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Comprehensive Research Report: RIC7 (ROP-interactive CRIB motif-containing protein 7) in Arabidopsis thaliana

Gene and Protein Identity

RIC7 (gene locus At4g28560, UniProt accession F4JLB7) encodes a ROP-interactive CRIB motif-containing protein 7 in Arabidopsis thaliana (hong2016therop2ric7pathway pages 1-2). This protein belongs to the RIC family, a group of eleven structurally divergent proteins in Arabidopsis that function as downstream effectors of ROP (Rho of Plants) GTPases (feiguelman2018ropgtpasesstructurefunction pages 1-5). The identity of RIC7 has been confirmed through molecular studies showing it contains the characteristic CRIB (Cdc42/Rac-interactive binding) domain that mediates interaction with activated ROP proteins (hong2016therop2ric7pathway pages 1-2, feiguelman2018ropgtpasesstructurefunction pages 10-13).

Protein Family and Structural Features

The RIC Family in Arabidopsis

RIC proteins represent a plant-specific family of ROP effectors characterized by the presence of a CRIB domain (feiguelman2018ropgtpasesstructurefunction pages 10-13, smokvarska2021functionofmembrane pages 1-2). The CRIB domain is a conserved sequence motif found in effector proteins that interact with GTP-bound forms of Rho-family GTPases (smokvarska2021functionofmembrane pages 1-2). In plants, CRIB domains are found not only in RIC proteins but also in certain ROPGAPs (ROP GTPase-activating proteins), where they enhance binding between ROPs and ROPGAPs and are required for subcellular localization (feiguelman2018ropgtpasesstructurefunction pages 10-13).

Arabidopsis possesses eleven RIC proteins (RIC1-11) that have evolved to control various ROP GTPase-dependent pathways through functional diversification (hong2016therop2ric7pathway pages 1-2). While some RIC family members like RIC1, RIC3, and RIC4 regulate cytoskeletal organization during processes such as pollen tube growth and cell morphogenesis (feiguelman2018ropgtpasesstructurefunction pages 1-5), RIC7 represents a specialized branch involved in guard cell signaling rather than the cytoskeletal functions typical of other RIC proteins (feiguelman2018ropgtpasesstructurefunction pages 1-5).

Domain Architecture

The defining feature of RIC7 is its CRIB motif, which enables direct interaction with activated (GTP-bound) ROP proteins (hong2016therop2ric7pathway pages 1-2, feiguelman2018ropgtpasesstructurefunction pages 10-13). This domain is essential for RIC7 to function as a molecular effector that transduces ROP signaling to downstream cellular processes. The CRIB domain binds preferentially to the active conformation of ROPs, ensuring signal specificity (smokvarska2021functionofmembrane pages 1-2).

Primary Molecular Function

Role as a Negative Regulator of Stomatal Movement

The primary function of RIC7 is to negatively regulate stomatal movements in Arabidopsis guard cells (hong2016therop2ric7pathway pages 1-2, hong2016therop2ric7pathway pages 3-4). Genetic and biochemical studies have established RIC7 as a key component that fine-tunes both light-induced stomatal opening and abscisic acid (ABA)-induced stomatal closure.

Knockout studies using the ric7-1 mutant (which harbors a T-DNA insertion in the second intron) demonstrated that loss of RIC7 function leads to accelerated light-induced stomatal opening (hong2016therop2ric7pathway pages 3-4). After 1-2 hours of light irradiation, ric7-1 mutant stomata opened approximately 18% more widely than wild-type stomata (hong2016therop2ric7pathway pages 3-4). Conversely, plants overexpressing RIC7 under the control of the 35S promoter exhibited slower light-induced stomatal opening compared to wild-type plants (hong2016therop2ric7pathway pages 3-4). Complementation lines expressing genomic RIC7 driven by its native promoter in the ric7-1 background restored wild-type stomatal opening kinetics, confirming that the rapid opening phenotype was due to RIC7 loss of function (hong2016therop2ric7pathway pages 3-4).

RIC7 also suppresses ABA-induced stomatal closure. The ric7-1 knockout mutant exhibited faster ABA-induced stomatal closure than wild-type, with differences evident as early as 30 minutes after ABA treatment (hong2016therop2ric7pathway pages 3-4). RIC7 overexpression lines showed slower ABA-induced closure (hong2016therop2ric7pathway pages 3-4). These results indicate that RIC7 functions as a negative regulator of both stomatal opening and closure, similar to its upstream regulator ROP2 (hong2016therop2ric7pathway pages 8-9).

Mechanistic Basis: The ROP2-RIC7-Exo70B1 Signaling Module

RIC7 does not function as a substrate-specific enzyme or transporter. Instead, it operates as a signaling adaptor protein that links ROP2 GTPase activity to the vesicle trafficking machinery required for stomatal movement (hong2016therop2ric7pathway pages 8-9, hong2016therop2ric7pathway pages 9-10).

Interaction with ROP2: RIC7 functions downstream of ROP2, a plant Rho-type GTPase that negatively regulates stomatal movements (hong2016therop2ric7pathway pages 1-2, hong2016therop2ric7pathway pages 8-9). Previous work established that RIC7 binds to activated ROP2 in vitro, positioning it as a ROP2 effector (hong2016therop2ric7pathway pages 1-2). The interaction between RIC7 and active ROP2 is mediated by the CRIB domain (hong2016therop2ric7pathway pages 1-2).

Direct Binding to Exo70B1: A critical discovery was the identification of Exo70B1 (exocyst subunit Exo70 family protein B1) as a direct interaction partner of RIC7 (hong2016therop2ric7pathway pages 4-5). Multiple experimental approaches confirmed this interaction:

• Yeast two-hybrid assays demonstrated that RIC7 binds to Exo70B1 (hong2016therop2ric7pathway pages 4-5)
• In vitro pull-down assays showed that GST-fused Exo70B1 directly pulls down MBP-fused RIC7, whereas free GST does not (hong2016therop2ric7pathway pages 4-5)
• Bimolecular fluorescence complementation (BiFC) assays in tobacco leaves confirmed the RIC7-Exo70B1 interaction in vivo (hong2016therop2ric7pathway pages 4-5)

Importantly, BiFC assays revealed that Exo70B1 does not bind directly to ROP2 (hong2016therop2ric7pathway pages 4-5), indicating that RIC7 serves as a linker protein connecting ROP2 to the exocyst machinery. This differs from yeast and animal systems where Rho GTPases often bind directly to Exo70 proteins (hong2016therop2ric7pathway pages 8-9). The requirement for a linker protein may reflect the absence of conserved C-terminal Rho-binding motifs in plant Exo70 proteins (hong2016therop2ric7pathway pages 8-9).

Functional Relationship with Exo70B1: RIC7 and Exo70B1 have opposing functions in stomatal regulation. While RIC7 is a negative regulator of light-induced stomatal opening, Exo70B1 is a positive regulator (hong2016therop2ric7pathway pages 5-7, hong2016therop2ric7pathway pages 9-10). The exo70b1-1 and exo70b1-2 knockout mutants showed retarded light-induced stomatal opening, with stomata opening approximately 14% less than wild-type after 1 hour of light (hong2016therop2ric7pathway pages 5-7). Genetic epistasis analysis using the ric7-1/exo70b1-1 double knockout mutant demonstrated that the double mutant phenotype resembled exo70b1-1 rather than ric7-1, indicating that Exo70B1 functions downstream of RIC7 (hong2016therop2ric7pathway pages 7-8).

The proposed mechanism is that RIC7 binding inhibits Exo70B1 function, thereby restraining vesicle trafficking and limiting the rate of stomatal opening (hong2016therop2ric7pathway pages 9-10). This creates a brake mechanism that prevents excessive stomatal opening and associated water loss while still allowing efficient CO₂ uptake for photosynthesis (hong2016therop2ric7pathway pages 8-9, hong2016therop2ric7pathway pages 9-10).

Subcellular Localization and Dynamic Regulation

RIC7 exhibits stimulus-dependent subcellular localization, which is critical for its regulatory function (hong2016therop2ric7pathway pages 7-8, hong2016therop2ric7pathway pages 5-7).

Localization Under Dark Conditions: In guard cells maintained in darkness, CFP-tagged RIC7 localizes predominantly to the nucleus (hong2016therop2ric7pathway pages 7-8, hong2016therop2ric7pathway pages 5-7). Under these conditions, YFP-tagged Exo70B1 shows a punctate pattern in the cytosol and nucleus (hong2016therop2ric7pathway pages 7-8).

Light-Induced Relocalization: Upon light irradiation (2 hours of white light), a subset of RIC7 relocates from the nucleus to the plasma membrane region, where it co-localizes with Exo70B1 (hong2016therop2ric7pathway pages 7-8, hong2016therop2ric7pathway pages 5-7). Fluorescence intensity profiles confirmed co-localization of YFP-Exo70B1 and CFP-RIC7 at the plasma membrane in light-treated guard cells (hong2016therop2ric7pathway pages 7-8).

ROP2 Activity-Dependent Localization: The relocalization of RIC7 to the plasma membrane is dependent on ROP2 activity. When co-expressed with constitutively active ROP2 (CA-ROP2), RIC7 and Exo70B1 both localize to the plasma membrane region even in the absence of light (hong2016therop2ric7pathway pages 7-8). In contrast, when co-expressed with a dominant-negative ROP2 (DN-ROP2), RIC7 remains primarily nuclear and Exo70B1 maintains its punctate cytoplasmic/nuclear distribution without plasma membrane enrichment (hong2016therop2ric7pathway pages 7-8). These findings indicate that light-mediated ROP2 activation triggers the recruitment of both RIC7 and Exo70B1 to the plasma membrane, where RIC7 can then modulate Exo70B1 function (hong2016therop2ric7pathway pages 7-8, hong2016therop2ric7pathway pages 9-10).

Biological Processes and Signaling Pathways

Guard Cell Function and Gas Exchange

RIC7 is highly expressed in stomatal guard cells, as demonstrated by RIC7 promoter::β-glucuronidase reporter analysis (hong2016therop2ric7pathway pages 3-4). Guard cells regulate stomatal apertures to balance CO₂ uptake for photosynthesis against water loss through transpiration. Stomatal opening requires increases in guard cell volume driven by ion accumulation and water influx, necessitating expansion of the plasma membrane and reorganization of the vacuolar membrane (hong2016therop2ric7pathway pages 9-10).

The ROP2 Signaling Pathway in Guard Cells

ROP2 (also known as RAC2) is a member of the plant-specific ROP subfamily of Rho GTPases and functions as a negative regulator of stomatal movements (hong2016therop2ric7pathway pages 1-2, feiguelman2018ropgtpasesstructurefunction pages 1-5). Like all small GTPases, ROP proteins cycle between an inactive GDP-bound state and an active GTP-bound state. This cycle is regulated by guanine nucleotide exchange factors (GEFs) that promote GDP release and GTP binding, and GTPase-activating proteins (GAPs) that enhance GTP hydrolysis (feiguelman2018ropgtpasesstructurefunction pages 1-5, smokvarska2021functionofmembrane pages 1-2).

In the context of stomatal regulation, light perception by phototropins and chlorophylls activates signaling pathways that ultimately modulate ROP2 activity (hong2016therop2ric7pathway pages 9-10). The current model proposes that light-induced stomatal opening activates positive regulatory pathways but simultaneously engages negative regulatory mechanisms, including the ROP2-RIC7-Exo70B1 pathway, to prevent excessive opening (hong2016therop2ric7pathway pages 8-9, hong2016therop2ric7pathway pages 9-10).

Vesicle Trafficking and Membrane Remodeling

The functional interaction between RIC7 and Exo70B1 implicates vesicle trafficking as a critical regulatory target in stomatal movement (hong2016therop2ric7pathway pages 9-10). Exo70B1 is a component of the exocyst complex, an eight-subunit complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that mediates tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to fusion (hong2016therop2ric7pathway pages 1-2, hong2016therop2ric7pathway pages 2-3).

In guard cells, Exo70B1 co-localizes with Exo84B, another exocyst component, supporting its role in exocyst function (hong2016therop2ric7pathway pages 7-8). However, Exo70B1 shows minimal co-localization with Golgi or trans-Golgi network/early endosome (TGN/EE) markers (only 3-4% overlap with Golgi marker ST-GFP and 11-12% with TGN/EE marker YFP-SYP61), suggesting involvement in non-conventional trafficking pathways or exocyst-positive organelle (EXPO)-mediated exocytosis (hong2016therop2ric7pathway pages 8-9).

The proposed mechanism is that Exo70B1 facilitates the trafficking of ion transporters and membrane materials to the plasma membrane and vacuolar membrane during stomatal opening (hong2016therop2ric7pathway pages 9-10). RIC7 binding to Exo70B1 inhibits this trafficking function, thereby limiting the rate of membrane expansion and ion transporter delivery, which in turn restrains the rate of stomatal opening (hong2016therop2ric7pathway pages 9-10). This provides a mechanism for plants to fine-tune stomatal aperture in response to environmental conditions.

Integration with ABA Signaling

RIC7 also modulates ABA-induced stomatal closure, although the precise mechanism differs from its role in light-induced opening (hong2016therop2ric7pathway pages 3-4, hong2016therop2ric7pathway pages 8-9). The observation that ric7 mutants exhibit faster ABA-induced closure suggests that RIC7 normally acts to dampen or slow the closure response (hong2016therop2ric7pathway pages 3-4). This may reflect a general role for RIC7 in modulating the kinetics of membrane trafficking events during stomatal movements, regardless of the directional stimulus.

Previous work has established that ROP proteins and ABA signaling form negative feedback loops (feiguelman2018ropgtpasesstructurefunction pages 1-5). ABA signaling suppresses ROP activation by promoting degradation of ROPGEFs, while ROP signaling suppresses ABA responses by physical interactions between ROPs (particularly ROP10 and ROP11) and ABI1/ABI2 PP2C phosphatases, negative regulators of ABA signaling (feiguelman2018ropgtpasesstructurefunction pages 1-5). RIC7's role in this network likely involves transducing ROP2 signals that modulate the kinetics of stomatal responses to both positive (light) and negative (ABA) stimuli.

Experimental Evidence

The characterization of RIC7 is supported by multiple lines of experimental evidence employing diverse methodologies:

Genetic Evidence:
- T-DNA insertion mutant ric7-1 (GK_062G02) with insertion in the second intron (hong2016therop2ric7pathway pages 1-2, hong2016therop2ric7pathway pages 3-4)
- RIC7 overexpression lines (OX1, OX3, OX8) under 35S promoter control (hong2016therop2ric7pathway pages 3-4)
- Complementation lines expressing genomic RIC7 under native promoter in ric7-1 background (hong2016therop2ric7pathway pages 3-4)
- Double mutant ric7-1/exo70b1-1 for epistasis analysis (hong2016therop2ric7pathway pages 7-8)

Biochemical Evidence:
- Yeast two-hybrid screens identifying Exo70B1 as RIC7-interacting protein (hong2016therop2ric7pathway pages 4-5)
- In vitro GST pull-down assays demonstrating direct RIC7-Exo70B1 interaction (hong2016therop2ric7pathway pages 4-5)
- Quantitative RT-PCR confirming gene expression levels in mutants and transgenics (hong2016therop2ric7pathway pages 2-3)

Cell Biological Evidence:
- Bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana confirming RIC7-Exo70B1 interaction in vivo (hong2016therop2ric7pathway pages 4-5)
- Transient expression of fluorescently tagged proteins in Vicia faba guard cells via particle bombardment (hong2016therop2ric7pathway pages 2-3, hong2016therop2ric7pathway pages 7-8)
- Confocal microscopy analysis of subcellular localization under different conditions (dark, light, with CA-ROP2, with DN-ROP2) (hong2016therop2ric7pathway pages 7-8, hong2016therop2ric7pathway pages 5-7)
- Co-localization studies with organelle markers (hong2016therop2ric7pathway pages 8-9)

Physiological Evidence:
- Stomatal aperture measurements showing altered light-induced opening kinetics in ric7 mutants and overexpression lines (hong2016therop2ric7pathway pages 3-4)
- ABA-induced stomatal closure assays demonstrating RIC7's role in closure kinetics (hong2016therop2ric7pathway pages 3-4)
- Promoter::β-glucuronidase reporter analysis revealing RIC7 expression patterns (hong2016therop2ric7pathway pages 3-4)

Comparison with Other RIC Family Members

The RIC family exhibits functional diversification, with different members regulating distinct cellular processes (feiguelman2018ropgtpasesstructurefunction pages 1-5). While RIC7 is specialized for guard cell signaling and vesicle trafficking regulation, other RIC proteins have well-characterized roles in cytoskeletal regulation:

• RIC1 regulates microtubule-mediated cell morphogenesis and localizes to the pollen tube tip (hong2016therop2ric7pathway pages 1-2, feiguelman2018ropgtpasesstructurefunction pages 1-5)
• RIC3 and RIC4 regulate cortical actin filament organization in growing pollen tubes (hong2016therop2ric7pathway pages 1-2, feiguelman2018ropgtpasesstructurefunction pages 1-5)
• RIC171 in barley (a RIC family ortholog) is involved in pathogen defense responses, where it accumulates at sites of fungal attack (feiguelman2018ropgtpasesstructurefunction pages 20-23)

This functional diversity reflects the evolution of the RIC family to mediate different downstream outputs of ROP signaling in different cellular contexts, despite sharing the common CRIB domain that enables ROP interaction (feiguelman2018ropgtpasesstructurefunction pages 1-5).

Current Understanding and Knowledge Gaps

The characterization of RIC7 published by Hong et al. in 2016 remains the most comprehensive functional study of this protein (hong2016therop2ric7pathway pages 1-2). Recent reviews (2020-2022) on ROP signaling pathways provide broader context for understanding RIC proteins as ROP effectors (feiguelman2018ropgtpasesstructurefunction pages 1-5, smokvarska2021functionofmembrane pages 1-2), but RIC7-specific studies remain limited compared to other RIC family members like RIC1, RIC3, and RIC4.

Key remaining questions include:
1. The precise molecular mechanism by which RIC7 binding inhibits Exo70B1 function
2. Whether RIC7 interacts with other exocyst components or trafficking machinery
3. The structural basis of RIC7-Exo70B1 interaction
4. Potential roles of RIC7 in other cell types beyond guard cells
5. The evolutionary origin and conservation of RIC7 function in other plant species

Summary

Table: This table compiles the verified identity, molecular function, localization, pathway role, and supporting evidence for Arabidopsis RIC7. It is useful as a concise annotation-ready summary centered on the experimentally supported ROP2-RIC7-Exo70B1 guard-cell signaling module.

RIC7 (At4g28560, UniProt F4JLB7) is a ROP-interactive CRIB motif-containing protein that functions as a negative regulator of stomatal movements in Arabidopsis thaliana. As a member of the eleven-protein RIC family, RIC7 serves as a downstream effector of ROP2 GTPase, transducing signals to the vesicle trafficking machinery through direct interaction with exocyst subunit Exo70B1. RIC7 exhibits dynamic subcellular localization, moving from the nucleus to the plasma membrane region in response to light stimulation or ROP2 activation. By binding to and inhibiting Exo70B1, RIC7 restrains excessive stomatal opening, providing a brake mechanism that balances CO₂ uptake against water loss. This function has been validated through comprehensive genetic, biochemical, and cell biological approaches, including analysis of knockout mutants, overexpression lines, protein-protein interaction assays, and subcellular localization studies. RIC7 represents a specialized branch of RIC protein evolution, diverging from the cytoskeletal regulatory functions of other family members to perform a unique role in guard cell physiology and gas exchange regulation.

Primary Reference: Hong, D., Jeon, B.W., Kim, S.Y., Hwang, J.-U., & Lee, Y. (2016). The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis. New Phytologist, 209(2), 624-635. doi:10.1111/nph.13625

Publication Date: October 2015 (published online), January 2016 (print)

DOI/URL: https://doi.org/10.1111/nph.13625

References

1. (hong2016therop2ric7pathway pages 1-2): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

2. (feiguelman2018ropgtpasesstructurefunction pages 1-5): Gil Feiguelman, Ying Fu, and Shaul Yalovsky. Rop gtpases structure-function and signaling pathways1[open]. Plant Physiology, 176:57-79, Nov 2018. URL: https://doi.org/10.1104/pp.17.01415, doi:10.1104/pp.17.01415. This article has 227 citations and is from a highest quality peer-reviewed journal.

3. (feiguelman2018ropgtpasesstructurefunction pages 10-13): Gil Feiguelman, Ying Fu, and Shaul Yalovsky. Rop gtpases structure-function and signaling pathways1[open]. Plant Physiology, 176:57-79, Nov 2018. URL: https://doi.org/10.1104/pp.17.01415, doi:10.1104/pp.17.01415. This article has 227 citations and is from a highest quality peer-reviewed journal.

4. (smokvarska2021functionofmembrane pages 1-2): Marija Smokvarska, Yvon Jaillais, and Alexandre Martinière. Function of membrane domains in rho-of-plant signaling. Plant Physiology, 185:663-681, Jan 2021. URL: https://doi.org/10.1093/plphys/kiaa082, doi:10.1093/plphys/kiaa082. This article has 54 citations and is from a highest quality peer-reviewed journal.

5. (hong2016therop2ric7pathway pages 3-4): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

6. (hong2016therop2ric7pathway pages 8-9): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

7. (hong2016therop2ric7pathway pages 9-10): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

8. (hong2016therop2ric7pathway pages 4-5): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

9. (hong2016therop2ric7pathway pages 5-7): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

10. (hong2016therop2ric7pathway pages 7-8): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

11. (hong2016therop2ric7pathway pages 2-3): Daewoong Hong, Byeong Wook Jeon, Soo Young Kim, Jae‐Ung Hwang, and Youngsook Lee. The rop2-ric7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit exo70b1 in arabidopsis. The New phytologist, 209 2:624-35, Oct 2016. URL: https://doi.org/10.1111/nph.13625, doi:10.1111/nph.13625. This article has 72 citations.

12. (feiguelman2018ropgtpasesstructurefunction pages 20-23): Gil Feiguelman, Ying Fu, and Shaul Yalovsky. Rop gtpases structure-function and signaling pathways1[open]. Plant Physiology, 176:57-79, Nov 2018. URL: https://doi.org/10.1104/pp.17.01415, doi:10.1104/pp.17.01415. This article has 227 citations and is from a highest quality peer-reviewed journal.

Artifacts

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Citations

1. feiguelman2018ropgtpasesstructurefunction pages 1-5
2. smokvarska2021functionofmembrane pages 1-2
3. feiguelman2018ropgtpasesstructurefunction pages 10-13
4. feiguelman2018ropgtpasesstructurefunction pages 20-23
5. open
6. https://doi.org/10.1111/nph.13625
7. https://doi.org/10.1111/nph.13625,
8. https://doi.org/10.1104/pp.17.01415,
9. https://doi.org/10.1093/plphys/kiaa082,

AIGR Gene Hypothesis Deep Research — Final Report

Gene: RIC7 / F4JLB7 (Arabidopsis thaliana, locus At4g28560)

Hypothesis under review: ProtNLM2 prediction of protein kinase activity (GO:0016301) and protein phosphorylation (GO:0016310)

Executive Judgment

Verdict: REFUTED (over-annotation / computational misassignment).

The ProtNLM2 prediction that F4JLB7 has protein kinase activity (GO:0016301) and participates in protein phosphorylation (GO:0016310) is not supported by the protein's sequence, domain architecture, or predicted structure. F4JLB7 (UniProt accession F4JLB7, locus At4g28560, 450 aa) is a secreted/membrane leucine-rich-repeat (LRR) protein of the receptor-like protein (RLP) type. It carries an N-terminal signal peptide followed by an extracellular LRR solenoid and no protein-kinase catalytic domain of any kind. The diagnostic active-site machinery required for phosphotransfer — the VAIK β3 lysine, the HRD catalytic loop, the DFG Mg²⁺-binding motif, and the APE activation-segment anchor, arranged in the canonical ordered triad — is absent. Because the essential HRD catalytic aspartate is missing, the protein is mechanistically incapable of catalyzing phosphotransfer, independent of any homology argument.

Two independent lines of evidence converge on this conclusion. First, the domain architecture from UniProt/InterPro/Pfam consists exclusively of LRR signatures (Pfam PF00560 ×3, PF13855; InterPro IPR001611, IPR032675, IPR052941; Gene3D 3.80.10.10 ribonuclease-inhibitor/LRR horseshoe fold; SUPFAM SSF52058 L-domain-like), with no PF00069 protein-kinase domain and no InterPro protein-kinase signature anywhere in the 450-residue sequence. Second, a direct motif scan of the sequence recovered 9 canonical LRR cores but found no ordered kinase catalytic triad; the isolated "DFG" (position 149, inside PEDFGSV) and a "GNGFHG" hit (position 186) are coincidental tripeptides embedded within the LRR solenoid, not part of a folded catalytic cleft. The AlphaFold DB model (mean pLDDT 84.7, "confident") is fully accounted for by the LRR fold, leaving no room for a cryptic kinase lobe.

The most likely origin of the ProtNLM2 error is frequency/similarity bias: LRR ectodomains are shared between non-catalytic receptor-like proteins (RLPs) and the far more numerous LRR receptor-like Ser/Thr kinases (LRR-RLKs) such as ERECTA. A language model trained on protein names readily over-generalizes "kinase" onto an LRR ectodomain even when the cytoplasmic kinase moiety is entirely absent. In fact, the FunFam annotation for F4JLB7 maps its LRR region to the ERECTA LRR-RLK ectodomain (3.80.10.10:FF:000041) — an ectodomain-only homology that explains, but does not justify, the kinase call. A secondary complication is a gene-symbol collision: F4JLB7 is labeled "RIC7," yet the genuine, functionally characterized RIC7 of the literature is a CRIB-motif ROP2 effector encoded by the adjacent locus At4g28556 (UniProt Q1G3K8 / A0A1P8B8E3) — also a non-catalytic protein. Under either identity, the kinase terms should be removed.

Key Findings

Finding 1 — F4JLB7 lacks any protein-kinase catalytic domain; the kinase prediction is refuted

The domain architecture of F4JLB7 (450 aa) as recorded in UniProt/InterPro is: N-terminal signal peptide (residues 1–22) followed by an extracellular leucine-rich-repeat region. The LRR assignments are extensive and mutually corroborating across databases:

Crucially, no protein-kinase Pfam (PF00069) and no InterPro protein-kinase domain is present anywhere in the sequence. A direct motif scan confirmed the diagnostic failure at the residue level: the HRD catalytic loop is absent (the essential catalytic aspartate is missing), the APE motif is absent, and there is no ordered VAIK → HRD → DFG catalytic triad. The only superficially "kinase-like" tripeptides — a lone "DFG" at position 149 (within the sequence context PEDFGSV) and "GNGFHG" at position 186 — are isolated coincidences inside the LRR solenoid, not part of an assembled catalytic cleft. In contrast, 9 canonical LRR core motifs ([LIVF]xxLxLxx[NCT]xL) were detected, exactly matching the Pfam LRR calls. UniProt keywords for the entry are Leucine-rich repeat, Signal, Glycoprotein, Membrane — the keyword set of a secreted/membrane LRR protein, not an enzyme.

The FunFam annotation maps the F4JLB7 LRR ectodomain to the LRR receptor-like Ser/Thr kinase ERECTA (FunFam 3.80.10.10:FF:000041). This is an ectodomain homology only: it reflects the fact that RLPs and LRR-RLKs share the same extracellular LRR scaffold, while F4JLB7 has no accompanying cytoplasmic kinase domain. F4JLB7 is therefore a receptor-LIKE protein (RLP), not a receptor kinase.

Finding 2 — Kinase-eligibility diagnostic: F4JLB7 cannot catalyze phosphotransfer

A formal kinase-eligibility test applied to the 450-aa sequence returned an unambiguous negative:

The HRD aspartate is the catalytic base that deprotonates the substrate hydroxyl during phosphotransfer; its absence makes the reaction chemically impossible. Independent of any homology or naming argument, F4JLB7 therefore cannot be a functional protein kinase. The AlphaFold DB model of F4JLB7 (450 residues, mean pLDDT 84.7 — a confident model) has a sequence that is fully accounted for by the LRR domain assignments, leaving no unmodeled region that could harbor a cryptic kinase lobe. This structural check rules out the possibility that a kinase domain is present but undetected by sequence-based tools.

Finding 3 — Gene-symbol collision: F4JLB7 is not the genuine RIC7 of the literature

The UniProt record for F4JLB7 carries the gene symbol "RIC7" (evidence ECO:0000313, propagated from TAIR/EMBL). However, F4JLB7's architecture — signal peptide + extracellular LRRs, 450 aa, glycoprotein, membrane — contains no CRIB motif. A sequence search for the CRIB core signature (ISxP…FxHxxHVG; "HVG"; "FxHxxH") returned no match in F4JLB7. The genuine RIC7 (ROP-interactive CRIB-motif-containing protein 7) characterized in the literature is a small CRIB-motif ROP2 effector/adaptor, not an LRR protein and not a kinase.

Resolving the identity conflict, there are three Arabidopsis UniProt entries carrying the symbol RIC7 mapping to two neighboring loci:

The genuine RIC7 (Q1G3K8) sequence contains the CRIB signature "KHVAHIGWDGP." F4JLB7 maps to a different, adjacent locus (At4g28560) and carries only LRR domains. The neighboring genomic position of At4g28556 and At4g28560 is consistent with automated gene-symbol carry-over onto the LRR gene F4JLB7. Importantly, none of the three entries contains an HRD kinase catalytic loop or any protein-kinase Pfam/InterPro signature — so the kinase prediction is wrong under either interpretation of "RIC7."

Finding 4 — Curated GO annotations for F4JLB7 are consistent with a receptor-like protein, not a kinase

The existing curated GO annotations for F4JLB7 are: apical plasma membrane (GO:0016324, IDA:TAIR), pollen tube growth (GO:0009860, IMP:TAIR), signaling receptor activity (GO:0038023, IBA), and signal transduction (IC). This annotation profile is coherent for a membrane-anchored LRR receptor-like protein involved in cell-surface signaling and pollen tube growth. It provides no support for catalytic kinase activity; "signaling receptor activity" is a non-catalytic molecular function. The ProtNLM2 kinase terms are therefore inconsistent with the experimentally supported (IDA/IMP) annotations already present.

Mechanistic Model / Interpretation

The evidence assembles into a clear picture of a computational over-annotation driven by ectodomain homology and a gene-symbol collision:

   ProtNLM2 name-based prediction
   ─────────────────────────────
   "RIC7" + LRR ectodomain  ──►  predicts "kinase activity"  ✗ (over-generalization)
                            │
                            ▼
   ┌──────────────────────────────────────────────────────────────┐
   │  ACTUAL F4JLB7 (At4g28560, 450 aa)                            │
   │                                                              │
   │  [Signal peptide 1–22]──[ extracellular LRR solenoid ]──[TM] │
   │        secreted/          horseshoe fold (3.80.10.10)         │
   │        membrane           9 LRR cores; PF00560×3, PF13855     │
   │                                                              │
   │  NO kinase lobe · NO VAIK · NO HRD · NO DFG · NO APE          │
   │  → receptor-LIKE protein (RLP), non-catalytic                 │
   └──────────────────────────────────────────────────────────────┘

   Why the error happens:
     LRR ectodomain is SHARED between
(a) non-catalytic RLPs  ← F4JLB7 is here
(b) LRR receptor kinases (e.g., ERECTA)  ← has cytoplasmic kinase
     Name/frequency model sees LRR → guesses "kinase" from the abundant
     (b) class, ignoring that the cytoplasmic kinase domain is ABSENT.

Two distinct problems compound one another:

1. Ectodomain-homology bias. F4JLB7's LRR region genuinely resembles the ectodomain of LRR-RLKs like ERECTA (captured by FunFam 3.80.10.10:FF:000041). A language-model annotator that keys on the LRR ectodomain — the part shared with kinases — but does not verify the presence of a cytoplasmic catalytic domain will spuriously assign kinase activity. F4JLB7 is a receptor-like protein (RLP): it has the LRR sensing module but not the kinase signaling module.

2. Gene-symbol carry-over. The "RIC7" label was propagated onto F4JLB7 (At4g28560) from the neighboring locus At4g28556, whose product is the genuine CRIB-motif ROP2 effector RIC7. This adds noise but does not rescue the kinase claim: the genuine RIC7 is an adaptor/scaffold (it binds Exo70B1 downstream of ROP2), also entirely non-catalytic.

Under either identity — mislabeled LRR RLP (At4g28560) or genuine CRIB ROP effector (At4g28556) — there is no kinase. The catalytic-residue test is decisive: no HRD aspartate ⇒ no phosphotransfer chemistry ⇒ not a kinase.

Evidence Matrix

GO Curation Implications

Lead (requires curator verification): REMOVE the ProtNLM2 kinase terms.

• GO:0016301 (kinase activity, MF) — Remove / do not accept. No kinase catalytic domain, no HRD catalytic aspartate, no PF00069. This is a computational (ProtNLM2) prediction contradicted by domain architecture and predicted structure. It should not be propagated.
• GO:0016310 (phosphorylation, BP) — Remove / do not accept. This BP term is downstream of the refuted MF and has no independent support.

Terms that are better supported and should be considered for retention (curator to verify against primary TAIR evidence):

• GO:0038023 (signaling receptor activity, MF) — plausible for an LRR receptor-like protein; already present (IBA). This is the more informative non-catalytic MF and is preferable to a generic "protein binding" fallback.
• GO:0016324 (apical plasma membrane, CC) — supported by IDA:TAIR; retain.
• GO:0009860 (pollen tube growth, BP) — supported by IMP:TAIR; retain.

Gene-symbol caveat for the curator: verify whether the "RIC7" symbol on F4JLB7 (At4g28560) is a carry-over from the adjacent locus At4g28556 (genuine RIC7 = Q1G3K8/A0A1P8B8E3). If so, the symbol assignment on F4JLB7 should be reviewed, because it can further mislead automated annotators. Note that fixing the symbol does not change the kinase verdict — neither locus encodes a kinase.

Mechanistic Scope

The immediate molecular function being tested is protein-kinase catalysis: the ability to transfer the γ-phosphate of ATP to a substrate hydroxyl (Ser/Thr/Tyr). This is a direct gene-product activity claim (GO:0016301) with an associated process claim (GO:0016310, protein phosphorylation).

• Direct gene-product activity (tested here): F4JLB7's own capacity to catalyze phosphotransfer — refuted at the residue level (no HRD, no ordered triad) and at the domain/structure level (LRR-only architecture, confident AlphaFold LRR fold).
• What F4JLB7 most plausibly does instead: act as a cell-surface LRR receptor-like protein (ectodomain sensing / signaling receptor activity), localized to the apical plasma membrane and involved in pollen tube growth — a non-catalytic signaling role.
• Downstream / not relevant to the MF claim: the genuine RIC7 (At4g28556) participates in ROP2-mediated stomatal regulation as an adaptor binding Exo70B1; these are phenotypic/pathway roles of a different protein and do not bear on F4JLB7's molecular function except to underscore that "RIC7" is not a kinase.

Conflicts and Alternatives

1. Gene-symbol / paralog confusion (primary alternative). The strongest complicating factor is that "RIC7" denotes two different things: (a) the LRR protein F4JLB7 (At4g28560), and (b) the genuine CRIB-motif ROP2 effector (At4g28556, Q1G3K8). This does not create a kinase in either case, but a curator must be aware that literature about "RIC7" (PMID:26451971, PMID:33586611) refers to the CRIB protein, not F4JLB7.
2. Ectodomain homology to LRR-RLKs. F4JLB7's LRR region resembles the ERECTA kinase ectodomain (FunFam FF:000041). One could misread this as evidence of kinase membership; in reality it is shared extracellular scaffolding, with the catalytic module absent. This is the likely proximate cause of the ProtNLM2 error.
3. Cryptic-domain possibility (excluded). A theoretical alternative — a divergent kinase domain undetectable by Pfam HMMs — is excluded by the AlphaFold model: the entire 450-aa sequence is accounted for by the LRR fold with no unmodeled region.
4. Pseudokinase possibility (excluded). Some "kinases" are catalytically dead pseudokinases that retain the fold but lose HRD/DFG. F4JLB7 is not even a pseudokinase — it has no kinase fold at all, only an LRR solenoid. So neither active kinase nor pseudokinase applies.

Limitations and Knowledge Gaps

• No direct biochemical assay of F4JLB7. The refutation is bioinformatic (sequence motifs, domain HMMs, AlphaFold geometry). While the absence of an HRD aspartate is decisive for catalysis, there is no in vitro kinase assay explicitly demonstrating lack of activity for this specific protein. Why it matters: curators may prefer experimental confirmation; resolution: an in vitro kinase assay would be definitive but is almost certainly unnecessary given the complete absence of a catalytic domain.
• Experimental characterization of F4JLB7 specifically is sparse. The curated IDA/IMP annotations (apical PM, pollen tube growth) indicate some experimental work, but the primary papers behind them were not retrieved in this investigation. Why it matters: the positive (non-kinase) function of F4JLB7 is less firmly established than its non-kinase status; resolution: retrieve the TAIR-cited primary references for GO:0016324 and GO:0009860.
• Gene-symbol provenance not fully traced. The inference that "RIC7" was carried over onto At4g28560 from At4g28556 is based on genomic adjacency and architecture mismatch, not a documented curation event. Why it matters: affects the symbol-correction recommendation; resolution: check TAIR/EMBL submission history for At4g28560.
• Transmembrane topology not explicitly recomputed here. UniProt keywords include "Membrane," and the schematic assumes a single-pass RLP topology; a hydropathy/TM prediction would confirm the anchor. Why it matters: strengthens the RLP model and the CC annotation; resolution: run TMHMM/DeepTMHMM on the sequence.

Discriminating Tests

1. Residue-level catalytic scan (done): confirm absence of VAIK Lys, HRD Asp, DFG, APE. This alone distinguishes kinase from non-kinase and is the most efficient test — already negative.
2. HMM domain scan vs PF00069 (done): no protein-kinase Pfam hit — confirms no kinase fold.
3. AlphaFold structural inspection (done): confirm the model is entirely LRR with no kinase bilobal fold — no cryptic domain.
4. Transmembrane/topology prediction (recommended): DeepTMHMM to confirm single-pass membrane RLP topology consistent with GO:0016324.
5. Reciprocal-best-hit / orthology check (recommended): confirm F4JLB7 orthologs are LRR RLPs, not RLKs, across Brassicaceae, cementing the RLP (not RLK) classification.
6. Symbol/locus disambiguation (recommended): cross-check At4g28560 vs At4g28556 in TAIR to formally document the RIC7 symbol collision.

Proposed Follow-up Experiments / Actions (Curation Leads)

Candidate action changes (require curator verification):
- Reject/remove GO:0016301 (kinase activity) on F4JLB7 — computational ProtNLM2 prediction refuted by architecture, motifs, and structure.
- Reject/remove GO:0016310 (protein phosphorylation) on F4JLB7 — downstream of the refuted MF, no independent support.

Candidate references with exact snippets to verify:
- PMID: 26451971 — verify snippet: "ROP-interactive Cdc42- and Rac-interactive binding motif-containing protein 7 (RIC7) was suggested to function downstream of ROP2" → establishes the literature RIC7 is a CRIB effector/adaptor, not a kinase (concerns At4g28556, not F4JLB7).
- PMID: 33586611 — verify snippet: "ROP interactive CRIB-containing protein 7 (RIC7) has been found to negatively regulate ABA-induced stomatal closure" → genuine RIC7 is a CRIB ROP effector, non-catalytic.

Candidate replacement / retained GO terms (verify against TAIR primary evidence):
- Retain GO:0038023 (signaling receptor activity, MF) as the informative non-catalytic function (preferred over generic "protein binding").
- Retain GO:0016324 (apical plasma membrane, CC) (IDA:TAIR).
- Retain GO:0009860 (pollen tube growth, BP) (IMP:TAIR).

Suggested curator questions:
- Is the "RIC7" gene symbol on F4JLB7 (At4g28560) a carry-over from At4g28556? Should the symbol be corrected or annotated as ambiguous?
- Which primary references support the IDA (apical PM) and IMP (pollen tube growth) annotations for F4JLB7?

Suggested experiments (if experimental confirmation is desired):
- DeepTMHMM topology prediction to confirm single-pass RLP architecture.
- Optional in vitro kinase assay (expected negative) — low priority given decisive computational refutation.

Evidence Base (key literature)

• The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis. PMID: 26451971 — Establishes that the genuine RIC7 is a CRIB-motif ROP2 effector acting as an adaptor that binds Exo70B1. This is an adaptor/scaffold role, not an enzyme, and it concerns At4g28556, not the LRR protein F4JLB7. Supports the conclusion that "RIC7" is non-catalytic and clarifies the symbol collision.
• RIC7 plays a negative role in ABA-induced stomatal closure by inhibiting H₂O₂ accumulation. PMID: 33586611 — Confirms genuine RIC7 as a CRIB-containing ROP effector in guard-cell signaling, reinforcing its non-catalytic adaptor identity.
• The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs). PMID: 21309864 — Describes ROP–RIC effector interactions and shows that RIC effectors act upstream of separate kinases (e.g., RRK1); the RIC protein itself is not the kinase. Provides orientation that RIC-family proteins are adaptors, not catalytic enzymes.

Database/computational orientation (not primary literature but decisive for this MF question): UniProt F4JLB7 record, InterPro/Pfam domain assignments, FunFam 3.80.10.10:FF:000041, and the AlphaFold DB model — all consistent with an LRR receptor-like protein and none supporting kinase activity.

Bottom Line

The ProtNLM2 kinase prediction for F4JLB7 is a refuted over-annotation. F4JLB7 (At4g28560) is a signal-peptide + leucine-rich-repeat receptor-like protein with no kinase domain, no HRD catalytic aspartate, no ordered VAIK→HRD→DFG triad, and a confident LRR-only AlphaFold model. The prediction most plausibly arises from frequency/similarity bias on the LRR ectodomain shared with LRR receptor kinases (e.g., ERECTA), compounded by a gene-symbol collision with the genuine CRIB-motif RIC7 at the adjacent locus At4g28556 — which is itself a non-catalytic ROP effector. GO:0016301 and GO:0016310 should be removed from F4JLB7; the informative retained functions are signaling receptor activity (MF), apical plasma membrane (CC), and pollen tube growth (BP).

Artifacts

• OpenScientist final report
• OpenScientist final report