Lycoris plants are known for their diverse flower colors, but the molecular mechanisms behind these variations remain unclear. In this study, we first used the CIELAB system to precisely measure flower color. We objectively defined the petals of Lycoris sprengeri as blue-purple (Bp) and compared them with the white petals of Lycoris longituba (W) and the red petals of Lycoris radiata var. pumila (R). Metabolomic analysis showed that specific kaempferol glycosides, including kaempferol-3-O-sophoroside and lonicerin, accumulated significantly in the blue-purple petals. Transcriptomic analysis revealed that genes related to flavonoid biosynthesis were generally more active in the colored petals (Bp and R). However, different expression patterns of key hydroxylase genes created a metabolic split. Specifically, the blue-purple petals showed high expression of LrF3'5'H (directing synthesis toward delphinidin) and LrFLS (promoting kaempferol accumulation), whereas the red petals mainly expressed LrF3'H (leading to cyanidin synthesis). Further investigation identified LrWRKY70 as a core transcription factor highly correlated with these flavonoid pathway genes. Crucially, we discovered a new long non-coding RNA, LncRNA401, located downstream of the LrWRKY70 antisense strand. It showed a strong positive correlation with LrWRKY70. Functional verification through transient overexpression demonstrated that LncRNA401 significantly increased the expression of LrWRKY70. This, in turn, broadly activated downstream flavonoid biosynthesis genes, including LrCHS, LrF3'5'H, LrFLS, and LrDFR. This cascade ultimately promoted the synthesis of anthocyanins and kaempferol derivatives, resulting in the unique blue-purple phenotype. Our results reveal a novel LncRNA401-LrWRKY70 regulatory module. This module plays a key role in metabolic reprogramming for flower color formation in Lycoris, providing important insights into plant secondary metabolism and valuable targets for breeding specific flower colors.

Abstract Understanding how plants adapt to extreme cold and ripen fruit is a key question in plant biology. Lonicera caerulea (blue honeysuckle), a cold-tolerant berry species native to the northern temperate zone, is valued as a ‘noble fruit’ for its rich nutrient profile, and serves as an ideal model for studies due to its unique ability to thrive in low temperatures and ripen fruit during early spring. Here, we generated a chromosome-level genome assembly for wild L. caerulea from the Changbai Mountain region. We integrated it with transcriptomic and metabolomic analyses to explore its molecular cold adaptation mechanisms. We found that L. caerulea achieves cold tolerance and fruit ripening through dynamic regulation of two important metabolic pathways. Under cold stress, the LcODO1/LcMYB15–LcSLAS module promotes iridoids accumulation, while the LcbHLH62/LcMYB3R5–LcF3GT module enhances anthocyanin biosynthesis-jointly increasing plant cold resistance. During fruit maturation, metabolite profiles show dynamic reprogramming: anthocyanin levels rise in both pulp and peel, while the content of bitter iridoids markedly decreases in the pulp. This work provides a comprehensive genomic framework for understanding cold adaptation mechanisms, offering valuable gene resources and a theoretical foundation for improving cold resistance and fruit quality in breeding programs.

To investigate the effect of sorbitol feeding on immune responses and its underlying mechanism, this study employed crabapple (Malus micromalus), a species native to China, as the experimental material. Detached leaves were subjected to sorbitol feeding experiments, with water feeding as the control. The dynamic changes in lesion area and cell membrane permeability were measured across four groups of leaves following feeding and fungal inoculation, and transcriptome sequencing was performed on these four groups. The results revealed that compared to the control group, leaves fed with sorbitol prior to inoculation exhibited significantly reduced lesion areas and decreased cell membrane permeability. Transcriptome and KEGG pathway analyses indicated that differentially expressed genes were significantly enriched in the phenylpropanoid biosynthesis pathway. HPLC results demonstrated that quercetin and isorhamnetin contents increased significantly after sorbitol feeding, and both compounds were shown to inhibit the mycelial growth of Alternaria alternata, thereby enhancing the disease resistance of crabapple leaves. Among the associated genes, MmF3H1, which regulates the synthesis of quercetin and isorhamnetin, showed the highest level of enrichment. Co-expression trend analysis and qPCR results revealed that MmNAC17 and MmbZIP1 exhibited strong co-expression relationships with MmF3H1 and responded to sorbitol regulation. Functional validation was conducted by constructing overexpression and silencing vectors for MmNAC17/MmbZIP1 and performing transient transformation, thereby elucidating the molecular and biochemical mechanisms underlying the response of M. micromalus to A. alternata. This study provides a theoretical reference for breeding crabapple varieties with enhanced resistance to leaf spot disease.

For the automation of Agrobacterium-mediated genetic transformation of tissue-cultured plantlets, accurate leaf vein segmentation is essential. The thin, low-contrast structure of leaf veins frequently leads to fragmented segmentation outputs, despite the proposal of various methodologies for vein segmentation. To address this issue, we propose Direction-Aware U-Net (DA-UNet), an improved U-Net architecture that incorporates a Direction-Aware Context Pooling (DACPool) module and Topology-aware Segmentation loss (TopoSeg loss). The DACPool module explicitly exploits vein orientation to aggregate directional contextual information, while the TopoSeg loss jointly optimizes pixel-level accuracy and topological continuity. DA-UNet achieves efficient leaf vein segmentation with improved continuity and structural integrity, according to evaluations on the self-constructed Tissue-Cultured Plantlet Vein Dataset 2025 (TCPVD2025). Comparative experiment results show that the improved model outperforms PSPNet, DeepLabV3+, U-Net, TransUNet, Swin-UNet, CCNet, and SegNeXt, as evidenced by Recall, Dice, and CONNECT scores of 71.35%, 69.08%, and -2.25, while maintaining competitive Precision of 66.98%. Ablation experiment results provide further evidence for the efficacy of the TopoSeg loss and the DACPool module. The results demonstrate the effectiveness of the proposed vein segmentation framework for generating outputs that are both accurate and structurally consistent, thus enabling reliable automated processes for plant genetic transformation.

Objective This study investigated differences in flavonoid-related gene expression and metabolite profiles in the flesh of red-fleshed grape berries and their self-pollinated progenies, aiming to elucidate the metabolic accumulation characteristics of flavonoid biosynthesis, and provide insights into transcriptional regulation, so as to lay a theoretical foundation for improving flavonoid contents in grape berries and breeding new grape varieties. Method The red-fleshed grape germplasm Zhongshanhongyu (ZSHY), its self-pollinated progenies Nan 19 (N19) and Nan 30 (N30), and Muscat (MGX) were used as materials. Four pairwise comparison groups (ZSHY_vs_MGX, N30_vs_ZSHY, N19_vs_MGX, and N30_vs_N19) were established for targeting flavonoid metabolomics alongside transcriptome sequencing (RNA-seq) of berry flesh. Enrichment analyses were subsequently conducted to characterize flavonoid metabolites accumulation, identify key structural genes, and dissect the transcriptional regulatory networks involved in flavonoid biosynthetic pathway. Result Based on targeted flavonoid metabolomics technology, a total of 104 differentially accumulated flavonoid metabolites were detected, and 62 core metabolites exhibiting significant differences were further identified. Notably, most flavonols and flavanols accumulated to substantially higher levels in ZSHY and N19 than in N30 and MGX. KEGG enrichment analysis revealed that these differential metabolites were predominantly enriched in flavonoid biosynthesis pathways, particularly responsible for anthocyanin, flavone, and flavonol biosynthesis. Transcriptome profiling identified 758 differentially expressed genes (DEGs), which were significantly overrepresented in flavonoid and phenylpropanoid biosynthesis pathways. Furthermore, 30 core DEGs directly involved in flavonoid biosynthesis were screened, whose expression patterns were broadly consistent with the metabolite accumulation profiles, showing high transcript abundance in ZSHY and N19, but low expression in N30 and MGX. In addition, 22 transcription factors (TFs) significantly correlated with flavonoid structural genes were identified, mainly belonging to the WRKY, MYB, and ERF families. Conclusion Significant differences existed in flavonoid metabolism between the red-fleshed grape and its self-pollinated progenies. The elevated flavonoid accumulation in the flesh of ZSHY and N19 was closely associated with the upregulation of the key structural genes in the flavonoid pathway, and the identified TFs might play important regulatory roles in this process.

Rapid climate shifts in high-latitude regions profoundly impact the geographical distribution boundaries and molecular adaptive strategies of cold-tolerant plants. Lonicera caerulea, known for its excellent cold tolerance, provides an ideal model for exploring the molecular mechanisms of climate adaptation and trait formation. To elucidate the ecological and molecular mechanisms of climate adaptation in L. caerulea, we integrated species distribution modeling with multi-tissue transcriptome profiling. Distribution modeling identified temperature and precipitation as primary constraints on its current range, with projections suggesting a significant poleward expansion under future warming scenarios. At the molecular level, we identified 19 LcSWEET genes exhibiting functional differentiation. Rather than listing specific candidates, our findings highlight that certain LcSWEET members are transcriptionally activated during fruit maturation, while others are significantly upregulated in response to cold stress, underscoring their dual roles in plant reproduction and ecological adaptation. This study revealed that the LcSWEET gene family exhibits functional diversification in tissue-specific expression and low-temperature stress response. It provides molecular candidates that may inform future studies on plant adaptive response under climate change and lays a theoretical foundation for germplasm conservation and high-quality breeding of L. caerulea.

With rising demand for food safety and sustainable packaging, the creation of antimicrobial and environmentally sustainable materials has become a critical research focus. In this study, a polyvinyl alcohol (PVA) film incorporated with visible-light-responsive graphitic carbon nitride (g-C3N4) was fabricated, exhibiting remarkable antimicrobial performance. The composite film achieved photocatalytic antimicrobial efficiencies of 99.99 % against Acinetobacter baumannii, 88.26 % against Staphylococcus aureus, and 98.33 % against Escherichia coli. The g-C3N4 was treated via wet ball milling, which significantly enhanced the film's mechanical properties, water vapor impermeability, UV resistance, and antimicrobial performance while reducing hydrophilicity. Additionally, the film demonstrated notable effectiveness in strawberry preservation, extending shelf life to 96 h under ambient conditions, compared to 48 and 72 h for polyethylene (PE) and pure PVA films, respectively. Importantly, the recyclable composite retained its mechanical integrity and antimicrobial efficiency after multiple reuse cycles. This study provides innovative insights into the development of efficient, sustainable, and environmentally conscious food packaging materials.

Soil salinization significantly limits agricultural yields; therefore, it is crucial to analyze grape salt tolerance mechanisms to better strategically place grape industries in saline–alkali soils. This study employed four grape varieties, including ’Kyoho’ , ’Beibinghong’ , ’Thompson Seedless’ and ’Muscat Hamburg’ to investigate salt stress. The results indicated that ’Kyoho’ grapes had the highest salt tolerance, while ’Beibinghong’ showed the lowest tolerance. Furthermore, ’Thompson Seedless’ and ’Muscat Hamburg’ were classified as moderately salt-tolerant and salt-sensitive varieties, respectively. Moreover, transcriptome sequencing was performed to evaluate the molecular mechanisms of grape salt tolerance using leaves of the four varieties at 0, 1, 3, and 5 days post-salt stress. The results showed that VvMYB308 and VvCHI2 are key enzymes associated with flavonoid synthesis and respond to salt stress in grapes. Similarly, physiological analysis indicated substantially increased Quercitrin levels in the salt-tolerant’Kyoho’variety under salt stress. Subcellular localization analysis showed that VvMYB308 resides in the nucleus and interacts with the VvCHI2 promoter. Similarly, transient transformation assays revealed that VvMYB308 positively modulates salt tolerance in grape tissue-cultured seedlings. This study indicated that the VvMYB308-VvCHI2 pathway modulates grape salt tolerance by promoting Quercitrin synthesis. These findings provide theoretical evidence for the breeding of salt-tolerant grape varieties and their cultivation in saline‐alkali soils.

Sorbitol is a major primary metabolite in Malus species, serving as the primary product of photosynthesis and the main form of carbon translocation. It also functions as a signaling molecule that regulates plant growth, development, and stress responses. Fungal foliar diseases are widespread in Malus and can cause substantial economic losses, yet the molecular mechanism underlying sorbitol-mediated disease resistance remains poorly understood. This study focused on Malus resistance to Alternaria alternata Mr1 and found that sorbitol markedly increased the accumulation of flavonoids, including phlorizin, phloretin, and catechin, enhancing disease resistance. Based on our previous transcriptome data, transcriptome analysis identified the chromatin remodeling factor LFR as a susceptibility-associated gene. LFR was transcriptionally downregulated by sorbitol and negatively regulated flavonoid biosynthesis. This study further identified the transcription factor MYBR1, whose expression showed a negative correlation with that of LFR. MYBR1 was activated by sorbitol and facilitated flavonoid accumulation, and its overexpression restored resistance to Alternaria alternata Mr1 in plants with high LFR expression. Collectively, sorbitol suppresses LFR expression, relieving its inhibitory effect on MYBR1, which in turn enhances flavonoid accumulation and boosts resistance to Alternaria alternata Mr1 in Malus.

Cold damage poses a significant challenge to the cultivation of soft-seeded pomegranate varieties, hindering the growth of the pomegranate industry. The genetic basis of cold tolerance in pomegranates has remained elusive, largely due to the lack of high-quality genome assemblies for cold-tolerant varieties and comprehensive population-scale genomic studies. In this study, we addressed these challenges by assembling a high-quality chromosome-level reference genome for 'Sanbai', a pomegranate variety renowned for its freezing resistance, achieving an impressive contig N50 of 15.93 Mb. This robust assembly, enhanced by long-read sequencing of 38 pomegranate accessions, facilitated the identification of 14 239 polymorphic structural variants, revealing their critical roles in genomic diversity and population differentiation related to cold tolerance. Of particular significance was the discovery of a ~ 5.4-Mb inversion on chromosome 1, which emerged as an important factor affecting cold tolerance in pomegranate. Moreover, through the integration of bulked segregant analysis, differential selection analysis, and genetic transformation techniques, we identified and validated the interaction between the PgNAC12 transcription factor and PgCBF1, disclosing their pivotal roles in response to cold stress. These findings mark a significant advancement in pomegranate genomics, offering novel insights into the genetic mechanisms of cold tolerance and providing valuable resources for the genetic improvement of soft-seeded pomegranate varieties.

Blue honeysuckle (Lonicera caerulea L.) is an important cash crop growing in freezing regions with high freezing tolerance. Previous studies have shown that reactive oxygen species (ROS) play an important role in plant defense against low-temperature stress, however, the regulatory mechanism of freezing resistance network formed during the long evolutionary evolution of blue honeysuckle is not clear. By treating blue honeysuckles at -4 °C for 0 h, 12 h, 24 h, and 48 h, it was found that the contents of ROS and malondialdehyde increased as well as the activities of antioxidant enzymes were enhanced. Analysis of the transcriptome data revealed a large number of differentially expressed genes enriched in the glutathione metabolic pathway. Among them, LcGST, LcGSS, LcGGT, LcGSR, LcGPX genes were differentially expressed and enriched in the glutathione synthesis pathway under low-temperature stress in blue honeysuckle. The transient overexpression of transcription factor LcERF107 demonstrated the transcriptional regulation of glutathione metabolism pathway genes and its ability to increase the activities of antioxidant enzymes such as CAT, APX, SOD and POD to increase the scavenging capacity of reactive oxygen species to improve the tolerance to low-temperature stress. In this study, we demonstrated that the transcription factor LcERF107 activated the expression of genes related to oxygen glutathione metabolism and significantly enhanced the activities of antioxidant enzymes, which led to the accumulation of ROS and reactive oxygen species in the body, thus improving the freezing tolerance of blue honeysuckles under low-temperature stress.

As the focus on green chemistry intensifies, researchers are progressively looking to incorporate biodegradable and environmentally friendly solvents. Given the prevalent use of inorganic solvents in conventional methods for detecting selenium content, this study utilized a mixture design approach to create four deep eutectic solvents (DESs). The elements of the DESs consisted of six different compounds: guanidine hydrochloride, fructose, glycerol, citric acid, proline, and choline chloride. The synthesized deep eutectic solvents (DESs) exhibited a uniform and transparent appearance. The ideal ratios for each DES were established based on their density and viscosity measurements, leading to the formulations of DES1 (34% guanidine hydrochloride, 21% fructose, 45% water), DES2 (23% guanidine hydrochloride, 32% glycerol, 45% water), DES3 (27.5% citric acid, 27.5% proline, 45% water), and DES4 (30% choline chloride, 25% citric acid, 45% water). The characterization of the deep eutectic solvents (DESs) was performed using nuclear magnetic resonance (NMR) spectroscopy and infrared (IR) spectroscopy, which confirmed the molecular formation of each DES. Following this, the DESs were applied as extraction solvents in a process involving ultrasonic-assisted microextraction (UAE) combined with inductively coupled plasma mass spectrometry (ICP-MS) to assess the selenium levels in selenium-rich rice. The results were benchmarked against traditional microwave-assisted acid digestion (TM-AD), revealing selenium recovery rates ranging from 85.5% to 106.7%. These results indicate that UAE is an effective method for extracting selenium from selenium-rich rice, thereby establishing a solid data foundation for the environmentally friendly analysis of selenium content in rice.

Flavonoid hyperoside boosts okra reproduction; its role in pigeon pea, with poor seed set, remains unclear. We found that hyperoside increases pigeon pea seed set by promoting pollen tube growth, a process that benefits from long noncoding RNAs (lncRNAs). Two lncRNAs, lnc187 and lnc999, are regulated by hyperoside and synergize to regulate target genes. Among them, lnc187 is the key effector; its loss may abolish downstream function. These lncRNAs up-regulate CcGPP (GRAS phosphorylase) kinase expression by binding to its promoter and acting as a scaffold to connect MRP23 and RNA polymerase while simultaneously inhibiting CcGDP (GRAS dephosphorylase) phosphatase activity by binding to key protein sites. Genetic evidence also confirms the relationship between lnc187/lnc999, MRP23, and the downstream CcGPP/CcGDP. This study clarifies the flavonoid hyperoside triggered regulatory axis where lnc187/lnc999 promotes hyperphosphorylated CcGRAS to regulate pollen tube growth and seed set in pigeon pea, offering insights into reproductive development research for high-value woody species.

Sorbitol, a main photosynthate and transport carbohydrate in all tree fruit species in Rosaceae, acts as a signal controlling resistance against Alternaria (A.) alternata in apple by altering the expression of the MdNLR16 resistance gene via the MdWRKY79 transcription factor. However, it is not known if N6-methyladenosine (m6A) methylation of the mRNAs of these genes participates in the process. Here, we found that decreased sorbitol synthesis in apple leaves leads to a transcriptome-wide reduction in the m6A modification, with fewer transcripts containing two or more methylation sites. We identified two methyltransferases, MdVIR1 and MdVIR2, that respond to sorbitol and A. alternata inoculation and positively control resistance to A. alternata. MdVIR1 and MdVIR2 act on MdWRKY79 and MdNLR16 mRNAs, and the resulting m6A modification stabilizes their mRNAs and improves translation efficiency. These data identify that m6A modification through MdVIR1 and MdVIR2 methyltransferases is essential for sorbitol-controlled resistance to A. alternata.

Sorbitol is an important primary metabolite that serves as both a carbon source and signal to pathogens. The leaf diseases caused by Alternata alternata are particularly serious in crabapple (Malus micromalus). Here, we found that sorbitol can enhance the resistance of crabapple to A. alternata R1 by increasing the content of flavonoid catechin. Nanomaterials as an emerging technology tool can efficiently deliver lncRNA to target cells. Here, we found nanoencapsulated lncRNA809 (SPc/lncRNA809) exhibits significant resistance to R1strain. To elucidate the effect of SPc/lncRNA809 on flavonoids catechin synthesis, we observed the expression of lncRNA809 was consistent with that of MmNAC17 which regulates the synthesis of catechin and both could jointly respond to sorbitol. MmNAC17 induced the accumulation of catechin in vivo by directly activating the expression of catechin synthase genes MmF3H and MmLAR. Correspondingly, overexpression of lncRNA809 significantly upregulated the expression of MmNAC17 and enhanced the disease resistance. This study reveals for the first time that sorbitol positively regulates the expression of MmNAC17 through lncRNA809, promoting the accumulation of catechin via the expression of MmF3H and MmLAR, ultimately improving the defense response of M. micromalus. This research provides a crucial foundation for the establishment and application of sorbitol-based signaling regulatory networks.

The olive tree is an important oil woody plant with high economic value, yet it is vulnerable to the attack of numerous fungi. The successful control of olive fungal diseases requires a comprehensive understanding of the disease resistance mechanisms in plants. Here, we isolated Alternaria alternata from the diseased leaves of olive plants, and screened a resistant ("Leccino") and susceptible ("Manzanilla de Sevilla") cultivar from eight olive cultivars to explore their resistance mechanisms. Transcriptomic and metabolomic analyses identified the flavonoid biosynthesis as a key defense pathway against A. alternata. Five important transcription factors associated with flavonoid biosynthesis were also determined. The overexpression of OeWRKY40 significantly enhanced the disease resistance of the susceptible cultivar and upregulated the expression of genes involved in flavonoid biosynthesis and the accumulation of related metabolites. LUC assays further proved that OeWRKY40 can activate the expression of OeC4H. These results help to better clarify the molecular mechanisms of flavonoid biosynthesis against A. alternata. Our study provides key information for further exploration of the molecular pathways of olive plants and their resistance to fungi, an important factor for molecular breeding and utilization of resistant cultivars.

MAIN CONCLUSION:Integrated transcriptome and physiological analysis of apricot leaves after Fusarium solani treatment. In addition, we identified core transcription factors and flavonoid-related synthase genes which may function in apricot disease resistance. Apricot (Prunus armeniaca) is an important economic fruit species, whose yield and quality of fruit are limited owing to its susceptibility to diseases. However, the molecular mechanisms underlying the response of P. armeniaca to diseases is still unknown. In this study, we used physiology and transcriptome analysis to characterize responses of P. armeniaca subjected to Fusarium solani. The results showed increasing malondialdehyde (MDA) content, enhanced peroxidase (POD) and catalase (CAT) activity during F. solani infestation. A large number of differentially expressed genes (DEGs), which included 4281 upregulated DEGs and 3305 downregulated DEGs, were detected in P. armeniaca leaves exposed to F. solani infestation. Changes in expression of transcription factors (TFs), including bHLH, AP2/ERF, and WRKY indicated their role in triggering pathogen-responsive genes in P. armeniaca. During the P. armeniaca response to F. solani infestation, the content of total flavonoid was changed, and we identified enzyme genes associated with flavonoid biosynthesis. Ectopic overexpression of PabHLH15 and PabHLH102 in Nicotiana benthamiana conferred elevated resistance to Fspa_1. Moreover, PabHLH15 and PabHLH102 positively interact with the promoter of flavonoid biosynthesis-related genes. A regulatory network of TFs regulating enzyme genes related to flavonoid synthesis affecting apricot disease resistance was constructed. These results reveal the potential underlying mechanisms of the F. solani response of P. armeniaca, which would help improve the disease resistance of P. armeniaca and may cultivate high-quality disease-resistant varieties in the future.

Flavonoids are important secondary metabolites in the plant growth and development process. As a medicinal plant, pigeon pea is rich in secondary metabolites. As a flavonoid, there are few studies on the regulation mechanism of naringenin in plant stress resistance. In our study, we found that naringenin can increase the pigeon pea???s ability to tolerate salt and influence the changes that occur in flavonoids including naringenin, genistein and biochanin A. We analyzed the transcriptome data after 1 mM naringenin treatment, and identified a total of 13083 differentially expressed genes. By analyzing the metabolic pathways of these differentially expressed genes, we found that these differentially expressed genes were enriched in the metabolic pathways of phenylpropanoid biosynthesis, starch and sucrose metabolism and so on. We focused on the analysis of flavonoid biosynthesis related pathways. Among them, the expression levels of enzyme genes CcIFS, CcCHI and CcCHS in the flavonoid biosynthesis pathway had considerably higher expression levels. By counting the number of transcription factors and the binding sites on the promoter of the enzyme gene, we screened the transcription factors CcMYB62 and CcbHLH35 related to flavonoid metabolism. Among them, CcMYB62 has a higher expression level than the others. The hairy root transgene showed that CcMYB62 could induce the upregulation of CcCHI, and promote the accumulation of naringenin, genistein and biochanin A. Our study revealed the molecular mechanism of naringenin regulating flavonoid biosynthesis under salt stress in pigeon pea, and pro-vided an idea for the role of flavonoids in plant resistance to abiotic stresses.

Aluminium (Al) toxicity decreases crop production in acid soils in general, but many crops have evolved complex mechanisms to resist it. However, our current understanding of how plants cope with Al stress and perform Al resistance is still at the initial stage. In this study, the citrate transporter CcMATE35 was identified to be involved in Al stress response. The release of citrate was increased substantially in CcMATE35 over-expression (OE) lines under Al stress, indicating enhanced Al resistance. It was demonstrated that transcription factor CcNFYB3 regulated the expression of CcMATE35, promoting the release of citrate from roots to increase Al resistance in pigeon pea. We also found that a Long noncoding RNA Targeting Citrate Synthase (CcLTCS) is involved in Al resistance in pigeon pea. Compared with controls, overexpression of CcLTCS elevated the expression level of the Citrate Synthase gene (CcCS), leading to increases in root citrate level and citrate release, which forms another module to regulate Al resistance in pigeon pea. Simultaneous overexpression of CcNFYB3 and CcLTCS further increased Al resistance. Taken together, these findings suggest that the two modules, CcNFYB3-CcMATE35 and CcLTCS-CcCS, jointly regulate the efflux and synthesis of citrate and may play an important role in enhancing the resistance of pigeon pea under Al stress.

As a multifunctional hormone-like molecule, melatonin exhibits a pleiotropic role in plant salt stress tolerance. While actin cytoskeleton is essential to plant tolerance to salt stress, it is unclear if and how actin cytoskeleton participates in the melatonin-mediated alleviation of plant salt stress. Here, we report that melatonin alleviates salt stress damage in pigeon pea by activating a kinase-like protein, which interacts with an actin-depolymerizing factor. Cajanus cajan Actin-Depolymerizing Factor 9 (CcADF9) has the function of severing actin filaments and is highly expressed under salt stress. The CcADF9 overexpression lines (CcADF9-OE) showed a reduction of transgenic root length and an increased sensitivity to salt stress. By using CcADF9 as a bait to screen an Y2H library, we identified actin depolymerizing factor-related phosphokinase 1 (ARP1), a novel protein kinase that interacts with CcADF9. CcARP1, induced by melatonin, promotes salt resistance of pigeon pea through phosphorylating CcADF9, inhibiting its severing activity. The CcARP1 overexpression lines (CcARP1-OE) displayed an increased transgenic root length and resistance to salt stress, whereas CcARP1 RNA interference lines (CcARP1-RNAi) presented the opposite phenotype. Altogether, our findings reveal that melatonin-induced CcARP1 maintains F-actin dynamics balance by phosphorylating CcADF9, thereby promoting root growth and enhancing salt tolerance.