2 research outputs found
Redox regulation of Salicylic acid biosynthesis by S-nitrosylation
A general outcome of plant-pathogen interactions is the oxidative and nitrosative
burst, characterized for the accumulation of reactive oxygen and nitrogen species
(ROS and RNA) which coordinate signalling cascades. Among RNS, Nitric oxide (NO)
stands as a key signalling molecule in different physiological processes including
plant immunity. An established mechanism for the transfer of NO bioactivity is S-nitrosylation
(SNO), the reversible binding of a NO molecule to the thiol group of a
susceptible cysteine, allowing specific proteins to respond to changes in the cellular
REDOX state.
The first genetic evidence about the role of NO in plant immunity was noted in
Arabidopsis thaliana plants carrying a loss-of-function mutation of the S-nitrosoglutathione
(GSNO) reductase 1 (GSNOR1) gene. This mutation resulted in
increased total cellular S-nitrosylation and compromised basal, non-host and R-gene
mediated immunity, and impaired synthesis and accumulation of the immune activator
salicylic acid (SA). SA plays a pivotal role in the regulation of basal and systemic
resistance. It is mainly produced by the activity of the enzyme Isochorismate Synthase
1 (ICS1) in response to pathogens.
To date, significant progress has been achieved in understanding the mechanisms by
which S-nitrosylation regulates SA signalling. However, the molecular mechanisms
by which NO regulates SA biosynthesis remains elusive.
To investigate if NO mediates transcriptional or posttranscriptional regulation over
ICS1 expression we generated the reporter line ICS1::GUS. Our data suggest that
ICS1 is subjected to transcriptional repression by S-nitrosylation. In agreement with
previous observations about inhibition of the DNA-binding of SARD1 to the ICS1
promoter upon S-nitrosylation of SARD1 at Cysteine 438. We observed a significant
reduction in the binding affinity of recombinant wild type (WT) SARD1 but not in
SARD1 with a Cystein 438 to Serine mutation. To expand this observations and
investigate the biological relevance of S-nitrosylation of Cys438 we generated
transgenic lines expressing a C-terminal HA and Nano luciferase SARD1 and
SARD1C438S fusion proteins.
We showed that SARD1 can be S-nitrosylated in vivo. We did not observe any
difference in local immunity against Pseudomonas syringae infection between the WT
and C438S lines. Interestingly, the SARD1C438S lines showed impaired activation of
systemic acquired resistance (SAR) compared to the WT. In addition, we observed
that SARD1 protein level follows a circadian rhythm after SA treatment, which was
impaired in the C438S mutant, suggesting that S-nitrosylation of SARD1 is necessary
for optimal activation of SAR. It is possible that S-nitrosylation of SARD1 coordinates
protein-protein interactions between SARD1 and other SAR activators.
We developed a strategy to express and purify recombinant SARD1 for structural
studies. Solving the three-dimensional structure of SARD1 can foster our
understanding on the molecular interactions behind the regulation of SARD1. Finally,
we designed a forward mutant screening to search for second-site mutations that can
suppress the gsnor1 phenotype, which we speculate could be related with a novel
mechanism for GSNO-turnover or NO metabolism. Collectively, our work can
contribute to integrate NO cues in the regulation of SA biosynthesis and suggests a
role for S-nitrosylation of SARD1 in systemic immunity
Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches
Abstract 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. Graphical Abstrac