Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0009734 auxin-activated signaling pathway	IEA GO_REF:0000043	REMOVE	Summary: SPKW (GO_REF:0000043) keyword-derived annotation, present only in the Sept 2025 snapshot and removed from current GOA. It originates from the UniProt keyword "Auxin signaling pathway", which was attached because of the protein's historical name "Auxin-regulated protein parA". parA is a downstream auxin-responsive gene, not a component of the auxin signal-transduction pathway. Reason: This is an over-annotation that conflates "transcriptionally induced by auxin" with "component of the auxin signaling pathway". parA was isolated as an early auxin-induced transcript in cultured tobacco protoplasts [PMID:2594768], and its promoter contains a cis-acting auxin-responsive element [PMID:2236015]. Being an auxin-responsive target gene is distinct from being part of the GO:0009734 pathway, which is defined as the series of events from binding of auxin to a receptor through downstream output and is reserved for receptor/signal-transduction components (TIR1/AFB receptors, SCF-TIR1, Aux/IAA repressors, ARF transcription factors). parA is a tau-class plant GST and a downstream effector, not a signaling component. The keyword-to-GO mapping propagated the misleading "auxin" keyword into a pathway-component term. GOA's removal of this annotation when it retired the keyword2GO (GO_REF:0000043) pipeline was JUSTIFIED: a correct annotation was not lost. If a process term capturing the auxin relationship is desired, the appropriate term is "response to auxin" (GO:0009733), which covers auxin-responsive gene expression without implying signal-transduction-component status (see the NEW entry below). Proposed replacements: response to auxin Supporting Evidence: PMID:2594768 The mRNA of the par gene is approximately 900 bases long and its accumulation was detected in cultured mesophyll protoplasts as early as 30 min after the addition of 2,4-dichlorophenoxyacetic acid to the culture medium. The par mRNA was not detected in leaves or freshly prepared protoplasts or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid. PMID:2236015 deletion of a 111-base-pair (bp) direct repeat in the 5' flanking sequences of the par gene abolished the auxin-induced GUS activity, it is deduced that in the 111-bp direct repeat of the par gene promoter is localized an auxin-responsive region, which regulates auxin-mediated activation of transcription.
GO:0004364 glutathione transferase activity	IEA GO_REF:0000120	ACCEPT	Summary: Family-level molecular-function annotation (InterPro IPR045074 Tau-class GST signature, RHEA:16437, EC 2.5.1.18, PANTHER PTN004250175). parA has the canonical two-domain GST fold and a glutathione-binding site, and the closely related parB/Nt103 was directly shown to be a functional GST [PMID:1729717]. A direct assay of the parA protein detected GST activity, although at a low level (<1/30 that of parB) [PMID:7767232]. Reason: parA belongs to the plant tau (U) class of the GST superfamily (PANTHER PTHR11260:SF756; CDD GST_N_Tau/GST_C_Tau; InterPro tau-class signatures). Tau and phi classes are the plant-specific GST classes that catalyse glutathione-dependent conjugation reactions [PMID:21425939]. GST activity has been directly detected for the parA recombinant protein, so this generic molecular-function term is supported, even though parA's activity is weak relative to its paralog parB and its preferred physiological substrate is unknown (hence UniProt names it "Probable glutathione S-transferase"). The IEA term is at an appropriate level of generality for a family-based assignment. Supporting Evidence: PMID:7767232 we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST. PMID:1729717 When we introduced expression vector pKK233-2, which retains parB cDNA, into Escherichia coli, we could detect GST activity in the parB gene product. file:TOBAC/PARA/PARA-deep-research-falcon.md Therefore, enzyme-function annotation for P25317 as an active EC 2.5.1.18 catalyst should be phrased as “probable” and supported by (i) family placement and (ii) proteomics annotation rather than an unambiguous parA-only enzymology experiment
GO:0005737 cytoplasm	IEA GO_REF:0000118	KEEP AS NON CORE	Summary: TreeGrafter (GO_REF:0000118) phylogenetic transfer of the cytoplasm location from the PTHR11260 GST family. Plant GSTs are predominantly cytosolic enzymes [PMID:11897031], so this is the family-typical location. However, the one direct study of parA protein localization reported parA in the nucleus of tobacco protoplasts and BY-2 cells, contrasting with conventional cytoplasmic GSTs including parB [PMID:7767232]. Reason: The cytoplasm assignment is a reasonable family-level call: plant GSTs "are predominantly expressed in the cytosol" [PMID:11897031] and parA has no predicted targeting/transit peptide. However, there is a genuine experimental discrepancy: immunofluorescence and confocal imaging reported the parA protein confined to the nucleus (excluded from the nucleolus), unlike the cytoplasmic parB GST [PMID:7767232]. Because the cellular location of parA is not robustly established and the only direct evidence argues against a purely cytoplasmic localization, this term is retained (it is not clearly wrong for a GST-fold protein) but marked as non-core rather than accepted as a confirmed core localization. The nuclear-localization observation is from a single 1995 study using overexpressed/tagged protein and could be artefactual, so neither cytoplasm nor nucleus can be asserted with confidence. Supporting Evidence: PMID:11897031 GSTs are predominantly expressed in the cytosol, where their GSH-dependent catalytic functions include the conjugation and resulting detoxification of herbicides PMID:7767232 Immunofluorescence studies using an antibody against parA protein revealed that the subcellular location of parA protein is the nucleus in cultured tobacco mesophyll protoplasts, while conventional GSTs' including the parB product were primarily located in the cytoplasm. file:TOBAC/PARA/PARA-deep-research-falcon.md the plastid-preparation detection supports association with plastid fractions but does not alone prove true plastid residency (common issue in organelle proteomics); together with a nuclear-localization report, it underscores that P25317 localization is not settled
GO:0006749 glutathione metabolic process	IEA GO_REF:0000120	ACCEPT	Summary: Family-level biological-process annotation accompanying the glutathione transferase activity call (InterPro IPR045074, PANTHER PTN004250175). Glutathione transferases use glutathione as cosubstrate/coenzyme in conjugation reactions, which constitutes glutathione-utilizing metabolism. Reason: As a tau-class GST that uses glutathione as a cosubstrate (the EC 2.5.1.18 reaction RX + glutathione = an S-substituted glutathione + a halide anion + H+), parA participates in glutathione metabolism. Plant GSTs are central to glutathione-dependent conjugation chemistry [PMID:21425939, PMID:11897031]. This generic process term is the standard partner of the GST molecular-function annotation and is appropriate at the IEA family-inference level, consistent with the directly demonstrated (if weak) GST activity of the parA protein [PMID:7767232]. Supporting Evidence: PMID:21425939 the numerous and plant-specific phi (F) and tau (U) classes are largely responsible for catalyzing glutathione-dependent reactions with xenobiotics, notably conjugation leading to detoxification PMID:7767232 we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST.
GO:0009733 response to auxin	IEP PMID:2594768 Isolation of an auxin-regulated gene cDNA expressed during t...	NEW	Summary: parA is one of the best-characterized auxin-responsive genes in tobacco. Its mRNA accumulates within 30 min of 2,4-D treatment in cultured mesophyll protoplasts and is absent without auxin [PMID:2594768], and a defined cis-acting auxin-responsive element in its promoter is required for this induction [PMID:2236015]. Reason: This term replaces the incorrect retired "auxin-activated signaling pathway" (GO:0009734) annotation. "Response to auxin" (GO:0009733) correctly captures the well-documented biology of parA: it is a downstream auxin-responsive target gene, transcriptionally induced by auxin, without implying that it is a component of the auxin signal-transduction pathway. The evidence is expression-based (auxin-dependent mRNA accumulation and a mapped auxin-responsive promoter element), so IEP is the appropriate evidence code. Supporting Evidence: PMID:2594768 The mRNA of the par gene is approximately 900 bases long and its accumulation was detected in cultured mesophyll protoplasts as early as 30 min after the addition of 2,4-dichlorophenoxyacetic acid to the culture medium. The par mRNA was not detected in leaves or freshly prepared protoplasts or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid. PMID:2236015 a chimeric gene, in which a reporter gene for bacterial beta-glucuronidase (GUS) was placed downstream of the 5' flanking sequences of the par gene ... elicited auxin-regulated expression of GUS activity.
GO:0009617 response to bacterium	IEP PMID:7948901 Structural organization of str 246C and str 246N, plant defe...	NEW	Summary: parA is identical in sequence to str246C, a defense-related gene whose transcripts accumulate in tobacco leaves upon inoculation with the bacterial pathogen Pseudomonas (Ralstonia) solanacearum, in both compatible and incompatible interactions [PMID:7948901]. Reason: The independent isolation of parA as the pathogen-inducible defense gene str246C documents that parA is transcriptionally responsive to bacterial infection. This is a distinct, experimentally supported aspect of parA biology not captured by any current or retired annotation. "Response to bacterium" (GO:0009617) accurately reflects the expression-based evidence (IEP). It also illustrates that parA is a general stress-inducible tau GST rather than an auxin-pathway-specific gene. Supporting Evidence: PMID:7948901 We have previously identified a cDNA clone, pNt246, whose corresponding transcripts accumulate in leaves in response to inoculation by compatible and incompatible isolates of the phytopathogenic bacterium Pseudomonas solanacearum file:TOBAC/PARA/PARA-deep-research-falcon.md Deletion analysis identifies a bacterial response element (BRE) between −184 and −101 required for infection inducibility

Core Functions

parA is a plant tau-class glutathione S-transferase that catalyses glutathione-dependent conjugation chemistry (EC 2.5.1.18). It has the canonical two-domain GST fold with a glutathione-binding site and detectable glutathione transferase activity, although its activity is weak relative to its paralog parB and its preferred physiological substrate is not established.

Molecular Function:

glutathione transferase activity

Directly Involved In:

glutathione metabolic process

Supporting Evidence:

PMID:7767232
we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST.
PMID:21425939
the numerous and plant-specific phi (F) and tau (U) classes are largely responsible for catalyzing glutathione-dependent reactions with xenobiotics, notably conjugation leading to detoxification
file:TOBAC/PARA/PARA-deep-research-falcon.md
P25317 is best annotated as a **stress-responsive GST-family protein** that likely participates in **GSH-dependent detoxification/redox buffering** and possibly **ligand binding/transport of hydrophobic molecules** under auxin and defense signaling

parA is a stress- and hormone-responsive gene whose defining, best-supported biology is transcriptional induction. It is rapidly induced by auxin (2,4-D) via a defined auxin-responsive promoter element and, as the defense gene str246C, is induced by bacterial pathogen infection. This places parA among the auxin-inducible / stress-inducible plant tau GSTs rather than among components of the auxin signal-transduction pathway.

Molecular Function:

glutathione transferase activity

Directly Involved In:

response to auxin response to bacterium

Supporting Evidence:

PMID:2594768
The par mRNA was not detected in leaves or freshly prepared protoplasts or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid.
PMID:7948901
a cDNA clone, pNt246, whose corresponding transcripts accumulate in leaves in response to inoculation by compatible and incompatible isolates of the phytopathogenic bacterium Pseudomonas solanacearum
file:TOBAC/PARA/PARA-deep-research-falcon.md
induced by **auxin** and **auxin + cytokinin** and is also a multiple-stimulus response promoter, activated by elicitors and infection

References

file:TOBAC/PARA/PARA-deep-research-falcon.md

Falcon deep-research report: Functional annotation of Nicotiana tabacum PARA / parA / STR246C (UniProt P25317).

Confirms that UniProt P25317 corresponds to tobacco parA, which is sequence-identical to the defense gene str246C, and maps parA to the auxin-regulated tobacco type-III (tau-class) GST subfamily Nt114 (older nomenclature GST3-1).
The "auxin-regulated" designation reflects transcriptional induction: the str246C/parA promoter is induced by auxin (2,4-D) via a defined auxin-responsive cis-element, and is independently a multi-stimulus response promoter activated by wounding (within 30 min), elicitors and bacterial infection - supporting the conclusion that parA is a downstream auxin/stress-responsive target gene, not a component of the auxin signal-transduction pathway.
Direct GST enzymology in the retrieved corpus is demonstrated for the paralogs parB and the Nt103/Nt107 isozymes (GST1-1/GST2-1), not for parA itself; parA-specific catalytic activity is weak, so EC 2.5.1.18 / GST activity for P25317 is best annotated as "probable" on the basis of family placement plus weak directly-detected activity.
Subcellular localization of parA remains unsettled: a protoplast immunostaining study reported nuclear localization, while BY-2 proteomics detected P25317 in plastid preparations and whole-cell 2-DE; the plastid-fraction detection may reflect contamination, so neither cytoplasm nor nucleus can be asserted with confidence.
A 2024 synthesis on plant GSTs (Micic et al.) notes that GST substrate promiscuity and the difficulty of detecting endogenous glutathione conjugates mean the physiological substrate of a probable GST such as parA cannot be assigned from sequence similarity alone.

GO_REF:0000043

Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping

SwissProt keyword-derived (SPKW) annotations present in the Sept 2025 goa_uniprot_gcrp snapshot but removed from the current GOA release after GOA retired the keyword2GO pipeline for cellular organisms.
The SPKW annotation auxin-activated signaling pathway (GO:0009734) for parA was derived from the UniProt keyword "Auxin signaling pathway", itself based on the historical protein name "Auxin-regulated protein"; it conflates auxin-responsiveness with auxin-pathway-component status and its removal was justified.

GO_REF:0000118

TreeGrafter-generated GO annotations

Cytoplasm localization was transferred from the PTHR11260 GST family; this is the family-typical location for cytosolic plant GSTs.

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

Glutathione transferase activity and glutathione metabolic process were inferred from the InterPro tau-class GST signature (IPR045074), the RHEA reaction RHEA:16437, EC 2.5.1.18 and PANTHER family membership.

PMID:2594768

Isolation of an auxin-regulated gene cDNA expressed during the transition from G0 to S phase in tobacco mesophyll protoplasts.

parA (par) was isolated as an auxin-regulated cDNA from cultured tobacco mesophyll protoplasts; its 220-aa product had no detectable homology to known proteins at the time of isolation.
par mRNA accumulates within 30 min of 2,4-D addition and is absent in leaves, freshly prepared protoplasts, or protoplasts without auxin, establishing parA as a rapidly auxin-induced gene.

PMID:2236015

Location of the cis-acting auxin-responsive region in the promoter of the par gene from tobacco mesophyll protoplasts.

A 111-bp direct repeat in the parA promoter constitutes an auxin-responsive cis-element required for auxin-mediated transcriptional activation.

PMID:7948901

Structural organization of str 246C and str 246N, plant defense-related genes from Nicotiana tabacum.

The genomic gene str246C is sequence-identical to parA.
str246C/parA transcripts accumulate in tobacco leaves on inoculation with the bacterial pathogen Pseudomonas (Ralstonia) solanacearum, in both compatible and incompatible interactions, identifying parA as a defense/stress-responsive gene.

PMID:1729717

parB: an auxin-regulated gene encoding glutathione S-transferase.

parB (Nt103), a close paralog of parA isolated from the same auxin-induced tobacco protoplast system, has clear sequence homology to glutathione S-transferases (EC 2.5.1.18).
Recombinant parB expressed in E. coli has GST activity, and GST activity increases in tobacco protoplasts cultured with 2,4-D, directly linking the par GST family to auxin-induced glutathione transferase function.

PMID:7767232

Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products.

The parA recombinant protein has only a low level of GST activity, below 1/30 that of the parB GST.
Immunofluorescence and confocal microscopy localized parA protein to the nucleus (excluded from the nucleolus) of tobacco protoplasts and BY-2 cells, in contrast to conventional cytoplasmic GSTs such as parB.
The authors proposed that parA may have a function distinct from a conventional cytosolic GST, possibly related to transcription, although this remains speculative and based on a weak similarity to an E. coli RNA-polymerase-binding protein.

PMID:21425939

Multiple roles for plant glutathione transferases in xenobiotic detoxification.

The plant-specific phi (F) and tau (U) GST classes catalyse glutathione-dependent conjugation of xenobiotics, leading to detoxification; plant GSTs use a catalytic serine rather than the tyrosine of mammalian GSTs.
Endogenous roles of plant GSTs are less well defined and include signaling, counteracting oxidative stress, and transport of secondary metabolites.

PMID:11897031

Plant glutathione transferases.

Soluble plant GSTs (EC 2.5.1.18) form a large gene family divided into phi, tau, theta, zeta and lambda classes; tau and phi are plant-specific and the most numerous.
Plant GSTs are predominantly cytosolic and use glutathione as a cosubstrate or coenzyme for conjugation, peroxide reduction, isomerization and non-catalytic ligandin functions.

Suggested Experiments

Experiment: Purify recombinant parA protein and assay activity against a panel of standard GST substrates (CDNB), organic hydroperoxides (GPOX activity), and candidate physiological substrates, comparing kinetics to parB to determine whether parA is a catalytically competent GST or a divergent ligandin/regulatory protein.

Hypothesis: parA retains GST-fold but has weak/altered catalysis and may function primarily as a ligand-binding (ligandin) protein rather than a conjugating enzyme.

Experiment: Re-examine parA subcellular localization using a native-promoter fluorescent-protein fusion or immuno-electron microscopy in stably transformed tobacco, under both auxin and pathogen induction, to resolve the cytoplasm-versus-nucleus discrepancy.

Hypothesis: The previously reported nuclear localization of parA reflects a genuine, possibly stress-regulated, partitioning distinct from cytosolic GSTs such as parB.

Experiment: Generate parA loss-of-function and overexpression lines in tobacco and challenge them with auxin, oxidative stress, herbicides, and Ralstonia solanacearum to test whether parA contributes to detoxification, oxidative stress tolerance, or disease resistance.

Hypothesis: parA functions as a stress-inducible detoxification/antioxidant tau GST contributing to defense responses rather than to auxin signal transduction.

Deep Research

Falcon

(PARA-deep-research-falcon.md)

Research Report: Functional Annotation of Nicotiana tabacum **PARA / parA / STR246C** (UniProt P25317) Falcon Edison Scientific Literature 44 citations 1 artifacts 2026-05-21T10:46:46.056935

## Context ID: pqac-00000046 The requested figures and table containing kinetic and inhibition data for GST1-1 and GST2-1 have been extracted. **Summary of Nume (image/png)

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Research Report: Functional Annotation of Nicotiana tabacum PARA / parA / STR246C (UniProt P25317)

Executive summary

UniProt P25317 corresponds to the tobacco gene historically described as parA and also isolated as str246C (STR246C), i.e., STR246C ≡ parA in Nicotiana tabacum. (froissard1994structuralorganizationof pages 1-3). The literature around “parA” contains important historical ambiguity: some studies treat parA/STR246C as a defense/auxin-regulated gene with GST-like homology, while other work in the same era reports parA protein nuclear localization and discusses it as a possible transcription-related factor, and places GST enzymatic activity more clearly on parB and on other auxin-regulated tobacco GSTs (Nt103/Nt107/Nt114 family) (takahashi1993functionsandmodulations pages 3-5). Later GST-focused work and proteomics resources nevertheless consistently annotate P25317 as a probable glutathione S-transferase (GST) and group it with the auxin-regulated tobacco Nt114 subfamily (often referred to as GST3-1 in older nomenclature) (droog199524dichlorophenoxyaceticacidand pages 4-6, laukens2004constructionofa pages 5-7, baginsky2004proteomeanalysisof pages 5-6).

Given this, the most defensible functional annotation for P25317 is: an auxin- and stress/pathogen-responsive GST-family protein (tau/type III plant GST context), implicated in cellular redox/detoxification processes and stress signaling; subcellular localization remains uncertain across studies, with nuclear localization reported in one protoplast study and proteomic detection in plastid preparations that may reflect true association or contamination (takahashi1993functionsandmodulations pages 3-5, baginsky2004proteomeanalysisof pages 5-6).

1. Identity verification and disambiguation (critical)

1.1. Gene/protein mapping and aliases

STR246C is identical to parA at the nucleotide sequence level in tobacco: Froissard et al. report the genomic clone str246C is identical to parA, with an ORF interrupted by an intron (froissard1994structuralorganizationof pages 1-3).
In the auxin-regulated tobacco GST literature, parA is stated to be identical to the Nt114 subfamily, while parC is nearly identical to Nt107 (droog199524dichlorophenoxyaceticacidand pages 4-6).

1.2. Separation from similarly named genes (parB, parC; Nt103/Nt107/Nt114)

Takahashi et al. (1993) explicitly treat parA and parB as distinct genes and report GST-like homology and CDNB GST activity for parB, not parA (takahashi1993functionsandmodulations pages 3-5).
Droog et al. (1995) focus on auxin-regulated type III GST isozymes and map parA → Nt114 within that GST family context (droog199524dichlorophenoxyaceticacidand pages 4-6).

Implication for annotation: the symbol “parA” is used in multiple related contexts (auxin-regulated gene family; defense-related str genes; GST isozyme naming). For UniProt P25317, the best-supported mapping in the retrieved corpus is parA/STR246C ≡ Nt114-associated GST-like protein, but older reports create ambiguity about whether “parA” was always used consistently for the GST-coding member versus a nuclear protein. This uncertainty should be explicitly recorded (takahashi1993functionsandmodulations pages 3-5, droog199524dichlorophenoxyaceticacidand pages 4-6).

2. Key concepts and definitions (current understanding)

2.1. What is a plant glutathione S-transferase (GST)?

Plant GSTs (EC 2.5.1.18) are typically dimeric enzymes that catalyze glutathione (GSH) conjugation (glutathionylation) to electrophilic substrates, supporting detoxification and stress tolerance; they also include non-catalytic “ligandin” binding roles and other catalytic activities (e.g., peroxidase-like activities) depending on class and isoform (marrs1996thefunctionsand pages 3-5, droog1997plantglutathionestransferases pages 1-2).

2.2. Why functional annotation is hard (2024 perspective)

A 2024 synthesis emphasizes that most plant GSTs show high substrate promiscuity, many candidate substrates can react spontaneously with GSH, and endogenous glutathione conjugates (GS-conjugates) are rarely detected because they can be transient/low abundance or rapidly metabolized—so knockouts often lack clear phenotypes/chemotypes (Micic et al., 2024; https://doi.org/10.1098/rstb.2023.0365; accepted 2024-08-06; published 2024-09 issue) (micic2024overlookedandmisunderstood pages 1-2, micic2024overlookedandmisunderstood pages 4-5, micic2024overlookedandmisunderstood pages 10-11).

Relevance to P25317: even if P25317 is a GST-family member, identifying its in vivo substrate(s) may be difficult without dedicated metabolomics of GS-conjugates and spatially resolved expression/localization (micic2024overlookedandmisunderstood pages 1-2, micic2024overlookedandmisunderstood pages 10-11).

3. Molecular function: enzymatic reaction and substrate specificity

3.1. Canonical GST reaction (general)

GSTs catalyze nucleophilic attack by the sulfur of GSH on electrophilic substrates, forming GSH conjugates that can be further transported/sequestered and metabolized (marrs1996thefunctionsand pages 3-5).

3.2. Evidence for enzymatic activity in the auxin-regulated tobacco GST family containing Nt114/parA

Droog et al. (1995) demonstrate that auxin-regulated tobacco isozymes GST1-1 (Nt103) and GST2-1 (Nt107) are functional GST enzymes in vitro using the model substrate CDNB and GSH, following Michaelis–Menten kinetics (Plant Physiology; 1995-04; https://doi.org/10.1104/pp.107.4.1139) (droog199524dichlorophenoxyaceticacidand pages 3-4).

Kinetic statistics (from Droog et al. 1995):
* Apparent Km(GSH): ~0.40 ± 0.15 mM (for both GST1-1 and GST2-1) (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61).
* Apparent Km(CDNB): ~0.85 ± 0.25 mM (GST1-1) and ~0.20 ± 0.15 mM (GST2-1) (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61).

Inhibition / ligand interaction statistics:
* 2,4-D competitively inhibits GST activity toward CDNB with apparent Ki ~80 ± 40 μM (GST1-1) and ~200 ± 100 μM (GST2-1) (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61).
* Ethacrynic acid is a potent inhibitor with apparent Ki ~5 μM (droog199524dichlorophenoxyaceticacidand pages 4-6, droog199524dichlorophenoxyaceticacidand media 50a1fd61).
* Structure–activity: inhibitory analogs generally required ≥1 chlorine atom (droog199524dichlorophenoxyaceticacidand pages 6-7).

Mapping to parA/P25317: Droog et al. state parA is identical to Nt114 (GST3-1 in older naming), placing P25317 in the same auxin-regulated type-III GST context (droog199524dichlorophenoxyaceticacidand pages 4-6). However, the retrieved excerpts provide detailed enzymology mainly for Nt103 and Nt107; they do not provide a dedicated kinetic panel for Nt114/parA itself.

3.3. Direct activity evidence for parA protein specifically

Within retrieved sources, direct in vitro GST activity is shown for parB (CDNB assay) and for GST1-1/GST2-1 isozymes (Nt103/Nt107), while parA-specific enzymology is not directly demonstrated in the excerpts (takahashi1993functionsandmodulations pages 3-5, droog199524dichlorophenoxyaceticacidand pages 3-4). Therefore, enzyme-function annotation for P25317 as an active EC 2.5.1.18 catalyst should be phrased as “probable” and supported by (i) family placement and (ii) proteomics annotation rather than an unambiguous parA-only enzymology experiment (droog199524dichlorophenoxyaceticacidand pages 4-6, laukens2004constructionofa pages 5-7).

4. Regulation, biological processes, and pathways

4.1. Auxin responsiveness and promoter architecture (str246C/parA)

Gough et al. (1995) show the str246C promoter (2.1 kb 5′ flank fused to GUS) is induced by auxin and auxin + cytokinin and is also a multiple-stimulus response promoter, activated by elicitors and infection (Molecular & General Genetics; 1995-05; https://doi.org/10.1007/bf00293200) (gough1995developmentalandpathogeninduced pages 1-2).

Quantitative induction examples (Gough et al. 1995):
* 5 μM 2,4-D: GUS activity 34.8 (units per their assay table) (gough1995developmentalandpathogeninduced pages 8-9).
* 5 μM auxin + cytokinin: 47.3 (gough1995developmentalandpathogeninduced pages 8-9).
* Induction timing: auxin (±cytokinin) induction is obvious at ~6 h and maximal 12–24 h (gough1995developmentalandpathogeninduced pages 8-9).

Takahashi et al. (1993) map an auxin-responsive region in the parA promoter to a 111-bp direct repeat (and identify a nuclear protein binding a 17-bp sequence in that repeat), though they note additional complexity in stable transgenics (Journal of Plant Research; 1993-12; https://doi.org/10.1007/bf02345981) (takahashi1993functionsandmodulations pages 3-5).

4.2. Pathogen and wound responsiveness (str246C/parA)

Rapid wound induction: wounding triggers str246C promoter expression within 30 min and peaks around 2 h (gough1995developmentalandpathogeninduced pages 8-9).

Infection/elicitor induction and promoter elements:
* Deletion analysis identifies a bacterial response element (BRE) between −184 and −101 required for infection inducibility (gough1995developmentalandpathogeninduced pages 10-14).
* The BRE contains multiple motifs including HS elements, a TGA-1 binding site, and a variant AuxRe B (gough1995developmentalandpathogeninduced pages 10-14).
* Infection- and hormone-inducibility can be separated, consistent with partly independent signaling pathways (gough1995developmentalandpathogeninduced pages 14-15).

WRKY/W-box connection: A tobacco repetitive element study notes a W-box (TTGAC) around ~1.4 kb upstream in the par/str246C promoter and reports par/str246C transcript is “rapidly and dramatically induced” during TMV-triggered hypersensitive response after temperature shift (Plant Science; 2001-09; https://doi.org/10.1016/S0168-9452(01)00454-X) (yang2001afamilyof pages 5-8).

4.3. Relation to glutathione metabolism and detoxification pathways

GSTs are central components of glutathione-dependent detoxification and broader defense/redox pathways; induction by hormones (auxin), SA, wounding, and pathogens fits this paradigm (marrs1996thefunctionsand pages 3-5, zaal1996auxinsensitiveelementsfrom pages 1-2). However, specific endogenous substrates for P25317 remain unverified in retrieved sources.

5. Subcellular localization and cellular context

5.1. Nuclear localization report

Takahashi et al. (1993) provide direct experimental evidence of nuclear localization for parA protein in tobacco mesophyll protoplasts (antibody staining after 6 h culture with auxin), suggesting possible transcription-related involvement (takahashi1993functionsandmodulations pages 3-5).

5.2. Proteomics detection in plastid preparations and whole-cell proteome

Two proteomics resources detect P25317 protein:
* BY-2 plastid proteome: P25317 is listed in plastid preparations with 3 peptides identified (Baginsky et al., 2004; Journal of Proteome Research; 2004-10; https://doi.org/10.1021/pr0499186) (baginsky2004proteomeanalysisof pages 5-6).
* BY-2 2-DE database: spot 45 annotated as P25317 “Probable glutathione S-transferase parA,” with 4 peptides, experimental MW 27.3 kDa and theoretical MW 25.2 kDa, potential function “Redox status” (Laukens et al., 2004; PROTEOMICS; 2004-03; https://doi.org/10.1002/pmic.200300614) (laukens2004constructionofa pages 5-7).

Interpretation: the plastid-preparation detection supports association with plastid fractions but does not alone prove true plastid residency (common issue in organelle proteomics); together with a nuclear-localization report, it underscores that P25317 localization is not settled and may be context-dependent or reflect family member confusion (takahashi1993functionsandmodulations pages 3-5, baginsky2004proteomeanalysisof pages 5-6).

6. Recent developments (prioritize 2023–2024)

Direct 2023–2024 studies on tobacco P25317 specifically were not retrieved in this run; therefore “recent developments” are addressed through state-of-the-field 2024 literature on plant GST functional annotation and 2024 applied studies illustrating GST roles.

6.1. 2024 authoritative synthesis: GST functions and annotation strategies

Micic et al. (2024) argue GST functional discovery should be driven by detection of endogenous GS-conjugates, combined with transcriptomics, transport inhibition, and spatial metabolomics, because single-gene knockouts often lack clear phenotypes due to redundancy and promiscuity (https://doi.org/10.1098/rstb.2023.0365) (micic2024overlookedandmisunderstood pages 1-2, micic2024overlookedandmisunderstood pages 10-11).

Implication for P25317 annotation: high confidence assignment of in vivo substrate(s) for P25317 likely requires direct GS-conjugate profiling and integrative multi-omics, not sequence similarity alone (micic2024overlookedandmisunderstood pages 1-2, micic2024overlookedandmisunderstood pages 2-3).

6.2. 2024 applied evidence: GST-mediated detoxification and stress tolerance

A 2024 rice study demonstrates that differing basal/inducible GST activity contributes to tolerance to the allelochemical DIMBOA, and that overexpression of two GST genes enhances resistance (BMC Plant Biology; 2024-02; https://doi.org/10.1186/s12870-024-04802-5) (zhang2024; not tobacco-specific but supports GST application logic) (shi2024genomewideidentificationand pages 1-2).

Although not directly about P25317, this type of work reflects current real-world implementation patterns: GSTs are targeted for engineering stress tolerance and xenobiotic resilience, consistent with how auxin/stress-inducible GSTs like parA/STR246C promoters and proteins are used/considered (gough1995developmentalandpathogeninduced pages 1-2, zaal1996auxinsensitiveelementsfrom pages 1-2).

7. Current applications and real-world implementations

7.1. Use of stress-/pathogen-inducible promoters (str246C)

Because str246C promoter is strongly inducible by pathogens/elicitors and other stimuli, it has been used (and is discussed) as a candidate promoter for transgenic applications requiring inducible expression rather than constitutive expression (Smirnova et al., 2015; “Promoters for Transgenic Horticultural Plants”; https://doi.org/10.1007/978-4-431-55251-2_12) (paper retrieved but not deeply evidence-extracted in this run). The primary experimental basis for inducibility comes from Gough et al. promoter-GUS work (gough1995developmentalandpathogeninduced pages 1-2, gough1995developmentalandpathogeninduced pages 8-9).

7.2. Herbicide/auxin analog interactions and detoxification hypotheses

Droog et al. propose that GST affinity toward 2,4-D and related chlorinated compounds could reflect roles in detoxification/metabolism/transport of auxin-like substances, and more generally highlight that plant GSTs are implicated in herbicide/pesticide detoxification (droog199524dichlorophenoxyaceticacidand pages 6-7, droog199524dichlorophenoxyaceticacidand pages 4-6). This connects auxin-regulated GSTs to agrichemical stress contexts.

8. Expert opinions and authoritative interpretations

Marrs (1996; Annual Review; 1996-06; https://doi.org/10.1146/annurev.arplant.47.1.127) frames plant GSTs as multifunctional enzymes involved in xenobiotic detoxification, oxidative stress, and ligand binding; the review lists tobacco parA/Nt114 as part of plant GST catalogs and notes possible nuclear localization for parA (marrs1996thefunctionsand pages 11-13, marrs1996thefunctionsand pages 10-11).
Droog (1997; Journal of Plant Growth Regulation; 1997-05; https://doi.org/10.1007/PL00006984) positions tobacco auxin-regulated GSTs in the broader plant GST classification discussion and explicitly lists auxin-regulated tobacco genes including Nt114/parA in a GST classification context (droog1997plantglutathionestransferases pages 2-3).
Micic et al. (2024) highlight that substrate promiscuity and missing GS-conjugate detection can mislead functional inferences—cautioning against overconfident substrate assignments without metabolite evidence (micic2024overlookedandmisunderstood pages 1-2, micic2024overlookedandmisunderstood pages 10-11).

9. Evidence-backed statistics and data highlights

9.1. Enzyme kinetics and inhibition (auxin-regulated tobacco GST isozymes)

From Droog et al. (1995) (Plant Physiology; 1995-04):
* Km(GSH) ≈ 0.40 ± 0.15 mM (GST1-1 and GST2-1) (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61)
* Km(CDNB) ≈ 0.85 ± 0.25 mM (GST1-1), 0.20 ± 0.15 mM (GST2-1) (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61)
* Ki(2,4-D) ≈ 80 ± 40 μM (GST1-1), 200 ± 100 μM (GST2-1) (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61)

9.2. Quantitative promoter inducibility (str246C promoter)

From Gough et al. (1995) (Mol Gen Genet; 1995-05):
* Wounding: induction detectable within 30 min; peak ~2 h (gough1995developmentalandpathogeninduced pages 8-9)
* Auxin 5 μM (2,4-D): GUS 34.8; auxin+cytokinin: 47.3 (gough1995developmentalandpathogeninduced pages 8-9)
* Salicylic acid 0.07 mM: GUS 9.8 at 24 h (gough1995developmentalandpathogeninduced pages 8-9)

9.3. Proteomics detection metrics for P25317

BY-2 plastid prep: 3 peptides for P25317 (baginsky2004proteomeanalysisof pages 5-6)
BY-2 whole-cell 2-DE spot: 4 peptides, MWexp 27.3 kDa, MWth 25.2 kDa (laukens2004constructionofa pages 5-7)

10. Synthesis: most likely function and working model

10.1. Most likely primary function

Based on (i) repeated annotation of P25317 as “probable GST”, (ii) inclusion in auxin-regulated Nt114 GST family context, (iii) strong induction by auxin and defense stimuli, and (iv) GST-family roles in redox and xenobiotic detoxification, P25317 is best annotated as a stress-responsive GST-family protein that likely participates in GSH-dependent detoxification/redox buffering and possibly ligand binding/transport of hydrophobic molecules under auxin and defense signaling (droog199524dichlorophenoxyaceticacidand pages 4-6, gough1995developmentalandpathogeninduced pages 1-2, marrs1996thefunctionsand pages 3-5).

10.2. Localization and pathway uncertainties

Two non-exclusive explanations should be maintained in annotation notes:
1) P25317 is a GST-like protein with canonical cytosolic GST roles; nuclear staining may reflect regulated translocation or antibody cross-reactivity among family members in protoplasts.
2) The “parA” designation in early protoplast work may have conflated closely related family members; later GST literature mapping parA→Nt114 supports GST identity, but exact correspondence between early nuclear-localization experiments and Swiss-Prot P25317 requires sequence-level confirmation not available in the retrieved excerpts (takahashi1993functionsandmodulations pages 3-5, droog199524dichlorophenoxyaceticacidand pages 4-6).

11. Practical recommendations for future functional validation (aligned with 2024 best practices)

To refine functional annotation of P25317, the strongest next steps (per 2024 GST functional discovery guidance) would be:
* GS-conjugate metabolomics under inducing conditions (auxin, pathogen elicitors, CuSO4), comparing wild type vs knockdown/CRISPR, to identify candidate endogenous substrates (micic2024overlookedandmisunderstood pages 1-2, micic2024overlookedandmisunderstood pages 10-11).
* Subcellular localization using fluorescently tagged P25317 (genomic context; native promoter) to resolve nuclear vs cytosolic/plastid association (takahashi1993functionsandmodulations pages 3-5, baginsky2004proteomeanalysisof pages 5-6).
* Recombinant enzymology for the exact P25317 sequence against standard GST panels (CDNB and candidate endogenous electrophiles/lipid peroxidation products) to confirm EC 2.5.1.18 activity and define substrate preferences.

Key sources (URLs, dates)

Droog et al. 1995-04. Plant Physiology 107:1139–1146. “2,4-Dichlorophenoxyacetic Acid… inhibit two auxin-regulated type-III tobacco GSTs.” https://doi.org/10.1104/pp.107.4.1139 (droog199524dichlorophenoxyaceticacidand pages 3-4, droog199524dichlorophenoxyaceticacidand media 50a1fd61)
Gough et al. 1995-05. Molecular and General Genetics 247:323–337. “Developmental and pathogen-induced activation… str246C.” https://doi.org/10.1007/bf00293200 (gough1995developmentalandpathogeninduced pages 8-9, gough1995developmentalandpathogeninduced pages 10-14)
Takahashi et al. 1993-12. Journal of Plant Research 106:357–367. “Functions and modulations of auxin-regulated genes.” https://doi.org/10.1007/bf02345981 (takahashi1993functionsandmodulations pages 3-5)
Micic et al. 2024 (received 2024-03-15; accepted 2024-08-06; in Sep 2024 issue). Phil. Trans. R. Soc. B 379:20230365. https://doi.org/10.1098/rstb.2023.0365 (micic2024overlookedandmisunderstood pages 1-2)
Baginsky et al. 2004-10. Journal of Proteome Research 3(6):1128–1137. https://doi.org/10.1021/pr0499186 (baginsky2004proteomeanalysisof pages 5-6)
Laukens et al. 2004-03. PROTEOMICS 4:720–727. https://doi.org/10.1002/pmic.200300614 (laukens2004constructionofa pages 5-7)

Evidence note / limitations

No 2023–2024 primary studies specifically re-characterizing tobacco P25317 were retrieved here; recent content is therefore provided as field-level updates on plant GST functional annotation and applications, while P25317-specific experimental evidence primarily comes from 1993–2004 literature and proteomics. The core ambiguity (parA nuclear-localization vs GST-family assignment) remains unresolved without direct sequence mapping of the exact experimental reagents used in early studies to UniProt P25317 (takahashi1993functionsandmodulations pages 3-5, droog199524dichlorophenoxyaceticacidand pages 4-6).

References

(froissard1994structuralorganizationof pages 1-3): Didier Froissard, Clare Gough, Pierre Czernic, Michel Schneider, Alain Toppan, Dominique Roby, and Yves Marco. Structural organization of str 246c and str 246n, plant defense-related genes from nicotiana tabacum. Plant Molecular Biology, 26:515-521, Oct 1994. URL: https://doi.org/10.1007/bf00039563, doi:10.1007/bf00039563. This article has 19 citations and is from a peer-reviewed journal.
(takahashi1993functionsandmodulations pages 3-5): Yohsuke Takahashi, Sarahmi Ishida, and Toshiyuki Nagata. Functions and modulations of auxin-regulated genes. Journal of Plant Research, 106:357-367, Dec 1993. URL: https://doi.org/10.1007/bf02345981, doi:10.1007/bf02345981. This article has 7 citations and is from a peer-reviewed journal.
(droog199524dichlorophenoxyaceticacidand pages 4-6): FNJ. Droog, PJJ. Hooykaas, and B. J. van der Zaal. 2,4-dichlorophenoxyacetic acid and related chlorinated compounds inhibit two auxin-regulated type-iii tobacco glutathione s-transferases. Plant Physiology, 107:1139-1146, Apr 1995. URL: https://doi.org/10.1104/pp.107.4.1139, doi:10.1104/pp.107.4.1139. This article has 132 citations and is from a highest quality peer-reviewed journal.
(laukens2004constructionofa pages 5-7): Kris Laukens, Peter Deckers, Eddy Esmans, Harry Van Onckelen, and Erwin Witters. Construction of a two‐dimensional gel electrophoresis protein database for the nicotiana tabacum cv. bright yellow‐2 cell suspension culture. PROTEOMICS, 4:720-727, Mar 2004. URL: https://doi.org/10.1002/pmic.200300614, doi:10.1002/pmic.200300614. This article has 34 citations and is from a peer-reviewed journal.
(baginsky2004proteomeanalysisof pages 5-6): Sacha Baginsky, Asim Siddique, and Wilhelm Gruissem. Proteome analysis of tobacco bright yellow-2 (by-2) cell culture plastids as a model for undifferentiated heterotrophic plastids. Journal of proteome research, 3 6:1128-37, Oct 2004. URL: https://doi.org/10.1021/pr0499186, doi:10.1021/pr0499186. This article has 84 citations and is from a peer-reviewed journal.
(marrs1996thefunctionsand pages 3-5): Kathleen A. Marrs. The functions and regulation of glutathione s-transferases in plants. Annual review of plant physiology and plant molecular biology, 47:127-158, Jun 1996. URL: https://doi.org/10.1146/annurev.arplant.47.1.127, doi:10.1146/annurev.arplant.47.1.127. This article has 1874 citations.
(droog1997plantglutathionestransferases pages 1-2): F. Droog. Plant glutathione s-transferases, a tale of theta and tau. Journal of Plant Growth Regulation, 16:95-107, May 1997. URL: https://doi.org/10.1007/pl00006984, doi:10.1007/pl00006984. This article has 217 citations and is from a domain leading peer-reviewed journal.
(micic2024overlookedandmisunderstood pages 1-2): Nikola Micic, Asta Holmelund Rønager, Mette Sørensen, and Nanna Bjarnholt. Overlooked and misunderstood: can glutathione conjugates be clues to understanding plant glutathione transferases? Philosophical Transactions of the Royal Society B: Biological Sciences, Sep 2024. URL: https://doi.org/10.1098/rstb.2023.0365, doi:10.1098/rstb.2023.0365. This article has 19 citations and is from a domain leading peer-reviewed journal.
(micic2024overlookedandmisunderstood pages 4-5): Nikola Micic, Asta Holmelund Rønager, Mette Sørensen, and Nanna Bjarnholt. Overlooked and misunderstood: can glutathione conjugates be clues to understanding plant glutathione transferases? Philosophical Transactions of the Royal Society B: Biological Sciences, Sep 2024. URL: https://doi.org/10.1098/rstb.2023.0365, doi:10.1098/rstb.2023.0365. This article has 19 citations and is from a domain leading peer-reviewed journal.
(micic2024overlookedandmisunderstood pages 10-11): Nikola Micic, Asta Holmelund Rønager, Mette Sørensen, and Nanna Bjarnholt. Overlooked and misunderstood: can glutathione conjugates be clues to understanding plant glutathione transferases? Philosophical Transactions of the Royal Society B: Biological Sciences, Sep 2024. URL: https://doi.org/10.1098/rstb.2023.0365, doi:10.1098/rstb.2023.0365. This article has 19 citations and is from a domain leading peer-reviewed journal.
(droog199524dichlorophenoxyaceticacidand pages 3-4): FNJ. Droog, PJJ. Hooykaas, and B. J. van der Zaal. 2,4-dichlorophenoxyacetic acid and related chlorinated compounds inhibit two auxin-regulated type-iii tobacco glutathione s-transferases. Plant Physiology, 107:1139-1146, Apr 1995. URL: https://doi.org/10.1104/pp.107.4.1139, doi:10.1104/pp.107.4.1139. This article has 132 citations and is from a highest quality peer-reviewed journal.
(droog199524dichlorophenoxyaceticacidand media 50a1fd61): FNJ. Droog, PJJ. Hooykaas, and B. J. van der Zaal. 2,4-dichlorophenoxyacetic acid and related chlorinated compounds inhibit two auxin-regulated type-iii tobacco glutathione s-transferases. Plant Physiology, 107:1139-1146, Apr 1995. URL: https://doi.org/10.1104/pp.107.4.1139, doi:10.1104/pp.107.4.1139. This article has 132 citations and is from a highest quality peer-reviewed journal.
(droog199524dichlorophenoxyaceticacidand pages 6-7): FNJ. Droog, PJJ. Hooykaas, and B. J. van der Zaal. 2,4-dichlorophenoxyacetic acid and related chlorinated compounds inhibit two auxin-regulated type-iii tobacco glutathione s-transferases. Plant Physiology, 107:1139-1146, Apr 1995. URL: https://doi.org/10.1104/pp.107.4.1139, doi:10.1104/pp.107.4.1139. This article has 132 citations and is from a highest quality peer-reviewed journal.
(gough1995developmentalandpathogeninduced pages 1-2): Clare Gough, Pascale Hemon, Maurice Tronchet, Christophe Lacomme, Yves Marco, and Dominique Roby. Developmental and pathogen-induced activation of an msr gene, str246c, from tobacco involves multiple regulatory elements. Molecular and General Genetics MGG, 247:323-337, May 1995. URL: https://doi.org/10.1007/bf00293200, doi:10.1007/bf00293200. This article has 34 citations.
(gough1995developmentalandpathogeninduced pages 8-9): Clare Gough, Pascale Hemon, Maurice Tronchet, Christophe Lacomme, Yves Marco, and Dominique Roby. Developmental and pathogen-induced activation of an msr gene, str246c, from tobacco involves multiple regulatory elements. Molecular and General Genetics MGG, 247:323-337, May 1995. URL: https://doi.org/10.1007/bf00293200, doi:10.1007/bf00293200. This article has 34 citations.
(gough1995developmentalandpathogeninduced pages 10-14): Clare Gough, Pascale Hemon, Maurice Tronchet, Christophe Lacomme, Yves Marco, and Dominique Roby. Developmental and pathogen-induced activation of an msr gene, str246c, from tobacco involves multiple regulatory elements. Molecular and General Genetics MGG, 247:323-337, May 1995. URL: https://doi.org/10.1007/bf00293200, doi:10.1007/bf00293200. This article has 34 citations.
(gough1995developmentalandpathogeninduced pages 14-15): Clare Gough, Pascale Hemon, Maurice Tronchet, Christophe Lacomme, Yves Marco, and Dominique Roby. Developmental and pathogen-induced activation of an msr gene, str246c, from tobacco involves multiple regulatory elements. Molecular and General Genetics MGG, 247:323-337, May 1995. URL: https://doi.org/10.1007/bf00293200, doi:10.1007/bf00293200. This article has 34 citations.
(yang2001afamilyof pages 5-8): Peizhen Yang and Zhixiang Chen. A family of dispersed repetitive dna sequences in tobacco contain clusters of w-box elements recognized by pathogen-induced wrky dna-binding proteins. Plant Science, 161:655-664, Sep 2001. URL: https://doi.org/10.1016/s0168-9452(01)00454-x, doi:10.1016/s0168-9452(01)00454-x. This article has 18 citations and is from a peer-reviewed journal.
(zaal1996auxinsensitiveelementsfrom pages 1-2): B. J. van der Zaal, FNJ. Droog, F. J. Pieterse, and PJJ. Hooykaas. Auxin-sensitive elements from promoters of tobacco gst genes and a consensus as-1-like element differ only in relative strength. Plant Physiology, 110:79-88, Jan 1996. URL: https://doi.org/10.1104/pp.110.1.79, doi:10.1104/pp.110.1.79. This article has 47 citations and is from a highest quality peer-reviewed journal.
(micic2024overlookedandmisunderstood pages 2-3): Nikola Micic, Asta Holmelund Rønager, Mette Sørensen, and Nanna Bjarnholt. Overlooked and misunderstood: can glutathione conjugates be clues to understanding plant glutathione transferases? Philosophical Transactions of the Royal Society B: Biological Sciences, Sep 2024. URL: https://doi.org/10.1098/rstb.2023.0365, doi:10.1098/rstb.2023.0365. This article has 19 citations and is from a domain leading peer-reviewed journal.
(shi2024genomewideidentificationand pages 1-2): Hongli Shi, Ke Wan, Bingde Dou, Yanyan Ren, Lihuan Huo, Chao Zhang, Shuning Yue, Zhongling Li, Huan Guo, and Jiakun Dai. Genome-wide identification and expression analysis of the glutathione transferase gene family and its response to abiotic stress in rye (secale cereale). BMC Genomics, Nov 2024. URL: https://doi.org/10.1186/s12864-024-11080-w, doi:10.1186/s12864-024-11080-w. This article has 7 citations and is from a peer-reviewed journal.
(marrs1996thefunctionsand pages 11-13): Kathleen A. Marrs. The functions and regulation of glutathione s-transferases in plants. Annual review of plant physiology and plant molecular biology, 47:127-158, Jun 1996. URL: https://doi.org/10.1146/annurev.arplant.47.1.127, doi:10.1146/annurev.arplant.47.1.127. This article has 1874 citations.
(marrs1996thefunctionsand pages 10-11): Kathleen A. Marrs. The functions and regulation of glutathione s-transferases in plants. Annual review of plant physiology and plant molecular biology, 47:127-158, Jun 1996. URL: https://doi.org/10.1146/annurev.arplant.47.1.127, doi:10.1146/annurev.arplant.47.1.127. This article has 1874 citations.
(droog1997plantglutathionestransferases pages 2-3): F. Droog. Plant glutathione s-transferases, a tale of theta and tau. Journal of Plant Growth Regulation, 16:95-107, May 1997. URL: https://doi.org/10.1007/pl00006984, doi:10.1007/pl00006984. This article has 217 citations and is from a domain leading peer-reviewed journal.

Artifacts

## Context ID: pqac-00000046 The requested figures and table containing kinetic and inhibition data for GST1-1 and GST2-1 have been extracted. **Summary of Nume

Citations

froissard1994structuralorganizationof pages 1-3
takahashi1993functionsandmodulations pages 3-5
marrs1996thefunctionsand pages 3-5
gough1995developmentalandpathogeninduced pages 1-2
gough1995developmentalandpathogeninduced pages 8-9
gough1995developmentalandpathogeninduced pages 10-14
gough1995developmentalandpathogeninduced pages 14-15
yang2001afamilyof pages 5-8
baginsky2004proteomeanalysisof pages 5-6
laukens2004constructionofa pages 5-7
shi2024genomewideidentificationand pages 1-2
droog1997plantglutathionestransferases pages 2-3
micic2024overlookedandmisunderstood pages 1-2
droog1997plantglutathionestransferases pages 1-2
micic2024overlookedandmisunderstood pages 4-5
micic2024overlookedandmisunderstood pages 10-11
zaal1996auxinsensitiveelementsfrom pages 1-2
micic2024overlookedandmisunderstood pages 2-3
marrs1996thefunctionsand pages 11-13
marrs1996thefunctionsand pages 10-11
https://doi.org/10.1098/rstb.2023.0365;
https://doi.org/10.1104/pp.107.4.1139
https://doi.org/10.1007/bf00293200
https://doi.org/10.1007/bf02345981
https://doi.org/10.1016/S0168-9452(01
https://doi.org/10.1021/pr0499186
https://doi.org/10.1002/pmic.200300614
https://doi.org/10.1098/rstb.2023.0365
https://doi.org/10.1186/s12870-024-04802-5
https://doi.org/10.1007/978-4-431-55251-2_12
https://doi.org/10.1146/annurev.arplant.47.1.127
https://doi.org/10.1007/PL00006984
https://doi.org/10.1007/bf00039563,
https://doi.org/10.1007/bf02345981,
https://doi.org/10.1104/pp.107.4.1139,
https://doi.org/10.1002/pmic.200300614,
https://doi.org/10.1021/pr0499186,
https://doi.org/10.1146/annurev.arplant.47.1.127,
https://doi.org/10.1007/pl00006984,
https://doi.org/10.1098/rstb.2023.0365,
https://doi.org/10.1007/bf00293200,
https://doi.org/10.1016/s0168-9452(01
https://doi.org/10.1104/pp.110.1.79,
https://doi.org/10.1186/s12864-024-11080-w,

📚 Additional Documentation

Notes

(PARA-notes.md)

PARA (Nicotiana tabacum) — research notes

UniProt: P25317 (GSTXA_TOBAC). "Probable glutathione S-transferase parA";
AltNames "Auxin-regulated protein parA" and "STR246C protein". EC 2.5.1.18.
220 aa, GST superfamily, tau (U) class. PANTHER PTHR11260:SF756 ("GLUTATHIONE
S-TRANSFERASE PARA-RELATED"). 220 aa, two-domain GST architecture (GST N-terminal
4-83; GST C-terminal 89-209). Reference proteome UP000790787, chromosome 2.
Evidence level PE 2 (transcript level) — no purified-protein characterization of
parA itself.

This review is part of the SPKW-PLANTS retrospective subproject: assessing whether
GOA's retirement of the keyword-to-GO (GO_REF:0000043) pipeline removed a correct
annotation or an over-annotation. The retired annotation here is
auxin-activated signaling pathway (GO:0009734).

The par gene family — discovery and identity

The par ("protoplast auxin-regulated") genes were isolated by Takahashi & Nagata
from cultured tobacco mesophyll protoplasts as genes induced very rapidly by
2,4-dichlorophenoxyacetic acid (2,4-D, a synthetic auxin) during the G0→S transition.

parA was the founding clone [PMID:2594768 "A cDNA clone for an auxin-regulated
gene was isolated from a tobacco mesophyll protoplast cDNA library by differential
screening"; "The mRNA of the par gene ... its accumulation was detected in
cultured mesophyll protoplasts as early as 30 min after the addition of
2,4-dichlorophenoxyacetic acid to the culture medium. The par mRNA was not
detected in leaves or freshly prepared protoplasts or in protoplasts in the
absence of 2,4-dichlorophenoxyacetic acid"]. At the time of isolation the authors
found "No significant homology to other known proteins" — the GST relationship
was recognised only later.
parB was isolated from the same system and was the first par gene whose product
was directly shown to be a GST PMID:1729717. parB is also known as Nt103. parB GST activity is robustly
induced by 2,4-D PMID:1729717.
parC is another member of the family; PANTHER places parC (P49332/Q03666) in
subfamily PTHR11260:SF726 ("PARC-RELATED"), distinct from parA's SF756. parA, parC
and Nicotiana plumbaginifolia MSR-1 (P50471) share the PARA-related subfamily/
closely related subfamilies — all tau-class plant GSTs.

So the par family is a cluster of auxin-inducible (and stress-inducible) tau-class
plant GSTs. The naming "auxin-regulated" refers to transcriptional induction by
auxin, established by promoter-reporter work: deletion of a 111-bp direct repeat
in the parA promoter abolished auxin-induced expression, localising a cis-acting
auxin-responsive region [PMID:2236015 (abstract) "deletion of a 111-base-pair (bp)
direct repeat in the 5' flanking sequences of the par gene abolished the
auxin-induced GUS activity ... an auxin-responsive region, which regulates
auxin-mediated activation of transcription"].

parA = str246C: a pathogen/stress defense gene

The genomic clone str246C is sequence-identical to parA PMID:7948901. str246C transcripts
accumulate in leaves on inoculation with the bacterial pathogen Pseudomonas
(Ralstonia) solanacearum PMID:7948901.
str246C/parA is therefore both auxin-inducible AND pathogen/defense-inducible —
classic behaviour of stress-responsive plant tau GSTs, which are induced by a wide
range of stimuli (auxin, herbicides, pathogens, oxidative stress, heavy metals).

Is parA a "conventional" GST? — the key functional ambiguity

A direct study of the parA protein itself found important departures from a typical
cytosolic GST PMID:7767232:
- Enzyme activity: "we detected a low level of GST activity in the parA products,
whose value was below 1/30 of that of parB products encoding tobacco ... GST."
So parA has measurable but very weak GST activity relative to parB.
- Localization: "the subcellular location of parA protein is the nucleus in
cultured tobacco mesophyll protoplasts, while conventional GSTs including the
parB product were primarily located in the cytoplasm." Confocal imaging of BY-2
cells showed "the parA product was confined to the nucleus, but was excluded
from the nucleolus."
- The authors note "exon/intron organization of the parA family was appreciably
different from that of conventional GSTs including parB" and that "the parA
protein is much more similar to a 24-kDa protein of Escherichia coli that is
reported to bind to RNA polymerase."
- Conclusion: "These different characteristics of parA compared with the
conventional GSTs indicate that parA protein would have distinct functions, such
as involvement in transcription, rather than functioning as a conventional GST."

Caveats: this is a single 1995 study; the "24-kDa E. coli RNA polymerase-binding
protein" comparison is weak by modern standards, and nuclear localization of an
overexpressed/tagged protein in protoplasts can be artefactual. UniProt nonetheless
retains "Probable glutathione S-transferase" (the name carries "Probable"), retains
EC 2.5.1.18 (a generic GST reaction RX + GSH), and assigns the "cytoplasm" CC term
by TreeGrafter family inference rather than experiment. The honest position: parA
has GST-family sequence/structure (tau class) and weak catalytic activity, but its
true physiological substrate and primary cellular role are not established. The
modern GOA molecular-function (glutathione transferase activity, IEA from
InterPro/RHEA/EC/PANTHER) and process (glutathione metabolic process) annotations
are family-level inferences that are reasonable but not experimentally proven for
parA specifically.

Plant tau-class GST biology (general)

Plant GSTs (EC 2.5.1.18) are a large, plant-specific-rich superfamily; phi (F) and
tau (U) classes are plant-specific and most numerous PMID:11897031. They use GSH as cosubstrate/
coenzyme; functions include "conjugation and resulting detoxification of herbicides,
the reduction of organic hydroperoxides formed during oxidative stress and the
isomerization of maleylacetoacetate" plus non-catalytic ligandin roles "binding
flavonoid natural products in the cytosol prior to their deposition in the vacuole"
and roles "as components of ultraviolet-inducible cell signaling pathways"
PMID:11897031. "GSTs are predominantly expressed in the cytosol" PMID:11897031.
The tau/phi classes "are largely responsible for catalyzing glutathione-dependent
reactions with xenobiotics, notably conjugation leading to detoxification"; plant
GSTs use "a catalytic serine in place of a tyrosine residue" PMID:21425939.
Endogenous roles are "less well defined ... linked to a diverse range of functions,
including signaling, counteracting oxidative stress, and detoxifying and
transporting secondary metabolites" PMID:21425939.

Auxin-INDUCED ≠ component of the auxin signaling pathway

Critical distinction for this review. The bona fide auxin signal-transduction
pathway (GO:0009734, "auxin-activated signaling pathway") consists of: auxin
perception by the TIR1/AFB F-box receptors, auxin-promoted degradation of Aux/IAA
repressors via the SCF^TIR1 ubiquitin ligase, and de-repression of ARF
transcription factors. parA/parB/parC are downstream auxin-responsive target
genes — their promoters contain auxin-responsive cis-elements and they are
transcriptionally induced when auxin signaling is active. A downstream
auxin-responsive metabolic enzyme is not a "component" of the signaling pathway.
The GO term that correctly captures "this gene is transcriptionally induced by
auxin" is "response to auxin" (GO:0009733), which is a parent that includes
auxin-responsive gene expression without implying signal-transduction-component
status. GO:0009734 (auxin-activated signaling pathway) is reserved for receptors/
Aux-IAA/ARF/SCF-TIR1 components.

The SPKW pipeline (GO_REF:0000043) generated GO:0009734 purely from the UniProt
keyword "Auxin signaling pathway", which UniProt had attached on the basis of the
historical name "Auxin-regulated protein". This is a textbook "expression ≠
function" / "downstream effector mislabeled as pathway component" over-annotation.

Summary judgement

parA is a tobacco tau-class plant GST (GST superfamily, PTHR11260:SF756),
EC 2.5.1.18; "Probable" because parA's own catalytic activity is weak (<1/30 of
parB) and its physiological substrate is unknown PMID:7767232.
parA = str246C: auxin-inducible AND pathogen/defense-inducible
[PMID:2594768; PMID:7948901].
The retired SPKW annotation "auxin-activated signaling pathway" (GO:0009734) is an
over-annotation derived from the historical "auxin-regulated" name. parA is a
downstream auxin-responsive gene, not a TIR1/Aux-IAA/ARF signaling component.
GOA's removal of this annotation when retiring the keyword2GO pipeline was
JUSTIFIED. If a process term is warranted it is "response to auxin" (GO:0009733).
The current GOA GST/glutathione-metabolism/cytoplasm IEA annotations are
family-level inferences. GST activity and glutathione metabolic process are
acceptable (parA is a tau-class GST with detectable activity). The "cytoplasm"
TreeGrafter annotation is the family-typical location and is reasonable as a
general call, but note the one direct study reports parA protein in the nucleus
PMID:7767232 — a discrepancy worth flagging.

📄 View Raw YAML

id: P25317
gene_symbol: PARA
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:4097
  label: Nicotiana tabacum
description: >
  parA (also known as str246C) is a tobacco (Nicotiana tabacum) member of the
  plant tau (U) class of glutathione S-transferases (GST superfamily; PANTHER
  PTHR11260:SF756, "glutathione S-transferase parA-related"). It was originally
  isolated as an auxin-regulated cDNA induced very rapidly (within ~30 min) by
  2,4-dichlorophenoxyacetic acid during the G0-to-S transition of cultured
  tobacco mesophyll protoplasts (Takahashi & Nagata). The "auxin-regulated"
  designation refers to transcriptional induction by auxin via a cis-acting
  auxin-responsive promoter element, not to participation in the auxin
  signal-transduction machinery. The same gene was independently isolated as
  str246C, a defense-related gene whose transcripts accumulate upon infection
  by the bacterial pathogen Pseudomonas (Ralstonia) solanacearum, so parA is
  both auxin-inducible and stress/pathogen-inducible, typical behaviour of
  stress-responsive plant tau GSTs. parA has the canonical two-domain GST fold
  (GST N-terminal thioredoxin-like and GST C-terminal helical domains) with a
  glutathione-binding site, and is assigned EC 2.5.1.18. However, the parA
  protein itself shows only weak GST activity (<1/30 that of the closely
  related parB/Nt103 GST) and one study reported nuclear localization for parA,
  prompting the suggestion that parA may have a function distinct from a
  conventional cytosolic detoxification GST. Its name therefore carries
  "Probable", and its physiological substrate and primary role remain
  uncertain. Best-supported view: parA is a tau-class plant GST whose
  established biology is transcriptional responsiveness to auxin and to biotic
  stress.
existing_annotations:
# --- SPKW keyword-mapping annotation (GO_REF:0000043) ---
# Present in the Sept 2025 goa_uniprot_gcrp snapshot (go-db plant.ddb); REMOVED
# from the current (2026) GOA release when GOA retired the keyword2GO pipeline
# for cellular organisms. Reviewed retrospectively to assess whether removal was
# justified. TRUE SPKW-unique (closure-filtered).
- term:
    id: GO:0009734
    label: auxin-activated signaling pathway
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  retired: true
  review:
    summary: >
      SPKW (GO_REF:0000043) keyword-derived annotation, present only in the
      Sept 2025 snapshot and removed from current GOA. It originates from the
      UniProt keyword "Auxin signaling pathway", which was attached because of
      the protein's historical name "Auxin-regulated protein parA". parA is a
      downstream auxin-responsive gene, not a component of the auxin
      signal-transduction pathway.
    action: REMOVE
    reason: >
      This is an over-annotation that conflates "transcriptionally induced by
      auxin" with "component of the auxin signaling pathway". parA was isolated
      as an early auxin-induced transcript in cultured tobacco protoplasts
      [PMID:2594768], and its promoter contains a cis-acting auxin-responsive
      element [PMID:2236015]. Being an auxin-responsive target gene is distinct
      from being part of the GO:0009734 pathway, which is defined as the series
      of events from binding of auxin to a receptor through downstream output
      and is reserved for receptor/signal-transduction components (TIR1/AFB
      receptors, SCF-TIR1, Aux/IAA repressors, ARF transcription factors).
      parA is a tau-class plant GST and a downstream effector, not a signaling
      component. The keyword-to-GO mapping propagated the misleading "auxin"
      keyword into a pathway-component term. GOA's removal of this annotation
      when it retired the keyword2GO (GO_REF:0000043) pipeline was JUSTIFIED:
      a correct annotation was not lost. If a process term capturing the
      auxin relationship is desired, the appropriate term is "response to
      auxin" (GO:0009733), which covers auxin-responsive gene expression
      without implying signal-transduction-component status (see the NEW entry
      below).
    proposed_replacement_terms:
    - id: GO:0009733
      label: response to auxin
    supported_by:
    - reference_id: PMID:2594768
      supporting_text: >
        The mRNA of the par gene is approximately 900 bases long and its
        accumulation was detected in cultured mesophyll protoplasts as early as
        30 min after the addition of 2,4-dichlorophenoxyacetic acid to the
        culture medium. The par mRNA was not detected in leaves or freshly
        prepared protoplasts or in protoplasts in the absence of
        2,4-dichlorophenoxyacetic acid.
    - reference_id: PMID:2236015
      supporting_text: >
        deletion of a 111-base-pair (bp) direct repeat in the 5' flanking
        sequences of the par gene abolished the auxin-induced GUS activity, it
        is deduced that in the 111-bp direct repeat of the par gene promoter is
        localized an auxin-responsive region, which regulates auxin-mediated
        activation of transcription.
# --- Current GOA annotations (2026 release) ---
- term:
    id: GO:0004364
    label: glutathione transferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: >
      Family-level molecular-function annotation (InterPro IPR045074 Tau-class
      GST signature, RHEA:16437, EC 2.5.1.18, PANTHER PTN004250175). parA has
      the canonical two-domain GST fold and a glutathione-binding site, and the
      closely related parB/Nt103 was directly shown to be a functional GST
      [PMID:1729717]. A direct assay of the parA protein detected GST activity,
      although at a low level (<1/30 that of parB) [PMID:7767232].
    action: ACCEPT
    reason: >
      parA belongs to the plant tau (U) class of the GST superfamily
      (PANTHER PTHR11260:SF756; CDD GST_N_Tau/GST_C_Tau; InterPro tau-class
      signatures). Tau and phi classes are the plant-specific GST classes that
      catalyse glutathione-dependent conjugation reactions [PMID:21425939].
      GST activity has been directly detected for the parA recombinant protein,
      so this generic molecular-function term is supported, even though parA's
      activity is weak relative to its paralog parB and its preferred
      physiological substrate is unknown (hence UniProt names it "Probable
      glutathione S-transferase"). The IEA term is at an appropriate level of
      generality for a family-based assignment.
    supported_by:
    - reference_id: PMID:7767232
      supporting_text: >
        we detected a low level of GST activity in the parA products, whose
        value was below 1/30 of that of parB products encoding tobacco
        (Nicotiana tabacum L.) GST.
    - reference_id: PMID:1729717
      supporting_text: >
        When we introduced expression vector pKK233-2, which retains parB cDNA,
        into Escherichia coli, we could detect GST activity in the parB gene
        product.
    - reference_id: file:TOBAC/PARA/PARA-deep-research-falcon.md
      supporting_text: >
        Therefore, enzyme-function annotation for P25317 as an active EC 2.5.1.18
        catalyst should be phrased as **“probable”** and supported by (i) family
        placement and (ii) proteomics annotation rather than an unambiguous
        parA-only enzymology experiment
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IEA
  original_reference_id: GO_REF:0000118
  review:
    summary: >
      TreeGrafter (GO_REF:0000118) phylogenetic transfer of the cytoplasm
      location from the PTHR11260 GST family. Plant GSTs are predominantly
      cytosolic enzymes [PMID:11897031], so this is the family-typical
      location. However, the one direct study of parA protein localization
      reported parA in the nucleus of tobacco protoplasts and BY-2 cells,
      contrasting with conventional cytoplasmic GSTs including parB
      [PMID:7767232].
    action: KEEP_AS_NON_CORE
    reason: >
      The cytoplasm assignment is a reasonable family-level call: plant GSTs
      "are predominantly expressed in the cytosol" [PMID:11897031] and parA has
      no predicted targeting/transit peptide. However, there is a genuine
      experimental discrepancy: immunofluorescence and confocal imaging
      reported the parA protein confined to the nucleus (excluded from the
      nucleolus), unlike the cytoplasmic parB GST [PMID:7767232]. Because the
      cellular location of parA is not robustly established and the only direct
      evidence argues against a purely cytoplasmic localization, this term is
      retained (it is not clearly wrong for a GST-fold protein) but marked as
      non-core rather than accepted as a confirmed core localization. The
      nuclear-localization observation is from a single 1995 study using
      overexpressed/tagged protein and could be artefactual, so neither
      cytoplasm nor nucleus can be asserted with confidence.
    supported_by:
    - reference_id: PMID:11897031
      supporting_text: >
        GSTs are predominantly expressed in the cytosol, where their
        GSH-dependent catalytic functions include the conjugation and resulting
        detoxification of herbicides
    - reference_id: PMID:7767232
      supporting_text: >
        Immunofluorescence studies using an antibody against parA protein
        revealed that the subcellular location of parA protein is the nucleus in
        cultured tobacco mesophyll protoplasts, while conventional GSTs'
        including the parB product were primarily located in the cytoplasm.
    - reference_id: file:TOBAC/PARA/PARA-deep-research-falcon.md
      supporting_text: >
        the plastid-preparation detection supports association with plastid
        fractions but does not alone prove true plastid residency (common issue
        in organelle proteomics); together with a nuclear-localization report,
        it underscores that **P25317 localization is not settled**
- term:
    id: GO:0006749
    label: glutathione metabolic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: >
      Family-level biological-process annotation accompanying the glutathione
      transferase activity call (InterPro IPR045074, PANTHER PTN004250175).
      Glutathione transferases use glutathione as cosubstrate/coenzyme in
      conjugation reactions, which constitutes glutathione-utilizing
      metabolism.
    action: ACCEPT
    reason: >
      As a tau-class GST that uses glutathione as a cosubstrate (the EC 2.5.1.18
      reaction RX + glutathione = an S-substituted glutathione + a halide anion
      + H+), parA participates in glutathione metabolism. Plant GSTs are central
      to glutathione-dependent conjugation chemistry [PMID:21425939,
      PMID:11897031]. This generic process term is the standard partner of the
      GST molecular-function annotation and is appropriate at the IEA
      family-inference level, consistent with the directly demonstrated (if
      weak) GST activity of the parA protein [PMID:7767232].
    supported_by:
    - reference_id: PMID:21425939
      supporting_text: >
        the numerous and plant-specific phi (F) and tau (U) classes are largely
        responsible for catalyzing glutathione-dependent reactions with
        xenobiotics, notably conjugation leading to detoxification
    - reference_id: PMID:7767232
      supporting_text: >
        we detected a low level of GST activity in the parA products, whose
        value was below 1/30 of that of parB products encoding tobacco
        (Nicotiana tabacum L.) GST.
# --- Proposed new annotation ---
- term:
    id: GO:0009733
    label: response to auxin
  evidence_type: IEP
  original_reference_id: PMID:2594768
  review:
    summary: >
      parA is one of the best-characterized auxin-responsive genes in tobacco.
      Its mRNA accumulates within 30 min of 2,4-D treatment in cultured
      mesophyll protoplasts and is absent without auxin [PMID:2594768], and a
      defined cis-acting auxin-responsive element in its promoter is required
      for this induction [PMID:2236015].
    action: NEW
    reason: >
      This term replaces the incorrect retired "auxin-activated signaling
      pathway" (GO:0009734) annotation. "Response to auxin" (GO:0009733)
      correctly captures the well-documented biology of parA: it is a
      downstream auxin-responsive target gene, transcriptionally induced by
      auxin, without implying that it is a component of the auxin
      signal-transduction pathway. The evidence is expression-based
      (auxin-dependent mRNA accumulation and a mapped auxin-responsive promoter
      element), so IEP is the appropriate evidence code.
    supported_by:
    - reference_id: PMID:2594768
      supporting_text: >
        The mRNA of the par gene is approximately 900 bases long and its
        accumulation was detected in cultured mesophyll protoplasts as early as
        30 min after the addition of 2,4-dichlorophenoxyacetic acid to the
        culture medium. The par mRNA was not detected in leaves or freshly
        prepared protoplasts or in protoplasts in the absence of
        2,4-dichlorophenoxyacetic acid.
    - reference_id: PMID:2236015
      supporting_text: >
        a chimeric gene, in which a reporter gene for bacterial
        beta-glucuronidase (GUS) was placed downstream of the 5' flanking
        sequences of the par gene ... elicited auxin-regulated expression of
        GUS activity.
- term:
    id: GO:0009617
    label: response to bacterium
  evidence_type: IEP
  original_reference_id: PMID:7948901
  review:
    summary: >
      parA is identical in sequence to str246C, a defense-related gene whose
      transcripts accumulate in tobacco leaves upon inoculation with the
      bacterial pathogen Pseudomonas (Ralstonia) solanacearum, in both
      compatible and incompatible interactions [PMID:7948901].
    action: NEW
    reason: >
      The independent isolation of parA as the pathogen-inducible defense gene
      str246C documents that parA is transcriptionally responsive to bacterial
      infection. This is a distinct, experimentally supported aspect of parA
      biology not captured by any current or retired annotation. "Response to
      bacterium" (GO:0009617) accurately reflects the expression-based
      evidence (IEP). It also illustrates that parA is a general
      stress-inducible tau GST rather than an auxin-pathway-specific gene.
    supported_by:
    - reference_id: PMID:7948901
      supporting_text: >
        We have previously identified a cDNA clone, pNt246, whose corresponding
        transcripts accumulate in leaves in response to inoculation by
        compatible and incompatible isolates of the phytopathogenic bacterium
        Pseudomonas solanacearum
    - reference_id: file:TOBAC/PARA/PARA-deep-research-falcon.md
      supporting_text: >
        Deletion analysis identifies a **bacterial response element (BRE)**
        between **−184 and −101** required for infection inducibility
references:
- id: file:TOBAC/PARA/PARA-deep-research-falcon.md
  title: >
    Falcon deep-research report: Functional annotation of Nicotiana tabacum
    PARA / parA / STR246C (UniProt P25317).
  findings:
  - statement: >
      Confirms that UniProt P25317 corresponds to tobacco parA, which is
      sequence-identical to the defense gene str246C, and maps parA to the
      auxin-regulated tobacco type-III (tau-class) GST subfamily Nt114
      (older nomenclature GST3-1).
  - statement: >
      The "auxin-regulated" designation reflects transcriptional induction:
      the str246C/parA promoter is induced by auxin (2,4-D) via a defined
      auxin-responsive cis-element, and is independently a multi-stimulus
      response promoter activated by wounding (within 30 min), elicitors and
      bacterial infection - supporting the conclusion that parA is a downstream
      auxin/stress-responsive target gene, not a component of the
      auxin signal-transduction pathway.
  - statement: >
      Direct GST enzymology in the retrieved corpus is demonstrated for the
      paralogs parB and the Nt103/Nt107 isozymes (GST1-1/GST2-1), not for parA
      itself; parA-specific catalytic activity is weak, so EC 2.5.1.18 / GST
      activity for P25317 is best annotated as "probable" on the basis of
      family placement plus weak directly-detected activity.
  - statement: >
      Subcellular localization of parA remains unsettled: a protoplast
      immunostaining study reported nuclear localization, while BY-2 proteomics
      detected P25317 in plastid preparations and whole-cell 2-DE; the
      plastid-fraction detection may reflect contamination, so neither
      cytoplasm nor nucleus can be asserted with confidence.
  - statement: >
      A 2024 synthesis on plant GSTs (Micic et al.) notes that GST substrate
      promiscuity and the difficulty of detecting endogenous glutathione
      conjugates mean the physiological substrate of a probable GST such as
      parA cannot be assigned from sequence similarity alone.
- id: GO_REF:0000043
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
  findings:
  - statement: >
      SwissProt keyword-derived (SPKW) annotations present in the Sept 2025
      goa_uniprot_gcrp snapshot but removed from the current GOA release after
      GOA retired the keyword2GO pipeline for cellular organisms.
  - statement: >
      The SPKW annotation auxin-activated signaling pathway (GO:0009734) for
      parA was derived from the UniProt keyword "Auxin signaling pathway",
      itself based on the historical protein name "Auxin-regulated protein";
      it conflates auxin-responsiveness with auxin-pathway-component status and
      its removal was justified.
- id: GO_REF:0000118
  title: TreeGrafter-generated GO annotations
  findings:
  - statement: >
      Cytoplasm localization was transferred from the PTHR11260 GST family;
      this is the family-typical location for cytosolic plant GSTs.
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings:
  - statement: >
      Glutathione transferase activity and glutathione metabolic process were
      inferred from the InterPro tau-class GST signature (IPR045074), the RHEA
      reaction RHEA:16437, EC 2.5.1.18 and PANTHER family membership.
- id: PMID:2594768
  title: >
    Isolation of an auxin-regulated gene cDNA expressed during the transition
    from G0 to S phase in tobacco mesophyll protoplasts.
  findings:
  - statement: >
      parA (par) was isolated as an auxin-regulated cDNA from cultured tobacco
      mesophyll protoplasts; its 220-aa product had no detectable homology to
      known proteins at the time of isolation.
  - statement: >
      par mRNA accumulates within 30 min of 2,4-D addition and is absent in
      leaves, freshly prepared protoplasts, or protoplasts without auxin,
      establishing parA as a rapidly auxin-induced gene.
- id: PMID:2236015
  title: >
    Location of the cis-acting auxin-responsive region in the promoter of the
    par gene from tobacco mesophyll protoplasts.
  findings:
  - statement: >
      A 111-bp direct repeat in the parA promoter constitutes an auxin-responsive
      cis-element required for auxin-mediated transcriptional activation.
- id: PMID:7948901
  title: >
    Structural organization of str 246C and str 246N, plant defense-related
    genes from Nicotiana tabacum.
  findings:
  - statement: >
      The genomic gene str246C is sequence-identical to parA.
  - statement: >
      str246C/parA transcripts accumulate in tobacco leaves on inoculation with
      the bacterial pathogen Pseudomonas (Ralstonia) solanacearum, in both
      compatible and incompatible interactions, identifying parA as a
      defense/stress-responsive gene.
- id: PMID:1729717
  title: 'parB: an auxin-regulated gene encoding glutathione S-transferase.'
  findings:
  - statement: >
      parB (Nt103), a close paralog of parA isolated from the same auxin-induced
      tobacco protoplast system, has clear sequence homology to glutathione
      S-transferases (EC 2.5.1.18).
  - statement: >
      Recombinant parB expressed in E. coli has GST activity, and GST activity
      increases in tobacco protoplasts cultured with 2,4-D, directly linking the
      par GST family to auxin-induced glutathione transferase function.
- id: PMID:7767232
  title: >
    Expression of the auxin-regulated parA gene in transgenic tobacco and
    nuclear localization of its gene products.
  findings:
  - statement: >
      The parA recombinant protein has only a low level of GST activity, below
      1/30 that of the parB GST.
  - statement: >
      Immunofluorescence and confocal microscopy localized parA protein to the
      nucleus (excluded from the nucleolus) of tobacco protoplasts and BY-2
      cells, in contrast to conventional cytoplasmic GSTs such as parB.
  - statement: >
      The authors proposed that parA may have a function distinct from a
      conventional cytosolic GST, possibly related to transcription, although
      this remains speculative and based on a weak similarity to an E. coli
      RNA-polymerase-binding protein.
- id: PMID:21425939
  title: Multiple roles for plant glutathione transferases in xenobiotic detoxification.
  findings:
  - statement: >
      The plant-specific phi (F) and tau (U) GST classes catalyse
      glutathione-dependent conjugation of xenobiotics, leading to
      detoxification; plant GSTs use a catalytic serine rather than the tyrosine
      of mammalian GSTs.
  - statement: >
      Endogenous roles of plant GSTs are less well defined and include
      signaling, counteracting oxidative stress, and transport of secondary
      metabolites.
- id: PMID:11897031
  title: Plant glutathione transferases.
  findings:
  - statement: >
      Soluble plant GSTs (EC 2.5.1.18) form a large gene family divided into
      phi, tau, theta, zeta and lambda classes; tau and phi are plant-specific
      and the most numerous.
  - statement: >
      Plant GSTs are predominantly cytosolic and use glutathione as a
      cosubstrate or coenzyme for conjugation, peroxide reduction, isomerization
      and non-catalytic ligandin functions.
core_functions:
- description: >
    parA is a plant tau-class glutathione S-transferase that catalyses
    glutathione-dependent conjugation chemistry (EC 2.5.1.18). It has the
    canonical two-domain GST fold with a glutathione-binding site and detectable
    glutathione transferase activity, although its activity is weak relative to
    its paralog parB and its preferred physiological substrate is not
    established.
  molecular_function:
    id: GO:0004364
    label: glutathione transferase activity
  directly_involved_in:
  - id: GO:0006749
    label: glutathione metabolic process
  supported_by:
  - reference_id: PMID:7767232
    supporting_text: >
      we detected a low level of GST activity in the parA products, whose value
      was below 1/30 of that of parB products encoding tobacco (Nicotiana
      tabacum L.) GST.
  - reference_id: PMID:21425939
    supporting_text: >
      the numerous and plant-specific phi (F) and tau (U) classes are largely
      responsible for catalyzing glutathione-dependent reactions with
      xenobiotics, notably conjugation leading to detoxification
  - reference_id: file:TOBAC/PARA/PARA-deep-research-falcon.md
    supporting_text: >
      P25317 is best annotated as a **stress-responsive GST-family protein**
      that likely participates in **GSH-dependent detoxification/redox
      buffering** and possibly **ligand binding/transport of hydrophobic
      molecules** under auxin and defense signaling
- description: >
    parA is a stress- and hormone-responsive gene whose defining, best-supported
    biology is transcriptional induction. It is rapidly induced by auxin (2,4-D)
    via a defined auxin-responsive promoter element and, as the defense gene
    str246C, is induced by bacterial pathogen infection. This places parA among
    the auxin-inducible / stress-inducible plant tau GSTs rather than among
    components of the auxin signal-transduction pathway.
  molecular_function:
    id: GO:0004364
    label: glutathione transferase activity
  directly_involved_in:
  - id: GO:0009733
    label: response to auxin
  - id: GO:0009617
    label: response to bacterium
  supported_by:
  - reference_id: PMID:2594768
    supporting_text: >
      The par mRNA was not detected in leaves or freshly prepared protoplasts
      or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid.
  - reference_id: PMID:7948901
    supporting_text: >
      a cDNA clone, pNt246, whose corresponding transcripts accumulate in
      leaves in response to inoculation by compatible and incompatible isolates
      of the phytopathogenic bacterium Pseudomonas solanacearum
  - reference_id: file:TOBAC/PARA/PARA-deep-research-falcon.md
    supporting_text: >
      induced by **auxin** and **auxin + cytokinin** and is also a
      multiple-stimulus response promoter, activated by elicitors and infection
proposed_new_terms: []
suggested_questions:
- question: >
    Does the parA protein have a genuine physiological glutathione transferase
    substrate (e.g. a xenobiotic, herbicide, oxidized lipid, or secondary
    metabolite), or is its weak GST activity vestigial?
- question: >
    Is the reported nuclear localization of parA a real feature with functional
    significance, or an artefact of overexpression/tagging in protoplasts?
- question: >
    Why is parA induced by both auxin and bacterial pathogens - does it have a
    role in linking growth-hormone signaling to stress/defense responses, as is
    seen for other stress-inducible plant tau GSTs?
suggested_experiments:
- description: >
    Purify recombinant parA protein and assay activity against a panel of
    standard GST substrates (CDNB), organic hydroperoxides (GPOX activity), and
    candidate physiological substrates, comparing kinetics to parB to determine
    whether parA is a catalytically competent GST or a divergent
    ligandin/regulatory protein.
  hypothesis: >
    parA retains GST-fold but has weak/altered catalysis and may function
    primarily as a ligand-binding (ligandin) protein rather than a conjugating
    enzyme.
- description: >
    Re-examine parA subcellular localization using a native-promoter
    fluorescent-protein fusion or immuno-electron microscopy in stably
    transformed tobacco, under both auxin and pathogen induction, to resolve the
    cytoplasm-versus-nucleus discrepancy.
  hypothesis: >
    The previously reported nuclear localization of parA reflects a genuine,
    possibly stress-regulated, partitioning distinct from cytosolic GSTs such as
    parB.
- description: >
    Generate parA loss-of-function and overexpression lines in tobacco and
    challenge them with auxin, oxidative stress, herbicides, and Ralstonia
    solanacearum to test whether parA contributes to detoxification, oxidative
    stress tolerance, or disease resistance.
  hypothesis: >
    parA functions as a stress-inducible detoxification/antioxidant tau GST
    contributing to defense responses rather than to auxin signal transduction.

PARA

Gene Description

Existing Annotations Review

Core Functions

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

Research Report: Functional Annotation of Nicotiana tabacum PARA / parA / STR246C (UniProt P25317)

Executive summary

1. Identity verification and disambiguation (critical)

1.1. Gene/protein mapping and aliases

1.2. Separation from similarly named genes (parB, parC; Nt103/Nt107/Nt114)

2. Key concepts and definitions (current understanding)

2.1. What is a plant glutathione S-transferase (GST)?

2.2. Why functional annotation is hard (2024 perspective)

3. Molecular function: enzymatic reaction and substrate specificity

3.1. Canonical GST reaction (general)

3.2. Evidence for enzymatic activity in the auxin-regulated tobacco GST family containing Nt114/parA

3.3. Direct activity evidence for parA protein specifically

4. Regulation, biological processes, and pathways

4.1. Auxin responsiveness and promoter architecture (str246C/parA)

4.2. Pathogen and wound responsiveness (str246C/parA)

4.3. Relation to glutathione metabolism and detoxification pathways

5. Subcellular localization and cellular context

5.1. Nuclear localization report

5.2. Proteomics detection in plastid preparations and whole-cell proteome

6. Recent developments (prioritize 2023–2024)

6.1. 2024 authoritative synthesis: GST functions and annotation strategies

6.2. 2024 applied evidence: GST-mediated detoxification and stress tolerance

7. Current applications and real-world implementations

7.1. Use of stress-/pathogen-inducible promoters (str246C)

7.2. Herbicide/auxin analog interactions and detoxification hypotheses

8. Expert opinions and authoritative interpretations

9. Evidence-backed statistics and data highlights

9.1. Enzyme kinetics and inhibition (auxin-regulated tobacco GST isozymes)

9.2. Quantitative promoter inducibility (str246C promoter)

9.3. Proteomics detection metrics for P25317

10. Synthesis: most likely function and working model

10.1. Most likely primary function

10.2. Localization and pathway uncertainties

11. Practical recommendations for future functional validation (aligned with 2024 best practices)

Key sources (URLs, dates)

Evidence note / limitations

Artifacts

Citations

📚 Additional Documentation

Notes

PARA (Nicotiana tabacum) — research notes

The par gene family — discovery and identity

parA = str246C: a pathogen/stress defense gene

Is parA a "conventional" GST? — the key functional ambiguity

Plant tau-class GST biology (general)

Auxin-INDUCED ≠ component of the auxin signaling pathway

Summary judgement

📄 View Raw YAML