Arabidopsis Transformation Research Articles

Expression and in vitro function of anti-cancer mAbs in transgenic Arabidopsis thaliana

The anti-colorectal cancer monoclonal antibody CO17-1A (mAb CO), which recognizes the tumor-associated antigen EpCAM, was expressed in transgenic Arabidopsis plants. PCR and western blot analyses showed the insertion and expression of heavy chain (HC)/HC fused to the KDEL ER retention modif (HCK) and light chain (LC) of mAb CO and mAb CO with HCK (mAb COK) in Arabidopsis transformants. Both plant-derived mAbP CO and mAbP COK were purified from a biomass of approximately 1,000 seedlings grown in a greenhouse. In sandwich ELISA, both mAbP CO showed a slightly higher binding affinity for the target, EpCAM, compared to mAbM CO. In cell ELISA, both mAbsP COs showed binding affinity to the human colorectal cancer cell line SW480. Furthermore, mAbM CO, mAbP CO, and mAbP COK exhibited dose and time-dependent regression effects on SW480 cells in vitro. In summation, both mAbP CO and mAbP COK, expressed in Arabidopsis, recognized the target antigen EpCAM and showed anti-proliferative activity against human colorectal cancer cells.

Function analysis of ZmNAC33, a positive regulator in drought stress response in Arabidopsis

Drought significantly affects plant growth and has devastating effects on crop production, NAC transcription factors respond to abiotic stresses by activating gene expression. In this study, a maize NAC transcription factor, ZmNAC33, was cloned and characterized its function in Arabidopsis. Transient transformation in Arabidopsis leaves mesophyll protoplasts and trans-activation assays in yeast showed that ZmNAC33 was localized in the nucleus and had transactivation activity. qRT-PCR analysis showed that ZmNAC33 in maize was induced by drought, high salinity and abscisic acid (ABA) stress. Promoter analysis identified multiple stress-related cis-acting elements in the promoter region of ZmNAC33. In ZmNAC33 transgenic Arabidopsis, germination rates were higher than in wild type plants under ABA and osmotic stress at the germination stage, and overexpression lines exhibited higher survival rates and higher antioxidant enzyme activities compared with wild type under drought stress. These results indicate that ZmNAC33 actes as a positive regulator in drought tolerance in plants.

Comparative Analysis of miRNA Abundance Revealed the Function of Vvi-miR828 in Fruit Coloring in Root Restriction Cultivation Grapevine (Vitis vinifera L.).

Root restriction cultivation leads to early maturation and quality improvement, especially in the anthocyanin content in grapevine. However, the molecular mechanisms that underlie these changes have not been thoroughly elucidated. In this study, four small RNA libraries were constructed, which included the green soft stage (GS) and ripe stage (RS) of ‘Muscat’ (Vitis vinifera L.) grape berries that were grown under root restriction (RR) and in traditional cultivation (no root restriction, CK). A total of 162 known miRNAs and 14 putative novel miRNAs were detected from the four small RNA libraries by high-throughput sequencing. An analysis of differentially expressed miRNAs (DEMs) revealed that 13 miRNAs exhibited significant differences in expression between RR and CK at the GS and RS stages, respectively. For different developmental stages of fruit, 23 and 34 miRNAs showed expression differences between the GS and RS stages in RR and CK, respectively. The expression patterns of the eight DEMs and their targets were verified by qRT-PCR, and the expression profiles of target genes were confirmed to be complementary to the corresponding miRNAs in RR and CK. The function of Vvi-miR828, which showed the down regulated expression in the RS stage under root restriction, was identified by gene transformation in Arabidopsis. The anthocyanin content significantly decreased in transgenic lines, which indicates the regulatory capacity of Vvi-miR828 in fruit coloration. The miRNA expression pattern comparison between RR and CK might provide a means of unraveling the miRNA-mediated molecular process regulating grape berry development under root restricted cultivation.

Expression of the cassava nitrate transporter NRT2.1 enables Arabidopsis low nitrate tolerance

The cassava grows well on low-nutrient soils because of its high-affinity to absorb nitrate. However, the molecular mechanisms by which cassava adapts itself to this environment remain elusive, although we have cloned a putative gene named MeNRT2.1 which has a crucial role in high-affinity nitrate transporter from cassava seeding. Here, the expression pattern of MeNRT2.1 was further assessed using the GUS activity driven by MeNRT2.1 promoter in Arabidopsis transformation plants. The GUS activity was monitored over time following the reduction of nitrate supply. The GUS gene expression not only peaked in roots after 12 h in 0.2mM nitrate media, but also stained stems and leaves. Arabidopsis plants with overexpression of MeNRT2.1 increased the biomass compared to the wild type on rich nitrogen (N-full) media. However, chlorate sensitivity analysis showed that Arabidopsis plants expressing MeNRT2.1 were more susceptable to chlorate than wild type. Significantly, after growing for 15 days on media containing 0.2mM nitrate concentration, wild-type plants became yellowor died, while the transgenic MeNRT2.1 Arabidopsis plants maintained normal growth. With significant increases in the amount of 15NO- 3 uptake in roots, the MeNRT2.1 plants also increased the contents of chlorophyll and nitrate reductase. Taken together, these results demonstrate that MeNRT2.1 has an important role in adaptation to low nitrate concentration as a nitrate transporter.

High temperature causes breakdown of S haplotype-dependent stigmatic self-incompatibility in self-incompatible Arabidopsis thaliana

Commercial seeds of Brassicaceae vegetable crops are mostly F1 hybrids, the production of which depends on self-incompatibility during pollination. Self-incompatibility is known to be weakened by exposure to elevated temperatures, which may compromise future breeding and seed production. In the Brassicaceae, self-incompatibility is controlled by two genes, SRK and SCR, which function as female and male determinants of recognition specificity, respectively. However, the molecular mechanisms underlying the breakdown of self-incompatibility under high temperature are poorly understood. In this study, we examined the self-incompatibility phenotypes of self-incompatible Arabidopsis thaliana SRK-SCR transformants under normal (23 °C) and elevated (29 °C) temperatures. Exposure to elevated temperature caused defects in the stigmatic, but not the pollen, self-incompatibility response. In addition, differences in the response to elevated temperature were observed among different S haplotypes. Subcellular localization revealed that high temperature disrupted the targeting of SRK to the plasma membrane. SRK localization in plants transformed with different S haplotypes corresponded to their self-incompatibility phenotypes, further indicating that defects in SRK localization were responsible for the breakdown in the self-incompatibility response at high temperature. Our results provide new insights into the causes of instability in self-incompatibility phenotypes.

A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang.

Many Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB123 and AcbHLH42, that regulate tissue-specific anthocyanin biosynthesis in the inner pericarp of Actinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified five MYB and three bHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB123 and AcbHLH42, is required for activating promoters of AcANS and AcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression of AcMYB123 and AcbHLH42. RNA interference repression of AcMYB123, AcbHLH42, AcF3GT1 and AcANS in 'Hongyang' fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays in Nicotiana tabacum leaves orActinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co-expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription ofAcF3GT1 and AcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB123 or AcbHLH42 are closely related to TT2 or TT8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB123 and AcbHLH42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.

Identification and validation of organ-preferential genes and analysis of corresponding upstream tissue-specific promoters in wheat

Tissue/organ-specific promoters are important tools in genetic engineering and crop molecular breeding. They are well characterized in dicots, such as Arabidopsis, tobacco, and tomato, but not sufficiently in monocots, especially in wheat. In this study, the genes specifically expressed in seven different tissues, including coleoptile, root, leaf, pistil, anther, embryo, and endosperm were identified through analyzing the public transcriptome data from a wheat microarray using the ROKU method. The expression patterns of selected genes were validated by reverse transcription polymerase chain reaction. The results showed that these selected genes were expressed specifically or preferentially in each representative tissue/organ. Moreover, the function of their promoters was verified by transient expression in wheat or stable transformation in Arabidopsis. The results showed that these promoters can efficiently and predominantly drive uidA (β-glucuronidase) reporter gene expression in different tissues. Due to their tissue-specific nature, these promoters can be used as potential candidates in plant genetic engineering.

Generating Transgenic Arabidopsis Plants for Functional Analysis of Pathogen Effectors and Corresponding R Proteins.

Inducible expression of a pathogen effector has been proven to be a powerful strategy for dissecting its virulence and avirulence functions. However, leaky expression of some effector proteins can cause drastic physiological changes, such as growth retardation, accelerated senescence, and sterility. Unfortunately, leaky expression from current inducible vectors is unavoidable. To overcome these problems, a highly efficient Arabidopsis transformation protocol is described here, which allows the generation of hundreds to over a thousand T1 plants for selecting appropriate lines. In addition, since transgenic silencing is frequently observed, a principle for screening stable transgenic plants is also introduced.

Factors affecting the efficiency of Rhizobium rhizogenes root transformation of the root parasitic plant Triphysaria versicolor and its host Arabidopsis thaliana

BackgroundRhizobium rhizogenes transformation is commonly used to generate transgenic roots traditionally called hairy roots, for both investigative and commercial applications. While fertile plants can be regenerated from transgenic roots, the transgenic roots are more typically used directly, either to investigate root biology or to produce valuable secondary metabolites. Hairy roots have been particularly useful for genetic studies of rhizosphere interactions; including the recognition of host plant roots by the roots of parasitic angiosperms.ResultsIn this manuscript we analyzed various environmental, nutritional and procedural conditions for their effects on transformation of the model hemi-parasitic plant Triphysaria versicolor and Arabidopsis thaliana, one of its hosts. We first examined the effects of media, gelling agents and co-incubation times on Triphysaria root transformation and determined that while all three affected transformation rates, the media were the most significant. Once those primary conditions were fixed, we examined the roles of seedling age, explant type, acetosyringone, temperature and illumination on Triphysaria hairy root transformation rates. Using the optimized procedure approximately 70% of Triphysaria seedlings developed transgenic roots as judged by expression of YFP. These conditions were then used to transform Arabidopsis and similar transformation rates were obtained.ConclusionsAnalyses of root transformation factors provides a method recovering transgenic roots from both parasitic plants and their hosts at high frequency. In addition to providing an effective in vitro approach for genetic investigations of parasitic plant-host plant interactions, these results are applicable to genetic studies of non-parasitic plants as well.

Broad-specificity amino acid racemase, a novel non-antibiotic selectable marker for transgenic plants

The broad-specificity amino acid racemase (Bsar) from Pseudomonas putida catalyzes the racemization of various amino acids, offering a flexible and feasible platform to develop a new non-antibiotic selectable marker system for plant transformation. In the present study, we demonstrated that a Bsar variant, Bsar-R174K, that is useful as a selectable marker gene in Arabidopsis and rice that were susceptible to l-lysine and D-alanine. The introduction of wild-type Bsar, Bsar-R174K or Bsar-R174A into E. coli lysine or asparagine auxotrophs was able to rescue the growth of these microorganisms in minimal media supplemented with selectable amino acid enantiomers. The transformation of Arabidopsis with Bsar or Bsar variants based on d-alanine selection revealed that Bsar-R174K had the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). Whereas, l-lysine-based selection exhibited lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis revealed normal growth properties. In addition, Bsar-R174K transgenic rice was obtained on l-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice revealed morphologically normal phenotypes comparable with their wild-type counterparts. This study presents the first report of broad range amino acid racemase Bsar-R174K as a non-antibiotic selectable marker system applied in transgenic plants.

Using CRISPR/Cas9 System to Introduce Targeted Mutation in Arabidopsis.

Gene editing enables the modification of genetic information in a targeted manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing approach that has been widely used in both the animal and plant studies. Plant transcription factors play critical roles in plant development. Recent studies have used the CRISPER/Cas9 system to disrupt transcription factors and cis-elements in plants. In this chapter, we describe a protocol of using the CRISPR/Cas9 system to generate transcription factor mutants in the model plant species Arabidopsis thaliana. Our protocol contains five different steps: (1) adaptor primers design, (2) cloning of sgRNA expression cassettes, (3) Golden Gate cloning, (4) E. coli and Agrobacterium transformation, and (5) Arabidopsis transformation.

ZmFKBP20-1 improves the drought and salt tolerance of transformed Arabidopsis

FK506-binding proteins (FKBPs), which belong to the peptidyl-prolyl cis/trans isomerase superfamily, are involved in plant response to abiotic stresses. A number of FKBP family genes have been isolated in plants, but little has been reported of FKBP genes in maize. In this study, a drought-induced FKBP gene, ZmFKBP20-1, was isolated from maize and was characterized for its role in stress responses using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes, and physiological parameter analysis. During drought and salt stresses, ZmFKBP20-1 transgenic Arabidopsis plants exhibited enhanced tolerance, which was concomitant with the altered expression of stress/ABA-responsive genes, such as COR15a, COR47, ERD10, RD22, KIN1, ABI1, and ABI2. The resistance characteristics of ZmFKBP20-1 overexpression were associated with a significant increase in survival rate. These results suggested that ZmFKBP20-1 plays a positive role in drought and salt stress responses in Arabidopsis and provided new insights into the mechanisms of FKBP in response to abiotic stresses in plants.

Ectopic expression of IiFUL isolated from Isatis indigotica could change the reproductive growth of Arabidopsis thaliana

The coding sequence of IiFUL in Isatis indigotica was isolated and was used in transformation of Arabidopsis. IiFUL overexpressing Arabidopsis plants exhibited early flowering phenotype, accompanied with the reduction of flower number and the production of terminal flower on the top of the main stems. In development process, the flowers located on the top of the main stems generated a lot of variations in phenotype, including abnormal swelling of pistil, withering and numerical change of stamens and petals, appearance of stigmatoid tissues and naked ovules at the margin or inside of sepals. Besides, secondary flower could be formed within the flowers on the top of the main stems. These observations illustrated that IiFUL mainly affected the development of inflorescence meristems and pistils, but its ectopic expression could also disturb the normal growth of other floral organs. Moreover, the fertile siliques produced by the lateral inflorescences of IiFUL overexpressing Arabidopsis plants showed indehiscent phenotype, and the shape of the cauline leaves was changed significantly. The results of quantitative real-time PCR revealed that higher transcriptional levels of IiFUL could be detected in flowers and silicles of I. indigotica. In comprehensive consideration of the previous reports about the dehiscence phenotype of Arabidopsis siliques and the fact that the siliques of IiFUL overexpressing Arabidopsis plants were indehiscent in the present work, it can be speculated that high expression of IiFUL in pericarp is likely the reason why the silicles of I. indigotica possess an indehiscent phenotype.

Development of iFOX-hunting as a functional genomic tool and demonstration of its use to identify early senescence-related genes in the polyploid Brassica napus.

SummaryFunctional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway‐compatible plant gene expression vector containing a methoxyfenozide‐inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium‐mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full‐length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl‐1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napus cDNA was identified as a homolog of an Arabidopsis acyl‐CoA binding protein (ACBP) gene designated BnACBP1‐like. The early senescence phenotype conferred by BnACBP1‐like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA‐Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1‐like expression. Our results demonstrate the utility of iFOX‐Hunting as a tool for gene discovery and functional characterization of Brassica napus genome.

Subcellular Localization of Arabidopsis Pathogenesis-Related 1 (PR1) Protein.

The Arabidopsis thaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.

Overexpression of ginseng UGT72AL1 causes organ fusion in the axillary leaf branch of Arabidopsis

BackgroundGlycosylation of natural compounds increases the diversity of secondary metabolites. Glycosylation steps are implicated not only in plant growth and development, but also in plant defense responses. Although the activities of uridine-dependent glycosyltransferases (UGTs) have long been recognized, and genes encoding them in several higher plants have been identified, the specific functions of UGTs in planta remain largely unknown. MethodsSpatial and temporal patterns of gene expression were analyzed by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and GUS histochemical assay. In planta transformation in heterologous Arabidopsis was generated by floral dipping using Agrobacterium tumefaciens (C58C1). Protein localization was analyzed by confocal microscopy via fluorescent protein tagging. ResultsPgUGT72AL1 was highly expressed in the rhizome, upper root, and youngest leaf compared with the other organs. GUS staining of the promoter: GUS fusion revealed high expression in different organs, including axillary leaf branch. Overexpression of PgUGT72AL1 resulted in a fused organ in the axillary leaf branch. ConclusionPgUGT72AL1, which is phylogenetically close to PgUGT71A27, is involved in the production of ginsenoside compound K. Considering that compound K is not reported in raw ginseng material, further characterization of this gene may shed light on the biological function of ginsenosides in ginseng plant growth and development. The organ fusion phenotype could be caused by the defective growth of cells in the boundary region, commonly regulated by phytohormones such as auxins or brassinosteroids, and requires further analysis.

High throughput selection of antibiotic-resistant transgenic Arabidopsis plants

Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation.

A salt-stress-regulator from the Poplar R2R3 MYB family integrates the regulation of lateral root emergence and ABA signaling to mediate salt stress tolerance in Arabidopsis

The roles of most MYB transcription factors (TFs) in the poplar remain unclear. Here, we demonstrate that PtrSSR1, a salt-stress-regulator in the Populus trichocarpa R2R3 MYB gene family, mediates the tolerance of transgenic Arabidopsis plants to salt stress. The transcripts of PtrSSR1 could be induced by salt stress rapidly in poplar. Subcellular localization and yeast assays indicated that PtrSSR1 encoded a nuclear protein with transactivation activity. The Arabidopsis transformants overexpressing PtrSSR1 clearly displayed lateral root emergence (LRE) inhibition compared with wild-type (Wt) under normal conditions; while upon NaCl treatment, the transformants showed improved tolerance, and the LRs emerged faster from salt-induced inhibition. A strong correlation could exist between the LRE mediated by PtrSSR1 and abscisic acid (ABA), mainly because the transformants displayed more sensitivity to exogenous ABA during both seed germination and LRE, and had a distinctly increased level of endogenous ABA. Furthermore, several ABA- and salt-related genes, such as NCED3, ABI1 and CBL1, were up-regulated. Thus, our results suggest that elevation in the endogenous ABA content bring alteration of plant LR development, and that the poplar R2R3 MYB TF PtrSSR1 vitally improve salt stress tolerance by integrating the regulation of LRE and ABA signaling in Arabidopsis.

Genome-wide identification and expression analysis of polyamine oxidase genes in upland cotton (Gossypium hirsutum L.)

Many reports showed that polyamine oxidase (PAO) genes play crucial roles in various abiotic and biotic stresses response. In this study, 12 putative PAO genes (GhPAO1–GhPAO12) were identified in upland cotton (Gossypium hirsutum L.) genome using the released genome database. A total of 349 cis-acting factors involved in hormone and stress response were predicted in 1.5-kb promoter regions at the upstream region of GhPAOs. These GhPAOs can be divided into four major groups when compared with their counterparts in Arabidopsis thaliana. Transcriptional level of GhPAOs were detected by qRT-PCR showing the highest levels in roots. Although GhPAOs exhibited various responses to NaCl treatment, the highest expression level was observed from 3 or 6 h after treatment. To further elucidate GhPAOs functions, GhPAO3 were selected for genetic transformation in Arabidopsis. The content of spermine was significantly decreased in transgenic Arabidopsis lines, whereas the content of putrescine was enhanced, which indicates a potential role of GhPAO3 in the conversion of spermidine and spermine. Discrepancy of salt resistance were observed in germination and seedling stage, which might be controlled by H2O2 concentration threshold. This study provides a valuable insight of the PAO genes in upland cotton response to abiotic stress.

Characterization of S-adenosylmethionine Synthetase Gene from Red Algae, Pyropia tenera

S-adenosylmethionine (SAM) is widely involved in variety of biosynthetic processes. It participates in the methionine (Met) metabolic pathway. SAM synthetase (EC 2.5.1.6) is the main enzyme that creates SAM from Met and adenosine triphosphate. SAMS genes are highly conserved in different species. We have isolated SAMS genes from red algae, Pyropia tenera. It has been reported that SAMS genes can confer abiotic stress-related response and growth of plant organs in other species. To elucidate the function of PtSAMS gene in evolutionary perspective, we analyzed SAMS gene using bioinformatics tools and plant transformation of Arabidopsis.