Plant membrane steroid binding proteins (MSBPs) belong to the membrane-associated progesterone receptors (MAPRs) present in all eukaryotic kingdoms. Plant MSBP proteins have been shown to regulate the function of cytochrome P450 enzymes, bind different steroidal compounds and confer salt tolerance. However, the exact molecular function of plant MSBPs remains elusive. Here we perform phylogenetic analysis of the six MAPR genes encoded in the Sorghum bicolor genome. Of these, four group into a distinct MSBP clade characterized by being N-terminally membrane anchored followed by a cytochrome b5 domain and an extended disordered C-terminal. Biophysical of SbMSBP1 demonstrates that this protein can bind heme, which leads to dimerization potentially through a heme-heme stacking mechanism. We further show using untargeted proteomics that MSBPs are upregulated in both root and shoot tissue upon exposure to salt stress. Based on weighted gene co-expression network analysis (WGCNA) we find that SbMSBP1 abundance clusters with ER remodeling and vesicle transport proteins. We further show that overexpression of SbMSBP1 in Sorghum bicolor protoplasts and tobacco results in formation of structures consistent with organized smooth endoplasmic reticulum (OSER). Our data indicates that SbMSBP1 functions to remodel ER membranes, which may be directly linked to a functional role in stress resilience towards both biotic and abiotic stresses and furthermore could serve as a useful tool for metabolic engineering of ER-scaffolded biosynthetic pathways.

The liverwort Marchantia polymorpha is a key model organism for understanding land plant evolution, development, and gene regulation. To support the growing demand for high-quality genomic resources, we present MarpolBase, a comprehensive and integrated genome database that hosts newly assembled, high-accuracy reference genomes for both the male Tak-1 and female Tak-2 accessions, designated as ver. 7.1 reference genomes. These new assemblies, generated using PacBio HiFi long-read sequencing, represent nearly telomere-to-telomere chromosome-level genomes, with improvements in assembly continuity, annotation accuracy, and structural resolution-especially for repeat-rich regions and sex chromosomes. MarpolBase offers not only access to genome sequences and gene annotations but also provides a unified platform for data exploration, comparative analysis, and community-driven gene nomenclature for Marchantia polymorpha. It includes keyword-searchable gene pages with structural and functional annotations, expression data integration, genome browser visualization, and online analytical and utility tools. By unifying genome assembly, annotation, nomenclature, and analysis tools in a single platform, MarpolBase serves as a central resource for functional genomics and evolutionary studies in M. polymorpha, and a model for future plant genome databases. The genomic resources of MarpolBase are freely available at https://marchantia.info.

Parasitic flowering plants are often seen as keystone species due to the broad influence they exert on communities worldwide. Positive and negative effects associated with parasitic plant infestation have been documented for a variety of species in multiple locations and under different experimental conditions. However, the impact of the different drivers of climate change on these plants has only recently begun to be analyzed in more detail. In this context, most studies have dealt with modelling future distribution ranges of parasite species and assessing potential ecological impacts. Building on this work, this review discusses studies that have employed a more mechanistic approach to investigate different aspects of parasitic plant physiology under climate change. Considering results obtained for both hemi- and holo-parasites, I hypothesize that, in the presence of conditions that improve parasite performance, such as reduced intraspecific competition or increased diversity of host species, elevated levels of atmospheric CO2 can partially alleviate the negative impact of parasitism on host growth. However, this reduction of negative impacts is potentially hampered by other drivers of climate change, such as extreme high temperatures and severe drought events. Future research should strive to analyze the combined impact of different components of climate change simultaneously, preferably considering a wider diversity of parasitic plant species.

Although microalgae such as diatoms and haptophytes have been studied to optimize fucoxanthin production, the complete biosynthetic pathway of fucoxanthin remains unclear. In this study, we subjected the haptophyte Tisochrysis lutea cells to heavy-ion beam irradiation to induce random mutations and obtained two greenish strains, GR1 and GR2, following exposures to 45 Gy and 100 Gy, respectively. The GR1 strain exhibited slow growth, whereas GR2 showed growth comparable to the wild-type strain. Neither GR1 nor GR2 accumulated fucoxanthin; instead, both strains accumulated fucoxanthin biosynthetic intermediates, haptoxanthin and phaneroxanthin, and harbored 74 and 148 mutation sites, respectively. As expected, higher radiation doses resulted in a greater number of mutations. Over 80% of these mutations consisted of short nucleotide insertions, primarily 4 to 8 bp in length. Additionally, mutations were identified in orthologs of the ZEP1 and CRTISO5 genes known in the diatom Phaeodactylum tricornutum to encode enzymes that convert haptoxanthin to phaneroxanthin and phaneroxanthin to fucoxanthin in GR1 and GR2 strains, respectively. The loss of fucoxanthin decreased photosynthetic capacity to some extent. However, the amounts of chlorophyll a and c did not change, suggesting that haptoxanthin and phaneroxanthin functioned as photosynthetic accessory pigments in the light-harvesting antennae. Because the genomic analysis results aligned with those from pigment analysis, our findings demonstrate that ZEP1 and CRTISO5 in T. lutea cells are involved in fucoxanthin biosynthesis and support the broader application of heavy-ion beam irradiation in fundamental microalgal research.

NITRILASEs (NITs) are enzymes that have been identified across kingdoms. Nitrilases are industrially important hydrolases widely used in the production of valuable chemicals and medicines. In plants, nitrilases are phylogenetically divided into two groups: NIT1 and NIT4. The NIT1 (NIT1-3) subfamily detoxifying nitriles is specific to the Brassicaceae and catalyze the conversion of indole-3-acetonitrile (IAN), derived from indole glucosinolates (IGs) or indole-3-acetaldoxime (IAOx), into indole-3-acetic acid (IAA), the principal auxin, which provides an evolutionary advantage since it's a growth hormone. The NIT1 subfamily has been implicated in the catabolism of indole acetamide (IAM), although this has yet to be confirmed in planta. NIT4 appears to function in cyanide detoxification and exhibits strong specificity toward β-cyanoalanine. Additionally, it is hypothesized that NIT4, as well as enzymes of the NIT1 subfamily, might be involved in phenylacetic acid (PAA) formation from phenylacetonitrile/benzyl cyanide (PAN/BnCN). Crop plants, such as Zea mays and Oryza sativa, have been used to study NITs sporadically, consequently, our understanding of the role of nitrilases is primarily derived from studies of the model plant Arabidopsis thaliana, including single or sparse multiple mutants, reporter lines, or overexpressing lines. This review mainly focuses on the NIT1 subfamily, which plays a role in root and flower development. However, NITs expression and activity have primarily been demonstrated under plant stress conditions, biotic and abiotic stress, such as saline, drought, sulfate deficiency, thermomorphogenesis, during which NIT-dependent auxin biosynthesis is activated. In addition, the role of NITs has been confirmed in morphogenetic processes in in vitro cultures, highlighting their role in stress-induced developmental reprogramming.

Single-cell RNA sequencing (scRNA-seq) enables high-resolution transcriptome analysis, enabling the study of cellular heterogeneity beyond bulk transcriptomics. However, plant science lags animal science in the field largely due to limited sssssssssmarker genes. This study developed PscOA (http://sdau.biodb.com.cn/pscoa/), a plant scRNA-seq marker gene database with 39 347 marker genes. PscOA integrates BLAST for homology-based marker gene mining, and SCSA for cell type annotation, complemented by visualization tools. Case studies in A. thaliana and P. alba demonstrate the potential of cell type annotation in PscOA. Leveraging A. thaliana marker genes from PscOA, we predict 258 potential markers in Nicotiana tabacum, showcasing its marker gene discovery potential for specific species. Differential expression analysis under stress reveals common and diverse strategies at the single-cell level, offering insights into plant cell type diversity and function. Altogether, PscOA serves as a valuable repository for original scRNA-seq analysis in plant science, deepening the understanding of plant cellular transcriptome.

Mesquite (Prosopis laevigata, Fabaceae) is a legume widely distributed in Mexico, infested by the mistletoe Psittacanthus calyculatus, Loranthaceae) a parasitic plant that absorbs water and nutrients from the mesquite. For parasitism to become established, the mistletoe must evade host defenses. To date, these defenses have only been studied in hosts with advanced parasitic infestations, but the initial defense of mesquite against these infestations remains unknown. The objective of this work was to investigate the early stress responses of mesquite trees after the first physical contact during infection by P. calyculatus. To do so, we first artificially inoculated mistletoe seeds and placed them on mesquite branches to initiate the parasitism. Mistletoe inoculations induced the early production of the phytohormones salicylic acid and jasmonic acid. These changes were accompanied by higher cell wall invertase activity, which precedes the increase in sucrose, glucose, and fructose. H₂O₂ formation and superoxide dismutase and peroxidase activity were also induced, while catalase activity decreased. Analyses revealed the presence of phenolic compounds as a defensive response against the mistletoe and, finally, elevated phenylalanine ammonia lyase activity after mistletoe inoculation. These data suggest that host trees recognize the presence of parasitic plants and trigger immune signaling responses well before haustorium formation. This is the first step toward understanding the interaction between the host tree and the mistletoe at beginning of parasitism.

Excess light during photosynthesis induces harmful reactive oxygen species. As a defense mechanism, plants possess rapidly reversible energy-dependent quenching (qE), in which excitation energy in the photosystem II antenna is dissipated as heat when absorbed light is in excess. In the Viridiplantae, qE is regulated by two key proteins: LHCSR and PsbS. LHCSR is widely conserved in green algae and bryophytes, early-diverging land plants. In contrast, PsbS functions as the major qE regulator in vascular plants, reflecting an evolutionary shift from LHCSR to PsbS. Despite its importance in vascular plants, the function of PsbS remains poorly understood in green algae, especially in streptophyte algae, the closest relatives of land plants. To examine PsbS activity in streptophyte algae, we focused on Chlorokybus cerffii and Klebsormidium nitens, which represent early-diverging lineages in Streptophyta. We expressed their PsbS genes in the PsbS-deficient Arabidopsis thaliana mutant npq4, along with PsbS genes from A. thaliana and Chlamydomonas reinhardtii. All PsbS genes complemented the npq4 mutant phenotype with varying degrees of efficiency depending on the protein expression levels. Moreover, the qE efficiencies per unit of PsbS protein in the algal PsbS transformants were equal to or higher than those of AtPsbS transformants. The results suggest that PsbS activity as a qE regulator was already established in the common ancestor of streptophytes prior to land plant colonization.

Comprising one of the largest plant gene families, MYB genes are major regulators of growth and development across plant tissues. Their evolutionary history is complex with recurrent gain and loss of the MYB domains, sometimes within the same multi-domained gene, creating a reticulate phylogenetic history, with various parts of the same gene having conflicting phylogenetic histories. Multiple MYB genes may co-operate or compete, thus constituting an on/off switch regulating transcription of downstream genes. We determined the phylogenetic origin of the multi-domained MYB regulators called DIVARICATA (DIV) genes, their cofactors the DRIF genes, and their competitors the LFG genes. We report that DIV arose through the fusion of two simpler MYB genes that resulted in a gene with three MYB domains (MYBA-MYB1-MYB2). The MYBA domain was later lost through a non-gradual process resulting in the two-domained MYB1-MYB2 DIV genes in green plants (including flowering plants). Further truncation of the MYB1 domain resulted in LFG genes that have only the MYB2 domain. The MYBA and the MYB2 domains were derived from the SHAQKY clade of MYB domains; the MYB1 domain and the MYBD domain of DRIF were derived from the clade associated with the SANT2 domain of ZUO1/ZRF genes. We discuss how the duplication and truncation of DIV has been repeatedly recruited in the evolution of on/off switches. Components of the DIV-based regulatory network, and their close homologs, are present in a diversity of eukaryotes suggesting that their interaction may be ancestral to a large group of eukaryotes.

Xylan, one of the most abundant hemicelluloses in plant cell walls, consists of β-(1 → 4)-linked xylosyl (Xyl) residues and often contains a conserved Reducing End Sequence (RES) in dicots and gymnosperms, comprising β-d-Xylp-(1 → 3)-α-l-Rhap-(1 → 2)-α-d-GalpA-(1 → 4)-d-Xylp. This tetrasaccharide has been proposed to function as a priming module ('primer hypothesis') or a termination signal ('terminator hypothesis') in xylan biosynthesis, yet its precise biochemical role remains unclear. Here, we examined whether the RES-containing oligosaccharide with one additional Xyl residue at the non-reducing end (X-RES) acts as a priming acceptor for IRREGULAR XYLEM10 (IRX10) proteins from a dicot, Arabidopsis thaliana, and a grass, Setaria viridis. Both recombinant AtIRX10L and SvIRX10 utilized fluorescently labeled X-RES and the canonical primer Xyl5 as acceptor substrates. Time-course analyses revealed that X-RES promoted a more efficient transition of +X1 to +X2 product, i.e. with minimal accumulation of +X1 product and enhanced formation of +X2 and longer products, suggesting that the RES motif facilitates seamless elongation. Consistent with these substrate-dependent differences, docking simulations showed that X-RES and its elongated form (X2-RES) bound more stably to the predicted IRX10 active site than the corresponding linear oligosaccharides Xyl5 and Xyl6. Moreover, the ability of SvIRX10 to recognize X-RES, despite RES motif not yet being detected in grass xylan, suggests that the RES-primed elongation may represent an ancestral substrate recognition in grasses. Our findings identify a structurally unique RES-containing oligosaccharide that functions as a primer in vitro, thereby extending current understanding of acceptor substrate flexibility in xylan biosynthesis. (242 words).

Cytoplasmic male sterility (CMS) is a trait wherein plants cannot develop normal male organs because of mitochondrial genes. In potatoes (Solanum tuberosum), reports on the relevant mitochondrial genes remain scarce. Many potato cultivars express pollen sterility caused by mitochondria, thereby limiting their use as male parents in breeding. Therefore, identifying the causal genes is crucial for potato breeding. In this study, we focused on the T/β cytoplasm type, the most prevalent cytoplasm of potato worldwide, to explore mitochondrial genes involved in CMS in potatoes. We identified a novel gene, open reading frame 320 (orf320) from potato with T/β type cytoplasm by comparing the mitochondrial genomes. The accumulation level of orf320 transcripts was drastically reduced in the anthers of a fertile potato cultivar compared with those of a sterile cultivar. Functional analysis of tomatoes showed that overexpression of orf320 with a mitochondrial transit peptide induced male sterility phenotype accompanying abnormal anther development and pollen abortion. Furthermore, an investigation of orf320 in 124 potato cultivars revealed that this gene is tightly associated with the T/β type cytoplasm and is absent from cultivars with other cytoplasm types. These findings provide evidence that orf320 is a candidate CMS-causing gene in male sterility of T/β type cytoplasm, offering valuable insights for future potato breeding.

Serine/arginine-rich (SR) proteins are essential splicing factors in animals, where their mutations often cause widespread splicing defects and carcinogenesis. The plant SR subfamily proteins are homologous to the well-studied human serine/arginine-rich splicing factor 1 (SRSF1), but their roles remain unclear. Here, we characterize the Arabidopsis SR subfamily genes: SR30, SR34, SR34a and SR34b. We show that GFP-tagged SR30, SR34 and SR34a co-localized with the spliceosomal protein U1-70K in speckled nuclear structures. To explore their physiological roles, we constructed a series of multiple mutants. Interestingly, the quadruple mutant displayed delayed flowering under long-day conditions but accelerated flowering under short-day conditions. Under long days, SR30, SR34 and SR34a function redundantly, as delayed flowering was observed only when all three were simultaneously disrupted. Under short day, SR34a plays a predominant role, being both necessary and sufficient to maintain normal flowering. RNA-seq and qPCR analysis revealed altered splicing of multiple flowering time regulators, including CONSTANS (CO) and FLOWERING LOCUS C (FLC). Particularly, increased production of an inhibitory CO isoform correlated with delayed flowering under long days, which was rescued by CO.1 overexpression, suggesting the phenotype was linked to CO missplicing. Overall, our findings uncover the roles of SR subfamily genes in floral transition, highlighting the physiological significance of splicing regulation in plants.

Obligate parasitic plants often establish symplastic connections with their hosts. This symplastic continuity is mediated by plasma membrane-lined channels referred to as interspecific secondary plasmodesmata, which develop at the interface between the parasite and its host. However, the molecular mechanisms underlying the formation of these interspecific secondary plasmodesmata remain unclear. In this mini review, we summarize current knowledge of plasmodesmata biogenesis at diverse cellular boundaries with distinct developmental origins, including those at graft junctions and between mesophyll and bundle sheath cells. Based on the literature review, we hypothesize that the formation of interspecific secondary plasmodesmata involves three key events: cell wall thinning, membrane rearrangement, and metabolic regulation. Finally, we discuss future research directions to elucidate the molecular basis of interspecific secondary plasmodesmata formation.

Abscisic acid (ABA) is a kind of plant hormone that alleviates drought stress in many plant species. DNA methylation also plays an important role in plant drought stress tolerance. However, the relationship between ABA and DNA demethylase is unclear. Here, the action mechanism of tomato DEMETER-LIKE protein 2 (DML2) in ABA-mediated drought stress resistance was studied by mutating SlDML2. We found that the mutation of SlDML2 weakened plant growth conditions under drought stress. Additionally, the content of chlorophylls, the osmoregulatory substances (proline, soluble sugar, and soluble protein), and flavonoid were lower in sldml2-1 mutant under drought circumstances when compared with wild-type (WT) plants. However, these effects of the mutated SlDML2 couldn't be reversed by the application of ABA. Furthermore, the expression levels of SlPYL1 and SlSnRK2.4 in ABA signaling pathway were downregulated in sldml2-1 mutants under drought stress compared with WT plants. Moreover, exogenous ABA reduced the DNA methylation level and the transcriptional abundances of its regulated genes by altering SlDML2-drived DNA demethylation under drought stress. The study here declared the important role of SlDML2 in ABA-improved plant drought tolerance, which may facilitate studies concerning ABA and DNA demethylation in the future.

Camelina sativa is an ancient native oilseed species characterized by broad environmental adaptability, low-input requirements and tolerance to multiple stresses. Its potential use in agroecological transition with double cropping systems could be improved by breeding a shorter life cycle. However, this strategy should not compromise its resilience to stresses as well as its metabolite profiles and plasticity. The impact of flowering time on drought stress response and seed quality was evaluated in six camelina edited mutants, carrying combinatory mutations on the flowering time genes SVP, TFL1, LHP1, ELF3 and FLC and leading to a range of flowering precocity and shoot architecture changes. We characterized the phenotype of these mutants in response to early and late drought and showed that their flowering time was not strongly altered contrary to branching and yield. Untargeted metabolomic demonstrated that in contrary to the lipidomic profile, the plasticity of the specialized metabolite was strongly modulated by drought in all genotypes. Specialized metabolite profile of the mutant seeds showed distinct pattern in response to drought with constitutive stress response of the bushy mutants in control condition including differences in antioxidant content such as glutathione, isoquercetrin, and coumaroyl quinic acid. Metabolite profiling in leaves also showed specific metabolic signatures of some mutants but with lower metabolite diversity than in seeds. Including additional genotypes with distinct flowering time, we identified metabolites correlating with this trait, such as vitamin B2 and kynurenic acid in seeds. These metabolites could be used as predictive markers of flowering time.

Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because 7 CRISPR-Cas3 components (6 Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39-71% of transformed calli by PCR analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the PAM sequence was estimated as 21-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice; base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.

Phosphorus is an essential nutrient critical for plant growth and development, yet its availability in soil is often limited. Consequently, most land plants establish symbiotic relationships with arbuscular mycorrhizal fungi (AMF) to enhance phosphate uptake. Strigolactones (SLs) function as rhizosphere signaling molecules that promote AMF symbiosis, distinct from their role as phytohormones regulating various plant functions. We previously identified an SL in Marchantia paleacea and demonstrated that the SLs primarily serve as rhizosphere signals rather than phytohormones in M. paleacea due to the absence of cognate receptors. In this study, we investigate the spatial localization of SL biosynthesis and secretion in M. paleacea. We find that SL biosynthesis genes are predominantly expressed in the basal region of the thallus compared to the distal region. Using Citrine driven by the promoter of MpaCCD8B, an SL biosynthesis gene, we show expression in smooth rhizoids and the ventral epidermis adjacent to these rhizoids, under phosphate-deficient conditions. When plants are cultured on medium, fluorescence is also detected in parenchymal cells, where AMF colonization occurs. In soil conditions, AMF colonization enhances MpaCCD8B expression in parenchymal cells, where AMF colonize. Furthermore, we assess SL secretion through germination assay of root parasitic plant seeds, revealing that exudates from the basal and midrib region exhibit the highest activity. These findings underscore that SLs are synthesized in the basal ventral tissues of M. paleacea and secreted into the rhizosphere, facilitating effective communication with AMF.