[Objective]To characterize the dynamic changes in sugar and organic acid composition across three key developmental stages in 14 major blueberry cultivars currently cultivated in China,which differ markedly in ripening period and flavor profile,and to elucidate the relationship between PEPC and organic acid metabolism in blueberry fruit,thereby providing theoretical support for blueberry production and flavor quality improvement.[Method]Fruits of 14 blueberry cultivars at three key ripening stages(green,pink,and blue)were used as materials.Soluble sugar and organic acid components were determined by HPLC,and the expression patterns of the PEPC gene were analyzed by qRT-PCR.[Result]During fruit ripening,the accumulation dynamics of TA and TSS varied among different blueberry cultivars and could be classified into three acid-accumulation patterns(continuous decline,stable-decline,and increase-decline)and three sugar-accumulation patterns(continuous increase,stable-increase,and decline-increase).Glucose and fructose were the major soluble sugar components in blueberry fruits,whereas citric acid,quinic acid,and malic acid were the predominant organic acids.Among them,quinic acid exhibited a relatively high coefficient of variation.Cluster analysis based on sugar and organic acid composition divided the 14 cultivars into four groups.In four cultivars,the major organic acids were citric acid,quinic acid,and malic acid;seven cultivars were dominated by citric acid and malic acid;and one cultivar was characterized primarily by citric acid and quinic acid.In these 12 cultivars,the proportions of glucose and fructose were relatively balanced.In contrast,the remaining two cultivars showed a clear predominance of quinic acid,with fructose content significantly higher than that of glucose.Total sugar content in blueberry fruits was significantly positively correlated with glucose and fructose contents,while total organic acid content was extremely significantly positively correlated with the contents of citric acid,quinic acid,oxalic acid,and lactic acid.In the three blueberry cultivars representing different acid-accumulation patterns,PEPC expression increased from the green fruit stage to the pink fruit stage,but was downregulated from the pink fruit stage to the blue fruit stage.During this latter period,the contents of malic acid and citric acid in the fruits also decreased correspondingly.[Conclusion]During blueberry fruit development,the variation patterns of TA and TSS were closely associated with cultivar characteristics.The major soluble sugar components were glucose and fructose,while the predominant organic acids were citric acid,quinic acid,and malic acid.Based on organic acid composition,the cultivars could be categorized into two types:high-citric-acid and high-quinic-acid types.In three blueberry cultivars representing different acid-accumulation patterns,PEPC expression was downregulated from the pink fruit stage to the blue fruit stage,which was consistent with the declining trends of malic acid and citric acid contents,indicating that PEPC is involved in the metabolism of organic acids in blueberry fruits.

Proanthocyanidins (PAs), polymers of flavan-3-ols, are crucial for the sensory quality and stress defense of mulberry (Morus spp.). The biosynthesis of their monomers, catechin and epicatechin, are catalyzed by leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), respectively, representing key rate-limiting steps that determine PA composition and abundance. In this study, systematic functional analyses including phylogenetic analysis, quantified spatio-temporal expression profiles during fruit development (S1-S4 stages), in vitro enzymatic assay, knock-down using Virus-Induced Gene Silencing (VIGS) in mulberry leaves and heterologous overexpression in Arabidopsis thaliana were conducted to reveal their roles in proanthocyanidin biosynthesis in mulberry. Results showed that MaLAR (969 bp) and MaANR (1014 bp) were successfully cloned and phylogenetically conserved. Spatio-temporal expression analysis revealed distinct patterns: MaLAR expression continuously increased, reaching highest expression level at the fully ripe stage (S4), whereas MaANR showed highest expression level at the color-turning stage (S2). In vitro enzymatic assays confirmed that MaLAR catalyzed the formation of catechin from leucoanthocyanidin, and MaANR catalyzed the formation of epicatechin from anthocyanidin. VIGS-mediated silencing of either gene in mulberry leaves led to significant reduction in total PA content. Conversely, heterologous overexpression of MaLAR or MaANR in Arabidopsis resulted in significant increase of PA in the seed coat. Our findings confirm that MaLAR and MaANR are conserved, key positive regulators of PA biosynthesis in mulberry. The differential expression patterns during fruit ripening suggest they play distinct, temporally regulated roles in determining the final PA composition and content. These results provide an important theoretical basis and represent important targets for the metabolic engineering and molecular breeding of mulberry for improved fruit quality.

This study established an integrated methodological framework for the efficient extraction and comprehensive analysis of flavonoids from mulberry (Morus alba L.). A high-performance liquid chromatography (HPLC) method was developed to simultaneously separate and identify 13 flavonoid components, including rutin, quercetin, kaempferol, and naringin, as well as their derivatives. Using seven representative flavonoids as standards, the ultrasonic-assisted extraction process was systematically optimized through single-factor and orthogonal experimental designs, followed by response surface methodology. The optimal extraction conditions were determined as follows: 80 % ethanol (v/v), an extraction time of 60 min, a solid-liquid ratio of 1:2.5 (g mL(-)& sup1;), and a temperature of 40 degrees C, conducted over two cycles. This optimized protocol achieved a high total flavonoid extraction yield of 84.96 %. The newly established HPLC protocol, employing a C18 column with gradient elution and dual-wavelength detection, demonstrated excellent stability and reproducibility in detecting target compounds across multiple mulberry samples. This approach features simple operation and high time efficiency. The optimized parameters suggest a profile compatible with potential scale-up, though direct evidence from pilot-scale tests and economic analysis would be required to confirm its practical industrial feasibility. Nonetheless, it provides a valuable methodological reference for the efficient extraction of natural active substances.

Heat stress transcription factors (HSFs) are central regulators of thermotolerance in plants. Although the HSF gene family has been well characterized in numerous species, its characterization in Morus alba remains ambiguous. In this study, 20 MaHSF members were identified and cloned from Morus alba, with systematically characterized. Phylogenetic analysis classified them into 3 groups and 12 subclasses, with gene duplication and synteny highlighting evolutionary conservation among perennial plants. Expression pattern analysis among three tissues and under high temperature showed variable expression trends, as several genes displayed tissue-preferential expression and were activated in response to heat. Heterologous expression of all 20 MaHSF proteins in yeast confirmed majority of the members exhibited thermoprotective functions. Arabidopsis HSFA6b functions as a positive regulator to high temperature stress, whereas the precise regulatory mechanism remains incompletely understood. The mulberry ortholog, MaHSF1, was strongly upregulated under heat stress and functionally validated in yeast for its role in thermotolerance. Functional analysis revealed that MaHSF1serves as a transcriptional activator, and was highly expressed in leaves. Transgenic Arabidopsis overexpressing MaHSF1 displayed enhanced heat tolerance, supported by reduced oxidative damage, elevated antioxidant enzyme activities, and increased proline accumulation. Spatiotemporal expression analysis in Arabidopsis further demonstrated that MaHSF1 was both developmentally regulated and heat-inducible. These findings revealed the molecular basis of HSF-mediated thermotolerance in mulberry and provided a candidate gene for improving heat resistance in plants.

Aspartokinase (LysC) has been proposed as a key enzyme involved in 1-deoxynojirimycin (1-DNJ) biosynthesis in mulberry, yet direct functional evidence remains limited. In this study, we cloned and functionally characterized MmLysC3, a candidate aspartokinase gene from mulberry (Morus multicaulis), and investigated its role in 1-DNJ biosynthesis. Domain architecture analysis confirmed the presence of both the conserved AA_kinase catalytic domain and two ACT regulatory domains, placing MmLysC3 within the class I aspartokinase family. Expression profiling revealed that MmLysC3 exhibits tissue-preferential expression, with higher transcript levels in buds and young leaves, spatially coinciding with active 1-DNJ accumulation sites. Correlation analysis across ten mulberry varieties demonstrated a significant positive association between LysC3 expression and 1-DNJ content (Spearman’s r = 0.6743, p = 9.04 × 10−5). Critically, transient overexpression of MmLysC3 in mulberry leaves resulted in a marked increase in 1-DNJ content, with the highest-accumulating line reaching approximately twice that of the control. These findings provide the first functional evidence that LysC3 acts as a positive rate-limiting enzyme in the aspartate-derived branch of the 1-DNJ biosynthetic pathway in mulberry, bridging the gap between transcriptomic predictions and functional validation.

Mulberry ( Morus alba L) leaves are the sole food source of the domesticated silkworm ( Bombyx mori ) and are rich in chlorogenic acid (ChA), a phenolic metabolite implicated in plant defense and silkworm performance. However, how herbivory-associated cues are integrated with ethylene (ET) signaling to regulate ChA accumulation remains elusive. Here, we found that silkworm feeding markedly enhanced ChA levels in mulberry leaves, accompanied by notable cascading changes in genes associated with the ethylene (ET) signaling pathway, particularly Morus alba L. ETHYLENE INSENSITIVE 2 ( MaEIN2 ), indicating that ET is involved in the stress response elicited by silkworm feeding. Virus-induced gene silencing (VIGS) of MaEIN2 significantly reduced ChA accumulation and downregulated downstream genes implicated in ChA biosynthesis. Moreover, MaEIN2 -silenced seedlings exhibited compromised resistance to A. tumefaciens harboring tobacco rattle virus (TRV)-based plasmids, supporting a role for MaEIN2 in biotic stress defense. Collectively, these results indicate that MaEIN2 mediates ET-dependent defense responses by modulating ChA accumulation in mulberry. Furthermore, herbivory-induced stress appears to operate through MaEIN2 -dependent ET signaling, which elevates ChA content potentially to accommodate silkworm developmental needs. This study provides new insights into the role of MaEIN2 in linking silkworm feeding with ChA accumulation in mulberry.

The preservation of biodiversity and the proper functioning of grassland ecosystems depend on our ability to understanding the effects of livestock grazing on soil characteristics and microbial communities. However, how soil microbial communities- including their diversity, composition, and network complexity-respond undergo change in response to increasing grazing intensity in typical grasslands remains unclear. To address this, a meticulously designed, ten-year controlled experiment was conducted in a typical grassland in Inner Mongolia to investigate changes in the soil microbial community under increasing grazing intensity. The results showed that grazing significantly affected the structure and composition of soil microorganisms, leading to a significant decrease in bacterial beta-diversity with increasing grazing intensity. In contrast, bacterial alpha-diversity significantly increased, while fungal alpha-diversity remained largely unchanged. However, higher grazing intensities reduced soil multifunctionality and network complexity, with stronger effects on bacteria. These findings enhance our understanding of how grazing intensity influences microbial communities and provide a theoretical basis for assessing grazing's impact on grassland soil ecosystems, thereby addressing a critical knowledge gap regarding its effects on subsurface ecosystems.

Grazing strongly influences soil microbial communities and their roles in ecosystem functioning. However, it remains unclear how moderate grazing mediates microbial network organization and functional pathways under contrasting environmental contexts, which is critical for understanding the mechanisms sustaining grassland ecosystem resilience. Here, we investigated soil bacterial and fungal communities in desert and meadow steppes of Inner Mongolia after seven years of moderate grazing using high-throughput amplicon sequencing, network topology analysis, and module-based functional prediction. Our results revealed pronounced steppe-specific responses. In meadow steppes, grazing significantly increased fungal network complexity, hub centrality, and functional redundancy, particularly enhancing carbon and energy cycling potentials. In contrast, desert steppes exhibited simplified bacterial-dominated networks, reduced modularity, and limited functional shifts, with a relative increase in pathogen-associated guilds. Partial Least Squares Path Modeling further demonstrated that microbial functions in meadow steppes were regulated primarily by plant diversity, whereas those in desert steppes were constrained by abiotic stressors such as soil moisture. These findings highlight distinct microbial strategies under grazing across arid and mesic ecosystems and underscore the importance of context-specific management to sustain belowground biodiversity and ecological resilience in temperate grasslands.

The Squamosa promoter-binding protein-like (SPL) family proteins plays pivotal roles in plant development and stress adaptation. In this study, we functionally characterized MaSPL8 in mulberry (Morus alba) and investigated its regulatory roles in biotic and abiotic stress responses. MaSPL8 encodes a 364-amino acid protein with a conserved SBP domain and lacks miR156/157 binding sites. Phylogenetic analysis confirmed its orthology to Arabidopsis AtSPL8, albeit with functional divergence. Downregulation of MaSPL8 via virus-induced gene silencing (VIGS) resulted in more susceptibility to Ciboria shiraiana infection, but significantly enhanced resistance to drought and salt stress, as evidenced by reduced oxidative damage, elevated proline accumulation, and increased antioxidant enzyme activities. Transcriptomic profiling of MaSPL8-silenced plants revealed enrichment of differentially expressed genes (DEGs) in brassinosteroid biosynthesis, jasmonic acid metabolism, and oxidative stress responses, suggesting hormone signaling interplay. Furthermore, bioinformatic predictions identified miR5658 and miR4221 as potential post-transcriptional regulators of MaSPL8. This study highlights MaSPL8 as a negative regulator of abiotic stress tolerance and positive regulator of biotic (C. shiraiana) stress tolerance in mulberry and provides insights into its integration with phytohormone pathways. Our findings underscore the evolutionary plasticity of SPL8 genes and propose MaSPL8 as a target for enhancing mulberry’s resilience in challenging environments.

MOTS-c is a mitochondrial peptide that plays a crucial role in regulating energy metabolism, gene expression, and immune processes. However, current research primarily focuses on mammals like humans and mice, with no reports on avian MOTS-c. This study aimed to identify and characterize MOTS-c coding sequences across major poultry species through bioinformatics analysis and experimental validation. The alignment results showed high sequence similarity in the MOTS-c coding regions between avian and mammalian species. However, a single nucleotide deletion was identified in avian sequences at the position corresponding to the fourth amino acid residue of mammalian homologs, resulting in divergent downstream amino acid sequences. Despite this deletion, several residues were conserved across species. Phylogenetic analysis of mRNA sequences grouped pigeons with mammals, while protein sequence analysis revealed that poultry and mammals form separate branches, highlighting the divergence between avian and mammalian MOTS-c sequences. Tissue expression profiling demonstrated widespread distribution of chicken MOTS-c across multiple tissues, with the highest expression levels in the heart. Fasting significantly reduced heart MOTS-c expression, suggesting potential metabolic regulatory functions. Functional analysis of MOTS-c in primary hepatocytes revealed significant enrichment of the ribosome, oxidative phosphorylation, and key signaling pathways (PI3K-AKT and JAK-STAT) following 24 hours of treatment. Western blot validation confirmed MOTS-c-mediated activation of the AKT signaling pathway. This study represents the first comprehensive characterization of avian MOTS-c, providing critical insights into its evolutionary conservation and its potential functional roles in gene expression and cellular metabolism. Our findings establish a foundation for further investigation into the functions of mitochondrial-encoded peptides in avian species.

The NAC (NAM/ATAF1/2/CUC2) transcription factors are pivotal regulators in plant development and stress responses. Despite the extensive studies on the NAC gene family across various plant species, the characterization of this gene family in mulberry (Morus atropurpurea) remains unexplored. Here, we conducted a genome-wide identification and characterization of the NAC gene family in M. atropurpurea. A total of 79 MaNAC genes were identified and classified into 20 subgroups, displaying an uneven distribution across the 14 chromosomes. The structural analysis found that most MaNAC genes possess at least three exons and contain the conserved NAC domain and characteristic motifs at the N-terminus. Eleven collinear gene pairs were identified in M. atropurpurea genome. Interspecies collinearity analysis demonstrated a closer evolutionary relationship between M. atropurpurea and Populus trichocarpa, supported by the identification of 116 collinear gene pairs. Expression profiling revealed dynamic changes in the transcript levels of most MaNAC genes during mulberry fruit maturation. Notably, the eight MaNAC members from the OsNAC7 subfamily exhibited tissue-specific expression patterns. A significant proportion of MaNAC genes displayed varying degrees of responsiveness to drought stress and sclerotium disease. MaNAC12, MaNAC32, MaNAC44 and MaNAC67 emerged as the most highly responsive candidates. Overexpression of MaNAC69 enhanced drought tolerance in Arabidopsis. These findings provide a robust foundation for future functional studies and mechanistic investigations into the roles of the NAC gene family in M. atropurpurea, offering insights into their contributions to development and stress adaptation.

Mulberry (Morus spp., Moraceae) is considered as a rapidly evolving plant species with enriched unique gene family members involved in flavonoid biosynthesis. Recently, transcriptional regulatory mechanism controlling flavonoid biosynthesis in mulberry has been extensively reported, yet studies on the regulation of flavonoid biosynthesis beyond the transcriptional level are limited. In present study, our in vivo and in vitro assays have shown that four MaCHI-fold proteins with different roles in flavonoid biosynthesis coordinated to flexibly regulate the influx for flavonoid biosynthesis. MaCHI2N was the dominant CHI to ensure the normal influx. The mulberry-specific type IB MaCHI1 worked as substitute and could also enhance the influx. The MaCHI2 alternative transcript product, MaCHI2D with low activity for naringin chalcone provided a "braker" for flavonoid biosynthesis by competing with MaCHI2N in transcription process. MaCHIL enhanced the influx for flavonoid biosynthesis by interacting with MaCHS1 and increasing the production of naringenin. Therefore, we proposed a transmission box model that presented the complicated regulation of influx for flavonoid biosynthesis based on the diversity of MaCHI-fold proteins and the homeostasis of naringenin chalcone and naringenin in mulberry. Our results reveal the unique regulatory mechanism of influx for flavonoids pathway beyond transcriptional level.

Flavonoids are an important type of bioactive substance contained in jujubes. Flavonol synthase (FLS) is a key enzyme for the synthesis of flavonoids such as flavonols and anthocyanins. To study the biological functions of FLS in jujubes, we cloned the ZjFLS gene; analyzed its physicochemical properties and evolutionary relationships; and then conducted an expression characteristic analysis, subcellular localization, prokaryotic expression and heterologous overexpression in Arabidopsis thaliana. The results showed that the length of ZjFLS is 951 bp, and it encodes 316 amino acids. A sequence analysis revealed that ZjFLS exhibited a high degree of conservation in evolution. The results of a qRT-PCR analysis indicated that the ZjFLS gene could be expressed in different tissues of jujube: the expression level was the highest in the leaves, followed by the flowers, and the lowest was in the fruits. Within these expression levels, it was higher in young leaves than in mature leaves and higher in the white-ripe-stage fruits than in the semi-red-stage fruits. Subcellular localization indicated that the ZjFLS gene was located in the nucleus, cytoplasmic matrix, and cytoplasmic membrane. Our research findings show that the ZjFLS protein can be induced and obtained in the prokaryotic expression system and successfully purified. It mainly exists in the form of inclusion bodies and has a relatively low content in the soluble supernatant. The total flavonoid content of Arabidopsis thaliana strains with a heterologous overexpression of the ZjFLS gene was significantly higher than that of the wild type, confirming that the ZjFLS gene can promote the biosynthesis of flavonoid substances.

Mulberry sclerotiniose is a devastating fungal disease of mulberry fruit and has been a limitation for the utility of mulberry fruits and the diversified development of sericulture. In the present study, we presented a workflow for screening candidate sclerotiniose-resistance genes and small secreted peptides (SSPs) based on a genome-wide annotation of SSPs and comparative transcriptome analysis of different mulberry varieties. A total of 1088 SSPs with expression evidence were identified and annotated in mulberry. A comprehensive analysis of the sclerotiniose-related RNA sequencing datasets showed that photosynthesis, plant hormone signaling, and metabolic pathways were the main pathways involved in the response to sclerotiniose. Fifty-two candidate sclerotiniose-response genes (SRGs), including 15 SSPs, were identified based on comparative transcriptome analysis. These SRGs are mainly involved in the hormone signaling pathway and cell wall biogenesis. Transient overexpression in tobacco and the knock-down of five SRGs affected the resistance against Ciboria shiraiana. MaMYB29, MaMES17, and MaSSP15 were primarily determined as negative regulators of plant resistance to C. shiraiana infection. Our results provide a foundation for controlling sclerotiniose in mulberry using genetic engineering and biological approaches such as spraying antifungal peptides.

The V-myb myeloblastosis viral oncogene homolog (MYB) family participate in various bioprocesses including development and abiotic stress responses. In the present study, we first report a 1R SHAQKYF-class MYB, MaMYBR30, in mulberry. Subcellular localization and sequence analysis indicated MaMYBR30 is located in the nucleus and belongs to a CCA-like subgroup with a conserved SHAQKYF motif. Expression profile analysis showed that MaMYBR30 is expressed in leaves and can be induced by drought and salt stress. The down-regulation of MaMYBR30 using virus-induced gene silence (VIGS) in mulberry and the overexpression of MaMYBR30 in Arabidopsis were induced to explore the function of MaMYBR30. The functional characterization of MaMYBR30 in vivo indicated that MaMYBR30 can positively regulate the resistance of mulberry to drought while negatively regulating the resistance of mulberry to salt stress. In addition, MaMYBR30 also affects flower development and reproductive growth, especially after exposure to salt stress. Weighted gene co-expression network analysis (WGCNA) primarily revealed the possible genes and signal pathways that are regulated by MaMYBR30. Our results also imply that complex molecular mechanisms mediated by MaMYBR30, including crosstalk of ion toxicity, phytohormone signal transduction, flowering development, and epigenetic modification, need to be further explored in the future.

Glutamine synthetase (GS) is a key enzyme involved in nitrogen metabolism. GS can be divided into cytosolic and plastidic subtypes and has been reported to respond to various biotic and abiotic stresses. However, little research has been reported on the function of GS in mulberry. In this study, the full length of MaGS2 was cloned, resulting in 1302 bp encoding 433 amino acid residues. MaGS2 carried the typical GS2 motifs and clustered with plastidic-subtype GSs in the phylogenetic analysis. MaGS2 localized in chloroplasts, demonstrating that MaGS2 is a plastidic GS. The expression profile showed that MaGS2 is highly expressed in sclerotiniose pathogen-infected fruit and sclerotiniose-resistant fruit, demonstrating that MaGS2 is associated with the response to sclerotiniose in mulberry. Furthermore, the overexpression of MaGS2 in tobacco decreased the resistance against Ciboria shiraiana, and the knockdown of MaGS2 in mulberry by VIGS increased the resistance against C. shiraiana, demonstrating the role of MaGS2 as a negative regulator of mulberry resistance to C. shiraiana infection.

This paper aims to evaluate the effects of polypropylene fibers on the water retention characteristics and tensile strength of phyllite residual soil, and to innovatively propose dual-peak soil water retention curve (SWRC) and tensile strength characteristics curve (TSCC) models, examining the influence of fiber content and initial void ratio across the full range of degree of saturation. Experimental results indicate that the SWRC of phyllite residual soil exhibits a distinct dual-step pattern, with a decrease in void ratio significantly enhancing water retention capacity. However, the addition of polypropylene fibers has a minimal impact on the SWRC, showing only slightly increases at the steps of the curve, with the SWRC at a fiber content of 0.2% being superior to that of 0.5%. The tensile strength displays a dual-peak variation trend across the full range of degree of saturation. At lower void ratio, the tensile strength of the soil is significantly improved. Moreover, the addition of polypropylene fibers markedly enhances the tensile strength, reaching an optimal point at a fiber content of 0.2%, after which the strength decreases due to fiber-soil particle aggregation and uneven fiber distribution. This research provides new insights into the performance of soil reinforced with fibers, thereby offering theoretical support for the practical application of reinforced soil in engineering projects.

Mulberry (Morus alba L.) is a significant economic tree species in China. The lignin component serves as a critical limiting factor that impacts both the forage quality and the conversion efficiency of mulberry biomass into biofuel. Cinnamoyl CoA reductase (CCR; EC 1.21.1.44) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95) are the key enzymes that catalyze the final two reductive steps in the biosynthesis of monolignols. In this study, we conducted a comprehensive functional analysis to validate the predominant CCR genes involved in monolignol biosynthesis. In this study, we initially validated the predominant CCR genes implicated in monolignol biosynthesis through an extensive functional analysis. Phylogenetic analysis, tissue-specific expression profiling and enzymatic assays indicated that MaCCR1 is the authentic CCR involved in lignin biosynthesis. Furthermore, the expression level of MaCCR1 exhibited a significant positive correlation with lignin content, and the down-regulation of MaCCR1 via virus-induced gene silencing resulted in altered lignin content in mulberry. The down-regulation of MaCCR1 and MaCAD3/4, both individually and concurrently, exhibited markedly different effects on lignin content and mulberry growth. Specifically, the simultaneous down-regulation of MaCCR1 and MaCAD3/4 significantly altered lignin content in mulberry, resulting in dwarfism of the plants. Conversely, the down-regulation of MaCAD3/4 alone not only decreased lignin content but also led to an increase in biomass. These findings offer compelling evidence elucidating the roles of MaCCRs in mulberry and identify specific target genes, thereby providing a crucial foundation for the genetic modification of lignin biosynthesis.

Human activities lead to an increase in greenhouse gases in the environment, among which carbon dioxide (CO2) is one of the most prominent, giving rise to global warming and climate change. Climate change, along with the resulting environmental degradation, is one of the most challenging difficulties faced by humanity in the twenty-first century. The forest ecosystem, with plants being its most important component, plays a pivotal role in regulating climate. Carbon sequestration and oxygen release (CSOR) by plants are major ecological service functions that play an important role in mitigating the negative impacts of the greenhouse effect and help to achieve carbon peaking and neutrality. The CSOR of mulberry (Morus spp.), a species of economic and ecological significance, is not yet understood. Six mulberry tree varieties were selected to evaluate their CSOR potential during summer. We took into consideration whole-plant diurnal assimilation amounts (P), carbon sequestration per unit leaf area (WCO2), oxygen release per unit leaf area (WO2), carbon sequestration per unit land area (PCO2), and oxygen release per unit land area (PO2). Zhongsang 1302 showed the greatest potential for CSOR among the six mulberry varieties. The PCO2 value of the Zhongsang 1302 variety was 1531.84 g·m−2 during summer, followed by Suhu 16 (1380.12 g·m−2), Husang 32 (1005.63 g·m−2), Zhongsang 9703 (990.01 g·m−2), Yu 711 (940.43 g·m−2), and Jin 10 (848.29 g·m−2). Moreover, the Pearson correlation and path analyses showed that photosynthetic rate (PN) and leaf area index (LAI) mainly affect the overall CSOR potential in mulberry. These findings not only enrich theoretical research on CSOR in mulberry, but also serve as an important reference for the use of different mulberry tree varieties in improving climate conditions and achieving carbon peaking and neutrality.

Phenotypic plasticity is the property of a given genotype to produce multiple phenotypes in response to changing environmental conditions. Understanding the genetic basis of phenotypic plasticity and establishing a predictive model is highly relevant for future agriculture under changing climate. Here, we investigated the genetic basis of leaf width plasticity in a tobacco MAGIC population across four different environments. Environmental changes not only resulted in differences in leaf width mean but also in leaf width plasticity. A total of 45 QTL were identified, including 14 QTL for leaf width mean and 43 for leaf width plasticity, with 12 overlap. Changes in the environment affected the magnitude of several QTL, thereby influencing phenotypic plasticity. We identified a QTL, qLW14, associated with leaf width plasticity and leaf width mean at Zhucheng, but had no significant impact at Guiyang, indicating that changes in the environment contributed to variations in leaf width plasticity. By integrating genetic diversity, environmental variation, and their interactions into a unified model, we were able to build a model for cross-environment predictions, and improved prediction accuracy by 7.2%. Overall, this study reveals complex genetic basis involving multiple alleles, and genotype interactions underlying variations of leaf width mean and plasticity. These findings contribute to assessing the role of plasticity in responding to climate or other environmental changes. ### Competing Interest Statement The authors have declared no competing interest.