The Plant Journal

Mitochondria play key roles in cellular energy metabolism within eukaryotic cells. As relics of endosymbiotic bacteria, most (but not all) mitochondria contain their own genome (mitogenome, mtDNA), as well as intrinsic biosynthetic machinery for making organelle RNAs and proteins. The expression of the mtDNA requires regulated metabolism of its transcriptome by nuclear-encoded factors. Post-transcriptional mtRNA modifications play a central role in the expression of the plant mitogenomes, and hence in organellar biogenesis and plant physiology. Despite extensive investigations, a full map of angiosperm mitochondrial transcriptomes, a prerequisite for the elucidation of the basic RNA biology of mitochondria, has not been reported yet. Using RNA-seq data, RT-PCR and bioinformatics, we sought to explore the gene expression profiles of land plant mitochondria. Here, we present the mitochondrial transcriptomic maps of two key Brassicaceae species, Arabidopsis thaliana (var Col-0) and cauliflower (Brassica oleracea var. botrytis). The revised transcriptome landscapes of Arabidopsis and cauliflower mitogenomes provide with more detail into mtRNA biology and processing in angiosperm mitochondria, and we expect that they would serve as a valuable resource for the plant organellar community. Accession numbersSequences are available at the Sequence Read Archive (accession no. PRJNA472433), for both Arabidopsis thaliana var. Col-0 mtRNA (SRA no. SRX4110179) and Brassica oleracea var. botrytis mtRNA (SRA no. SRX4110177).

SummaryMitochondria are semi-autonomous organelles that serve as hubs for aerobic energy metabolism. The biogenesis of the respiratory (OXPHOS) system relies on nuclear-encoded factors, which regulate the transcription, processing and translation of mitochondrial (mt)RNAs. These include proteins of primordial origin, as well as eukaryotic-type RNA-binding families recruited from the host genomes to function in mitogenome expression. Pentatricopeptide repeat (PPR) proteins constitute a major gene-family in angiosperms that is pivotal in many aspects of mtRNA metabolism, such as editing, splicing or stability. Here, we report the analysis of MITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1 (MSP1, At4g20090), a canonical mitochondria-localized PPR protein that is necessary for mitochondrial biogenesis and embryo-development. Functional complementation confirmed that the phenotypes result from a disruption of the MSP1 gene. As a loss-of-function allele of Arabidopsis MSP1 leads to seed abortion, we employed an embryo-rescue method for the molecular characterization of msp1 mutants. Our data show that msp1 embryo-development fails to proceed beyond the heart-torpedo transition stage as a consequence of a severe nad1 pre-RNA processing-defect, resulting in the loss of respiratory complex I (CI) activity. The maturation of nad1 involves the processing of three RNA-fragments, nad1.1, nad1.2 and nad1.3. Based on biochemical analyses and the mtRNA profiles in wild-type and msp1 plants, we concluded that through its association with a specific site in nad1.1, MSP1 facilitates the generation of its 3-terminus and stabilizes it -a prerequisite for nad1 exons a-b splicing. Our data substantiate the importance of mtRNA metabolism for the biogenesis of the respiratory machinery during early-plant development.

Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of NLR plant immune receptors has greatly advanced following the discovery of Arabidopsis ZAR1 resistosome. Here, we describe a novel vector system of 52 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. NbZAR1 triggers autoimmune cell death in N. benthamiana and this activity is dependent on a functional N-terminal 1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source plasmids form an additional experimental system to Arabidopsis for in planta resistosome studies.

O_LIPlant pigmentation secrets are among the oldest interests of plant scientists, with pigments such as chlorophyll, carotenoids, flavonoids, and betalains contributing to the diversity of hues in higher plants. Anthocyanins, a class of flavonoids responsible for vibrant shades of pink, red, and blue pigmentation, are almost ubiquitous in angiosperms but are replaced by betalains in some families in the order Caryophyllales. C_LIO_LIWe investigated anthocyanin pigmentation in Cucurbitaceae, one of the largest fruit and vegetable families, characterised by white and yellow flowers and red, orange, and green fruits predominantly pigmented by carotenoids. Using a comprehensive collection of 258 datasets representing 183 unique species across all 15 tribes of Cucurbitaceae, along with a phylogenomics approach, we observed a systematic absence of genes involved in anthocyanin and proanthocyanidin biosynthesis. Absence of the structural genes DFR, ANS, arGST, LAR, and ANR, along with the anthocyanin-related regulatory MYB genes, was consistently confirmed by synteny and phylogenetic analysis. C_LIO_LIThese results suggest that anthocyanin loss in angiosperms is more common than previously assumed. In light of this new discovery, we propose a stepwise loss of anthocyanin pigmentation in Cucurbitaceae, likely accompanied by a partial functional replacement with carotenoid pigmentation. C_LI

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the involved protein factors and the underlying molecular mechanisms are still not well understood. In this study, we report the function of the mitochondrial stability factor 3 (MTSF3) protein and we show that it is essential for accumulation of the mitochondrial nad2 transcript in Arabidopsis and not for the splicing of nad2 intron 2, as recently proposed (Marchetti et al., 2020). The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality but viable mtsf3 mutant plants could be generated by partial complementation with the developmentally-regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses reveal that mtsf3 rescued plants display growth retardation due to the specific destabilization of a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3-terminus of nad2. The destabilization of nad2 mRNA induces a significant decrease in complex I assembly and activity, and an overexpression of the alternative respiratory pathway. Our results support that the MTSF3 protein protects nad2 transcript from degradation by mitochondrial exoribonucleases by binding to its 3 extremity.

O_LI13C-Metabolic flux analysis (13C-MFA) have greatly contributed to revealing the regulation of plant metabolism. However, mass spectrometry (MS) approaches have hitherto been limited in their power to deduce flux information due to lack of positional information. C_LIO_LIHere we established an MS-based 13C-positional isotopomer labelling approach and performed a multi-species/cell-types analysis based on previous 13C-MFA to compare flux modes through the tricarboxylic acid (TCA) cycle and associated pathways in mesophyll (MCs) and guard cells (GCs). C_LIO_LIBoth cell types showed high 13C-enrichment in pyruvate. However, GCs and sink MCs, but not source MCs showed high 13C-incorporation into Glu/Gln following provision of 13C-sucrose. Only GCs showed higher 13C-enrichment in the carbon 1 atom of Gln, which is derived from PEPc-mediated CO2 fixation. Increased 13C-enrichment in the carbon 1 of Glu was also observed in both trxo1 and ntra ntrb mutants, but not in wild type Arabidopsis plants, following provision of 13C-glucose. C_LIO_LIOur results suggest that the mitochondrial thioredoxin system restricts the fluxes from PEPc and glycolysis to Glu in illuminated MCs and reveal that fluxes throughout the TCA cycle of GCs resemble those of sink MCs but operate different non-cyclic flux modes to support Gln synthesis in the light. C_LI

We explored the expression of the aldehyde dehydrogenase (ALDH) superfamily in two model plants, Physcomitrium patens (moss) and Hordeum vulgare (barley), under various stress conditions. The ALDH enzymes are crucial for oxidizing aldehydes to carboxylic acids and are involved in multiple metabolic pathways. We found significant differences in enzyme expression between moss and barley within the same ALDH families. We then focused on the ALDH5, ALDH10, and ALDH21 families, which are part of the {gamma}-aminobutyric acid (GABA) shunt, noting that the ALDH21 family is absent in barley. The kinetic properties of ALDH10 and ALDH5 enzymes were analyzed, revealing that PpALDH5F1 exhibits high specificity for succinic semialdehyde (SSAL), a product of GABA. The crystal structure of PpALDH5F1 identified key residues for SSAL binding. Knockout mutants of moss aldh5F2, aldh10A1, and aldh21A1 showed slightly smaller colonies than the wild-type. GABA and glutamate levels were elevated in aldh5F2 and aldh21A1 knockouts due to a partially blocked GABA shunt pathway, while aldh10A1 knockout showed no changes in GABA levels. Transcriptomic data revealed a link between several genes, including six upregulated glutathione-S-transferase genes in all three aldh knockouts, suggesting a direct compensatory mechanism for oxidative stress protection via conjugation of undegraded aldehydes to glutathione. HighlightMoss knockouts of GABA shunt-associated aldehyde dehydrogenases display slower growth, changes in levels of glutamate, glutamine and GABA, and result in upregulation of several unique glutathione-S-transferase genes.

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of plant responses. AC domains are intrinsic components of complex molecules with multiple functions, some of which are co-regulated by cAMP. Here we used an amino acid search motif based on annotated ACs in organisms across species to identify 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9-cis-epoxycarotenoid dioxygenase (NCED3, At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally for activity. We show that an NCED3 AC fragment can complement an AC deficient E. coli mutant and this rescue is nullified when key amino acids in the AC motif are mutated. AC activity was also confirmed by tandem liquid chromatography mass spectrometry (LC-MS/MS). Our results are consistent with a moonlighting role for mononucleotide cyclases in multi-domain proteins that have at least one other distinct molecular function such as catalysis or ion channel activation and promise to yield new insights into tuning mechanisms of ABA dependent plant responses. Finally, our search method can also be applied to discover ACs in other species including Homo sapiens. HighlightsO_LIAn adenylyl cyclase (AC) catalytic center motif identifies novel ACs in plants C_LIO_LIACs can moonlight in complex proteins with other enzymatic domains C_LIO_LIA 9-cis-epoxycarotenoid dioxygenase essential for abscisic acid synthesis contains an AC C_LIO_LIThis finding implicates cAMP in abscisic acid synthesis and signaling C_LI

The enzymatic steps involved in O_SCPLOWLC_SCPLOW-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and O_SCPLOWLC_SCPLOW-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and O_SCPLOWLC_SCPLOW-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and O_SCPLOWLC_SCPLOW-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration. One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-O_SCPLOWLC_SCPLOW-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

Heracleum sosnowskyi, belonging to a group of giant hogweeds, is a plant with large effects on ecosystems and human health. It is an invasive species that contributes to the deterioration of grassland ecosystems. The ability of H. sosnowskyi to produce linear furanocoumarins (FCs), photosensitizing compounds, makes it very dangerous. At the same time, linear FCs are compounds with high pharmaceutical value that are used in skin disease therapies. Despite this high importance, it has not been the focus of genetic and genomic studies. Here, we report a chromosome-scale assembly of the Sosnowskys hogweed genome. Genomic analysis revealed an unusually high number of genes (55 206) in the hogweed genome, in contrast to the 25-35 thousand found in most plants. However, we did not find any traces of recent whole genome duplications not shared with its confamiliar, Daucus carota (carrot), which has approximately thirty thousand genes. The analysis of the genomic proximity of duplicated genes indicates tandem duplications as a main reason for this increase. We performed a genome-wide search of the genes of the FC biosynthesis pathway and their expression in aboveground plant parts. Using a combination of expression data and phylogenetic analysis, we found candidate genes for psoralen synthase and experimentally showed the activity of one of them using a heterologous yeast expression system. These findings expand our knowledge on the evolution of gene space in plants and lay a foundation for further analysis of hogweed as an invasive plant and as a source of FCs.

Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the Northeastern regions of Africa. Although it is grown in a limited manner, B. carinata is of value as it is resistant/highly tolerant to most of the pathogens affecting cultivated Brassica species of the Us triangle that are grown worldwide as oilseed and vegetable crops. We report a chromosome-scale genome assembly of B. carinata accession HC20 using long-read Oxford Nanopore and Illumina sequencing and BioNano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the available long-read genome assemblies of the six species of the Us triangle and found a highly conserved gene number and collinearity suggesting that B. juncea (AABB), B. napus (AACC), and B. carinata are strict allopolyploids. We cataloged the nucleotide-binding and leucine-rich repeat immune receptor (NLR) repertoire of B. carinata resulting in the identification of 465 NLRs. We investigated the extent and nature of early generation genomic interactions between the subgenomes of B. carinata and B. juncea in interspecific crosses between the two species. We found that C chromosome additions are well tolerated, with homoeologous exchanges occurring between the A and C genomes. Based on the genomic interactions, we propose strategies to utilize the interspecific crosses for transferring disease resistance from B. carinata to B. juncea and other Brassica species.

Stinging nettle (Urtica dioica L.) is a widespread weed of economic significance with a dioecious mating system. Previously, we generated a high-quality genome assembly of a diploid female plant, which showed extreme levels of structural variation between haplotypes. Here, we present a chromosome-level, haplotype-resolved sequence of a diploid male plant; since the male is believed to be the heterogametic sex in Urtica dioica, this assembly represents a first step towards elucidating the control of sex determination in this species. This completely independently assembled genome also allows confirmation of previously reported features of the nettle genome, including (1) a high degree of structural variation between haplotypes, including large inversions, (2) the likely existence of polycentric centromeres, and (3) the presence of urticaceous "pain peptide" sequences. Chromosome 8 stands out for its multiple large, nested inversions and high levels of repetitive sequences, features that are often associated with sex determining regions (SDRs). This chromosome is therefore a candidate for further investigations to characterize the sex determination in nettle.

The regulation of gene expression in plant responses to drought has been thoroughly investigated in previous studies. Despite this, a detailed understanding of the cell type-specific regulatory mechanisms, encompassing multi-layered biological processes, is lacking. In this study, we report the use of single-nucleus multiomic analysis in Arabidopsis seedlings in response to drought stress. Our single-nuclei RNA (snRNA) analysis delineated 14 distinct clusters representing major root and shoot cell types and discovered new cell type-specific drought markers. Integration of snRNA with single-nuclei ATAC (snATAC) data in leaf epidermis, root endodermis, and guard cells revealed accessible chromatin regions (ACRs)-linked genes predominantly enriched in pathways responsive to drought, heat, and light. Motif enrichment analysis and gene regulatory network (GRN) inference highlighted key transcription factors (TFs) and regulatory networks related to ethylene signaling pathways in endodermis as well as circadian rhythms in both endodermis and guard cells. Pseudotime analysis identified critical transcriptomic progression from metabolic process to stress response within three cell types. Overall, this study elucidates drought-related regulatory mechanisms in Arabidopsis at single-cell resolution, providing valuable insights into the fundamental regulatory events involved in stress responses. It also serves as a reference for future single-cell multiomic investigations in crop plants. One Sentence SummarySingle cell multiomic analysis under drought stress

BackgroundRecent advances in assemblies of nearly gap-free, high-quality genomes have enabled detailed analysis of centromeres in large and highly repetitive crop genomes. Here, we analyse the hexaploid Avena sativa genome and its tetraploid (A. insularis) and diploid relatives (A. longiglumis, A. atlantica and A. eriantha). ResultsAvena centromeres are largely composed of retrotransposons belonging to three families, RLG_Ava, RLG_Cereba and RLG_Beth. Analysis of retrotransposon populations revealed striking differences in the centromere architecture between the A, C and D genome lineages. We identified distinct profiles of transposable element bursts for these lineages which include the emergence and disappearance of retrotransposon families and subfamilies. We identified multiple centromere shifts which occurred in the C genome lineage within the past [~] 4 myrs and retraced species divergences and polyploidization events in the Avena genus. Although the studied species are closely related, our data show that their centromeres have rapidly evolved different and distinct centromere architectures, for example through the spread of novel satellite repeats or activity bursts of different retrotransposon families and subfamilies. Additionally, we found that RLG_Ava and RLG_Cereba retrotransposons have been coexisting while simultaneously competing for the centromeric "niche" since the emergence of the Poaceae (grasses). ConclusionsOur comparative analyses provided detailed insight into centromere evolution across the Avena genus and revealed that composition and architecture of centromeres can vary greatly even between closely related species and different ploidy levels. Our findings emphasize the need for extended analyses of large genome species to improve our understanding of centromere evolution.

Arabidopsis thaliana Col-0 has plastid and mitochondrial genomes encoding for over one hundred proteins and several ORFs. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of such RNA editing at the protein level is largely unexplored due to the complexities of detection. This study first assembled the non-redundant set of identifiers, their correct protein sequences, and 452 predicted non-synonymous editing sites of which 56 are edited at lower frequency. Accumulation of edited and/or unedited proteoforms was then determined by searching [~]259 million raw MSMS spectra from ProteomeXchange as part of Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). All mitochondrial proteins and all except three plastid-encoded proteins (NDHG/NDH6, PSBM, RPS16), but none of the ORFs, were identified; we suggest that all ORFs and RPS16 are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical challenges of detection.167 predicted edit sites were detected at the proteome level. Minor frequency sites were indeed also edited at low frequency at the protein level. However, except for sites RPL5-22 and CCB382-124, proteins only accumulate in edited form (>98 -100% edited) even if RNA editing levels are well below 100%. This study establishes that RNA editing for major editing sites is required for stable protein accumulation.

Camelina sativa is an oilseed of the Brassicaceae, whose close relatives vary in ploidy number, providing a novel platform for studying plant genome evolution. The availability of diploid, tetraploid and hexaploid species of Camelina allow the evolutionary trajectory and fate of duplicated genes in the neopolyploid Camelina species to be elucidated. Here we report an improved assembly of the widely used C. sativa reference DH55 and three new genome assemblies of Camelina microcarpa; one tetraploid CN119243 (2n = 26), and two hexaploids with divergent chromosome numbers, Type 1 - CN119205 (2n=40) and Type 2 - CN120025 (2n=38). The tetraploid represents the first step in the evolutionary path to form C. sativa, while the hexaploids suggest three divergent lineages in the formation of higher ploidy Camelina species. The previously uncharacterized fourth subgenome found in C. microcarpa Type 2, although showing some homology to the C. sativa diploid progenitor genome, C. neglecta, showed numerous unique chromosomal rearrangements differentiating it from other subgenomes present in known Camelina species. Although this species was recently formed, the second subgenome showed gene expression dominance, which was in contrast to both 2n=40 Camelina species where the third subgenome was dominant. The expression dominance in Type 2 C. microcarpa contradicted the accepted two-step evolutionary process which led to the generation of related Brassicaceae species. However, the observed genome dominance in all Camelina species was negatively correlated with inter-subgenome chromatin interaction frequencies, suggesting that chromosome confirmation and proximity in the nucleus contributes to this mechanism of genome evolution. Despite the differences in genome structure, successful inter-specific hybridization provided evidence of chromosomal exchange between the divergent third sub-genomes of C. sativa and C. microcarpa Type 2, opening up a novel avenue to new diversity in the established oilseed. Key pointsO_LIAn improved genomic understanding of Camelina species and identification of distinct subgenome structures and relationships, which will facilitate strategies to increase the genetic diversity in C. sativa. C_LIO_LISubgenome evolution and subgenome dominance in polyploids is associated with chromosomal architecture and proximity in the nucleus. C_LIO_LIGenome assemblies representing all ploidy levels in the Camelina genus provide a unique and valuable platform for polyploid research. C_LI

Nicotiana benthamiana has long served as a crucial plant material extensively used in plant physiology research, particularly in the field of plant pathology, because of its high susceptibility to plant viruses. Additionally, it serves as a production platform to test vaccines and other valuable substances. Among its approximately 3.1 Gb genome, 57,583 genes have been annotated within a 61 Mb region. We created a comprehensive and easy-to-use platform to use transcriptomes for modern annotation. These tools allow to visualize gene expression profiles, draw molecular evolutionary phylogenetic trees of gene families, perform functional enrichment analyses, and facilitate output downloads. To demonstrate their utility, we analyzed the gene expression profiles of enzymes within the nicotine biosynthesis pathway, a secondary metabolic pathway characteristic of the Nicotiana genus. Using the developed tool, expression profiles of the nicotine biosynthesis pathway genes were generated. The expression patterns of eight gene groups in the pathway were strongly expressed in the roots and weakly expressed in leaves and flowers of N. benthamiana. The results were consistent with the established gene expression profiles in Nicotiana tabacum and provided insights into gene family composition and expression trends. The compilation of this database tool can facilitate genetic analysis of N. benthamiana in the future. Significance statementA tool was developed to visualize gene expression profiles, draw molecular evolutionary phylogenetic trees of gene families, perform functional enrichment analyses, and facilitate output downloads of Nicotiana benthamiana. The database developed using the tool can evolve into a comprehensive all-in-one analysis platform by continuously incorporating transcriptome data released to date, newly released RNA-seq data, and annotations in the future.

Riboflavin (RF) serves as a precursor to FMN and FAD, crucial cofactors in various metabolic processes. Strict regulation of cellular flavin homeostasis is imperative, yet information regarding the factors governing this regulation remains largely elusive. In this study, we first examined the impact of external flavin treatment on the Arabidopsis transcriptome to identify novel regulators of cellular flavin levels. Our analysis revealed alterations in the expression of 49 putative transcription factors. Subsequent reverse genetic screening highlighted a member of the Dehydration-Responsive Element Binding (DREB) family, AtDREB2G, as a potential regulator of cellular flavin levels. Knockout mutants of AtDREB2G (dreb2g) exhibited reduced flavin levels and decreased expression of RF biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of RF biosynthetic genes and elevated flavin levels. In wild-type plants, exposure to low temperatures and abscisic acid treatment stimulated enhanced flavin levels and upregulated the expression of RF biosynthetic genes, concomitant with the induction of AtDREB2G. Notably, these responses were significantly attenuated in dreb2g mutants. Our findings establish AtDREB2G as a novel positive regulator of flavin biosynthesis in Arabidopsis, particularly under conditions of low temperature and abscisic acid treatment.

The evolution of long-read sequencing technologies has advanced the development of genome assemblies that are now frequently presented as gap-free or nearly gapless, reflecting substantial improvements in sequencing accuracy and completeness. However, discrepancies between the genome size estimates derived from genome assemblies and flow cytometry create ambiguity regarding the accuracy of these complementary approaches. Accurate genome size estimation via flow cytometry relies on use of an internal standard with a genome of known size. Historically, the genome size of these standards was often calibrated against incomplete genome assemblies or non-plant genomes, such as the human male genome, which was previously considered to be 7 pg but is now known to be around 6.15 pg after 20 years of advancements. Calibrating plant references against non-plant standards is not recommended due to differential staining properties. Therefore, we recalibrated the size of five plant genomes commonly used as reference standards in flow cytometry, by utilizing a recent gapless, telomere-to-telomere (T2T) genome assembly of Nipponbare rice. Our results indicate a significant overestimation of around 20% in previous flow cytometry-based estimates for Pisum sativum and Nicotiana benthamiana, around 10% for Arabidopsis thaliana, and less than 5% for Sorghum bicolor, and Gossypium hirsutum. The close alignment of the recalibrated GS estimates to the reference genome assemblies and recalculated estimates from different studies confirms their suitability as reference standards for more accurate measurement of plant genome size.