Recombinant Arabidopsis thaliana Protein SCO1 homolog 2, mitochondrial (HCC2)

What is HCC2 and how is it related to SCO proteins?

HCC2 (Homolog of Copper Chaperone 2) in Arabidopsis thaliana is a homolog of the yeast copper chaperone Sco2p. It belongs to the HD2 family and contains a thioredoxin domain, though unlike its paralog HCC1, it lacks the copper-binding motif found in Sco1p and Sco2p. This structural difference suggests functional divergence despite their homology. HCC2 is localized to mitochondria and plays roles distinct from HCC1, with evidence pointing to its involvement in stress response mechanisms rather than direct copper delivery for cytochrome c oxidase (COX) assembly .

Where is HCC2 localized within plant cells?

HCC2 has been confirmed to localize specifically to mitochondria. This localization has been experimentally verified through confocal laser scanning microscopy (CLSM) using HCC2 proteins fused to monomeric RFP (mRFP) and co-expressed with mitochondria-targeted GFP (mt-GFP). The fluorescence signals from both perfectly overlapped, confirming mitochondrial localization. The HCC2-mRFP signals appeared as dot-like, fast-moving structures approximately 0.5 μm in diameter, consistent with the typical size of Arabidopsis mitochondria. No HCC2-mRFP signals were detected in any other cellular compartments, indicating exclusive mitochondrial targeting .

What are the established methods for generating and validating hcc2 knockout mutants?

Validated methodologies for generating hcc2 knockout mutants include T-DNA insertional mutagenesis targeting different regions of the HCC2 gene. Two successful approaches include targeting intron 1 (as in hcc2-1) or the junction between exon 3 and intron 3 (as in hcc2-2). Validation of knockout status requires RT-PCR analysis using primers that span the T-DNA insertion sites. Complete absence of HCC2 transcripts in homozygous lines confirms knockout status. Phenotypic validation should include assessment of primary shoot length after 31 days of growth and UV-B sensitivity testing. Complementation tests using the native HCC2 promoter driving HCC2 cDNA expression are essential to confirm that observed phenotypes are specifically caused by HCC2 disruption rather than secondary mutations or position effects .

How can HCC2 protein be effectively tagged for localization and interaction studies?

For effective tagging of HCC2 protein, multiple strategies have been validated:

• C-terminal fusion with the Snap-tag (19.6 kDa) under control of the native HCC2 promoter

  • Amplify the entire HCC2 coding sequence from A. thaliana cDNA

  • Use Gateway cloning technology with donor plasmids like pDONR P4r-P3r

  • Clone into a destination vector such as pGWB516

  • Select transformants on suitable antibiotics (sulfonamide and hygromycin)

  • Confirm by PCR using specific primers (e.g., Sco2E1F/Sco2E5R)

• C-terminal fusion with mRFP (27.3 kDa) under control of the 35S promoter

  • Amplify the HCC2 sequence from start codon to 28 nt before the stop codon

  • Clone into pENTR 3C vector and transfer to pGWB554

  • Transform into lines expressing mitochondrial-targeted GFP markers

  • Select on kanamycin and hygromycin

  • Validate by PCR and qRT-PCR analysis

Both tagging methods preserve HCC2 function, as demonstrated by complementation of knockout phenotypes.

What complementation strategies are effective for validating hcc2 mutant phenotypes?

Effective complementation of hcc2 mutants requires careful consideration of promoter choice and protein tagging. The most reliable approach involves using the native HCC2 promoter region to drive expression of the complete HCC2 coding sequence. For the complementation construct, the following methodology has been validated:

• Amplify the entire HCC2 coding sequence from A. thaliana cDNA

• Optionally fuse to a small tag (e.g., Snap-tag) that preserves protein function

• Place under control of the identical HCC2 promoter region used for expression studies

• Transform into homozygous hcc2 knockout lines

• Select transformants on appropriate antibiotics

• Confirm presence of the transgene by PCR

• Validate restoration of wild-type phenotypes (shoot length, UV-B sensitivity)

This approach successfully rescued both the reduced shoot length phenotype and UV-B sensitivity in hcc2 knockout lines, confirming the specific role of HCC2 in these processes .

How does HCC2 contribute to UV-B stress responses in Arabidopsis?

HCC2 plays a significant role in UV-B stress tolerance through mechanisms distinct from its paralog HCC1. When exposed to UV-B radiation, hcc2 knockout mutants display markedly increased sensitivity compared to wild-type plants. Conversely, wild-type plants respond to UV-B exposure by upregulating HCC2 transcript levels, indicating a specific adaptive response. Complementation of hcc2 knockouts with functional HCC2 fully rescues the UV-B-sensitive phenotype, confirming HCC2's direct involvement in this stress response.

The molecular mechanism may involve:

• Potential redox regulation through its thioredoxin domain

• Protection of mitochondrial proteins from UV-B-induced oxidative damage

• Signaling cascades that connect mitochondrial status to nuclear gene expression

Unlike HCC1, which primarily functions in copper delivery for cytochrome c oxidase assembly, HCC2 appears to have evolved specialized functions in stress response pathways. The absence of the copper-binding motif in HCC2 further supports its divergent function from typical SCO proteins .

What is the relationship between HCC2 and cytochrome c oxidase (COX) activity?

Despite being a homolog of SCO proteins that are typically involved in cytochrome c oxidase (COX) assembly, HCC2 shows surprisingly little impact on COX activity. Experimental measurements of COX activity in hcc2 knockout mutants revealed no significant difference compared to wild-type plants. This stands in stark contrast to hcc1 mutants, where disruption of even one HCC1 gene copy suppresses respiration by more than half compared to wild-type.

This functional divergence can be explained by the absence of the copper-binding motif in HCC2, which is present in HCC1 and critical for delivering copper to the catalytic center of COX. The data strongly suggest that HCC2 has evolved away from direct involvement in COX assembly toward specialized functions in stress response pathways, particularly UV-B tolerance. This represents a fascinating example of paralog divergence where structural differences (presence/absence of copper-binding motif) directly correlate with functional specialization .

How do HCC2 and HCC1 functions differ despite their homology?

Despite being homologs, HCC2 and HCC1 exhibit profound functional divergence in Arabidopsis:

This functional divergence likely evolved following gene duplication, with HCC1 retaining the ancestral function in copper delivery for respiration, while HCC2 neofunctionalized toward stress response roles. The lack of the copper-binding motif in HCC2 represents a key structural adaptation that underlies this functional shift .

How does HCC2 relate to other stress response factors in the HD2 family?

HCC2 functions within a broader network of HD2 family proteins involved in stress responses. Particularly noteworthy is its relationship with HD2A and HD2C, which coordinate to regulate drought stress responses and root growth. While HCC2 primarily responds to UV-B stress, the broader HD2 family shows diverse stress response functions:

The hd2a hd2c double mutant (Mac16) shows more severe phenotypes than single mutants, suggesting cooperative function. Similarly, future research might explore potential interactions between HCC2 and other HD2 family members under combined stress conditions. While HCC2's mitochondrial localization differs from the primarily nuclear HD2A/HD2C, all appear to be involved in transcriptional responses to environmental stresses, suggesting potential retrograde signaling mechanisms .

What experimental approaches can determine HCC2 protein interactions in mitochondria?

To effectively characterize HCC2 protein interactions within mitochondria, multiple complementary approaches should be considered:

• In vivo protein tagging and co-immunoprecipitation (Co-IP)

  • Use C-terminal tagging with small epitopes (Snap-tag has been validated)

  • Express under native promoter to maintain physiological levels

  • Isolate mitochondria using established gradient centrifugation methods

  • Perform Co-IP followed by mass spectrometry to identify interacting partners

• Bimolecular Fluorescence Complementation (BiFC)

  • Split fluorescent protein approach (as demonstrated for HD2 family proteins)

  • Co-express HCC2 fused to one fragment of YFP with potential interacting partners fused to complementary YFP fragment

  • Visualize interactions through restored fluorescence in mitochondria

  • Validate with appropriate controls (non-interacting proteins)

• Yeast two-hybrid (Y2H) assays with mitochondrial proteins

  • Use mature protein sequences (without transit peptides) for HCC2 and candidate interactors

  • Include controls for auto-activation

  • Validate positive interactions with in vivo methods

• Proximity-dependent biotin identification (BioID)

  • Fuse HCC2 to a promiscuous biotin ligase

  • Allow biotinylation of proximal proteins in intact mitochondria

  • Identify biotinylated proteins by streptavidin pulldown and mass spectrometry

These approaches can reveal whether HCC2 interacts with components of the respiratory chain, other stress response proteins, or forms part of larger protein complexes within mitochondria .

How can researchers effectively analyze HCC2 expression changes under different stress conditions?

For comprehensive analysis of HCC2 expression under varying stress conditions, the following methodological approach is recommended:

• Quantitative RT-PCR (qRT-PCR) analysis

  • Use validated primers (e.g., HCC2-RT-F/HCC2-RT-R) that span exon junctions

  • Include appropriate reference genes stable under stress conditions

  • Perform time-course experiments to capture expression dynamics

  • Apply multiple stress conditions (UV-B, drought, heat, cold, salinity)

• Translational reporter fusions

  • Generate HCC2 promoter:HCC2-GUS constructs for histochemical analysis

  • Create HCC2 promoter:HCC2-luciferase fusions for real-time monitoring

  • Image reporter expression in different tissues and under various stresses

• Western blot analysis of protein levels

  • Develop specific antibodies against HCC2 or use epitope-tagged versions

  • Compare transcript and protein levels to identify post-transcriptional regulation

  • Fractionate cells to determine if stress affects HCC2 localization

• Transcriptome analysis in wild-type vs. hcc2 mutants

  • Perform RNA-seq under normal and stress conditions

  • Identify genes differentially regulated in the absence of HCC2

  • Conduct pathway enrichment analysis to place HCC2 in broader stress response networks

These approaches should be integrated to obtain a comprehensive understanding of HCC2's role in stress responses, potentially revealing novel pathways and regulatory mechanisms .

What methodologies can determine if HCC2 has acquired novel functions beyond the ancestral SCO protein role?

To investigate potential neofunctionalization of HCC2 compared to ancestral SCO proteins, researchers should employ the following methodological approaches:

• Comparative structural analysis

  • Determine the three-dimensional structure of HCC2 using X-ray crystallography or cryo-EM

  • Compare with known structures of yeast Sco2p and bacterial SCO proteins

  • Identify structural adaptations beyond the missing copper-binding motif

• Heterologous complementation tests

  • Express Arabidopsis HCC2 in yeast sco2 mutants and assess rescue of phenotypes

  • Express yeast Sco2p in Arabidopsis hcc2 mutants to test functional conservation

  • Create chimeric proteins swapping domains between HCC2 and other SCO proteins

• Evolutionary analysis across plant species

  • Compare HCC2 sequences across diverse plant lineages

  • Identify sites under positive selection that may indicate functional adaptation

  • Correlate evolutionary changes with stress tolerance in different plant groups

• Metabolomics profiling

  • Compare metabolite profiles between wild-type and hcc2 mutants under normal and stress conditions

  • Identify metabolic pathways affected by HCC2 absence

  • Look for unique metabolic signatures distinct from respiratory deficiencies

This integrative approach will help determine whether HCC2 has evolved entirely new functions or retains aspects of ancestral SCO protein roles beyond direct involvement in cytochrome c oxidase assembly .

How might researchers explore the potential for HCC2 in engineering stress-tolerant plants?

To investigate HCC2's potential in developing stress-tolerant plants, researchers should consider these methodological approaches:

• Overexpression studies with tissue-specific and stress-inducible promoters

  • Generate transgenic lines with HCC2 under control of constitutive (35S), tissue-specific, and stress-inducible promoters

  • Assess tolerance to UV-B and other stresses in these lines

  • Measure growth parameters to identify potential trade-offs between stress tolerance and productivity

• Engineering HCC2 variants with enhanced function

  • Use structure-guided mutagenesis to modify the thioredoxin domain

  • Create chimeric proteins incorporating functional domains from other stress-response proteins

  • Test functional improvements in transgenic plants

• Multi-omics analysis of HCC2 overexpression lines

  • Conduct transcriptome, proteome, and metabolome analyses

  • Identify downstream pathways affected by HCC2 modulation

  • Develop markers for enhanced stress tolerance

• Field trials under various stress conditions

  • Test HCC2-modified plants under controlled field conditions

  • Assess real-world performance during UV-B exposure and other stresses

  • Measure yield components to determine agricultural relevance

This research direction could lead to the development of crops with enhanced tolerance to UV-B radiation and potentially other environmental stresses, contributing to sustainable agriculture under changing climate conditions .