BY-2 Lines Research Articles

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Expression of Arabidopsis WEE1 in tobacco induces unexpected morphological and developmental changes

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.

Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing

BackgroundPlant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines.ResultsHere we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana.ConclusionsOur results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

Transcriptome Profiling Identified Multiple Jasmonate ZIM-Domain Proteins Involved in the Regulation of Alkaloid Biosynthesis in Tobacco BY-2 Cells

Jasmonate (JA) zinc-finger expressed in inflorescence meristem (ZIM)-domain (JAZ) proteins are key regulators of the JA response in plants. Transcriptome profiling of tobacco BY-2 cells was used to identify 17 members of the NtJAZ family, which were divided into 12 distinct groups based on their predicted amino acid sequences and conserved domains. Transcript levels of eight of the NtJAZ groups increased rapidly upon JA treatment, whereas the remaining members did not show a significant response. The majority of JA-induced NtJAZs formed homo- and heteromers and interacted with NtMYC2a (but not NtERF189) in yeast two-hybrid assays. NtJAZ1, NtJAZ3b, NtJAZ7 and NtJAZ10 were localised in the nucleus and degraded rapidly via the 26S proteasome pathway following the treatment of BY-2 with MeJA. RNAi-induced silencing of NtJAZ1, NtJAZ3, NtJAZ7a and NtJAZ10 greatly reduced the levels of NtPMT transcripts and specifically decreased the nicotine content in the four RNAi transgenic BY-2 lines. The levels of transcripts encoding other nicotine biosynthesis enzymes, NtERF189 and NtMYC2a, and other NtJAZs exhibited different expression patterns in RNAi lines with or without MeJA treatment. Our results indicate that cross-talk occurs among different NtJAZs and forms a complex transcription regulatory scheme for JA-induced nicotine biosynthesis in tobacco.

Hybrid proline-rich proteins: novel players in plant cell elongation?

Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion.

Esterases as a marker for growth of BY-2 tobacco cells and early somatic embryos of the Norway spruce

Growth is one of the basic properties of biological systems. The methods which are commonly used for the determination of growth are usually difficult and not very accurate. In the present work we decided to use esterase activity as a growth marker in tobacco suspension culture (BY-2 line) and in early somatic embryos of Norway spruce (clone 2/32) grown on a semi-solid medium. Esterase activity correlates well with the classical growth characteristics of BY-2 and spruce early somatic embryos. Determination of esterase activity is based on spectrophotometric and spectrofluorimetric detection of reaction products, which arise from the enzymatic hydrolysis of two substrates (p -nitrophenyl acetate and fluorescein diacetate) by esterase. The spectrophotometric method enabled us to detect approximately 104 BY-2 cells and 25 spruce embryos whereas the more sensitive spectrofluorimetric method allowed us to detect approximately 800 BY-2 cells and 5 early somatic embryos of Norway spruce.

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line).

The rapid generation of H(2)O(2) by Cd(2+)-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H(2)O(2) has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H(2)O(2) induced by Cd(2+). These data suggest the involvement of an NADPH oxidase-like enzyme leading to H(2)O(2) production through O(2)(*-) dismutation by superoxide dismutase enzymes. To investigate the implication of Ca(2+) channels in a Cd(2+)-induced oxidative burst, different inhibitors of Ca(2+) channels were used. Only La(3+) totally inhibited the generation of H(2)O(2) induced by Cd(2+). However, verapamil and nifedipine, inhibitors of Ca(2+) channels, were not effective. Calmodulin or a Ca(2+)-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H(2)O(2) induced by Cd(2+), suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

Activation of the promoters of Arabidopsis genes for the branched-chain alpha-keto acid dehydrogenase complex in transgenic tobacco BY-2 cells under sugar starvation.

Sugar starvation exerted by sub-10 mM levels of sucrose on Arabidopsis T87 suspension-cultured cells triggered marked accumulation of the transcripts of genes for E1beta and E2 subunit of the branched-chain alpha-keto acid dehydrogenase complex. Similar levels of sugar starvation increased the luciferase activity in transgenic tobacco BY-2 lines expressing the Arabidopsis E1beta- or E2-promoter-luciferase fusion gene. These results indicate that sugar levels tightly regulate the E1beta and E2 promoter activity in the heterologous plant system. We further showed in the transgenic tobacco BY-2 lines that sugar-starvation-induced activation of the E1beta and E2 promoters was prevented by K-252a, an inhibitor of Ser/Thr protein kinase, and was enhanced by okadaic acid, an inhibitor of protein phosphatases. By contrast, the cauliflower mosaic virus 35S promoter activity in sugar-starved BY-2 cells was not significantly affected by K-252a and only slightly enhanced by okadaic acid. Taken together, we propose that transcriptional activation of genes for the branched-chain alpha-keto acid dehydrogenase complex and its modulation by specific protein kinases/phosphatases are of critical importance in branched-chain amino acid catabolism in plant cells under sugar starvation.

A ribosomal protein gene (rpl32) from tobacco chloroplast DNA is transcribed from alternative promoters: similarities in promoter region organization in plastid housekeeping genes.

Multiple transcriptional start sites have been identified in the tobacco plastid ribosomal protein gene rpl32 by RNA mapping and in vitro capping techniques. A promotor with a canonical -10 Pribnow Box (P1) produces a major transcript in leaf chloroplasts. Transcription is also driven from additional promoters in non-photosynthetic plastids from heterotrophically cultured cells (BY2 line). Among them, a second promoter located downstream (P2) generates the most prominent transcript in this type of cell. The absence of typical plastid promoter motifs upstream of this site and the higher steady-state level of the P2-derived transcript in BY2 cells suggest a distinct modulation of transcription. Mobility shift experiments also seem to indicate the existence of differences in protein-DNA binding between both kinds of plastids with respect to a DNA fragment including the sequence upstream from the P2 starting site. The structure of the rpl32 promoter region is discussed in relation to that of other plastid housekeeping genes encoding elements of the genetic machinery.

Effects of reduction of auxin and destruction of microtubules on cell wall proteins and cell morphology of the BY-2 line of tobacco cells

Variations of cell wall proteins and proteins in the medium associated with changes in cell morphology were investigated in the BY-2 line of cultured cells. BY-2 cells cultured in LS medium grew as long chains of cells, with the plane of division perpendicular to the longitudinal axis. Reduction in the levels of auxin in the medium resulted in inhibition of cell division and promotion of cell elongation. Levels of cell wall proteins in cell walls decreased and relative levels of cell wall proteins and proteins in the medium changed. Upon treatment with the anti-microtubule drug, propyzamide, cells expanded laterally. Level of cell wall proteins and relative levels of individual cell wall proteins did not change very much, but levels of proteins in the culture medium increased. In both cases, levels of acid and basic peroxidases in cell walls increased and isozyme patterns of these changed.

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 μm, and 60% of them ranged from 7 to 11 μrn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.