Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis.

Introduction: Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. Methods: We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Results: Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. Discussion: We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.

COL2-dependent photoperiodic floral induction in Nicotiana sylvestris seems to be lost in the N. sylvestris × N. tomentosiformis hybrid N. tabacum.

Introduction: Plants are sessile organisms that maximize reproductive success by adapting to their environment. One of the key steps in the reproductive phase of angiosperms is flower development, requiring the perception of multiple endogenous and exogenous signals integrated via a complex regulatory network. Key floral regulators, including the main transcription factor of the photoperiodic pathway (CONSTANS, CO) and the central floral pathway integrator (FLOWERING LOCUS T, FT), are known in many species. Methods and results: We identified several CO-like (COL) proteins in tobacco (Nicotiana tabacum). The NtCOL2a/b proteins in the day-neutral plant N. tabacum were most closely related to Arabidopsis CO. We characterized the diurnal expression profiles of corresponding genes in leaves under short-day (SD) and long-day (LD) conditions and confirmed their expression in phloem companion cells. Furthermore, we analyzed the orthologs of NtCOL2a/b in the maternal LD ancestor (N. sylvestris) and paternal, facultative SD ancestor (N. tomentosiformis) of N. tabacum and found that they were expressed in the same diurnal manner. NtCOL2a/b overexpression or knock-out using the CRISPR/Cas9 system did not support a substantial role for the CO homologs in the control of floral transition in N. tabacum. However, NsCOL2 overexpression induced flowering in N. sylvestris under typically non-inductive SD conditions, correlating with the upregulation of the endogenous NsFTd gene. Discussion: Our results suggest that NsFTd is transcriptionally regulated by NsCOL2 and that this COL2-dependent photoperiodic floral induction seems to be lost in N. tabacum, providing insight into the diverse genetics of photoperiod-dependent flowering in different Nicotiana species.

The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network.

Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.

The functionality of plant mechanoproteins (forisomes) is dependent on the dual role of conserved cysteine residues.

Forisomes are giant polyprotein complexes that undergo reversible conformational rearrangements from a spindle-like to a plug-like state in response to Ca2+ or changes in pH. They act as valves in the plant vasculature, and reproduce this function in vitro to regulate flow in microfluidic capillaries controlled by electro-titration. Heterologous expression in yeast or plants allows the large-scale production of tailor-made artificial forisomes for technical applications. Here we investigated the unexpected disintegration of artificial forisomes in response to Ca2+ following the deletion of the M1 motif in the MtSEO-F1 protein or the replacement of all four conserved cysteine residues therein. This phenomenon could be mimicked in wild-type forisomes under reducing conditions by adding a thiol alkylating agent. We propose a model in which reversible changes in forisome structure depend on cysteine residues with ambiguous redox states, allowing the formation of intermolecular disulfide bridges (confirmed by mass spectrometry) as well as noncovalent thiol interactions to connect forisome substructures in the dispersed state. This is facilitated by the projection of the M1 motif from the MtSEO-F1 protein as part of an extended loop. Our findings support the rational engineering of disintegrating forisomes to control the release of peptides or enzymes in microfluidic systems.

The tobacco phosphatidylethanolamine-binding protein NtFT4 simultaneously improves vitality, growth, and protein yield in human cells.

The production of biopharmaceutical proteins in mammalian cells by transient expression or stable transformation requires robust and viable cells. Cell line engineering must therefore balance improved cell growth and viability with high productivity. We tested the ability of nonmammalian phosphatidylethanolamine-binding proteins to enhance cell proliferation in monolayers and suspension cultures. The tobacco protein NtFT4 improved the proliferation of multiple human cell lines. Viable cell density is usually impaired by efficient transfection, but we found that the number of HEK-293TNtFT4 cells at the peak of protein expression was twice that of standard HEK-293T cells, and the antibody yield increased by approximately one-third. Improved growth and viability were observed in different cell lines, in different culture media, and also after transient transfection, suggesting the beneficial trait is consistent and transferable. Additional modifications could boost the productivity of high-density HEK-293TNtFT4 cells even further as we showed for a fluorescent marker protein and recombinant antibody expressed in monolayer cultures. The HEK-293TNtFT4 cell line provides a new human model platform that increases cell proliferation, also achieving a fundamental improvement in recombinant protein expression.

Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification.

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

Loss of function mutation of the Rapid Alkalinization Factor (RALF1)-like peptide in the dandelion Taraxacum koksaghyz entails a high-biomass taproot phenotype.

The Russian dandelion (Taraxacum koksaghyz) is a promising source of inulin and natural rubber because large amounts of both feedstocks can be extracted from its roots. However, the domestication of T. koksaghyz requires the development of stable agronomic traits such as higher yields of inulin and natural rubber, a higher root biomass, and an agronomically preferable root morphology which is more suitable for cultivation and harvesting. Arabidopsis thaliana Rapid Alkalinisation Factor 1 (RALF1) has been shown to suppress root growth. We identified the T. koksaghyz orthologue TkRALF-like 1 and knocked out the corresponding gene (TkRALFL1) using the CRISPR/Cas9 system to determine its impact on root morphology, biomass, and inulin and natural rubber yields. The TkRALFL1 knockout lines more frequently developed a taproot phenotype which is easier to cultivate and harvest, as well as a higher root biomass and greater yields of both inulin and natural rubber. The TkRALFL1 gene could therefore be suitable as a genetic marker to support the breeding of profitable new dandelion varieties with improved agronomic traits. To our knowledge, this is the first study addressing the root system of T. koksaghyz to enhance the agronomic performance.

The enzymes OSC1 and CYP716A263 produce a high variety of triterpenoids in the latex of Taraxacum koksaghyz.

Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz, thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol (1) and its ketone lup-19(21)-en-3-one (2). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, ß-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.

The Major Floral Promoter NtFT5 in Tobacco (Nicotiana tabacum) Is a Promising Target for Crop Improvement.

The FLOWERING LOCUS T (FT)-like gene family encodes key regulators of flower induction that affect the timing of reproduction in many angiosperm species. Agricultural research has therefore focused on such genes to improve the success of breeding programs and enhance agronomic traits. We recently identified a novel FT-like gene (NtFT5) that encodes a day-neutral floral activator in the model tobacco crop Nicotiana tabacum. However, further characterization is necessary to determine its value as a target for breeding programs. We therefore investigated the function of NtFT5 by expression analysis and mutagenesis. Expression analysis revealed that NtFT5 is transcribed in phloem companion cells, as is typical for FT-like genes. However, high levels of NtFT5 mRNA accumulated not only in the leaves but also in the stem. Loss-of-function mutants (generated using CRISPR/Cas9) were unable to switch to reproductive growth under long-day conditions, indicating that NtFT5 is an indispensable major floral activator during long-days. Backcrossing was achieved by grafting the mutant scions onto wild-type rootstock, allowing the restoration of flowering and pollination by a wild-type donor. The resulting heterozygous Ntft5- /NtFT5+ plants flowered with a mean delay of only ~2 days, demonstrating that one functional allele is sufficient for near-normal reproductive timing. However, this minor extension of the vegetative growth phase also conferred beneficial agronomic traits, including a >10% increase in vegetative leaf biomass on the main shoot and the production of more seeds. The agronomic benefits of the heterozygous plants persisted under various abiotic stress conditions, confirming that NtFT5 is a promising target for crop improvement to address the effects of climate change.

Taraxacum brevicorniculatum rubber elongation factor TbREF associates with lipid droplets and affects lipid turn-over in yeast.

A protein named TbREF that is localized on rubber particles of the rubber producing dandelion species Taraxacum brevicorniculatum was expressed in tobacco leaves and in yeast. TbREF fused to fluorescence proteins colocalized on globular, hydrophobic structures, most likely lipid droplets. Furthermore, triacylglycerol, sterol and total lipid content of TbREF expressing yeast was determined by photometric analyses of nile red stainings and GC-MS analyses. Therefore, yeast exposed an enhanced nile red fluorescence as well as an increased TAG and sterol content compared to wildtype and vector control. Altogether, these findings gave new insights into the putative function of TbREF that might be pushing rubber particle production due to its cytotoxic nature and/or shielding and preventing degradation of lipid droplets. Furthermore, these results highlight possible biotechnological applications regarding the accumulation of hydrophobic compounds in lipid droplet like structures.

Kinetics and modeling of cell growth for potential anthocyanin induction in cultures of Taraxacum officinale G.H. Weber ex Wiggers (Dandelion) in vitro

Background: Taraxacum officinale G.H. Weber ex Wiggers is a wild plant used in folk medicine to treat several diseases owing to bioactive secondary metabolites present in its tissue. The accumulation of such molecules in plant cells can occur as a response against abiotic stress, but these metabolites are often deposited in low concentrations. For this reason, the use of a biotechnological approach to improve the yields of technologically interesting bioactive compounds such as anthocyanins is a compelling option. This work focuses on investigating the potential of in vitro T. officinale cultures as an anthocyanin source. Results: To demonstrate the suitability of anthocyanin induction and accumulation in calluses under specific conditions, anthocyanin was induced in the T. officinale callus. A specific medium of 5.5% sucrose supplemented with 6-benzylaminopurine /1-naphthaleneacetic acid in a 10:1 ratio was used to produce an anthocyanin yield of 1.23 mg g-1 fw. An in vitro dandelion callus line was established from this experiment. Five mathematical models were then used to objectively and predictably explain the growth of anthocyanin-induced calluses from T. officinale. Of these models, the Richards model offered the most suitable representation of anthocyanin callus growth in a solid medium and permitted the calculation of the corresponding kinetic parameters. Conclusions: The findings demonstrate the potential of an in vitro anthocyanin-induced callus line from T. officinale as an industrial anthocyanin source.

The FT/FD-dependent initiation of flowering under long-day conditions in the day-neutral species Nicotiana tabacum originates from the facultative short-day ancestor Nicotiana tomentosiformis.

Photoperiod is an important external stimulus governing the precise timing of the floral transition in plants. Members of the FLOWERING LOCUS T (FT)-like clade of phosphatidylethanolamine-binding proteins induce this developmental process in numerous species by forming regulatory protein complexes with FD-like bZIP transcription factors. We identified several thus far unknown FT-like and FD-like genes in the genus Nicotiana and found that, even in the day-neutral species Nicotiana tabacum, floral initiation requires the photoperiod-dependent expression of several FT-like genes. Furthermore, floral promotion under long-day (LD) and short-day (SD) conditions is mediated by an FT-like protein (NtFT5) that originates from the genome of the paternal, facultative SD ancestor Nicotiana tomentosiformis. In contrast, its ortholog of the maternal LD ancestor Nicotiana sylvestris is not present in the genome of N. tabacum cv. SR1. Expression profiling in N. tabacum and its ancestors confirmed the relevance of these FT and FD orthologs in the context of polyploidization. We also found that floral inhibition by tobacco FT-like proteins is not restricted to SD conditions, highlighting the coincident expression of tobacco FT-like genes encoding floral activators and floral inhibitors. Multicolor bimolecular fluorescence complementation analysis revealed the preferential formation of FT/FD complexes that promote rather than inhibit flowering, which in concert with the regulation of NtFT and NtFD expression could explain how floral promotion overcomes floral repression during the floral transition in tobacco.

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis-1,4-isoprene) bodies.

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex-containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1-5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1-5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild-type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3-5 on the dispersity of artificial poly(cis-1,4-isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet-associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.

Characterization of five subgroups of the sieve element occlusion gene family in Glycine max reveals genes encoding non-forisome P-proteins, forisomes and forisome tails.

P-proteins are structural phloem proteins discussed to be involved in the rapid sealing of injured sieve elements. P-proteins are found in all dicotyledonous and some monocotyledonous plants, but additional crystalloid P-proteins, known as forisomes, have evolved solely in the Fabaceae. Both types are encoded by members of the sieve element occlusion (SEO) gene family, which comprises seven phylogenetic subgroups. The Fabaceae-specific subgroup 1 contains genes encoding forisome subunits in e.g. Medicago truncatula, Vicia faba, Dipteryx panamensis and Canavalia gladiata whereas basal subgroup 5 encodes P-proteins in Nicotiana tabacum (tobacco) and Arabidopsis thaliana. The function of remaining subgroups is still unknown. We chose Glycine max (soybean) as a model to investigate SEO proteins representing different subgroups in one species. We isolated native P-proteins to determine the SEO protein composition and analyzed the expression pattern, localization and structure of the G. max SEO proteins representing five of the subgroups. We found that subgroup 1 GmSEO genes encode forisome subunits, a member of subgroup 5 encodes a non-forisome P-protein and subgroup 2 GmSEO genes encode the components of forisome tails, which are present in a restricted selection of Fabaceaen species. We therefore present the first molecular characterization of a Fabaceae non-forisome P-protein and the first evidence that forisome tails are encoded by a phylogenetically-distinct branch of the SEO gene family.

Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

BACKGROUND: Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. METHODOLOGY/PRINCIPAL FINDINGS: We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. CONCLUSIONS/SIGNIFICANCE: We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast.

Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.

Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures.

Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×10(6) per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10µM naphthalene acetic acid, 2µM 2,4-dichlorophenoxyacetic acid, 2µM indole-3-butyric acid, 2µM gibberellic acid and 2µM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid.

P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing.

Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing.

A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.