Transgenic Arabidopsis Seeds Research Articles

Spatial and temporal activity of the foxtail millet (Setaria italica) seed-specific promoter pF128.

pF128 drives GUS specifically expressed in transgenic seeds of foxtail millet and Zea mays with higher activity than the constitutive CaMV35S promoter and the maize seed-specific 19Z promoter. Foxtail millet (Setaria italica), a member of the Poaceae family, is an important food and fodder crop in arid regions. Foxtail millet is an excellent C4 crop model owing to its small genome (~490 Mb), self-pollination and availability of a complete genome sequence. F128 was isolated from a cDNA library of foxtail millet immature seeds. Real-time PCR analysis revealed that F128 mRNA was specifically expressed in immature and mature seeds. The highest F128 mRNA level was observed 5 days after pollination and gradually decreased as the seed matured. Sequence analysis suggested that the protein encoded by F128 is likely a protease inhibitor/seed storage protein/lipid-transfer protein. The 1,053 bp 5' flanking sequence of F128 (pF128) was isolated and fused to the GUS reporter gene. The corresponding vector was then transformed into Arabidopsis thaliana, foxtail millet and Zea mays. GUS analysis revealed that pF128 drove GUS expression efficiently and specifically in the seeds of transgenic Arabidopsis, foxtail millet and Zea mays. GUS activity was also detected in Arabidopsis cotyledons. Activity of pF128 was higher than that observed for the constitutive CaMV35S promoter and the maize seed-specific 19 Zein (19Z) promoter. These results indicate that pF128 is a seed-specific promoter. Its application is expected to be of considerable value in plant genetic engineering.

Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA.

Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds.

Arabidopsis DREB2C modulates ABA biosynthesis during germination

Plant dehydration-responsive element binding factors (DREBs) are transcriptional regulators of the APETELA2/Ethylene Responsive element-binding Factor (AP2/ERF) family that control expression of abiotic stress-related genes. We show here that under conditions of mild heat stress, constitutive overexpression seeds of transgenic DREB2C overexpression Arabidopsis exhibit delayed germination and increased abscisic acid (ABA) content compared to untransformed wild-type (WT). Treatment with fluridone, an inhibitor of the ABA biosynthesis abrogated these effects. Expression of an ABA biosynthesis-related gene, 9-cis-epoxycarotenoid dioxygenase 9 (NCED9) was up-regulated in the DREB2C overexpression lines compared to WT. DREB2C was able to trans-activate expression of NCED9 in Arabidopsis leaf protoplasts in vitro. Direct and specific binding of DREB2C to a complete DRE on the NCED9 promoter was observed in electrophoretic mobility shift assays. Exogenous ABA treatment induced DREB2C expression in germinating seeds of WT. Vegetative growth of transgenic DREB2C overexpression lines was more strongly inhibited by exogenous ABA compared to WT. These results suggest that DREB2C is a stress- and ABA-inducible gene that acts as a positive regulator of ABA biosynthesis in germinating seeds through activating NCED9 expression.

A promoter analysis of MOTHER OF FT AND TFL1 1 (JcMFT1), a seed-preferential gene from the biofuel plant Jatropha curcas

MOTHER OF FT AND TFL1 (MFT)-like genes belong to the phosphatidylethanoamine-binding protein (PEBP) gene family in plants. In contrast to their homologs FLOWERING LOCUS T (FT)-like and TERMINAL FLOWER 1 (TFL1)-like genes, which are involved in the regulation of the flowering time pathway, MFT-like genes function mainly during seed development and germination. In this study, a full-length cDNA of the MFT-like gene JcMFT1 from the biodiesel plant Jatropha curcas (L.) was isolated and found to be highly expressed in seeds. The promoter of JcMFT1 was cloned and characterized in transgenic Arabidopsis. A histochemical β-glucuronidase (GUS) assay indicated that the JcMFT1 promoter was predominantly expressed in both embryos and endosperms of transgenic Arabidopsis seeds. Fluorometric GUS analysis revealed that the JcMFT1 promoter was highly active at the mid to late stages of seed development. After seed germination, the JcMFT1 promoter activity decreased gradually. In addition, both the JcMFT1 expression in germinating Jatropha embryos and its promoter activity in germinating Arabidopsis embryos were induced by abscisic acid (ABA), possibly due to two ABA-responsive elements, a G-box and an RY repeat, in the JcMFT1 promoter region. These results show that the JcMFT1 promoter is seed-preferential and can be used to control transgene expression in the seeds of Jatropha and other transgenic plants.

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2cisΔ9,12; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.

Strong seed-specific protein expression from the Vigna radiata storage protein 8SGα promoter in transgenic Arabidopsis seeds

Vigna radiata (mung bean) is an important crop plant and is a major protein source in developing countries. Mung bean 8S globulins constitute nearly 90% of total seed storage protein and consist of three subunits designated as 8SGα, 8SGα′ and 8SGβ. The 5′-flanking sequences of 8SGα′ has been reported to confer high expression in transgenic Arabidopsis seeds. In this study, a 472-bp 5′-flanking sequence of 8SGα was identified by genome walking. Computational analysis subsequently revealed the presence of numerous putative seed-specific cis-elements within. The 8SGα promoter was then fused to the gene encoding β-glucuronidase (GUS) to create a reporter construct for Arabidopsis thaliana transformation. The spatial and temporal expression of 8SGα∷GUS, as investigated using GUS histochemical assays, showed GUS expression exclusively in transgenic Arabidopsis seeds. Quantitative GUS assays revealed that the 8SGα promoter showed 2- to 4-fold higher activity than the Cauliflower Mosaic Virus (CaMV) 35S promoter. This study has identified a seed-specific promoter of high promoter strength, which is potentially useful for directing foreign protein expression in seed bioreactors.

A Rice OsAP23, Functioning as an AP2/ERF Transcription Factor, Reduces Salt Tolerance in Transgenic Arabidopsis

The AP2/ERF transcription factors of higher plants regulate transcriptional networks that control development and responses to biotic and abiotic stresses. In this study, the rice OsAP23 gene, which encodes an AP2/ERF transcription factor, was isolated from rice using a yeast-one-hybrid system. The OsAP23 transcription factor belongs to the B3 group of the ERF subfamily. Germination results for transgenic Arabidopsis plants hosting the OsAP23 gene revealed greater inhibition at the germination and seedling stages with increasing concentration of NaCl compared to the wild-type plants. Exogenous ABA also reduced the rate of germination of transgenic Arabidopsis seeds that over-express OsAP23 compared with wild-type Arabidopsis seeds. In addition, following exposure to high salt concentrations, several stress-responsive regulatory genes were induced to a significantly greater extent in the wild-type lines compared with lines that over-express OsAP23. These results suggested that OsAP23 might be a negative regulator of the salt stress response in higher plants.

The expression of wheat TaSTG gene can enhance salt tolerance in plants

By exploring microarray data in a salt-tolerant wheat mutant, RH8706-49, we identified a novel stress-related gene, named TaSTG (Triticum aestivum salt tolerance gene), which can be induced by salt stress in wheat. Transgenic rice and Arabidopsis plants overexpressing TaSTG were generated and proved to be more salt-tolerant than the wild-type control. Seeds of transgenic Arabidopsis exhibited an elevated germination rate under salt stress. Compared with those of the wild type, the levels of chlorophyll, soluble sugars and proline in transgenic Arabidopsis were significantly higher, whereas the levels of Na+ and malondialdehyde were significantly lower. Moreover, the expression profile of seven salt tolerance-related genes, alcohol dehydrogenase class-P (ADH1), cold-regulated protein 15a (COR15A), inositol polyphosphate 1-phosphatase FIERY1 (FRY1), Gamma-glutamyl phosphate reductase (P5CS1), desiccation-responsive rd29 (RD29B), U6 snRNA-associated Sm-like protein (SAD1), salt overly sensitive 3 (SOS3), in transgenic Arabidopsis suggests their possible interaction with TaSTG. Compared with those of the wild type, the expression levels of six of these genes in transgenic plants were significantly increased, whereas the expression of SOS3 was decreased. We propose that TaSTG may act upstream of the up-regulated genes, possibly by regulating plant responses to osmotic stress through phospholipases and CDPK pathway, consequently enhancing plant salt tolerance via proline accumulation.

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.

Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids.

An oleosin-fusion protein driven by the CaMV35S promoter is accumulated in Arabidopsis (Brassicaceae) seeds and correctly targeted to oil bodies

Oleosin-fusion technology is used to express desired proteins. It was developed based on the properties of oleosin; the heterologous protein gene is fused to the oleosin gene and the fusion gene is driven by a seed-specific promoter. We replaced the seed specific promoter with the CaMV35S promoter to dive a gfp-oleosin fusion gene in transformed Arabidopsis. The heterologous oleosin-fusion protein was mainly accumulated in the transgenic Arabidopsis seeds and correctly targeted to oil bodies. This provides an alternate choice of promoter in oleosin-fusion technology.

NnHSP17.5, a cytosolic class II small heat shock protein gene from Nelumbo nucifera, contributes to seed germination vigor and seedling thermotolerance in transgenic Arabidopsis

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.

Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis

Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. Although plant MTs have been intensively studied, their roles in seeds remain to be clearly established. Here, we report the isolation and characterization of NnMT2a, NnMT2b and NnMT3 from sacred lotus (Nelumbo nucifera Gaertn.) and their roles in seed germination vigor. The transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity, oxidative stresses and heavy metals. Analysis of transformed Arabidopsis protoplasts showed that NnMT2a-YFP and NnMT3-YFP were localized in cytoplasm and nucleoplasm. Transgenic Arabidopsis seeds overexpressing NnMT2a and NnMT3 displayed improved resistance to accelerated aging (AA) treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment. In addition, we showed that NnMT2a and NnMT3 conferred improved germination ability to NaCl and methyl viologen on transgenic Arabidopsis seeds. Taken together, these data demonstrate that overexpression of NnMT2a and NnMT3 in Arabidopsis significantly enhances seed germination vigor after AA treatment and under abiotic stresses.

The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds

Engineering of oilseed plants to accumulate unusual fatty acids (FAs) in seed triacylglycerol (TAG) requires not only the biosynthetic enzymes for unusual FAs but also efficient utilization of the unusual FAs by the host-plant TAG biosynthetic pathways. Competing pathways of diacylglycerol (DAG) and subsequent TAG synthesis ultimately affect TAG FA composition. The membrane lipid phosphatidylcholine (PC) is the substrate for many FA-modifying enzymes (desaturases, hydroxylases, etc.) and DAG can be derived from PC for TAG synthesis. The relative proportion of PC-derived DAG versus de novo synthesized DAG utilized for TAG synthesis, and the ability of each pathway to utilize unusual FA substrates, are unknown for most oilseed plants, including Arabidopsis thaliana. Through metabolic labeling experiments we demonstrate that the relative flux of de novo DAG into the PC-derived DAG pathway versus direct conversion to TAG is ∼14/1 in wild-type Arabidopsis. Expression of the Ricinus communis FA hydroxylase reduced the flux of de novo DAG into PC by ∼70%. Synthesis of TAG directly from de novo DAG did not increase, resulting in lower total synthesis of labeled lipids. Hydroxy-FA containing de novo DAG was rapidly synthesized, but it was not efficiently accumulated or converted to PC and TAG, and appeared to be in a futile cycle of synthesis and degradation. However, FA hydroxylation on PC and conversion to DAG allowed some hydroxy-FA to accumulate in sn-2 TAG. Therefore, the flux of DAG through PC represents a major bottleneck for the accumulation of unusual FAs in TAG of transgenic Arabidopsis seeds.

Cloning of a Vacuolar H+-pyrophosphatase Gene from the Halophyte Suaeda corniculata whose Heterologous Overexpression Improves Salt, Saline-alkali and Drought Tolerance in Arabidopsis

Salt, saline-alkali conditions, and drought are major environmental factors limiting plant growth and productivity. The vacuolar H(+)-translocating inorganic pyrophosphatase (V-H(+)-PPase) is an electrogenic proton pump that translocates protons into vacuoles in plant cells. Expression of V-H(+)-PPase increases in plants under a number of abiotic stresses, and is thought to have an important role in adaptation to abiotic stress. In this work, we report the isolation and characterization of the gene, ScVP, encoding a vacuolar inorganic pyrophosphatase (V-H(+)-PPase) from the halophyte, Suaeda corniculata. Semi-quantitative reverse transcription-polymerase chain reaction analysis showed that ScVP was induced in roots, stems and leaves under treatment with salt, saline-alkali and drought. Compared with wild-type (WT) Arabidopsis, transgenic plants overexpressing ScVP accumulated more Na(+) in leaves and roots, and showed increased tolerance to high salinity, saline-alkali and drought stresses. The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under the abiotic stresses. The root length of transgenic plants under salt stress was longer than that of WT plants. Furthermore, the rate of water loss during drought stress was higher in WT than in transgenic plants. Collectively, these results indicate that ScVP plays an important role in plant tolerance to salt, saline-alkali and drought stress.

Endoplasmic Reticulum-Located PDAT1-2 from Castor Bean Enhances Hydroxy Fatty Acid Accumulation in Transgenic Plants

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.

Construction of Plant Expression Constructs Harboring Full-length <i>Bt</i> Cry1Ac22 Toxin Gene and Truncated Functional Domains of <i>Bt</i> Cry1Ac22 Toxin and Arabidopsis Transformation

The full-length Bt toxin gene (3 534 bp) and truncated functional domains of Bt toxin gene (1 959 bp) of cry1Ac22 were amplified by PCR from Bt strain W015-1. The PCR products were ligated into plant expression vector pBI121 cutting off the s-glucuronidase gene to make the constructs of pBI121-Cry1Ac22F and pBI121-Cry1Ac22T. The constructs carrying with Kanamycin resistant marker were transferred into T-DNA vector and then validated by restricted enzyme digestion and PCR identification. Arabidopsis transformation with the transfered T-DNA vector were performed during the flowering stage mediated by Agrobacterium tumufaciens . Transgenic Arabidopsis seeds with positive Kanamycin resistance were harvested in this research that facilitate the understanding of Bt toxin functions in plant transgenic breeding.

Production of the 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1, a leading malaria vaccine antigen, in Arabidopsis thaliana seeds

Malaria is widely associated with poverty, and a low-cost vaccine against malaria is highly desirable for implementing comprehensive vaccination programmes in developing countries. Production of malaria antigens in plants is a promising approach, but its development has been hindered by poor expression of the antigens in plant cells. In the present study, we targeted plant seeds as a low-cost vaccine production platform and successfully expressed the Plasmodium falciparum 42-kDa fragment of merozoite surface protein 1 (MSP1₄₂), a leading malaria vaccine candidate, at a high level in transgenic Arabidopsis seeds. We overcame hurdles of transcript and protein instabilities of MSP1₄₂ in plants by synthesizing a plant-optimized MSP1₄₂ cDNA and either targeting the recombinant protein to protein storage vacuoles or fusing it with a stable plant storage protein. An exceptional improvement in MSP1₄₂ expression, from an undetectable level to 5% of total extractable protein, was achieved with these combined strategies. Importantly, the plant-derived MSP1₄₂ maintains its natural antigenicity and can be recognized by immune sera from malaria-infected patients. Our results provide a strong basis for the development of a plant-based, low-cost malaria vaccine.

Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis

Sterol C24 methyltransferase (SMT2) genes governing the pattern of phytosterols synthesized in higher plants have been studied in Glycine seedlings and wild-type and engineered Arabidopsis thaliana plants. The SMT2 genes of soybean (SMT2-1 and SMT2-2) previously cloned and characterized (Neelakandan et al. 2009) were shown to complement the SMT deficient cvp1 mutant Arabidopsis plants, consistent with their role in regulation of 24-alkyl sterol-controlled plant physiology. Further analysis of these genes showed that environmental cues, including dehydration, cold, and abscisic acid induced differential changes in transcript levels of the SMT2 during soybean seedling growth. Sterol analyses of transgenic Arabidopsis seeds originating in variant constructs of AtHMGR1, GmSMT1, and GmSMT2 engineered in seeds showed relevant modifications in the ratio of 24-methyl to 24-ethyl sterol in the direction of sitosterol formation. To provide insight into the structural features of the sterol gene that affects transcript regulation, the upstream promoter sequences of soybean SMT2 genes were cloned and characterized. Sequence analysis revealed several important cis-elements and transcription factor binding sites. The analysis of promoter-GUS fusions in transgenic Arabidopsis plants revealed shared and distinct expression features in different developmental stages and tissues. The data are interpreted to imply that SMT2 is an important contributor to normal plant growth and development.

A novel light‐dependent selection marker system in plants

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.