Transgenic Plant Lines Research Articles

CDC48 regulates immunity pathway in tobacco plants

The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.

Overexpression of AtMYB12 transcription factor simultaneously enhances quercetin-dependent metabolites in radish callus

The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens-mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12. Radish seeds were infected with the Agrobacterium suspension (0.8 OD600) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes (AtMYB12, CHS, F3H, and FLS) and antioxidant genes (GPX, APX, CAT, and SOD) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g−1 dry weight), flavonoid content (463 mg g−1 dry weight), and quercetin content (48.24 mg g−1 dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.

Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus

BackgroundSecondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression.ResultsWe overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants.ConclusionsOverall, saccharification efficiency was increased by 35–50%, 21–42%, and 8–39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

Root Hair Imaging Using Confocal Microscopy.

Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

OsWRKY97, an Abiotic Stress-Induced Gene of Rice, Plays a Key Role in Drought Tolerance.

Drought stress is one of the major causes of crop losses. The WRKY families play important roles in the regulation of many plant processes, including drought stress response. However, the function of individual WRKY genes in plants is still under investigation. Here, we identified a new member of the WRKY families, OsWRKY97, and analyzed its role in stress resistance by using a series of transgenic plant lines. OsWRKY97 positively regulates drought tolerance in rice. OsWRKY97 was expressed in all examined tissues and could be induced by various abiotic stresses and abscisic acid (ABA). OsWRKY97-GFP was localized to the nucleus. Various abiotic stress-related cis-acting elements were observed in the promoters of OsWRKY97. The results of OsWRKY97-overexpressing plant analyses revealed that OsWRKY97 plays a positive role in drought stress tolerance. In addition, physiological analyses revealed that OsWRKY97 improves drought stress tolerance by improving the osmotic adjustment ability, oxidative stress tolerance, and water retention capacity of the plant. Furthermore, OsWRKY97-overexpressing plants also showed higher sensitivity to exogenous ABA compared with that of wild-type rice (WT). Overexpression of OsWRKY97 also affected the transcript levels of ABA-responsive genes and the accumulation of ABA. These results indicate that OsWRKY97 plays a crucial role in the response to drought stress and may possess high potential value in improving drought tolerance in rice.

Control of erucic acid biosynthesis in Camelina (Camelina sativa) by antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistanceto abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elangase 1 (FAE1) gene encoded the enzyme β-ketoacyl COA synthase (KCS), which is a key factor in the biosynthesis of erucic acid. In this study, isolation and cloning of the FAE1 from the Camelina sativa were performed to prepare an antisense structure. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning FAE1 into the Bluescriprt II SK+ vector and sequencing, these fragments were used for the preparation of antisense structurein the pBI121 plant expression vector. The approved structurewas transferred to the camelina plant via the Agrobacterium-mediated method. Also, the conditions of tissue culture and gene transfer were optimized. Moreover, the erucic acid content of the immature seeds of T0 transgenic plants was analyzed with gas chromatography (GC). Results showed significant changes in erucic acid levels of two control plants (0.88%), while two lines of the RFAE1 transgenic plants showed a decrease of approximately 0% in erucic acid level. It can be concluded that the antisense structure can be effective in reducing erucic acid.

Foxtail millet SiCDPK7 gene enhances tolerance to extreme temperature stress in transgenic plants

Global climate change has resulted in increasingly harsh environments that have become the restriction factors for plants growth and development. Calcium-dependent protein kinases (CDPKs) in plants play critical roles in resisting various abiotic stresses, such as heat and cold stresses. Using the expression pattern analysis of foxtail millet CDPK family members under heat- and cold-treatments, respectively, and the SiCDPK7 was found to respond to extreme temperature stress and was selected for further study. The results suggested that overexpression of SiCDPK7 in Arabidopsis conferred tolerance to heat stress by increasing seedling survival rates and hypocotyl elongation compared with wild type (WT) plants, and also can enhance heat stress tolerance in foxtail millet. Analysis of physiological and biochemical indexes showed that SiCDPK7 transgenic plant lines had the markedly higher catalase (CAT) activity and the significantly lower malonaldehyde (MDA) content than WT plants. In addition, qRT-PCR analysis showed that the transcription levels of heat and cold stress-responsive genes were significantly increased in SiCDPK7 transgenic Arabidopsis and foxtail millet under stress conditions. This supporting evidence suggests that SiCDPK7 facilitated the extreme temperature tolerance capabilities in plants

Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00–07, S-00–11, S-00–16 and S-00–23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

Biotechnological and molecular genetic methods in grape breeding

The development of new grape varieties with improved quality and resistance against environmental factors using biotechnological methods is a pressing task in the context of import substitution policy implementation. Due to the high heterogeneity of grape varieties, techniques for regenerating plants from embryogenic callus by somatic embryogenesis have been developed only for a small number of genotypes. This work presents a versatile approach to the development of globular, heart, and torpedo embryos in various grape genotypes. Experiments were carried out using cultures of colchicine embryogenic cell suspensions and somatic globular embryos of the E-342 hybrid form in liquid media. The developed methodology allows the protocols of embryogenesis and polyploidy induction to be effectively improved. The versatility of the presented methodology for polyploidy induction and somatic embryogenesis in grape plants is ensured by a fourfold reduction in the concentration of inorganic nitrogen and vitamins in the NN and PG media and the addition of organic nitrogen and other biological active substances at the stages of embryogenic callus development, colchicination, and development of somatic embryoids in liquid media. The methods of indirect ploidy determination by the properties of the grape leaf epidermis were studied and implemented. Nine promising somaclones were preliminary selected based on a set of four features. It was established that full-fledged transgenic grape plants can only be obtained by the method of transformation, which involves the production of embryogenic callus from a leaf explant, its subsequent passage into a liquid culture to obtain a liquid embryo culture, and the transformation of this culture. In total, 300 leaf explants of the Libya variety and 1 100 globular embryos of the Podarok Magaracha variety were transformed. As a result, six callus kanamycin-resistant lines of the Libya variety and six lines of transgenic plants of the Podarok Magaracha variety were selected. Following genotyping based on nine nuclear microsatellite loci, seven unknown forms with a seedless phenotype were identified. Among the population of hybrid seedlings, hybrids having alleles associated with seedlessness were revealed using the VMC7f2 and p3_VvAGL11 MAS markers.

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Natural GMOs: a history of research

Agrobacterium mediated transformation underlies the production of most modern lines of transgenic plants. At the same time, in nature, plants are described that have been transformed by Agrobacterium without human intervention. They are called natural GMOs. Such plants were first described by White in 1983 [1] within the genus Nicotiana L., and the phenomenon of horizontal gene transfer from Agrobacterium to plants was considered unique for representatives of this genus for a long time. Only in 2012, another genus of natural GMOs was found. It was Linaria Mill. In 2019, the list of species of natural GMOs was increased by an order of magnitude and is constantly updated until now [2]. Several stages can be identified in the history of natural GMO research: 1. Description of individual examples of natural GMOs. 2. Estimation of the frequency of the horizontal gene transfer from Agrobacterium to plants based on the analysis of NGS data. 3. Description of the diversity of cT-DNA in terms of composition and copy number [3]. 4. Studies of the functions of individual pGMO genes [4]. 5. Phylogenetic studies of natural GMOs [5]. During the analysis of cT-DNA of natural GMOs, it became clear that they differ in the composition and intactness of transgenes, which can be interpreted from the point of view of the lack of a common function in all cT-DNAs. Genes that were not previously found in known Agrobacterium strains, are identifiedIn in cT-DNAs, indicating a greater biodiversity of Agrobacterium than previously thought [2]. Many cT-DNA genes are intact and expressed. For some it was possible to identify products [4]. All these discoveries lead us to understanding of the role of horizontal gene transfer from Agrobacterium to plants during their evolution. The work was performed using the equipment of the RC of St. Petersburg State University Development of Molecular and Cellular Technologies and Biobank with the support of the Ministry of Science and Higher Education of the RF in accordance with agreement No. 075-15-2022-322 dated 04/22/2022 on the provision of a grant in the form of a subsidy from the Federal budget of the Russian Federation for the creation and development of the world-class Scientific Center Agrotechnologies for the Future.

Overexpression of genes encoding enzymes involved in trehalose synthesis from Caragana korshinskii enhances drought tolerance of transgenic plants

Trehalose, which plays important roles in resistance to abiotic stresses and preservation of biological activity in plants, is synthesized by two key enzymes, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Therefore, the expressions of the TPS and TPP genes directly affect trehalose synthesis and stress resistance of plants. In this study, CkTPS and CkTPP from Caragana korshinskii were identified, and the role of trehalose synthesis in the adaptation of this desert plant to adverse conditions was investigated. Higher CkTPS and CkTPP expressions were observed in the roots, whereas expressions were much lower in leaves and stems, and their expressions were upregulated under drought stress. Histochemical analyses showed that β-glucuronidase expression driven by the CkTPS and CkTPP promoters was strongly induced by abiotic stresses and phytohormones, such as abscisic acid, gibberellin, methyl jasmonate, and mannitol, which suggests that trehalose synthesis may be regulated by various signaling pathways. To determine the functional mechanism underlying the role of trehalose synthesis in regulating drought response in plants, CkTPS and CkTPP were introduced into Arabidopsis. Compared to wild-type (WT) plants, these transgenic plants showed higher germination rate, survival, less damage, better shoot growth, and longer roots under drought stress. Moreover, transgenic plants had a significantly higher content of proline, chlorophyll, trehalose, and activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and lower malondialdehyde (MDA) content than WT controls. Double-transgenic plants carrying CkTPS and CkTPP showed better growth and stronger drought tolerance than either single transgenic plant line. These results provide a theoretical and experimental basis for further understanding the function and regulatory mechanism of CkTPS and CkTPP, as well as the possibility of their application for improving drought tolerance in crops through genetic engineering.

Overexpression of Chloroplast Triosephosphate Isomerase Marginally Improves Photosynthesis at Elevated CO2 Levels in Rice.

We recently suggested that chloroplast triosephosphate isomerase (cpTPI) has moderate control over the rate of CO2 assimilation (A) at elevated CO2 levels via the capacity for triose phosphate utilization (TPU) in rice (Oryza sativa L.) from its antisense-suppression study. In the present study, the effects of cpTPI overexpression on photosynthesis were examined in transgenic rice plants overexpressing the gene encoding cpTPI. The amounts of cpTPI protein in the two lines of transgenic plants were 4.8- and 12.1-folds higher than in wild-type plants, respectively. The magnitude of the increase approximately corresponded to the increase in transcript levels of cpTPI. A at CO2 levels of 100 and 120 Pa increased by 6-9% in the transgenic plants, whereas those at ambient and low CO2 levels were scarcely affected. Similar increases were observed for TPU capacity estimated from the CO2 response curves of A. These results indicate that the overexpression of cpTPI marginally improved photosynthesis at elevated CO2 levels via improvement in TPU capacity in rice. However, biomass production at a CO2 level of 120 Pa did not increase in transgenic plants, suggesting that the improvement in photosynthesis by cpTPI overexpression was not sufficient to improve biomass production in rice.

Induction of AmpC-Mediated β-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens

ABSTRACTThe remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here, we use a reverse-genetics approach to identify genes involved in ampicillin resistance, with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor, AmpR. Loss of the putative ampD orthologue atu2113 led to constitutive production of AmpC-dependent β-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent β-lactamase activity, and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 transforms plants with efficiency comparable to that of the wild type but can be cleared with sublethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium.IMPORTANCE Agrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in β-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit β-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

Effects of suppression of chloroplast phosphoglycerate kinase on photosynthesis in rice.

As chloroplast phosphoglycerate kinase (cpPGK) is one of the enzymes which has the highest capacity among the Calvin-Benson cycle enzymes, it has not been regarded as a determinant for photosynthetic capacity. However, it was reported that the rate of CO2 assimilation decreased under high irradiance and normal [CO2] levels in the Arabidopsis cpPGK-knockdown mutant, implying that cpPGK has a control over photosynthetic capacity at a normal [CO2] level. In the present study, the contribution of cpPGK to photosynthetic capacity was evaluated in transgenic rice plants with decreased amounts of cpPGK protein under high irradiance and various [CO2] levels. The gene encoding cpPGK was suppressed using RNA interference techniques. Two lines of transgenic plants, Pi3 and Pi5, in which the amount of cpPGK protein decreased to 21% and 76% of that in wild-type plants, respectively, were obtained. However, there was no substantial difference in the rates of CO2 assimilation between wild-type and transgenic plants. The rates of CO2 assimilation decreased only slightly at elevated [CO2] levels in the transgenic line Pi3 and did not differ between wild-type plants and the transgenic line Pi5, irrespective of [CO2] level. These results clearly indicate that cpPGK does not have a strong control over photosynthetic capacity at various [CO2] levels in rice.

CRISPR/Cas9-mediated knockout of a prolyl-4-hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection.

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the numberof peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.

The Dawn of Plant Molecular Biology: How Three Key Methodologies Paved the Way.

The adoption of Arabidopsis thaliana in the 1980s as a universal plant model finally enabled researchers to adopt and take full advantage of the molecular biology tools and methods developed in the bacterial and animal fields since the early 1970s. It further brought the plant sciences up to speed with other research fields, which had been employing widely accepted model organisms for decades. In parallel with this major development, the concurrent establishment of the plant transformation methodology and the description of the cauliflower mosaic virus (CaMV) 35S promoter enabled scientists to create robust transgenic plant lines for the first time, thereby providing a valuable tool for studying gene function. The ability to create transgenic plants launched the plant biotechnology sector, with Monsanto and Plant Genetic Systems developing the first herbicide- and pest-tolerant plants, initiating a revolution in the agricultural industry. Here I review the major developments over a less than 10-year span and demonstrate how they complemented each other to trigger a revolution in plant molecular biology and launch an era of unprecedented progress for the whole plant field. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Visualising the ionome in resistant and susceptible plant-pathogen interactions.

At the morphological and anatomical levels, the ionome, or the elemental composition of an organism, is an understudied area of plant biology. In particular, the ionomic responses of plant-pathogen interactions are scarcely described, and there are no studies on immune reactions. In this study we explored two X-ray fluorescence (XRF)-based ionome visualisation methods (benchtop- and synchrotron-based micro-XRF [µXRF]), as well as the quantitative inductively coupled plasma optical emission spectroscopy (ICP-OES) method, to investigate the changes that occur in the ionome of compatible and incompatible plant-pathogen interactions. We utilised the agronomically important and comprehensively studied interaction between potato (Solanum tuberosum) and the late blight oomycete pathogen Phytophthora infestans as an example. We used one late blight-susceptible potato cultivar and two resistant transgenic plant lines (only differing from the susceptible cultivar in one or three resistance genes) both in control and P. infestans-inoculated conditions. In the lesions from the compatible interaction, we observed rearrangements of several elements, including a decrease of the mobile macronutrient potassium (K) and an increase in iron (Fe) and manganese (Mn), compared with the tissue outside the lesion. Interestingly, we observed distinctly different distribution patterns of accumulation at the site of inoculation in the resistant lines for calcium (Ca), magnesium (Mg), Mn and silicon (Si) compared to the susceptible cultivar. The results reveal different ionomes in diseased plants compared to resistant plants. Our results demonstrate a technical advance and pave the way for deeper studies of the plant-pathogen ionome in the future.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Morphophysiological alterations in transgenic rice lines expressing PPDK and ME genes from the C4 model Setaria italica

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.