Stone cell formation, resulting from aberrant lignin deposition in parenchyma cells, is a key determinant of pear fruit quality. Although exogenous calcium application is known to inhibit lignin biosynthesis and stone cell development, the underlying molecular mechanism involving calcium sensor proteins remains poorly understood. This study aimed to elucidate the molecular pathway by which calcium signaling modulates lignin biosynthesis. Our findings demonstrate that the calmodulin-like protein PbCML46 significantly suppresses lignin accumulation. This function was validated through complementary approaches, including transient injection in pear fruit and stable overexpression in pear calli. A yeast two-hybrid screen revealed that PbCML46 specifically interacts with the bHLH transcription factor PbbHLH96, an interaction further confirmed by pull-down and luciferase complementation imaging (LCI) assays. Further yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and luciferase (LUC) reporter assays analysis showed that PbbHLH96 directly binds to the MYC cis-element in the PbCAD6 promoter and functions as a transcriptional repressor. Dual-luciferase reporter (DLR) and in vivo co-expression assays indicated that PbCML46 markedly enhances the repressive activity of PbbHLH96 on the PbCAD6 promoter, an effect strictly dependent on PbbHLH96. In conclusion, this study reveals a Ca2+-initiated regulatory cascade in which calcium signaling promotes the interaction between the sensor PbCML46 and the transcriptional repressor PbbHLH96, leading to cooperative suppression of the key lignin biosynthetic gene PbCAD6 and thereby negatively regulating lignin deposition in pear stone cells. These findings provide novel mechanistic insights into how calcium signaling improves fruit quality.

Breeding red-fleshed apples with enhanced health benefits is a primary objective of modern apple improvement, yet high acidity and small fruit size limit their commercial potential. Resolving trade-offs among flesh color, flavor, and appearance and dissecting their underlying genetic relationships remain major challenges. In this study, using the red-fleshed 'CSR6R6' (Malus sieversii) and the cultivated 'Royal Gala' (M. domestica) as parents, we developed an F1 population comprising 140 individuals and performed integrative large-scale multi-omics analyses, identifying 13,331,096 SNPs and 2,134 metabolites. Red flesh correlated positively with malic acid but negatively with fruit weight. A major QTL linked to red flesh was mapped, and six genes regulating anthocyanin and proanthocyanidin biosynthesis were functionally validated, including MdUGT89A2.1, MdUGT89A2.2, MdALMT4, MdALMT4-like, MdTT1, and MdLAR1. Notably, MdALMT4 and MdALMT4-like encode malate transporters that coordinate malate accumulation and anthocyanin biosynthesis, suggesting a genetic link between acidity and pigmentation. Integrative multi-omics analysis revealed a large-scale regulatory network, revealing gene-metabolite-phenotype interactions and accurately predicting pathways involving the anthocyanin regulator MdTT1 and the fruit shape gene MdMADS13. Leveraging this network, we identified opposing effects of red-flesh QTL haplotypes across traits, defining the genetic basis of phenotypic antagonism, and resolved 11 positive and 13 negative haplotypes affecting red flesh, malic acid, and fruit weight. Pseudo-backcrossing further demonstrated that aggregating positive haplotypes substantially mitigates trade-offs among flesh color, flavor, and appearance, enabling their concurrent improvement. These findings will advance omics-assisted apple breeding, offering potential strategies for genetic improvement of other perennial fruit trees.

Flavonoid compounds, including anthocyanins and proanthocyanidins, are significant secondary metabolites in plants and play crucial roles in various aspects of plant growth, development, and environmental stress responses. In the present study, we identified a key transcription factor from the NAC family, designated as MdNAC72-like, which had a strong correlation with the anthocyanin content during the apple fruit ripening process. Through techniques including yeast one-hybrid analysis, electrophoretic mobility shift assays, and luciferase reporter assays, we illustrated that MdNAC72-like directly interacts with the promoters of the MdMYB9, MdLAR, and MdUFGT genes. This interaction enhances their transcriptional activity, leading to a favorable impact on the biosynthesis of anthocyanins and proanthocyanidins in plants. Furthermore, utilizing yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays, we demonstrated that MdERF1B forms an interaction with MdNAC72-like, which in turn augments the transcriptional activation ability of MdNAC72-like on the downstream structural genes MdMYB9, MdLAR, and MdUFGT. In conclusion, MdNAC72-like presents significant research potential, and these results offer a theoretical framework for understanding the regulatory mechanisms governing anthocyanin and proanthocyanidin synthesis in apple.

Postharvest loss constitutes a critical issue during the storage period of pears. Based on previous studies, both 1-methylcyclopropene (1-MCP) treatment and modified atmosphere packaging (MAP) can delay fruit senescence and softening. To investigate whether the combination of these treatments achieves a superior storage effect, 'Shannong Su' pears were treated with 1 μL/L 1-MCP for 24 h, then packaged in sealed or perforated film bags or left unpackaged, and stored at 4.00 ± 0.50 °C for 120 days. Firmness, ethylene release, quality indicators, gene expression, and metabolite profiles were analyzed. Results showed that 1-MCP inhibited ethylene production and the expression of PbACS1/2 and PbACO1. The combined treatment (1-MCP + sealed film bag) achieved the lowest expression levels of nine key cell wall-degrading enzyme genes (PbXTH1/28, PbPL8/18, PbCGR3, PbPG, Pbα/β-GAL, Pbβ-GLU) and maintained higher firmness, antioxidant capacity, total sugar, protopectin, and cellulose content. Metabolomic analysis revealed that 1-MCP treatment altered the biosynthesis of plant secondary metabolites and fatty acids, suggesting it may influence the anabolic metabolism of defensive compounds and lipids in plants. Film bag packaging affected antioxidant stress responses, nitrogen metabolism, specific amino acid derivative metabolism, and cofactor biosynthesis, indicating that sealed film packaging may induce oxidative stress responses and activate specific defensive or protective metabolic pathways. In conclusion, the combined treatment preserves pear quality through synergistic inhibition of ethylene synthesis, suppression of cell wall-degrading genes, and modulation of metabolic pathways. This cost-effective method can reduce postharvest losses in 'Shannong Su' pears and guide the storage of other similar climacteric fruits.

Low temperature (LT) stress negatively impacts the yield and quality of key horticultural crops such as apples. While previous research has predominantly focused on white-fleshed apple varieties with limited cold tolerance, the mechanisms underlying low temperature responses in red-fleshed apples remain largely unexplored. In this study, we used red-fleshed apple seedlings that we developed previously to investigate the biochemical responses to varying temperature conditions. Notably, LT stress significantly enhanced the accumulation of anthocyanins, soluble sugars, soluble proteins, and other metabolites in the red-fleshed apple seedlings. Leveraging RNA-seq data, we identified a potential LT stress-responsive transcription factor, which we designated as MdbHLH51. Functional characterization revealed that overexpression of MdbHLH51 in ‘Orin’ calli significantly promoted anthocyanin accumulation and up-regulated the expression of all MdCBFs genes, thereby bolstering tolerance to cold stress. These findings provide valuable insights into the cold stress response mechanisms in red-fleshed apples, offering a theoretical foundation for the genetic breeding of cold-resistant red apple varieties.

Sugar beet ( Beta vulgaris L.) is one of the most important sugar crops worldwide. However, studies on sugar beet gene functions are still lagging compared to other crops due to the lack of an effective genetic manipulation. In this study, we generated an infectious clone of beet sever curly top virus (BSCTV) and engineered it into a VIGS vector. BSCTV- BvPDS can induce strong and persistent bleached PDS -silencing phenotype in 8 tested sugar beet varieties. We further efficiently silenced B. vulgaris MYB1 gene that regulating the betalain pathway in sugar beet by BSCTV-based VIGS system. Finally, we used this system to silence the effector CbNip1 of Cercospora beticola through cross-kingdom RNAi, resulting in reduced disease symptoms in sugar beet plants. Therefore, the BSCTV VIGS system can be used to study sugar beet-pathogen interactions. In summary, we have established an easy and efficiency BSCTV-based VIGS vector in sugar beet. This system will be an attractive tool for functional genomic studies for sugar beet. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 32270165 China Agricultural Industry Technology System, CARS-170304

Red-fleshed apples are characterized by high nutritional value due to their abundant anthocyanin content. However, their commercial development is often constrained by a decline in fresh-eating quality caused by excessive acidity. The molecular mechanism underlying the coordinated accumulation of anthocyanins and malic acid in apple flesh remains largely unclear. In this study, transcriptome analysis of an F1 hybrid population derived from a cross of Malus sieversii ‘CSR6R6’ and Malus domestica cv. ‘Royal Gala’ identified an R2R3-MYB transcription factor, MdMYB21, which was further confirmed as a key negative regulator of both metabolic pathways. Functional assays revealed that overexpressing MdMYB21 significantly inhibited the accumulation of anthocyanins and malic acid in apple calli and fruit tissues. Mechanistic investigations using chromatin immunoprecipitation sequencing (ChIP-Seq), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays demonstrated that MdMYB21 directly binds to promoters of core genes involved in anthocyanin synthesis and transport—including the glycosyltransferase gene MdAGT1 and glutathione S-transferase genes MdGSTU15, MdGSTU16, and MdGSTU22—and acts as a transcriptional repressor that inhibits their expression. These findings uncover a novel mechanism by which MdMYB21 coordinately regulates color and flavor metabolism in apple, providing an important genetic target for breeding new red-fleshed apple varieties with a desirable balance of visual and sensory quality.

Chloroplasts play a crucial role in plant immunity against invading microbes. However, whether photosynthetic metabolites from chloroplasts participate directly in host defenses remains poorly understood. Here, we uncovered that an Arabidopsis thaliana triose phosphate/phosphate translocator (TPT) in the inner membrane of the chloroplast envelope suppresses viral infection and evokes defense responses. AtTPT overexpression impairs virus accumulation in plants, and loss-of-function tpt-3 mutants exhibit an increased viral load. The antiviral activity of AtTPT requires its metabolite transport capacity, implying that it indeed functions through its metabolite(s). To this end, we found that glyceraldehyde 3-phosphate (GAP), one of the metabolites translocated by AtTPT, drastically enhances the expression of defense-related genes and induces defense signaling pathways. Moreover, AtTPT or GAP robustly impairs the proliferation of diverse phytopathogens. Therefore, we propose that AtTPT exports GAP to mediate broad-spectrum pathogen resistance, which provides insights into the mechanisms underlying chloroplast-mediated immunity induced by a photosynthetic metabolite.

Salt stress is an important abiotic stress affecting the growth and fruit quality of apple fruits. Although jasmonic acid (JA) hormones and WRKY transcription factors (TFs) have both been reported to be involved in plant salt stress responses, the molecular mechanisms by which JA-mediated WRKY TFs regulate salt stress in apples remain unclear. Here, we report the identification of a WRKY family TF from apple, MdWRKY9, and its involvement in apple salt tolerance by regulating the expression of Na+/H+ antiporters, MdNHX1, and MdSOS2. Furthermore, we show that the protein repressors MdJAZ5 and MdJAZ10 in the JA signaling pathway can both interact with MdWRKY9 to form a complex and inhibit its DNA-binding and transcriptional activation activity. The JA signal triggers the degradation of MdJAZ5 and MdJAZ10 proteins by the 26S proteasome, disrupting the JAZ-WRKY protein complex and thereby releasing MdWRKY9 to activate downstream gene expression, promoting salt tolerance in apples. These findings provide important insights into the molecular mechanism of the WRKY TFs in JA-mediated salt tolerance in plants.

A novel tri-segmented virus, tentatively named "Chenopodium trirhavirus 1" (CheTRV1), was identified in Chenopodium album in China using high-throughput sequencing combined with conventional Sanger sequencing after RT-PCR. The genome of CheTRV1 consists of three negative-sense single-stranded RNAs designated as RNA1, RNA2, and RNA3. RNAs 1-3 of CheTRV1 share 57.93-64.41%, 48.16-56.96%, and 38.12-42.22% nucleotide sequence identity with the corresponding segments of previously reported trirhaviruses. The 3'- and 5'-termini of the three segments are similar and exhibit partial inverse complementarity. The ORFs of CheTRV1 are separated by a conserved gene junction region with the consensus motif 3'-AAUUCUUUUGN(N)nUUC- 5'. RNA1 consists of 6749 nucleotides (nt) and encodes a single putative L protein. RNA2 is 4393 nt in length and contains four genes in the order 3'-N-P2-P3-P4-5'. The smaller RNA3 consists of 3897 nt and has three genes in the order 3'-P6-P7-P8-5'. Phylogenetic analysis and pairwise comparisons suggested that CheTRV1 is most closely related to Medicago trirhavirus 1, with 68.82% amino acid sequence identity in the L protein. This is the first report of the complete genome sequence of a tri-segmented rhabdovirus, and it expands our knowledge of rhabdovirus evolution.

'Shannongsu' pear is a new high-quality cultivar. To ascertain the storage characteristics of 'Shannongsu' pears at low temperatures (0 ± 0.5 °C), the following parameters were determined: fruit firmness, ethylene, aromatic compounds, sugar content, acidity, ascorbic acid, and the expression levels of ethylene-related genes and texture-softening genes. The firmness of 'Shannongsu' pears changed less than that of the control, decreasing by only 18.8% after 170 days of storage. Low temperatures suppressed the expression of key genes associated with PbACS1a and PbACO1. Moreover, the expression of key genes related to fruit softening (PbPG1, PbXET, PbPME, and Pbα-L-Af) was suppressed during storage at low temperatures and remained at low levels. Therefore, the low levels of ethylene biosynthesis and the expression of key genes involved in fruit softening might play a major role in the excellent storage characteristics of the 'Shannongsu' cultivar. After 170 days of storage, 'Shannongsu' pears did not show significant changes in key quality dimensions such as firmness, sugar, acid, sugar-acid ratio, and ascorbic acid content. Therefore, low temperatures could help maintain the freshness, flavor, and nutritional quality of the 'Shannongsu' pear. Our findings reveal for the first time the low-temperature storage characteristics of 'Shannongsu' pears, providing a new scientific theoretical basis for pear production and marketing.

The ubiquitin-26S proteasome system (UPS) is a conserved protein degradation process involved in plant growth and immunity. However, whether some UPS E3 ligases directly target plant viruses in the endoplasmic reticulum (ER) remains less understood. Here, we identify an E3 ubiquitin ligase Hmg-CoA reductase degradation 1 of Nicotiana benthamiana (NbHRD1) interacting with the triple gene block (TGB) movement proteins of beet necrotic yellow vein virus (BNYVV) in the ER. The TGB proteins are ubiquitinated by NbHRD1 and then degraded by the UPS. Consequently, overexpression of NbHRD1a significantly inhibits BNYVV infection, whereas silencing of NbHRD1 promotes BNYVV infection in N. benthamiana. Moreover, NbHRD1a mainly impairs BNYVV cell-to-cell movement, rather than virus replication. Interestingly, NbHRD1 also targets the TGB proteins of potato virus X for ubiquitination and virus inhibition. Collectively, our results demonstrate that NbHRD1 is an important antiviral component targeting plant viruses with TGB movement proteins.

Chloroplasts play a crucial role in plant immunity against invading microbes. However, it remains poorly understood whether photosynthetic metabolites from chloroplasts participate directly in host defenses. Here, we uncovered Arabidopsis thalinana triose phosphate/phosphate translocator (AtTPT), a known translocator for chloroplast inner membrane, plays an indispensable role in suppressing virus infection and evoking defense responses. Interestingly, overexpression of AtTPT impairs virus accumulation in plants, while loss-of-function tpt3 mutants exhibit an increased viral load. The antiviral activity of AtTPT requires its phosphate transport capacity, implying that it actually functions through its metabolite(s). To this end, we found that glyceraldehyde 3-phosphate (GAP), one of AtTPT’s translocated metabolites, can drastically enhance expression of defense-related genes and prominently induce defense signaling pathways. More excitingly, AtTPT or GAP robustly restricts the proliferation of multiple types of phytopathogens. Collectively, we propose that AtTPT exports GAP to mediate broad-spectrum resistance to pathogens, which provides new insights into the mechanism underlying the chloroplast-mediated immunity by a photosynthetic metabolite. ### Competing Interest Statement The authors have declared no competing interest.

microRNAs (miRNAs) influence many biological processes at the post-transcriptional level. However, the molecular characterization of miRNAs in the Myzus persicae response to Brassica yellows virus (BrYV) stress remains unclear. In this study, we present the results of miRNA profiling in Myzus persicae under two different treatments: treatment one (raised on turnip plants), and treatment two (raised on Arabidopsis thaliana). A total of 72 known and 113 novel mature miRNAs were identified in both non-viruliferous and viruliferous aphids, under treatment one. In treatment two, 72 known and 112 novel mature miRNAs were identified in BrYV-free aphids; meanwhile, 71 known and 115 novel miRNAs were identified in BrYV-carrying aphids. Moreover, eight upregulated and four downregulated miRNAs were identified in viruliferous aphids under treatment two, whereas only two miRNAs were differentially expressed under treatment one. These results indicated the relative BrYV level could influence miRNA expression in aphids. KEGG enrichment analysis showed the predicted genes targeted by differentially expressed miRNAs were primarily involved in Peroxisome, neuroactive ligand–receptor interaction, and metabolism of xenobiotics by cytochrome P450 pathways. Taken together, these findings reveal the effect of BrYV on miRNAs in Myzus persicae and provide key clues for further studies on the molecular mechanisms of BrYV transmission via aphids.

MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red-flesh phenotype due to a minisatellite insertion in its R-6 promoter, but R-6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull-down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB-bHLH-WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163-bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R-6 by structural variation analysis, contains a typical W-box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R-6 background. These findings reveal a novel regulatory role of the WRKY-containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants.

Wheat yellow mosaic virus (WYMV) causes severe viral wheat disease in Asia. The WYMV P1 protein encoded by RNA2 has viral suppressor of RNA silencing (VSR) activity to facilitate virus infection, however, VSR activity has not been identified for P2 protein encoded by RNA2. In this study, P2 protein exhibited strong VSR activity in Nicotiana benthamiana at the four-leaf stage, and point mutants P70A and G230A lost VSR activity. Protein P2 interacted with calmodulin (CaM) protein, a gene-silencing associated protein, while point mutants P70A and G230A did not interact with it. Competitive bimolecular fluorescence complementation and competitive co-immunoprecipitation experiments showed that P2 interfered with the interaction between CaM and calmodulin-binding transcription activator 3 (CAMTA3), but the point mutants P70A and G230A could not. Mechanical inoculation of wheat with in vitro transcripts of WYMV infectious cDNA clone further confirmed that VSR-deficient mutants P70A and G230A decreased WYMV infection in wheat plants compared with the wild type. In addition, RNA silencing, temperature, ubiquitination and autophagy had significant effects on accumulation of P2 protein in N. benthamiana leaves. In conclusion, WYMV P2 plays a VSR role in N. benthamiana and promotes virus infection by interfering with calmodulin-related antiviral RNAi defense.

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.

Potassium (K+) plays crucial roles in both plant development and immunity. However, the function of K+ in plant-virus interactions remains largely unknown. Here, we utilized Barley yellow striate mosaic virus (BYSMV), an insect-transmitted plant cytorhabdovirus, to investigate the interplay between viral infection and plant K+ homeostasis. The BYSMV accessory P9 protein exhibits viroporin activity by enhancing membrane permeability in Escherichia coli. Additionally, P9 increases K+ uptake in yeast (Saccharomyces cerevisiae) cells, which is disrupted by a point mutation of glycine 14 to threonine (P9G14T). Furthermore, BYSMV P9 forms oligomers and targets to both the viral envelope and the plant membrane. Based on the recombinant BYSMV-GFP (BYGFP) virus, a P9-deleted mutant (BYGFP Delta P9) was rescued and demonstrated infectivity within individual plant cells of Nicotiana benthamiana and insect vectors. However, BYGFP Delta P9 failed to infect barley plants after transmission by insect vectors. Furthermore, infection of barley plants was severely impaired for BYGFP-P9G14T lacking P9 K+ channel activity. In vitro assays demonstrate that K+ facilitates virion disassembly and the release of genome RNA for viral mRNA transcription. Altogether, our results show that the K+ channel activity of viroporins is conserved in plant cytorhabdoviruses and plays crucial roles in insect-mediated virus transmission. The viroporin of a plant cytorhabdovirus exhibits K+ channel activity and is essential for cross-kingdom virus transmission from insect vectors to host plants.

Apple (Malus domestica Borkh.) is among the most widely planted and economically valuable horticultural crops globally. Over time, the apple fruit's cut surface undergoes browning, and the degree of browning varies among different apple varieties. Browning not only affects the appearance of fruits but also adversely affects their taste and flavor. In the present study, we observed browning in different apple varieties over time and analyzed the expression of genes in the polyphenol oxidase gene family. The results indicated a strong correlation between the browning degree of the fruit and the relative expression of the polyphenol oxidase gene MdPPO2. With the MdPPO2 promoter as bait, the basic leucine zipper (bZIP) transcription factor MdbZIP44 was identified using the yeast single-hybrid screening method. Further investigation revealed that the overexpression of MdbZIP44 in ‘Orin’ callus could enhance the expression of MdPPO2 and promote browning of the callus. However, knocking out MdbZIP44 resulted in a callus with no apparent browning phenotype. In addition, our results confirmed the interaction between MdbZIP44 and MdbZIP11. In conclusion, the results indicated that MdbZIP44 can induce apple fruit browning by activating the MdPPO2 promoter. The results provide a theoretical basis for further clarifying the browning mechanism of apple fruit.