The Atlantic potato genome, annotation file, protein sequence and other information were obtained from potato database (). We used PFam (https://pfam.xfam.org/) to download the HMM model of the conserved domain (PF01370) in CCR protein (Jaina et al., 2020) to identify the potato CCR gene family genome-wide. The potato protein sequence was compared with Arabidopsis CCR protein sequence in NCBI database by BLASTP. By online website (http://eggnog-mapper.embl.de/), eggNOG HMMER3.0 (http://hmmer.org/download.html) (Jaina et al., 2013), the NCBI - CDD (Aron et al., 2015) of preliminary screening genes annotated and search, etc. After comprehensive analysis, we finally identified 10 StCCR genes, and these potato CCR genes were renamed StCCRs. According to the gene location relationship on the chromosome, they were identified as StCCR1 to StCCR10. For example, The gene ID Soltu.Atl.03_1G016980.1 has been named StCCR1. The amino acid number, molecular weight, isoelectric point and hydrophobic index of potato StCCR members were analyzed by ExPASy (https://www.expasy.org/) (Elisabeth et al., 2003). WoLF PSORT(https://wolfpsort.hgc.jp) was used to predict protein subcellular localization (Paul et al., 2007).

The start and end locations of all genes on each chromosome were obtained from the potato database (), and the physical location and chromosome division information of StCCR gene of Atlantic potato were obtained according to the position of 48 chromosomes. The collinearity analysis of StCCRs was performed using MCScanX (Wang et al., 2012). Possible segment duplication and tandem duplication events were defined based on the physical location of the chromosomes of genes (Panchy et al., 2016), and the ratio of non-synonymous substitution to synonymous substitution (Ka/Ks) of duplicate gene pairs was determined by KaKs_Calculator (Zhang et al., 2006). Dual Synteny Plot in TBtools was used to analyze the collinearity of StCCRs with other species (Arabidopsis, tobacco, tomato) (Tang et al., 2008). The genomes of species files and annotations, respectively in the Ensembl the Plants (https://plants.ensembl.org/index.html) and an Eggplant Genome The Database (http://eggplant.kazusa.or.jp/index.html) to download. TBtools software was used to visualize the results (Chen et al., 2020).

Download the GFF file of the potato gene structure from the potato database (). The StCCR gene structure (exon-intron) was defined by TBtools software, and the conserved motif of StCCR protein was obtained from the online MEME program (https://meme-suite.org/) (Bailey et al., 2009). Use the following parameters: minimum width set to 6 bp, maximum width set to 25 bp, and maximum number of motifs set to 10. The structure and conserved domain of the StCCR gene were visualized by TBtools.

In order to explore the evolutionary relationship of CCR gene family, multi-sequence comparison of CCR and other proteins of potato and Arabidopsis thaliana was performed using MEGA 7 (Sudhir et al., 2016) software, and the phylogenetic tree was constructed by Neighbor-joining method. using the maximum likelihood (ML) with 1000 bootstraps, other parameters default, to use online software Evolview v (https://www.evolgenius.info/evolview) to optimize the evolutionary tree.

The structure and conserved domain of StCCR gene were visualized by TBtools. The upstream 2000 bp sequence of the start codon of each StCCR gene was obtained from the potato genome. Through Plant CARE database (https://bioinformatics.PSB.Ugent.Be/webtools/plantcare/HTML/) of StCCR gene promoter sequence of cis element for on-line retrieval (Lescot, 2002).

The potatoes used in this study were of Atlantic variety. The original seed potatoes were provided by the College of Life Sciences of Northeast Agricultural University in China. They were planted in planting pots containing soil and vermiculite (in a ratio of 1:1) (dimensions: 40cm * 15cm * 15cm), and then placed in a laboratory incubator (temperature 22°C, humidity 60%, light cycle 16/8 hours, light intensity 10000 lx) for growth, with normal water and fertilizer management. To clarify the expression patterns of the StCCRs gene in different tissues of potatoes, as well as the expression patterns under various abiotic stresses and exogenous hormone stimulation, potato plants that had been growing normally for 30 days after emergence, with similar growth conditions, were selected. Samples were taken from the roots, above-ground stems, and leaves, wrapped in tin foil, and rapidly frozen in liquid nitrogen. They were then stored at -80°C for future use. According to researcher (Guan et al., 2020; Chang et al., 2024; Shi et al., 2025), taking the normally growing plants as the control group, the normal potato plants that had been growing normally for 30 days, those with good growth, and those with similar growth conditions were respectively subjected to high-temperature (42°C), low-temperature (4°C), salt (250 mmol/L NaCl), and drought (20% PEG6000) treatments. Compared with distilled water spray, leaves were sprayed with indole acetic acid (10 μmol/L IAA), gibberellin (50 μmol/L GA3) and abolic acid solution (10 μmol/L ABA), and then placed in a light incubator for culture. After treatment for 0,2,4,8,12,24 hours, samples were collected from leaves. Wrap with tin foil, freeze liquid nitrogen and store in a -80°C refrigerator for later use. All samples were taken for 3 replicates.

BIOER’s LineGene 9620 real-time fluorescent quantitative PCR system was used to perform qRT-PCR analysis on 10 genes. Q-StCCR Primer sequence ( Supplementary Table S9 ) was designed by prime-blast (Ye et al., 2012). Total RNA was extracted from each collected sample using the Total RNA Rapid Extraction kit (ER501-01, TransGen Biotech, Beijing, China). and reverse-transcribed to cDNA using TransScript^®One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311, TransGen Biotech, Beijing, China). qRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) kit. β-action was used as the internal reference gene (Tang et al., 2017), and three independent and repeated data were calculated by 2−^ΔΔCt method (Bustin et al., 2009). Analysis of variance (ANOVA) and drawing of gene expression heat maps were performed using GraphPad Prism 9 and TBtools.

Potato leaves were taken, total RNA was extracted and its integrity was detected. cDNA synthesis was performed using a reverse transcription kit as the template for reverse transcription. The StCCR6 gene sequence in potatoes was amplified by using the SnapGene software to design the primer StCCR6-1F/R, and it was cloned into the transient expression vector pCAMBIA2300-StCCR6-eGFP. The method of transient tobacco infection was referred to Mo et al. (2023). The localization of StCCR6-eGFP fluorescence signals was then observed using a laser confocal microscope (TCSSP8, Carl Zeiss, Germany).

Design primers will be in the SGN - VIGS (https://vigs.solgenomics.net/) online design StCCR6 best silent loci (VIGSStCCR6) amplification on pTRV2 carrier. The plasmids pTRV1, pTRV2, pTRV2-PDS and pTRV2-StCCR6 were transferred into Agrobacterium GV3101 together. The resuspended pTRV2, pTRV2-PDS and pTRV2-StCCR6 were respectively mixed with pTRV1 in equal volumes, and then left to stand in the dark at room temperature for 4 hours to obtain the TRV infection solution. VIGS transient infection was carried out on the leaves of 4-week-old potatoes. During the injection process, the main veins of the potatoes were avoided. After dark incubation for 2 days, they were transferred to the artificial culture room for normal management. The photobleaching symptoms of potato leaves after being treated with pTRV2-PDS infection were observed to confirm the successful infection. The StCCR6 gene-silenced plants were identified by qPCR method.

1. Materials: For plants that have been growing normally for 30 days, take the above-ground stem tissue from the same part and trim it into long strips that are 3 mm long and 1 mm wide. 2. Fixation: Pre-fixation: Fixation with 2.5% glutaraldehyde fixative for 2 hours; Rinse: Rinse 3 times with 0.1M phosphoric acid buffer solution (pH=6.8), each time for 15 minutes; Post-fixation: Fixation with 1% osmium acid fixative; Rinse: Rinse three times with 0.1M phosphate buffer solution (pH=6.8), each time for 15 minutes. 3. Dehydration: Stay in 50% ethanol for 15 minutes; Stay in 75% ethanol for 15 minutes; Stay in 90% ethanol for 15 minutes; Stay in 100% ethanol for 20 minutes; 100% ethanol +100% acetone = 1:1 Stay for 15 minutes; Stay in 50% acetone for 5 minutes. 4. Immersion: Pure acetone + embedding solution (1:1) for 1 day; Pure acetone + embedding solution (1:2) retention for 1 day; Pure acetone + embedding solution (1:3) retention for 1 day. 5. Embedding. 6. Aggregation: 3 days. 7. Repair the block. 8. Slicing with an ultra-thin slicer. 9. Electron microscope observation. The transmission electron microscope of Hitachi Scientific Instruments Beijing Co., LTD., model HT7800, has a high voltage of 80kV and magnifications of 2000, 5000 and 10000 times respectively. The thickness of the cell wall was measured using ImageJ.

The in vitro leaf inoculation method was adopted. Sterile filter paper was placed in the culture dish and it was soaked with sterile water. Excessive water can easily cause leaf rot. The lint-free cotton is soaked and then wrapped around the petioles to retain moisture. Inoculate 20 μl of the bacterial liquid of bacterial blight (Ralstonia solanacearum, RS) (OD₆₀₀ = 0.8) with a needle-free syringe at 1/4 of the surface of isolated leaves of healthy and disease-free silent and wild-type potatoes to allow the bacterial liquid to spread along the veins. Seal the petri dish and maintain humidity. Cultivate under room temperature and 16 h/d light conditions. The leaf conditions were recorded at 1, 2, 3, 4 and 5 days after pathogen treatment. The area of the lesion was statistically analyzed using ImageJ.

The overexpressed potato and wild-type potato leaves at 0 and 5 days after inoculation with the pathogen were used as materials. The contents of hydrogen peroxide (H₂O₂), superoxide anion (O2-), malondialdehyde (MDA), as well as the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined according to the specific steps and calculation methods provided in the kit manual of Suzhou Komeng Biotechnology Co., Ltd., and the kit was used for the measurement. Three independent and repeated data were analyzed using GraphPad Prism 9 for variance analysis (ANOVA).

Systematic genome-wide identification revealed 10 StCCR genes in Solanum tuberosum L. (Atlantic variety), phylogenetically classified and renamed as StCCR1-StCCR10 based on chromosomal localization ( Supplementary Table S1 ). The encoded proteins exhibited conserved physicochemical properties: polypeptide lengths ranged from 206 (StCCR8) to 354 amino acids (StCCR4), with molecular weights spanning 34.5 kDa (StCCR9) to 39.4 kDa (StCCR4). Theoretical isoelectric points (pI) varied between 5.27 (StCCR3) and 7.02 (StCCR7), while aliphatic indices (77.01–95.29) and the GRAVY (Grand Average of Hydropathy) value of StCCRs ranges from -0.289 (StCCR1) to -0.046 (StCCR9). The instability index of StCCRs ranges from 23.32 to 37.04 (> 40 indicates protein instability, < 40 indicates protein stability), and both are stable proteins. Subcellular prediction results showed that StCCRs were located in cytoplasm (3) and chloroplasts (7), respectively. In order to reveal the distribution of CCR gene, we compared its sequence with potato genome data and used genomic localization information for chromosome mapping. As shown in Figure 1 , a total of 10 StCCRs genes were unevenly distributed on 7 chromosomes of potato. Chromosomes chr3_1, chr03_3, chr03_4 contain two StCCR genes, while chromosomes chr06_2, chr8_1, chr08_3, ch8_4 contain only one StCCR gene. Chromosomal ideograms are scaled in megabases (Mb, left axis). Gene loci (red labels) denote physical positions of StCCR members across seven chromosomes. Gradient blue shading illustrates regional gene density, with darker tones corresponding to higher gene concentrations. Chromosome numbers are annotated adjacent to each ideogram.

Collinear analysis was performed on these identified StCCR genes, and it was found that 10 StCCR genes participated in 12 duplicate gene pairs ( Figure 2 ; Supplementary Table S2 ). Notably, StCCR2, StCCR4, StCCR6, and StCCR7 each participated in three duplication events, whereas other members engaged in two events. These results indicate that StCCRs is mainly amplified by fragment repetition during the evolution of Atlantic potato. The nonsynonymous substitution replacement rate (Ka) and synonymous replacement rate (Ks) are the basis for evaluating whether collinear genes are subjected to selection pressure. Ka/Ks value 1 indicates natural selection, Ka/Ks < 1 indicates purification selection, and Ka/Ks > 1 indicates positive selection (Zhang et al., 2006). Selection pressure analysis ( Supplementary Table S3 ) demonstrated Ka/Ks ratios ranging from 0.0347 to 0.8897 across duplicated pairs, consistent with strong purifying selection (Ka/Ks < 1). This evolutionary constraint indicates functional conservation within the StCCR family, though retained paralogs may exhibit conditional redundancy.StCCR gene synonym substitution is more favorable for potato evolution, and the low Ka/Ks ratio indicates that non-synonym mutations are largely excluded, a phenomenon commonly observed in functionally important and conserved genes, such as genes encoding metabolically critical enzymes or structural proteins. Depicted are concentric tracks representing genomic features (outer to inner): chromosome ideograms scaled in megabases (Mb), gene density heatmap (red = high density, blue = low), GC content histogram (height proportional to GC ratio), N-content dot plot (dot density reflecting unknown base frequency), and GC skew bars (blue = guanine excess, yellow = cytosine excess). Syntenic relationships among StCCR paralogs are highlighted by red connecting lines.

To investigate genomic expansion mechanisms and evolutionary relationships of StCCRs, we performed collinearity analysis between potato and four representative species (Arabidopsis thaliana, Solanum lycopersicum, Capsicum annuum, and Solanum melongena) using potato StCCR sequences as anchors ( Figure 3 ). Comparative synteny revealed stronger conservation within Solanaceae, with potato-tomato collinear pairs (26 pairs) significantly exceeding those of potato-pepper (15 pairs), potato-eggplant (13 pairs), and potato-Arabidopsis (8 pairs). Chromosomal distribution analysis identified conserved syntenic blocks predominantly localized to chr3, chr6, and chr8 in Solanaceae species, while potato-Arabidopsis collinear pairs clustered on chr1, chr3, and chr6 ( Supplementary Table S4 ). Gray lines indicate homologous genomic blocks in potato relative to other species. MCscanX was employed to identify collinear relationships between potato StCCRs and homologous genes in Arabidopsis thaliana, tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena), with syntenic gene pairs highlighted by red connectors. The analysis showed that these homologous gene pairs may have existed before the differentiation of ancestral lineages, collinearity retained genes are conserved and may have core functions, and they exist stably in collinearity blocks across species, which also implies that the Atlantic potato StCCRs gene family may also expand and evolve through gene replication.

To elucidate evolutionary relationships among potato CCR members, a neighbor-joining (NJ) phylogenetic tree was constructed based on nucleotide sequences. The 10 StCCRs clustered into two subfamilies: Subfamily I contained four genes, while Subfamily II comprised six members ( Figure 4A ). Conserved motifs in StCCR proteins were predicted using MEME, identifying 10 distinct motifs ( Figure 4B ; Supplementary Table S5 ). motif composition and distribution are relatively conservative among members of the same subgroup, and the type and number of motifs in the same subgroup are similar. All proteins contain motif1, 2, 3, and 5 motifs, among which there are conserved motifs specific to CCR proteins, and these motifs are also conserved during the evolution of potato CCR, which may be necessary for maintaining the function of CCR proteins. CCR members clustered in the same branch have similar motif composition and motif position. For example, motif6 and 8 appear in subfamily I, motif4 and 7 appear in subfamily II, and motif9 and 10 are specific to StCCR1, StCCR3 and StCCR5. Domain architecture analysis via NCBI CDD revealed that all StCCRs belong to the NADB_Rossmann superfamily, confirming conserved catalytic domains ( Figure 4C ). Gene structure comparisons demonstrated distinct intron-exon organizations: Subfamily II members contained five exons and four introns, while Subfamily I exhibited six exons and five introns except for StCCR7 ( Figure 4D ). Sequence similarity and high similarity in intron-exon structure among members of the two subfamily suggest that the potato cinnamyl CoA reductase gene may have undergone a gene replication event during evolution.

To elucidate evolutionary relationships within the CCR family, we constructed a phylogenetic tree using 61 CCR protein sequences from diverse species, including 10 StCCRs from Atlantic potato ( Figure 5 ). The analysis revealed four distinct clades: Clade I contained 33 CCRs from monocots, dicots, gymnosperms, and ferns, incorporating six StCCRs from Subfamily II (StCCR1/3/5/8/9/10). Clade II exclusively comprised three gymnosperm CCRs. Clade III included 19 CCRs, notably containing four StCCRs from Subfamily I (StCCR2/4/6/7) that clustered closely with two Solanaceae homologs (SlCCRs and PhCCR1) and showed evolutionary proximity to Arabidopsis CCRs in Clade IV. We speculate that these 4 StCCR proteins may be related to the real CCR function, and whether the genes of StCCR subfamily II have enzyme activity needs to be further proved by subsequent studies and experiments. Taxonomic groups are denoted by distinct markers: dicots (red triangles), monocots (blue circles), gymnosperms (green triangles), and ferns (black squares). Clade-specific coloration indicates evolutionary groupings: Clade I (red), Clade II (green), Clade III (yellow), and Clade IV (blue). Complete species designations and corresponding sequence accession numbers are provided in Supplementary Table S6 .

A conserved CCR signature motif related to NADP (H) binding and substrate catalysis was found in the amino acid sequence of most plant CCR proteins. This region can form a β-α-β structure in the secondary structure, and it is speculated that it may be the catalytic site of CCR, or the binding region of its cofactor NADPH, especially the two lysine residues on it may directly bind to the substrate. Conserved motifs G-X-X-G-X-A and D-X-X-D have been reported to be involved in NAD (P) binding and adenine pocket stable binding. In addition, NADP+ specific motifs R (X) 5K have been identified, which is A key structure that distinguishes CCR from other NAD (H) dependent SDRS (Pan et al., 2014; Chao et al., 2017, 2019).The comparison between two AtCCR proteins and 10 StCCR proteins showed ( Figure 6 ) that both AtCCR proteins and StCCR subfamily I proteins had a KNWYCYGK conserved motif. However, changes in the conserved motifs of subfamily II May affect protein binding to substrates and catalytic activity. This is similar to the situation of rice and maize (Joël Piquemal et al., 2010; Bai et al., 2003). The comparison between AtCCR/StCCR proteins showed that some of them had the NAD (P) binding motif GXXGXXA/G. The catalytic triplet Ser-Tyr-Lys has been confirmed as an active SDR residue (Chao et al., 2017), and the corresponding conserved motif Y-X-X-X-K has been identified in all AtCCR/StCCR proteins. The diversity of conserved motifs in StCCR proteins suggests that they may have different activities and biological functions. Functionally critical motifs are annotated: CCR signature motif (red boxes), NAD (P)-binding domains (black boxes), NADP specificity determinant R(X)5K (blue boxes), and catalytic triad residues Ser-Tyr-Lys (black squares).

Promoter cis-elements play a key role in the initiation of gene expression. In order to further investigate the response of StCCR gene family to abiotic stress, we analyzed the promoter cis-acting elements of StCCR family members (Oudelaar and Higgs, 2021). Through in-depth analysis of 2000 bp sequences upstream of each gene translation initiation site, it was found that promoters of StCCR gene family members contain multiple cis-acting elements. The results ( Figure 7 ) showed that the cis-acting elements in the promoter region of StCCR gene involved in stress response were mainly divided into photoresponsive elements, hormone-responsive elements, environmental stress-related elements and site-binding elements. The colored squares in Figure 7A represent the different types of cis-acting elements and their positions in each StCCR genes; Figure 7B shows the number of cis-acting components. Three types of elements involved in plant hormones, light response and environmental stress response are particularly abundant. All StCCR promoter sequences contain ARE response elements and light response elements related to anaerobic induction (for example, GT1-motif and some genes contain a large number of Box-4 cis-acting elements). Among the elements responding to plant hormones, StCCRs may be involved in plant responses to plant hormones such as methyl jasmonate, salicylic acid, ABA, auxin and gibberellin. ABRE responsive elements, AuxRR-core, P-box, CGTCA-motif and TGACG-motif related to jasmonic acid reaction, and TCA-element involved in salicylic acid reaction also exist in some StCCRs gene promoters. Abiotic stress-related elements include LTR, MYB binding sites MBS, defense and stress-related regulatory elements (TC-rich repeats). StCCR2, StCCR4 and StCCR6 also have Sp1 elements related to cell growth and differentiation. These results suggest that the expression of StCCRs may be regulated by light stimulation, anaerobic induction, plant hormones and abiotic stress during potato growth, and play an important role in protecting the plant against stress.

Gene expression serves as a critical determinant of gene function. To investigate the expression profiles of potato CCR genes across plant organs during growth stages, qRT-PCR was performed using the β-actin gene as an internal reference and root tissues as the baseline control ( Supplementary Table S7 ). The organ-specific expression analysis ( Figure 8 ) revealed that most StCCRs exhibited higher relative expression levels in stems and leaves compared to roots. Notably, Subfamily II members including StCCR3, StCCR5, StCCR8, and StCCR10 demonstrated pronounced leaf-predominant expression, while StCCR3 and StCCR5 showed elevated expression in stems, all of which were higher than those of Subfamily I genes. The fact that the CCR genes were more highly expressed in the stems and leaves of potato plants than in the roots suggests that lignin biosynthesis is more active in these above-ground tissues. CCR is a key enzyme in the phenylpropanoid pathway that produces lignin, a structural compound that strengthens cell walls. In short, higher CCR expression in stems and leaves means that these tissues invest more in structural support and defense, possibly due to their exposure to mechanical stress, pathogens, or the need for rigidity for upright growth and photosynthesis. Moreover, different StCCR genes have different functions in various organs of potatoes.

Abiotic stresses (drought, salinity, extreme temperatures) and hormonal stimuli constitute critical constraints on potato productivity. Building upon prior bioinformatics and cis-element analyses, we investigated StCCR responses to these challenges through qRT-PCR quantification under high-temperature (42°C), low-temperature (4°C), drought, salt, IAA, ABA, and GA3 treatments ( Supplementary Table S8 ). All seven treatments significantly modulated CCR transcript levels within 24 hours ( Figure 9 ). StCCR1/3/5 exhibited basal expression across most conditions, while StCCR4/6/10 demonstrated marked responsiveness. StCCR2/4/6/7 are the same subfamily with close evolutionary relationship, and the gene expression levels and trends of each gene in each treatment are similar in each group. StCCR8/9/10 as another group, the situation is similar. The expression patterns of the remaining genes were different due to different stress and genes. Thermal treatments elicited temporal-specific responses: StCCR6 maintained sustained activation throughout 2–24 h exposure, and the relative expression levels of StCCR8/9/10 at 42°C increased first and then decreased after 24 h treatment with StCCR2/4/8 at 4°C. The relative expression levels of StCCR2/4/7 at 42°C and StCCR3/5/7/10 at 4°C for 24 h showed a trend of continuous decline. The expression of CCR gene decreased after treatment with low temperature for a certain period of time. We speculated that CCR gene in plants gradually decreased with low temperature to reduce lignin synthesis and accumulation, so that water in plant cells could flow out more easily, and the influence of ice crystals caused by low temperature on cell structure could be reduced. Under drought treatment, the relative expression of StCCR1 continued to increase with the increase of treatment time, which was also one of the most significant responses of StCCR1 in many treatments. The relative expression levels of StCCR2/4/7 genes remained at a high level during 2–4 h, and then showed a downward trend, while the expression levels of StCCR8/9/10 in group 2 increased first and then decreased. Under salt stress, the expression levels of StCCR2/4/6 in subfamily I continued to increase and reached the highest level at 24 h, while in subfamily II, on the contrary, reached the lowest level at 24 h. Phytohormone responses revealed differential kinetics: IAA and ABA treatments upregulated both subclades, with StCCR2/4 showing pronounced time-dependent amplification. GA3 activated Subfamily II members (StCCR8/9/10). These findings highlight functional diversification among StCCRs, with StCCR4/6/10 emerging as key stress-responsive candidates through their consistent transcriptional alterations across multiple challenges.

To determine the subcellular localization of StCCR6, Agrobacterium-mediated transient transformation of Nicotiana benthamiana leaves was performed using the pCAMBIA2300-StCCR6-eGFP fusion construct, with the empty pCAMBIA2300-eGFP vector serving as the control. Transfected leaves were maintained at 26°C under low-light conditions for 48 h prior to fluorescence examination. The results ( Figure 10 ) showed that the empty pCAMBIA2300-eGFP construct displayed green fluorescence signals in the nucleus, cytoplasm, and cell membrane. In contrast, GFP fluorescence of the fusion protein pCAMBIA2300-StCCR6-eGFP appears in the cytoplasm.

Bioinformatic analysis combined with qRT-PCR profiling identified differential expression patterns among StCCR subfamily members under various stress conditions, with StCCR6 demonstrating particularly pronounced transcriptional regulation. To functionally characterize StCCR6, virus-induced gene silencing (VIGS) was implemented via Agrobacterium-mediated transformation using the tobacco rattle virus (TRV) vector system. Figure 11A illustrates experimental controls, with left panel showing asymptomatic pTRV2 empty vector plants and right panel displaying characteristic photobleaching in pTRV2-PDS positive controls (phytoene desaturase PDS serving as VIGS validation marker). The gene silencing efficiency of PTRV2-StCCR6 detected by qRT-PCR was 85.1%, 73.3%, 74.7%, 73.7%, 81.2%, 88.1%, respectively, and the gene expression level was lower than that of pTRV2 (control) plants. Subsequently, strains 1# and 6# with high selection efficiency were selected for subsequent experimental research ( Figure 11B ) and named VIGS-1 and VIGS-6.

Transmission electron microscopy (TEM) analysis of stem cross-sections revealed ultrastructural differences between silenced and WT plants ( Figure 11C ). Quantitative measurements demonstrated 14.8% (VIGS-1: 0.2395 μm) and 25.1% (VIGS-6: 0.2105 μm) reductions in cell wall thickness relative to WT plants (0.281 μm) ( Figure 11D ). By further measuring the lignin content of WT plants and silent plants, it was found that the lignin content of WT plants was higher than that of silent plants ( Figure 11E ).

Transcriptional profiling of phenylpropanoid pathway genes ( Figure 11F ) revealed differential regulation in silenced plants: StPAL, StC4H, StCCoAOMT, and StCOMT exhibited elevated expression in WT plants, whereas St4CL, StHCT, StC3H, and StCAD showed reduced expression compared to silenced lines. These results suggest StCCR6 modulates lignification through coordinated regulation of phenylpropanoid biosynthesis components.

Under adverse conditions, plants generate reactive oxygen species (ROS) that induce cellular toxicity, leading to structural damage and programmed cell death in plant tissues. Comparative analysis of 30-day-old StCCR6-silenced plants (pTRV2-StCCR6) and empty vector controls (pTRV2) revealed differential responses to bacterial challenge in controlled light conditions. Figure 12A demonstrates progressive leaf lesion development over a 5-day infection period, with pTRV2-StCCR6 lines exhibiting significantly enhanced susceptibility compared to controls. Quantitative measurements showed RS-inoculated WT plants developed lesions averaging 0.918 cm², while VIGS-1 and VIGS-6 silenced lines displayed 2.26-fold (2.075 cm²) and 2.02-fold (1.852 cm²) increases in necrotic area, respectively. These findings suggest StCCR6 silencing compromises potato resistance to RS infection, exacerbating foliar damage severity.

In order to evaluate oxidative damage, the contents and activities of H₂O₂, O^2-, SOD, POD, CAT and MDA in leaves of StCCR6-silenced plants and WT plants under RS stress were detected. The result is shown in Figure 12B . After inoculation, the accumulation of H₂O₂ and O^2- in potato leaf cells increased, and pTRV2-StCCR6 was higher than that in control plants. MDA is the product of membrane lipid peroxidation, and its content indirectly reflects the degree of oxidative damage of cells. Before inoculation, there was no difference in the content of silent plants and WT plants, but the MDA content of plants after inoculation was accumulated. Among them, VIGS-6 was significantly increased compared with WT, and VIGS-1 was not different from WT. SOD, POD and CAT are important antioxidant enzymes in plants. There was no significant difference in physiological indexes of potato leaves before inoculation. Due to the stress of RS bacteria, the contents of SOD, POD and CAT of silent plants were lower than those of WT except POD of VIGS-1.