) is involved in the biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic stress tolerance in plants. This study is to improve sweet potato, a GB accumulator, resistant to multiple abiotic stresses by promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet potato cultivar Sushu-2 via Agrobacterium-mediated transformation. The overexpression of SoBADH in the transgenic sweet potato improved tolerance to various abiotic stresses, including salt, oxidative stress, and low temperature. The increased BADH activity and GB accumulation in the transgenic plant lines under normal and multiple environmental stresses resulted in increased protection against cell damage through the maintenance of cell membrane integrity, stronger photosynthetic activity, reduced reactive oxygen species (ROS) production, and induction or activation of ROS scavenging by the increased activity of free radical-scavenging enzymes. The increased proline accumulation and systemic upregulation of many ROS-scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-scavenging system and proline biosynthesis via an integrative mechanism. This study demonstrates that the enhancement of GB biosynthesis in sweet potato is an effective and feasible approach to improve its tolerance to multiple abiotic stresses without causing phenotypic defects. This strategy for trait improvement in sweet potato not only stabilizes yield production in normal soils in unpredictable climates but also provides a novel germplasm for sweet potato production on marginal lands.     

Development of transgenic sweet potato with multiple virus resistance in South Africa (SA)

Multiple infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KwaZulu-Natal, South Africa. In order to address the problem of multiple virus infections and synergism, this study aimed to develop transgenic sweet potato (cv. Blesbok) plants with broad virus resistance. Coat protein gene segments of SPFMV, SPCSV, SPVG and SPMMV were used to induce gene silencing in transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring the expression cassette. Polymerase chain reaction and Southern blot analyses showed integration of the transgenes occurred in six of the 24 putative transgenic plants and that all plants seemed to correspond to the same transformation event. The six transgenic plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV-infected Ipomoea setosa Ker. Although virus presence was detected using nitrocellulose enzyme-linked immunosorbent assay, all transgenic plants displayed delayed and milder symptoms of chlorosis and mottling of lower leaves when compared to the untransformed control plants. These results warrant further investigation on resistance to virus infection under field conditions.     

Enhanced drought and salinity tolerance in transgenic potato plants with a BADH gene from spinach

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = −2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4–0.9 cm and fresh weight per plant increased by 17–29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.     

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 α(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.     

Functional Characterization of Dihydroflavonol-4-Reductase in Anthocyanin Biosynthesis of Purple Sweet Potato Underlies the Direct Evidence of Anthocyanins Function against Abiotic Stresses

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.     

Overexpression of barley nicotianamine synthase 1 confers tolerance in the sweet potato to iron deficiency in calcareous soil

Background and aims

Iron (Fe) is an essential micronutrient for all higher organisms. Fe is sparingly available in calcareous soils and Fe deficiency is a major agricultural problem worldwide. Nicotianamine (NA) is a metal chelator involved in metal translocation in plants. Sweet potato is an attractive crop that can grow in poor soil and thus is useful for planting in uncultivated soil. In addition, the sweet potato has recently been suggested as a source of bioethanol. Our aim is to increase NA concentration in sweet potato to ameliorate Fe deficiency.

Method

Sweet potato plants expressing the barley NA synthase 1 (HvNAS1) gene under the control of CaMV 35S promoter were produced by Agrobacterium-mediated transformation.

Results

The transgenic sweet potato exhibited tolerance to low Fe availability when grown in calcareous soil. The level of tolerance to low Fe availability was positively correlated with the HvNAS1 expression level. The NA concentration of the transgenic sweet potato leaves was up to 7.9-fold greater than that of the non-transgenic (NT) plant leaves. Furthermore, the Fe and zinc concentrations were 3- and 2.9-fold greater, respectively, in transgenic sweet potato than in NT plant leaves.

Conclusions

Our results suggest that increasing the NA concentration of sweet potato by overexpression of HvNAS1 could significantly improve agricultural productivity and energy source.

     

A betaine aldehyde dehydrogenase gene in quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>): structure,phylogeny, and expression pattern

Betaine aldehyde dehydrogenase (BADH) is widely considered as a key enzyme in glycine betaine metabolism in higher plants. Several paralogous genes encoding different isozymes of BADH have been identified and characterized in some plants; however, until now, only limited information is available about BADH genes in quinoa (Chenopodium quinoa). Here, we report the molecular cloning, structural organization, phylogenetic evolution, and expression profile of a BADH gene (CqBADH1) from quinoa. The translated putative CqBADH1 protein included five conserved features of the ALDH Family 10. Comparisons between the cDNA and genomic sequences revealed that the CqBADH1 gene contained 15 exons and 14 introns. Comparative screening of introns in homologous genes demonstrated that the number and position of the BADH introns were highly conserved among the BADH genes in Amaranthaceae plants and in other more distantly related plant species. A phylogenetic analysis showed that CqBADH1 had the closest relationship with a protein from Atriplex canescens and belonged to the ALDH10 family. Expression profile analyses indicated that CqBADH1 was expressed only in root, and showed time-dependent expression profiles under NaCl-stress condition. Moreover, in quinoa, NaCl stress led to increased levels of CqBADH1 mRNA accompanied by the accumulation of glycine betaine. This is the first study to describe a BADH gene in quinoa.     

Overexpression of <i>IbSINA5</i> Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Seven in absentia (SINA) family proteins play a central role in plant growth, development and resistance to abiotic stress. However, their biological function in plant response to cold stress is still largely unknown. In this work, a seven in absentia gene IbSINA5 was isolated from sweet potato. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato, with a predominant expression in fibrous roots, and was remarkably induced by cold, drought and salt stresses. Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus. Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants, which was associated with the up-regulated expression of IbCOR (cold-regulated) genes, increased proline production, and decreased malondialdehyde (MDA) and H₂O₂ accumulation in the leaves of transgenic plants. Furthermore, transient expression of IbCBF3, a C-repeat binding factor (CBF) gene, in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes. Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato, and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.     

Ectopic Expression of Sweet Potato Cysteine Protease SPCP3 Alters Phenotypic Traits and Enhances Drought Stress Sensitivity in Transgenic Arabidopsis Plants

In this report sweet potato cysteine protease SPCP3 cDNAs, with or without the corresponding granulin-like domain, were overexpressed in transgenic Arabidopsis plants. Transgenic Arabidopsis plants with ectopic expression of full-length SPCP3 exhibited slight promotion of earlier floral transition from vegetative to reproductive growth and a higher percentage of yellowing siliques per plant. Transgenic progeny seeds showed similar patterns of germination rates and germination curves but lower germination percentages compared to those of wild-type control seeds. During drought treatment, photochemical F _v/F _m values and relative water content of transgenic plants were significantly reduced compared to those of wild-type controls. Transgenic Arabidopsis plants with ectopic expression of sweet potato SPCP3 with or without the corresponding C-terminal granulin-like domain exhibited similar drought-stress sensitivity patterns. Drought stress also enhanced SPCP3 gene expression, photochemical F _v/F _m reduction, and wilting in sweet potato detached leaves. Based on these data, we conclude that sweet potato granulin-containing cysteine protease SPCP3 is a functional gene, and its ectopic expression alters phenotypic traits and enhances drought-stress sensitivity in transgenic Arabidopsis plants. The presence of the C-terminal granulin-like domain has no significant influence on SPCP3-mediated drought-stress sensitivity in transgenic Arabidopsis plants.     

甘薯草甘膦抗性基因EPSPS克隆和序列分析

EPSPS基因编码5-烯醇式丙酮酰莽草酸-3-磷酸合成酶,该酶是芳香族氨基酸合成的关键酶,该基因在细菌、真菌、藻类和植物中被广泛克隆和研究。EPSPS酶是草甘膦除草剂的靶点酶,过量表达EPSPS基因可以提高作物的草甘膦抗性。该研究根据甘薯基因组数据库设计引物,以‘广薯87’为材料提取RNA,通过RT-PCR方法扩增甘薯IbEPSPS基因,测序后进行生物信息学分析和表达分析。结果表明:(1)成功克隆获得甘薯IbEPSPS基因,该基因全长CDS为1569 bp,编码522个氨基酸,其中在第98~113、173~183位氨基酸序列具有2个EPSPS的保守结构域。(2)系统进化树分析结果表明,甘薯IbEPSPS基因与三裂叶薯(Ipomoea triloba)、打碗花(Calystegia hederacea)、田旋花(Convolvulus arvensis)和牵牛(Ipomoea nil)聚在一类,其中与三裂叶薯的亲缘关系最近。(3)实时荧光定量PCR分析结果表明,甘薯IbEPSPS基因在茎、叶和茎尖表达量较高,同时受到草甘膦胁迫后IbEPSPS基因表达量提高。该研究结果为进一步探讨甘薯IbEPSPS基因的功能及甘薯对草甘膦的耐药性机制奠定了基础。     

Isolation and characterization of BADH2 gene from in vitro propagated <Emphasis Type="Italic">Pandanus amaryllifolius</Emphasis> Roxb

The 2-acetyl-1-pyrroline (2AP) induced ‘basmati rice’ aroma is plausibly the highest among the plant kingdom in Pandanus amaryllifolius Roxb. The defective/truncated allelic forms of betaine aldehyde dehydrogenase (BADH2) gene is the major contributing factor behind the accumulation of this fragrance. The present study reports the isolation and characterization of the full-length BADH2 gene from P. amaryllifolius Roxb. The in silico analysis involving homology modelling and docking studies revealed the deficiency in catalytic efficiency of Pandanus BADH2 towards its substrate γ-aminobutyraldehyde (GAB-ald) as compared to the functional BADH2 gene of rice. We have also studied the tissue-specific expression of the BADH2 gene in different plant parts of in vivo donor and in vitro propagated P. amaryllifolius Roxb. In addition to the leaves, we are reporting histochemical localization of 2AP for the first time in the roots of P. amaryllifolius.     

Resequencing Reveals Different Domestication Rate for BADH1 and BADH2 in Rice (Oryza sativa)

BADH1 and BADH2 are two homologous genes, encoding betaine aldehyde dehydrogenase in rice. In the present study, we scanned BADHs sequences of 295 rice cultivars, and 10 wild rice accessions to determine the polymorphisms, gene functions and domestication of these two genes. A total of 16 alleles for BADH1 and 10 alleles for BADH2 were detected in transcribed region of cultivars and wild species. Association study showed that BADH1 has significant correlation with salt tolerance in rice during germination stage, the SNP (T/A) in exon 4 is highly correlated with salt tolerance index (STI) (P<10⁻⁴). While, BADH2 was only responsible for rice fragrance, of which two BADH2 alleles (8 bp deletion in exon 7 and C/T SNP in exon 13) explain 97% of aroma variation in our germplasm. Theses indicate that there are no overlapping functions between the two homologous genes. In addition, a large LD block was detected in BADH2 region, however, there was no large LD blocks in a 4-Mb region of BADH1. We found that BADH2 region only showed significant bias in Tajima’s D value from the balance. Extended haplotype homozygosity study revealed fragrant accessions had a large LD block that extended around the mutation site (8 bp deletion in exon 7) of BADH2, while both of the BADH1 alleles (T/A in exon 4) did not show large extended LD block. All these results suggested that BADH2 was domesticated during rice evolution, while BADH1 was not selected by human beings.     

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H₂O₂ elevation

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H₂O₂ amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H₂O₂, NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H₂O₂ elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H₂O₂ homeostasis in leaves caused by developmental cues and environmental stimuli.