Pei Yu

Eelgrass (Zostera marina L.) is a marine angiosperm exposed to substantial salinity fluctuations, yet the transcriptional mechanisms associated with its salinity adaptation responses remain incompletely understood. To characterize the NAC transcription factor family and its potential involvement in salinity responses, we performed genome-wide identification, phylogenetic, structural, duplication, promoter, and expression analyses in Z. marina, followed by quantitative reverse transcription PCR (qRT-PCR) validation. Sixty-three ZmNAC genes were identified and classified into six phylogenetic groups. Most encoded proteins contained a conserved N-terminal NAC domain but varied considerably in length, motif composition, gene structure, and predicted subcellular localization. Duplication analysis classified 55 genes as dispersed duplicates, seven as whole-genome duplication (WGD) or segmental duplicates, and one as a proximal duplicate. Promoter analysis identified 64 cis-acting element types associated mainly with light, phytohormone, and stress responses. RNA-seq analysis of roots, stems, and leaves after 24 h at NaCl concentrations of 0‰, 24‰, and 40‰ revealed distinct salinity- and tissue-dependent expression patterns among the ZmNAC family members. Expression patterns of four candidate genes in leaves, assessed by qRT-PCR, were largely consistent with the RNA-seq results. These findings reveal structural and expression diversification within the ZmNAC family and identify candidate genes for further functional investigation of salinity responses in eelgrass.

Pangenome-level gene family identification often applies sequence similarity clustering without phylogenetic or synteny information, which risks biologically misleading evolutionary inferences. Using five transcription factor families (bHLH, MYB, NAC, WRKY, MADS-box) across 401 rice pangenome accessions, we compared clustering strategies: OrthoFinder alone, cd-hit alone, MMseqs2 alone, and OrthoFinder-informed refinement by cd-hit or MMseqs2. Methods solely based on sequence similarity merged distinct orthogroups and generated fewer orthogroups than approaches incorporating graph-based orthology. Conflicting cluster assignments, measured against OrthoFinder, varied strongly among families, from approximately 14% in MADS-box to approximately 57% in MYB, and were associated with protein length differences. Core, shell, and cloud gene classifications shifted substantially depending on the method, especially in MYB, NAC, and WRKY families. Critically, Ka/Ks distributions for core genes were highly method-sensitive, with orthology-aware methods yielding more convergent and less variable estimates of selective pressure, whereas noncore gene estimates remained robust. These findings demonstrate that neglecting graph-based orthogroup inference inflates methodological artifacts. We recommend a two-step strategy: initial graph-based orthogroup delineation followed by sequence similarity refinement to balance evolutionary accuracy and resolution in pangenome-scale gene family studies.

Main conclusion Through integrated transcriptomic and metabolomic analyses, we systematically assessed the role of calcium signaling pathways in adaptation of eelgrass to high-salinity environments. Phenylpropanoid biosynthesis is a crucial metabolic pathway through which calcium signaling involves to salt adaptability of eelgrass. There is a close crosstalk between calcium signaling and nitric oxide in eelgrass. Zostera marina L. (eelgrass), a representative marine submerged angiosperm, exhibits unique traits for salt adaptation. In previous studies, we found that the calcium signaling pathway in eelgrass was activated under high salt conditions, but the specific role of it in adaptation of eelgrass to salt environments is still unclear. In this study, we utilized ethylene glycol tetraacetic acid to inhibit calcium signaling, thereby to find differentially expressed genes and differential accumulated metabolites in eelgrass. Through integrated transcriptomic and metabolomic analyses, we systematically assessed the role of calcium signaling pathways in adaptation of eelgrass to high-salinity environments. Specifically, calcium signaling in roots adjusts homeostasis through cell wall regulation and plant hormone signaling pathways, contributing to osmotic regulation and antioxidant defenses; In stems, calcium signaling primarily mediates ion transport and osmotic regulation; In leaves, the antioxidant defense system would be activated as a compensatory mechanism to alleviate salt stress damage after inhibiting calcium signaling. Notably, phenylpropanoid biosynthesis is a crucial metabolic pathway through which calcium signaling is involved in salt adaptability of eelgrass. Additionally, there is a close crosstalk between calcium signaling and nitric oxide in eelgrass: calcium signaling regulates the expression of nitric oxide synthase, while nitric oxide also influences the expression of several calcium sensor proteins during calcium signaling transduction. These studies provide valuable insights into the role of calcium signaling in eelgrass, contributing to the understanding of the evolutionary processes of marine higher plants, and offering a theoretical foundation for the improved cultivation of salt-tolerant terrestrial crops.

ASMT/COMT, as a key rate-limiting enzyme regulating melatonin biosynthesis, has garnered significant attention. This study investigates the evolutionary mechanisms of the ASMT/COMT gene family in melatonin biosynthesis. A total of 28 010 ASMT/COMT genes from 1052 species were identified through an integrated approach combining large-scale identifications and analyses. At the pan-genome level, we identified 5186, 336, 2137, and 1814 ASMT/COMT genes respectively in Triticum aestivum, Aegilops tauschii, diploid and tetraploid Solanum tuberosum haplotype genomes (247, 86, 670, and 96 orthologous gene groups). Expansion patterns of the ASMT/COMT gene family were explored through synteny networks in 104 Poaceae and 88 Solanaceae plants. Further investigation of copy number variation (CNV) in the 1052 species, along with a focused analysis of hexaploid wheat and its diploid progenitor Ae. tauschii, indicated a functional divergence linked to gene dosage. The catalytically efficient COMT is maintained at low-copy conditions, whereas the less active ASMT is amplified under high-copy conditions. Intriguingly, in polyploid potatoes, the total ASMT/COMT copy number was lower in tetraploids than in diploids, suggesting a distinct dosage balance mechanism operating in polyploids. In contrast, the melatonin receptor CAND2 consistently remained in a low-copy state, with no significant correlation to ASMT/COMT copy number. Expression analysis revealed that COMT is generally expressed at higher levels than ASMT, highlighting a compensatory relationship between gene dosage and transcriptional regulation. Collectively, our findings uncover a dosage balance mechanism that fine-tunes melatonin biosynthetic homeostasis through coordinated CNV and expression regulation, offering a new perspective on the evolution of metabolic enzymes.

Wheat (Triticum aestivum) is one of the most important cereal crops globally, with significant economic value. The Arabidopsis Tóxicos en Levadura (ATL) gene family, which comprises members of ubiquitin ligase enzymes (E3s), functions in substrate protein tagging during ubiquitin-mediated protein modification. Recent studies have demonstrated its involvement in stress responses. However, the ATL gene family in wheat remains poorly characterized. This study aimed to identify the members of the ATL gene family in wheat and investigate their roles under salt stress. We identified 334 TaATL genes in the wheat genome, all of which contain either RING-H2, RING U-box, or RAD18 superfamily domains, exhibiting a remarkably low proportion of intron-containing genes. The Ka/Ks (non-synonymous to synonymous substitution rate) analysis and cis-acting element analysis of the TaATL gene family indicate that its sequences are highly conserved and functionally constrained, suggesting that it may participate in abiotic stress responses through the ABA, MeJA, and MYB signaling pathways. Both RNA-seq analysis and RT-qPCR data demonstrated that the expression levels of the TaATL gene family were significantly upregulated under stress conditions, indicating their crucial roles in stress responses. This study demonstrates that the targeted regulation of stress-responsive signaling pathways mediated by superior TaATL gene family members can effectively enhance wheat salt tolerance, thereby providing a viable strategy for the development of high-yielding cultivars adapted to saline agricultural ecosystems.

As a subfamily of the PD-(D/E)XK nuclease superfamily, DUF506 family shows great potential in abiotic stress responding of higher plant, yet its clues of structure, evolution and functions remain largely unexplored due to their distant phylogenetic relationship with other nuclease families, especially in Triticum aestivum. In this study, 26 T. aestivum DUF506 genes (TaDUF506) were identified from genome-wide level through bioinformatic techniques. Phylogenetic and structural analyses revealed that TaDUF506 genes exhibit conserved motif and gene structure patterns intra-phylogenetic clusters but display significant divergence inter-clusters. Gene duplication identification showed that whole-genome duplication event (WGD) was the primary driver of TaDUF506 family expansion, while Ka/Ks analysis indicated that whole TaDUF506 family experienced purifying selection generally. Gene ontology analysis and protein-protein interaction prediction suggested that DUF506 plays a potential role in transcription regulation and nucleotide-excision generally. Promoter analyses highlighted an enrichment of hormone-responsive elements linked to salt stress in TaDUF1.3-3D TaDUF5.1-3A, with expression analysis demonstrated their significant upregulation under salt stress, suggesting the potential roles in stress responses. Altogether, our study advances the understanding of DUF506 gene family in higher plant from structural, evolutional and functional aspects, and thereby provides a foundation for the development of salt-tolerant wheat varieties.

Main conclusion This review highlights the sulfur transporters, key enzymes and their encoding genes involved in plant sulfur anabolism, focusing on their occurrence, chemistry, location, function, and regulation within sulfur assimilation pathways. Abstract Sulfur, a vital element for plant life, plays diverse roles in metabolism and stress response. This review provides a comprehensive overview of the sulfur assimilation pathway in plants, highlighting the intricate network of enzymes and their regulatory mechanisms. The primary focus is on the key enzymes involved: ATP sulfurylase (ATPS), APS reductase (APR), sulfite reductase (SiR), serine acetyltransferase (SAT), and O-acetylserine(thiol)lyase (OAS-TL). ATPS initiates the process by activating sulfate to form APS, which is then reduced to sulfite by APR. SiR further reduces sulfite to sulfide, a crucial step that requires significant energy. The cysteine synthase complex (CSC), formed by SAT and OAS-TL, facilitates the synthesis of cysteine, thereby integrating serine metabolism with sulfur assimilation. The alternative sulfation pathway, catalyzed by APS kinase and sulfotransferases, is explored for its role in synthesizing essential secondary metabolites. This review also delves into the regulatory mechanism of these enzymes such as environmental stresses, sulfate availability, phytohormones, as well as translational and post-translational regulations. Understanding the key transporters and enzymes in sulfur assimilation pathways and their corresponding regulation mechanisms can help researchers grasp the importance of sulfur anabolism for the life cycle of plants, clarify how these enzymes and their regulatory processes are integrated to balance plant life systems in response to changes in both external conditions and intrinsic signals.

Main conclusion The pearl millet gene PgPM19 inhibits seed dormancy by negatively regulating the ABA biosynthesis and ABA signaling pathways in response to salinity stress in Arabidopsis. Abstract Abscisic acid (ABA) plays a pivotal role in orchestrating plant stress responses and development. However, how the ABA signal is transmitted in response to stresses remains primarily uncertain, particularly in monocotyledonous plants. In this study, PgPM19, a gene whose expression is induced by drought, salinity, heat, and ABA in both leaf and root tissues, was isolated from pearl millet. The expression of PgPM19 in yeast cells did not influence their growth when subjected to mannitol, sorbitol, or NaCl stress. However, Arabidopsis plants overexpressing PgPM19 (PgPM19_OE plants) exhibited increased germination rates, greater fresh weights and longer roots under salinity stress during germination, compared to wild-type (WT) plants. Conversely, the pm19L1 (SALK_075435) mutant, featuring a transfer DNA insertion in a closely related PgPM19 homolog (AT1G04560) in Arabidopsis, demonstrated reduced germination rates and smaller fresh weights under salinity-stressed condition than did WT and PgPM19_OE plants. A pivotal ABA biosynthesis gene, NCED3, ABA signaling pathway genes, such as PYL6 and SnRK2.7, alongside downstream ABI genes and stress-responsive genes RAB28 and RD29, were downregulated in PgPM19_OE plants, as evidenced by both transcriptome analysis and quantitative reverse transcription-PCR. These findings raise the possibility that PgPM19 is involved in regulating seed germination by mediating ABA biosynthesis and signaling pathway in response to salinity stress in Arabidopsis. This study contributes to a better understanding of PgPM19 in response to salinity stress and establishes a foundation for unraveling the crosstalk of stress responses and ABA in Arabidopsis and other plant species.