5′ Regulatory region of ubiquitin 2 gene from Porteresia coarctata makes efficient promoters for transgene expression in monocots and dicots

Key message

Porteresia ubiquitin 5′ regulatory region drives transgene expression in monocots and dicots.

Abstract

Ubiquitin promoters are promising candidates for constitutive transgene expression in plants. In this study, we isolated and characterized a novel 5′ regulatory sequence of a ubiquitin gene from Porteresia coarctata, a stress-tolerant wild grass species. Through functional analysis in heterologous plant systems, we have demonstrated that full length (Port Ubi2.3) or truncated sequence (PD2) of the isolated regulatory fragment can drive constitutive expression of GUS in monocots and/or dicots. In silico analysis of Port Ubi2.3 has revealed the presence of a 640 bp core promoter region followed by two exons and two introns with numerous putative cis-acting sites scattered throughout the regulatory region. Transformation and expression studies of six different deletion constructs in rice, tobacco and sugarcane revealed that the proximal intron has an enhancing effect on the activity of the core promoter in both monocots and dicots, whereas, Port Ubi2.3 was able to render strong expression only in monocots. This regulatory sequence is quite distinct from the other reported ubiquitin promoters in structure and performs better in monocots compared to other commonly used promoters—maize Ubi1 and Cauliflower Mosaic Virus 35S.     

The relationship between PMI (manA) gene expression and optimal selection pressure in Indica rice transformation

Key message

An efficient mannose selection system was established for transformation of Indica cultivar IR58025B . Different selection pressures were required to achieve optimum transformation frequency for different PMI selectable marker cassettes.

Abstract

This study was conducted to establish an efficient transformation system for Indica rice, cultivar IR58025B. Four combinations of two promoters, rice Actin 1 and maize Ubiquitin 1, and two manA genes, native gene from E. coli (PMI-01) and synthetic maize codon-optimized gene (PMI-09) were compared under various concentrations of mannose. Different selection pressures were required for different gene cassettes to achieve corresponding optimum transformation frequency (TF). Higher TFs as 54 and 53 % were obtained when 5 g/L mannose was used for selection of prActin-PMI-01 cassette and 7.5 g/L mannose used for selection of prActin-PMI-09, respectively. TFs as 67 and 56 % were obtained when 7.5 and 15 g/L mannose were used for selection of prUbi-PMI-01 and prUbi-PMI-09, respectively. We conclude that higher TFs can be achieved for different gene cassettes when an optimum selection pressure is applied. By investigating the PMI expression level in transgenic calli and leaves, we found there was a significant positive correlation between the protein expression level and the optimal selection pressure. Higher optimal selection pressure is required for those constructs which confer higher expression of PMI protein. The single copy rate of those transgenic events for prActin-PMI-01 cassette is lower than that for other three cassettes. We speculate some of low copy events with low protein expression levels might not have been able to survive in the mannose selection.     

A strong root-specific expression system for stable transgene expression in bread wheat

Key message

A strong, stable and root-specific expression system was developed from a rice root-specific GLYCINE - RICH PROTEIN 7 promoter for use as an enabling technology for genetic manipulation of wheat root traits.

Abstract

Root systems play an important role in wheat productivity. Genetic manipulation of wheat root traits often requires a root-specific or root-predominant expression system as an essential enabling technology. In this study, we investigated promoters from rice root-specific or root-predominant expressed genes for development of a root expression system in bread wheat. Transient expression analysis using a GREEN FLUORESCENT PROTEIN (GFP) reporter gene driven by rice promoters identified six promoters that were strongly expressed in wheat roots. Extensive organ specificity analysis of three rice promoters in transgenic wheat revealed that the promoter of rice GLYCINE-RICH PROTEIN 7 (OsGRP7) gene conferred a root-specific expression pattern in wheat. Strong GFP fluorescence in the seminal and branch roots of wheat expressing GFP reporter driven by the OsGRP7 promoter was detected in epidermal, cortical and endodermal cells in mature parts of the root. The GFP reporter driven by the promoter of rice METALLOTHIONEIN-LIKE PROTEIN 1 (OsMTL1) gene was mainly expressed in the roots with essentially no expression in the leaf, stem or seed. However, it was also expressed in floral organs including glume, lemma, palea and awn. In contrast, strong expression of rice RCg2 promoter-driven GFP was found in many tissues. The GFP expression driven by these three rice promoters was stable in transgenic wheat plants through three generations (T1–T3) examined. These data suggest that the OsGRP7 promoter can provide a strong, stable and root-specific expression system for use as an enabling technology for genetic manipulation of wheat root traits.

     

Isolation and functional characterization of a novel rice constitutive promoter

Key message

A novel rice constitutive promoter (P _OsCon1 ) was isolated. The molecular mechanism of the promoter activity was investigated. P _OsCon1 could be used as an alternative constitutive promoter for crop transgenic engineering.

Abstract

Monocot constitutive promoter is an important resource for crop transgenic engineering. In this report, we isolated a novel promoter, Oscon1 promoter (P _OsCon1 ), from the 5′ upstream region of a constitutively expressed rice gene OsDHAR1. In P _OsCon1 ::GUS transgenic rice, we showed that P _OsCon1 had a broad expression spectrum in all tested tissues. The expression of the promoter was further analyzed in comparison with the previously characterized strong constitutive promoters. P _OsCon1 exhibited comparable activity to OsCc1, OsAct1 or ZmUbi promoters in most tissues, and more active than 35S promoter in roots, seeds, and calli. Further quantitative assays indicated that P _OsCon1 activity was not affected by developmental stages or by environmental factors. Further, 5′-deletions analysis indicated that the distinct regions might contribute to the strong expression of P _OsCon1 in different tissues. Overall, our results suggest that P _OsCon1 is a novel constitutive promoter, which could potentially use in transgenic crop development.     

Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress

Key message

Overexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses.

Abstract

Glutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10 days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death.     

Genome-wide identification and expression profiling of DNA methyltransferase gene family in maize

Key message

In this study, we identified eight DNA MTase genes in maize and the diversity of expression patterns of them was presented by EST mining, microarray and semi-quantitative expression profile analyses.

Abstract

DNA methylation plays a pivotal role in promoting genomic stability through diverse biological processes including regulation of gene expression during development and chromatin organization. Although this important biological process is mainly regulated by several conserved Cytosine-5 DNA methyltransferases encoded by a smaller multigene family in plants, investigation of the plant C5-MTase-encoding gene family will serve to elucidate the epigenetic mechanism diversity in plants. Recently, genome-wide identification and evolutionary analyses of the C5-MTase-encoding gene family have been characterized in multiple plant species including Arabidopsis, rice, carrot and wheat. However, little is known regarding the C5-MTase-encoding genes in the entire maize genome. Here, genome-wide identification and expression profile analyses of maize C5-MTase-encoding genes (ZmMETs) were performed from the latest version of the maize (B73) genome. Phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice) were categorized into four classes. Chromosomal location of these genes revealed that they are unevenly distributed on 6 of all 10 chromosomes with three chromosomal/segmental duplication events, suggesting that gene duplication played a key role in expansion of the maize C5-MTase-encoding gene family. Furthermore, EST expression data mining, microarray data and semi-quantitative expression profile analyses detected in the leaves by two different abiotic stress treatments have demonstrated that these genes had temporal and spatial expression pattern and exhibited different expression levels in stress treatments, suggesting that functional diversification of ZmMET genes family. Overall, our study will serve to present signification insights to explore the plant C5-MTase-encoding gene expression and function and also be beneficial for future experimental research to further unravel the mechanisms of epigenetic regulation in plants.     

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene

Key message

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin.

Abstract

The ubiquitin–26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA₃ conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.     

Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response

Key message

Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death.

Abstract

Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants.     

Xcc-facilitated agroinfiltration of citrus leaves: a tool for rapid functional analysis of transgenes in citrus leaves

Key message

Xanthomonas citri subsp. citri pretreatment before agroinfiltration could significantly promote transient expression in citrus leaves which were previously recalcitrant to agroinfiltration.

Abstract

Transient expression via agroinfiltration is widely used in biotechnology but remains problematic in many economically important plants. Xanthomonas citri subsp. citri (Xcc)-facilitated agroinfiltration was employed to promote transient protein expression in Valencia sweet orange leaves, which are recalcitrant to agroinfiltration. However, it is unclear whether Xcc-facilitated agroinfiltration has broad application, i.e., whether Xcc-facilitated agroinfiltration could be used on other citrus varieties. In addition, we intended to investigate whether Xcc-facilitated agroinfiltration could be used to hasten transgene function assays, e.g., Cre/lox system and Cas9/sgRNA system. In this report, Xcc-facilitated agroinfiltration was further exploited to enhance β-glucuronidase (GUS) expression in five citrus varieties. Xcc-facilitated agroinfiltration also significantly increased GFP expression in six citrus varieties tested. Both GUS and GFP assays indicated that Xcc-facilitated agroinfiltration had the best performance in grapefruit. After Xcc-facilitated agroinfiltration was carried out in grapefruit, protoplast analysis of the transformed cells indicated that there were more than 20 % leaf cells expressing GFP. In grapefruit, usefulness of Xcc-facilitated agroinfiltration was assayed in three case studies: (1) fast functional analysis of Cre/lox system, (2) the heat shock regulation of HSP70B promoter derived from Arabidopsis, and (3) Cas9/sgRNA-mediated genome modification.     

Heterologous expression of Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) enhanced chilling tolerance in transgenic eggplant (Solanum melongena L.)

Key message

Our study shows that the expression of AtCBF3 and AtCOR15A improved the chilling tolerance in transgenic eggplant.

Abstract

In an attempt to improve chilling tolerance of eggplant (Solanum melongena L) plants, Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) genes both driven by an Arabidopsis RESPONSIVE TO DESSICATION 29A promoter (AtRD29A) were transferred into the plants of eggplant cultivar Sanyueqie. Two independent homozygous transgenic lines were tested for their cold tolerance. The leaves of the transgenic plants in both lines withered much slower and slighter than the wild-type plants after exposure to cold stress treatment at 2 ± 1 °C. The gene expression of AtCBF3 and AtCOR15A was significantly increased as well as the proline content and the levels of catalase and peroxidase activities, while the relative electrical conductivity and the malondialdehyde content were remarkably decreased in the transgenic plants compared with the wild type at 4 ± 0.5 °C. The results showed that the expression of the exogenous AtCBF3 and AtCOR15A could promote the cold adaptation process to protect eggplant plants from chilling stress.     

Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench)

Key message

Sequence analysis and genetic mapping revealed that a 1,444 bp deletion causes a premature stop codon in SbBADH2 of sorghum IS19912. The non-function of SbBADH2 is responsible for fragrance in sorghum IS19912.

Abstract

2-acetyl-1-pyrroline (2AP) is a potent volatile compound causing fragrance in several plants and foods. Seeds of some varieties of rice, sorghum and soybean possess fragrance. The genes responsible for fragrance in rice and soybean are orthologs that correspond to betaine aldehyde dehydrogenase 2 (BADH2). Genotypes harboring fragrance in rice and soybean contain a premature stop codon in BADH2 which impairs the synthesis of full length functional BADH2 protein leading to the accumulation of 2AP. In this study, we reported an association between the BADH2 gene and fragrance in sorghum. An F₂ population of 187 plants developed from a cross between KU630 (non-fragrant) and IS19912 (fragrant) was used. Leaves of F₂ and F₃ progenies were evaluated for fragrance by organoleptic test, while seeds of F₂ plants were analyzed for 2AP. The tests consistently showed that the fragrance is controlled by a single recessive gene. Gene expression analysis of SbBADH1 and SbBADH2 in leaves of KU630 and IS19912 at various stages revealed that SbBADH1 and SbBADH2 were expressed in both accessions. Sequence comparison between KU630 and IS19912 revealed a continuous 1,444 bp deletion encompassing exon 12 to 15 of SbBADH2 in IS19912 which introduces a frameshift mutation and thus causes a premature stop codon. An indel marker was developed to detect polymorphism in SbBADH2. Bulk segregant and QTL analyses confirmed the association between SbBADH2 and fragrance.     

Different non-host resistance responses of two rice subspecies,japonica and indica,to Puccinia striiformis f. sp. tritici

Key message

Japonica and indica have different non-host resistance (NHR) abilities to Puccinia striiformis f. sp. tritici ( Pst ), and hydrogen peroxide (H ₂ O ₂ ) has a positive function in NHR to japonica against Pst.

Abstract

Non-host interactions between Puccinia striiformis f. sp. tritici (Pst) and two rice subspecies were characterized using 23 rice varieties, including 11 japonica and 12 indica. Results showed that the infected fungal structures were easily produced in the leaves of indica, whereas only several substomatal vesicles and primary infection hyphae were observed in the leaves of japonica. This result indicated that indica is less resistant or more susceptible to Pst than japonica. Hydrogen peroxide accumulated in the initial phase of japonica–Pst interaction but not in indica–Pst interaction. A set of reactive oxygen species (ROS)-related genes was also induced in response to Pst infection, suggesting that ROS activation is one of the major mechanisms of non-host resistance of rice to Pst.