Recent advances in paclitaxel biosynthesis and regulation.

Paclitaxel (PTX) is a high value plant natural product (PNP) derived from Taxus (yew) species. This plant secondary metabolite (PSM) and its derivatives constitute a cornerstone for the treatment of an increasing variety of cancers. New applications for PTX also continue to emerge, further promoting demand for this WHO designated essential medicine. Here we review recent advances in our understanding of PTX biosynthesis and its cognate regulation, which have been enabled by the development of transcriptomic approaches and the recent sequencing and annotation of three Taxus genomes. Collectively, this has resulted in the elucidation of two functional gene sets for PTX biosynthesis, unlocking new potential for the use of heterologous hosts to produce PTX. Knowledge of the PTX pathway also provides a valuable resource for understanding the regulation of this key PSM. Epigenetic regulation of PSM in plant cell culture (PCC) is a major concern for PTX production, given the loss of PSM production in long-term cell cultures. Recent developments aim to design tools for manipulating epigenetic regulation, potentially providing a means to reverse the silencing of PSM caused by DNA methylation. Exciting times clearly lie ahead for our understanding of this key PSM and improving its production potential.

An Engineered Heat-Inducible Expression System for the Production of Casbene in Nicotiana benthamiana.

Plants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene casbene in Nicotiana benthamiana, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from Arabidopsis thaliana Hsp101 or Glycine max GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV 35S, and the 5'UTR from the tobacco mosaic virus. The resulting four chimeric promoters fused to a green fluorescent protein (GFP) reporter showed that the variant Ara2 had the strongest fluorescent signal after heat shock. We next created a 4-gene cassette driven by the Ara2 promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into Nicotiana benthamiana. Metabolic analysis on the transgenic lines revealed that continuous heat outperforms heat shock, with up to 1 µg/mg DW of casbene detected after 32 h of uninterrupted 40 °C heat. These results demonstrate the potential of heat-inducible promoters as synthetic biology tools for metabolite production in plants.

Expanding roles for S-nitrosylation in the regulation of plant immunity.

Following pathogen recognition, plant cells produce a nitrosative burst resulting in a striking increase in nitric oxide (NO), altering the redox state of the cell, which subsequently helps orchestrate a plethora of immune responses. NO is a potent redox cue, efficiently relayed between proteins through its co-valent attachment to highly specific, powerfully reactive protein cysteine (Cys) thiols, resulting in formation of protein S-nitrosothiols (SNOs). This process, known as S-nitrosylation, can modulate the function of target proteins, enabling responsiveness to cellular redox changes. Key targets of S-nitrosylation control the production of reactive oxygen species (ROS), the transcription of immune-response genes, the triggering of the hypersensitive response (HR) and the establishment of systemic acquired resistance (SAR). Here, we bring together recent advances in the control of plant immunity by S-nitrosylation, furthering our appreciation of how changes in cellular redox status reprogramme plant immune function.

An activated form of NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to trigger cell death in rice.

A key event that follows pathogen recognition by a resistance (R) protein containing an NB-ARC (nucleotide-binding adaptor shared by Apaf-1, R proteins, and Ced-4) domain is hypersensitive response (HR)-type cell death accompanied by accumulation of reactive oxygen species and nitric oxide. However, the integral mechanisms that underlie this process remain relatively opaque. Here, we show that a gain-of-function mutation in the NB-ARC protein RLS1 (Rapid Leaf Senescence 1) triggers high-light-dependent HR-like cell death in rice. The RLS1-mediated defense response is largely independent of salicylic acid accumulation, NPR1 (Nonexpressor of Pathogenesis-Related Gene 1) activity, and RAR1 (Required for Mla12 Resistance 1) function. A screen for suppressors of RLS1 activation identified RMC (Root Meander Curling) as essential for the RLS1-activated defense response. RMC encodes a cysteine-rich receptor-like secreted protein (CRRSP) and functions as an RLS1-binding partner. Intriguingly, their co-expression resulted in a change in the pattern of subcellular localization and was sufficient to trigger cell death accompanied by a decrease in the activity of the antioxidant enzyme APX1. Collectively, our findings reveal an NB-ARC-CRRSP signaling module that modulates oxidative state, the cell death process, and associated immunity responses in rice.

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches.

In this study, several approaches were tested to optimise the production and recovery of the widely used anticancer drug Taxol® (paclitaxel) from culturable vascular stem cells (VSCs) of Taxus baccata, which is currently used as a successful cell line for paclitaxel production. An in situ product recovery (ISPR) technique was employed, which involved combining three commercial macro-porous resin beads (HP-20, XAD7HP and HP-2MG) with batch and semi-continuous cultivations of the T. baccata VSCs after adding methyl jasmonate (Me-JA) as an elicitor. The optimal resin combination resulted in 234 ± 23 mg of paclitaxel per kg of fresh-weight cells, indicating a 13-fold improved yield compared to the control (with no resins) in batch cultivation. This resin treatment was further studied to evaluate the resins' removal capacity of reactive oxygen species (ROS), which can cause poor cell growth or reduce product synthesis. It was observed that the ISPR cultivations had fourfold less intracellular ROS concentration than that of the control; thus, a reduced ROS concentration established by the resin contributed to increased paclitaxel yield, contrary to previous studies. These paclitaxel yields are the highest reported to date using VSCs, and this scalable production method could be applied for a diverse range of similar compounds utilising plant cell culture.

Feedback loop promotes sucrose accumulation in cotyledons to facilitate sugar-ethylene signaling-mediated, etiolated-seedling greening.

De-etiolation is indispensable for seedling survival and development. However, how sugars regulate de-etiolation and how sugars induce ethylene (ET) for seedlings to grow out of soil remain elusive. Here, we reveal how a sucrose (Suc) feedback loop promotes de-etiolation by inducing ET biosynthesis. Under darkness, Suc in germinating seeds preferentially induces 1-amino-cyclopropane-1-carboxylate synthase (ACS7; encoding a key ET biosynthesis enzyme) and associated ET biosynthesis, thereby activating ET core component ETHYLENE-INSENSITIVE3 (EIN3). Activated EIN3 directly inhibits the function of Suc transporter 2 (SUC2; a major Suc transporter) to block Suc export from cotyledons and thereby elevate Suc accumulation of cotyledons to induce ET. Under light, ET-activated EIN3 directly inhibits the function of phytochrome A (phyA; a de-etiolation inhibitor) to promote de-etiolation. We therefore propose that under darkness, the Suc feedback loop (Suc-ACS7-EIN3-|SUC2-Suc) promotes Suc accumulation in cotyledons to guarantee ET biosynthesis, facilitate de-etiolation, and enable seedlings to grow out of soil.

Perturbations in nitric oxide homeostasis promote Arabidopsis disease susceptibility towards Phytophthora parasitica.

Phytophthora species can infect hundreds of different plants, including many important crops, causing a number of agriculturally relevant diseases. A key feature of attempted pathogen infection is the rapid production of the redox active molecule nitric oxide (NO). However, the potential role(s) of NO in plant resistance against Phytophthora is relatively unexplored. Here we show that the level of NO accumulation is crucial for basal resistance in Arabidopsis against Phytophthora parasitica. Counterintuitively, both relatively low or relatively high NO accumulation leads to reduced resistance against P. parasitica. S-nitrosylation, the addition of a NO group to a protein cysteine thiol to form an S-nitrosothiol, is an important route for NO bioactivity and this process is regulated predominantly by S-nitrosoglutathione reductase 1 (GSNOR1). Loss-of-function mutations in GSNOR1 disable both salicylic acid accumulation and associated signalling, and also the production of reactive oxygen species, leading to susceptibility towards P. parasitica. Significantly, we also demonstrate that secreted proteins from P. parasitica can inhibit Arabidopsis GSNOR1 activity.

A Novel DUF569 Gene Is a Positive Regulator of the Drought Stress Response in Arabidopsis.

In the last two decades, global environmental change has increased abiotic stress on plants and severely affected crops. For example, drought stress is a serious abiotic stress that rapidly and substantially alters the morphological, physiological, and molecular responses of plants. In Arabidopsis, several drought-responsive genes have been identified; however, the underlying molecular mechanism of drought tolerance in plants remains largely unclear. Here, we report that the "domain of unknown function" novel gene DUF569 (AT1G69890) positively regulates drought stress in Arabidopsis. The Arabidopsis loss-of-function mutant atduf569 showed significant sensitivity to drought stress, i.e., severe wilting at the rosette-leaf stage after water was withheld for 3 days. Importantly, the mutant plant did not recover after rewatering, unlike wild-type (WT) plants. In addition, atduf569 plants showed significantly lower abscisic acid accumulation under optimal and drought-stress conditions, as well as significantly higher electrolyte leakage when compared with WT Col-0 plants. Spectrophotometric analyses also indicated a significantly lower accumulation of polyphenols, flavonoids, carotenoids, and chlorophylls in atduf569 mutant plants. Overall, our results suggest that novel DUF569 is a positive regulator of the response to drought in Arabidopsis.

Recent advances in the regulation of plant immunity by S-nitrosylation.

S-nitrosylation, the addition of a nitric oxide (NO) moiety to a reactive protein cysteine (Cys) thiol, to form a protein S-nitrosothiol (SNO), is emerging as a key regulatory post-translational modification (PTM) to control the plant immune response. NO also S-nitrosylates the antioxidant tripeptide, glutathione, to form S-nitrosoglutathione (GSNO), both a storage reservoir of NO bioactivity and a natural NO donor. GSNO and, by extension, S-nitrosylation, are controlled by GSNO reductase1 (GSNOR1). The emerging data suggest that GSNOR1 itself is a target of NO-mediated S-nitrosylation, which subsequently controls its selective autophagy, regulating cellular protein SNO levels. Recent findings also suggest that S-nitrosylation may be deployed by pathogen-challenged host cells to counteract the effect of delivered microbial effector proteins that promote pathogenesis and by the pathogens themselves to augment virulence. Significantly, it also appears that S-nitrosylation may regulate plant immune functions by controlling SUMOylation, a peptide-based PTM. In this context, global SUMOylation is regulated by S-nitrosylation of SUMO conjugating enzyme 1 (SCE1) at Cys139. This redox-based PTM has also been shown to control the function of a key zinc finger transcriptional regulator during the establishment of plant immunity. Here, we provide an update of these recent advances.

Regulating the regulator: nitric oxide control of post-translational modifications.

Nitric oxide (NO) is perfectly suited for the role of a redox signalling molecule. A key route for NO bioactivity occurs via protein S-nitrosation, and involves the addition of a NO moiety to a protein cysteine (Cys) thiol (-SH) to form an S-nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants that are linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post-translational modifications including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues.

A role for S-nitrosylation of the SUMO-conjugating enzyme SCE1 in plant immunity.

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, is emerging as a key modulator of eukaryotic immune function. In plants, a SUMO1/2-dependent process has been proposed to control the deployment of host defense responses. The molecular mechanism underpinning this activity remains to be determined, however. Here we show that increasing nitric oxide levels following pathogen recognition promote S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1, at Cys139. The SUMO-conjugating activities of both SCE1 and its human homolog, UBC9, were inhibited following this modification. Accordingly, mutation of Cys139 resulted in increased levels of SUMO1/2 conjugates, disabled immune responses, and enhanced pathogen susceptibility. Our findings imply that S-nitrosylation of SCE1 at Cys139 enables NO bioactivity to drive immune activation by relieving SUMO1/2-mediated suppression. The control of global SUMOylation is thought to occur predominantly at the level of each substrate via complex local machineries. Our findings uncover a parallel and complementary mechanism by suggesting that total SUMO conjugation may also be regulated directly by SNO formation at SCE1 Cys139. This Cys is evolutionary conserved and specifically S-nitrosylated in UBC9, implying that this immune-related regulatory process might be conserved across phylogenetic kingdoms.

Novel and conserved functions of S-nitrosoglutathione reductase in tomato.

Nitric oxide (NO) is emerging as a key signalling molecule in plants. The chief mechanism for the transfer of NO bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cellular S-nitrosylation, by depleting S-nitrosoglutathione (GSNO), the major cellular NO donor. Here we show that depletion of GSNOR function impacts tomato (Solanum lycopersicum. L) fruit development. Thus, reduction of GSNOR expression through RNAi modulated both fruit formation and yield, establishing a novel function for GSNOR. Further, depletion of S. lycopersicum GSNOR (SlGSNOR) additionally impacted a number of other developmental processes, including seed development, which also has not been previously linked with GSNOR activity. In contrast to Arabidopsis, depletion of GSNOR function did not influence root development. Further, reduction of GSNOR transcript abundance compromised plant immunity. Surprisingly, this was in contrast to previous data in Arabidopsis that reported that reducing Arabidopsis thaliana GSNOR (AtGSNOR) expression by antisense technology increased disease resistance. We also show that increased SlGSNOR expression enhanced pathogen protection, uncovering a potential strategy to enhance disease resistance in crop plants. Collectively, our findings reveal, at the genetic level, that some but not all GSNOR activities are conserved outside the Arabidopsis reference system. Thus, manipulating the extent of GSNOR expression may control important agricultural traits in tomato and possibly other crop plants.

Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases.

Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of EcoRI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the EcoRI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the de novo synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.

Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation.

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

Loss of NLRP3 Function Alleviates Murine Hepatic Graft-versus-Host Disease.

NLRP3 is associated with multiple risks in graft-versus-host disease, though unifying principles for these findings remain largely unknown. To explore the effects and mechanisms of the absence of NLRP3 function on hepatic graft-versus-host-disease, we established an allogeneic hematopoietic cell transplantation mice model by infusing bone marrow mononuclear cells and spleno-T cells of the BALB/c mouse into either NLRP3 knockout (NLRP3-/- ) or wild-type C57BL/6 mice. Elevated inflammatory cell infiltration, liver fibrosis, and secretions of alanine aminotransferase (ALT) and aspartate transaminase (AST), together with weight loss, were observed in C57BL/6 recipients after transplantation. However, moderate injury pathology was detected in the liver of NLRP3-/- recipients at day 14, which gradually improved over time. Likewise, proinflammatory cytokine IL-1ß, a downstream effecter of NLRP3 inflammasome activation, showed significantly lower expression (P < .05) in the liver of NLRP3-/- recipients relative to C57BL/6 recipients at day 7 and day 21. Moreover, compared with C57BL/6 recipients, the expression of both TNF-α and IL-1ß were decreased 3-fold and 4.7-fold, respectively, at day 21 in NLRP3-/- recipients. Interestingly, NLRP1a was expressed at a significantly reduced level in the liver of NLRP3-/- recipients (P < .001). Furthermore, systemic inflammation was analyzed by measuring the concentration of IL-1ß and adenosine triphosphate (ATP) in serum. The concentration of IL-1ß achieved a maximum at day 14, then decreased at day 21 and day 28 in NLRP3-/- recipients. In contrast, the concentration of IL-1ß in C57BL/6 recipients gradually increased from day 7 to day 28. ATP levels reduced from day 7 to day 28 in NLRP3-/- recipients, but were extremely high in C57BL/6 recipients from day 14 to day 28 (P < .01). The decreased levels of P2X7R were connected to less ATP in NLRP3-/- recipients at day 21 and day 28. In conclusion, NLRP3 knockout in recipients could significantly relieve liver injury after transplantation and block the NLRP3 inflammasome pathway, thus providing a promising strategy for the treatment of graft-versus-host disease prophylaxis.

Regulation of Anticancer Styrylpyrone Biosynthesis in the Medicinal Mushroom Inonotus obliquus Requires Thioredoxin Mediated Transnitrosylation of S-nitrosoglutathione Reductase.

The medicinal macrofungus Inonotus obliquus widely utilized as folk medicine in Russia and Baltic countries is a source of phenylpropanoid-derived styrylpyrone polyphenols that can inhibit tumor proliferation. Insights into the regulatory machinery that controls I. obliquus styrylpyrone polyphenol biosynthesis will enable strategies to increase the production of these molecules. Here we show that Thioredoxin (Trx) mediated transnitrosylation of S-nitrosoglutathione reductase (GSNOR) underpins the regulation of styrylpyrone production, driven by nitric oxide (NO) synthesis triggered by P. morii coculture. NO accumulation results in the S-nitrosylation of PAL and 4CL required for the synthesis of precursor phenylpropanoids and styrylpyrone synthase (SPS), integral to the production of styrylpyrone, inhibiting their activities. These enzymes are targeted for denitrosylation by Trx proteins, which restore their activity. Further, this Trx S-nitrosothiol (SNO) reductase activity was potentiated following S-nitrosylation of Trx proteins at a non-catalytic cysteine (Cys) residue. Intriguingly, this process was counterbalanced by Trx denitrosylation, mediated by Trx-dependent transnitrosylation of GSNOR. Thus, unprecedented interplay between Trx and GSNOR oxidoreductases regulates the biosynthesis of styrylpyrone polyphenols in I. obliquus.

Molecular and Biochemical Examination of Spraing Disease in Potato Tuber in Response to Tobacco rattle virus Infection.

Field-grown tubers of potato were examined for infection by Tobacco rattle virus (TRV) and consequent production of corky ringspot or spraing symptoms. A microarray study identified genes that are differentially expressed in tuber tissue in response to TRV infection and to spraing production, suggesting that hypersensitive response (HR) pathways are activated in spraing-symptomatic tubers. This was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of a selected group of HR-related genes and by histochemical staining of excised tuber tissue with spraing symptoms. qRT-PCR of TRV in different regions of the same tuber slice showed that nonsymptomatic areas contained higher levels of virus relative to spraing-symptomatic areas. This suggests that spraing formation is associated with an active plant defense that reduces the level of virus in the infected tuber. Expression of two of the same plant defense genes was similarly upregulated in tubers that were infected with Potato mop-top virus, a virus that also induces spraing formation.

Identification of S-Nitrosothiols by the Sequential Cysteine Blocking Technique.

Here, we describe a procedure for the identification of S-nitrosothiols that has been used in our laboratory to study the roles of protein S-nitrosylation in the immune responses of Arabidopsis thaliana and other organisms. It employs a modified version of the biotin-switch technique, which we termed the sequential cysteine blocking technique, encompassing the sequential redox-blocking of recombinant proteins followed by LC-MS/MS analysis.

Nitric oxide and S-nitrosoglutathione function additively during plant immunity.

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation. Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-of-function mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression. Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants. Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.