Plant and Cell Physiology

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

Iron (Fe) homeostasis is fundamental to plant growth and development and is centrally regulated by a well-conserved network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 regulates the expression of FER-like iron deficiency-induced transcription factor (FIT) and clade Ib bHLH38/39/100/101 genes, among others. However, its functional conservation remains unexplored, and some contradictory results have been reported regarding the effects of bHLH121 overexpression. Here, we investigated the evolutionary conservation of bHLH121 through complementary tests in the Arabidopsis bhlh121 mutant created by CRISPR/Cas9 gene editing. We also investigated the effects of ectopically expressing full-length, N-terminal, and C-terminal fragments of bHLH121 on plant responses to Fe deficiency in Arabidopsis. Transgenic plants overexpressing the full-length bHLH121 driven by the cauliflower mosaic virus 35S promoter (bHLH121 ox) resulted in shorter roots and more severe leaf chlorosis compared to wild-type plants under Fe limiting conditions. Moreover, overexpression of the N-terminal fragment (N121 ox) rendered plants highly sensitive to Fe deficiency, whereas overexpression of the C terminal fragment (C121 ox) did not. Consistent with the phenotypic observations, N121 ox plants accumulated lower Fe content, and the ferric chelate reductase activity was significantly reduced. In N121 ox plants, the induction of key Fe regulatory genes, especially FIT, was significantly inhibited under Fe deficiency. Confocal imaging showed that both N121-GFP and C121-GFP fusion proteins could localize to the nucleus. Electrophoretic mobility shift assay showed that both full-length bHLH121 and N121 could effectively bind the probes derived from the promoter of FIT. The present work reveals a conserved function of bHLH121 in Fe homeostasis across several dicot species and demonstrates that the N121 may possess a dominant nature in Fe deficiency responses.

Abstract: Picrotoxane-type sesquiterpene alkaloids, including dendrobine, are characteristic constituents of Dendrobium nobile, but the enzymes that produce their proposed skeletal precursors remain in completely characterized. Here, we combined tissue metabolite profiling with genome-guided screening and biochemical characterization of terpene synthases (TPSs). GC-MS analysis revealed variation in sesquiterpenoid profiles and dendrobine signals among tissues and developmental stages. Manual curation of the chromosome-level genome yielded 16 full-length TPS-a candidates. Functional screening in an FPP-supplying Escherichia coli system showed that three closely related genes on chromosome 1, DnTPS1~DnTPS3, produced copaborneol as their dominant product. The principal product of DnTPS1 was purified and identified by NMR spectroscopy, and a purified-enzyme assay confirmed its direct conversion of FPP to copaborneol. Fluorescence imaging in rice protoplasts and Nicotiana benthamiana leaves indicated predominantly cytosolic localization of DnTPS1. Guided by sequence comparisons and structural modeling, we tested 12 DnTPS1 variants and found that substitutions at W377 and I295 altered the relative distribution of sesquiterpene products. Phylogenetic and regional gene-order analyses placed DnTPS1~DnTPS3 in the same clade and identified related TPS candidates within corresponding genomic regions of six Dendrobium species. These findings establish that three D. nobile TPSs can form copaborneol and identify DnTPS1 residues that influence product selectivity, providing a basis for testing the proposed connection to dendrobine biosynthesis.

Plants have remarkable capacity to reconstruct entire organ systems from tissue explants. In Arabidopsis two-step tissue culture system, pluripotency regulators are specifically expressed in the middle-cell layer of the stratified callus tissue. However, regulatory mechanisms underlying the radial patterning of callus remained unclear. Here, we found that very-long-chain fatty acids (VLCFAs) synthesized in the epidermis-like outermost layer are essential for pluripotency acquisition and successful shoot regeneration. Our genetic and transcriptomic analyses revealed that the regulatory roles of VLCFAs on pluripotency acquisition involve inter-cell layer signaling in callus tissue, while they are at least partly independent of ATML1/PDF2 functions and cuticular wax synthesis in the outermost layer. VLCFAs spatially restrict procambium cell identity by non-cell-autonomously suppressing cytokinin signaling, thereby allowing for establishment of the middle-cell layer. We propose that the inhibitory relationships between layer-specific regulators underlie the intricate balance of cellular fate determination in pluripotent callus.

BackgroundN6-Methyladenosine (m6A) regulates RNA fate in flowering plants, but the repertoire and expression patterns of m6A-related genes remain incompletely characterized in liverworts. Here, we characterized these gene families in the liverwort Marchantia polymorpha within a comparative framework spanning diverse plant lineages. ResultsA comparative survey of 119 plant genomes using sequence similarity and conserved-domain criteria identified 18 candidate m6A-related loci in M. polymorpha, comprising seven writer-related genes, five AlkB homolog genes, and six YTH- or CPSF30-related genes. Across the surveyed genomes, canonical writer components generally had low copy numbers, whereas AlkB homolog and reader-related families showed greater copy-number variation. Phylogenetic and domain analyses supported the family assignments of the M. polymorpha candidates. Eight of the 18 candidate loci showed detectable collinearity with Marchantia quadrata. Putative promoter analysis identified motifs annotated as hormone-, light-, and stress-responsive, with hormone-responsive motifs accounting for the largest share of predicted sites. Public transcriptome analysis revealed variation in candidate gene expression across developmental stages and tissues. Quantitative reverse-transcription PCR showed higher transcript abundance for all six YTH- or CPSF30-related genes in thalli than in gemmae or gemma cups. Cold, heat, osmotic, salt, and dark treatments elicited gene-specific expression responses. Darkness increased transcript abundance of several candidates, including MpMTA, MpALKBH1A, and MpECT2. A predicted protein-association network suggested links between subsets of the candidates and RNA-processing proteins. ConclusionsThese findings define a candidate m6A-related gene repertoire in M. polymorpha and identify tissue- and stress-responsive genes for targeted functional studies of RNA methylation in this liverwort.

GOLDEN2-like (GLK) transcription factors are key regulators of chloroplast differentiation and photosynthetic gene expression. The classical yellow mutation in Japanese morning glory (Ipomoea nil) produces yellowish-green leaves, whereas an unstable allele, yellow-mutable, produces green somatic sectors on a yellowish-green background. The gene responsible for these mutations was identified as InGLK, which encodes a GOLDEN2-like transcription factor. The stable yellow mutant carried a 4-bp frameshift insertion in InGLK, whereas two yellow-mutable lines carried the Tpn1-family transposon Tpn12 in intron 5. Excision of Tpn12 in germinal revertants left short footprints and restored the green leaf phenotype. Genome searches identified InGLK as the sole GLK gene in I. nil. Pigment analysis of green somatic reversion sectors and yellowish-green background areas showed that most of the measured photosynthetic pigments were significantly reduced in the yellowish-green background, whereas the chlorophyll a/b ratio was unchanged. Chloroplasts in the yellowish-green tissue retained thylakoid-like membranes and starch granule-like structures but had less distinct grana-like stacks and sparse stromal lamellae-like structures. Wild-type-like chloroplast ultrastructure was restored in germinal revertants. These findings show that loss of function of a single-copy GLK gene broadly reduces photosynthetic pigment accumulation and alters chloroplast internal membrane organization. The yellow mutants of I. nil therefore provide a genetic system for examining non-redundant GLK function.

Background Strigolactones (SLs) regulate diverse aspects of plant development and have been implicated in promoting leaf senescence. However, senescence phenotypes associated with SL deficiency have not been consistently observed across species, suggesting that this function may be species- or context-dependent. Moreover, the contribution of endogenous SL biosynthesis to senescence in leafy vegetable crops remains unclear. Here, we investigated the role of the SL biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) in dark-induced leaf senescence in lettuce (Lactuca sativa). Results We found that endogenous SL biosynthesis plays a major role in dark-induced senescence in lettuce. SL pathway genes were induced during dark storage, while exogenous GR24 accelerated senescence and lettuce MAX1 (LsMAX1) complemented the delayed-senescence phenotype of the Arabidopsis max1 mutant. Consistent with these findings, CRISPR/Cas9-generated Lsmax1 mutants exhibited a pronounced stay-green phenotype during prolonged darkness, accompanied by strongly reduced induction of key senescence-associated genes. Despite this delayed visible senescence, Lsmax1 retained a substantial transcriptional response to dark storage. Strikingly, loss of LsMAX1 did not simply weaken the wild-type senescence program, but redirected part of the response toward a distinct stress-associated transcriptional state that was largely absent from wild type. Loss of LsMAX1 did not affect vegetative rosette architecture, although increased branching emerged after bolting. Conclusions Our findings establish MAX1-dependent SL biosynthesis as an important regulator of leaf senescence in lettuce and reveal a role that extends beyond controlling the rate of senescence. Rather than simply delaying the wild-type program, loss of LsMAX1 alters the transcriptional trajectory of senescence, favoring an alternative stress-associated state during prolonged darkness. The strong stay-green phenotype without detectable changes to vegetative rosette architecture further highlights SL biosynthesis as a potential target for extending postharvest longevity in lettuce and other leafy crops.

Plants possess a remarkable capacity for regeneration, which involves the redeployment of developmental programs and diverse regulatory mechanisms. However, how related regulators with overlapping functions are differentially deployed during regeneration remains poorly understood. The model liverwort Marchantia polymorpha provides a powerful experimental system for studying regeneration because it readily regenerates apical meristems from basal thallus fragments after removal of the original meristem, even without exogenous plant hormones. Here, we identify the R2R3-MYB transcription factor GEMMA CUP-ASSOCIATED MYB1-LIKE (MpGC1L), the closest paralog of the clonal propagation regulator MpGCAM1, as a positive regulator of regeneration. MpGC1L was rapidly induced at the cut site following meristem removal. Ectopic overexpression of MpGC1L caused the proliferation of undifferentiated cells, whereas Mpgc1l mutants showed delayed regeneration and reduced S-phase entry. Loss of MpGCAM1 alone had little effect on regeneration but markedly enhanced the Mpgc1l phenotype, indicating partially redundant functions. Transcriptome analysis of the double mutant revealed reduced induction of genes associated with ribosome biogenesis and the cell cycle. We next examined the relationship between MpGC1L and the known jasmonate- and auxin- related regeneration regulators, MpERF15 and MpLAXR. MpGC1L induction was retained in Mperf15 and Mplaxr mutants and was unaffected by OPDA or auxin treatment, whereas MpERF15 and MpLAXR were still induced in Mpgc1l Mpgcam1 double mutants. Thus, these regulators are not arranged in a simple linear transcriptional pathway. Our findings reveal that the paralogous MYB transcription factors MpGC1L and MpGCAM1 promote cell proliferation in distinct developmental contexts, thereby linking clonal propagation and wound-induced regeneration.

Although iron (Fe) is abundant in soils, its low solubility, particularly under alkaline conditions, often limits plant Fe acquisition. Under these conditions, coumarin-mediated Fe mobilization becomes increasingly important. Arabidopsis thaliana FE DEFICIENCY-INDUCED KELCH REPEAT PROTEIN 1 (FDK1, AT3G07720) is strongly induced by Fe deficiency and regulated by the central transcription factor FIT, but its function is unknown. We investigated FDK1 using genetic, physiological, metabolite, and transcriptomic analyses. Loss of FDK1 altered root growth, whereas this phenotype was restored in the FDK1-Ox overexpression line. Under alkaline Fe deficiency, growth medium from FDK1-Ox contained strongly increased levels of the Fe-mobilizing coumarins fraxetin and esculetin compared with wild type, whereas their induction was attenuated in fdk1-1. RNA-seq analysis linked FDK1 abundance to coumarin biosynthesis and iron-homeostasis gene expression. FIT and many FIT-associated genes were highly expressed in FDK1-Ox, whereas several upstream iron-homeostasis regulators, including bHLH Ib factors, were highest in fdk1-1. These findings identify FDK1 as a component of the alkaline Fe-deficiency response that promotes iron-mobilizing coumarin accumulation and is associated with coordinated regulation of iron-homeostasis genes.

Sporadically, all plastids produce dynamic tubular extensions in response to myriad stresses. Although the use of fluorescent proteins revealed these transient tubules to be stroma-filled and led to their being called stromules, the mechanism underlying tubule extension from a spheroidal plastid remained unknown. As plastids in viridiplantae have two bounding membranes the fluid stroma remains confined within the inner envelope membrane (IEM) while the outer envelope membrane (OEM) around the IEM forms the interface with the rest of the cytoplasm. Here, using a sucrose treatment for inducing tubules in the dark in transgenic Arabidopsis plants expressing different stroma and OEM-targeted fluorescent proteins we carried out comparative molecular-genetic and lipidomics analyses. Our data strongly suggest that plastid tubule formation occurs due to increase in bilayer-forming galactolipids such as digalactosyldiacylglycerol (DGDG) on the OEM. While the dynamic behaviour of stromules occurs within an external encasing OEM, stresses such as phosphate starvation and high light intensity cause the galactolipid-rich OEM-tubule projecting beyond the stromule to be shed into the cytoplasm. OEM shedding from long tubules with extended plastid outreach into the cell suggests their important role in dispersing plastid-derived, membrane-stabilizing, galactolipids to the rest of the cell to augment its resilience to stress.

Iron (Fe) deficiency is a major constraint for plant growth and triggers extensive physiological and transcriptional reprogramming to maintain Fe homeostasis. Here, we identify the glucosinolate-associated transcription factors MYB28 and MYB29 as previously unrecognized regulators of Arabidopsis thaliana adaptation to Fe deficiency. Across various growth systems, loss of MYB28 increased sensitivity to Fe deficiency, whereas the myb28myb29 double mutant displayed stronger chlorosis, reduced root growth and impaired biomass accumulation, indicating cooperative but unequal functions of these transcription factors. Despite their enhanced Fe-deficiency phenotype, double mutant plants accumulated higher Fe levels in roots and exhibited stronger induction of canonical Fe-deficiency responses, suggesting impaired Fe utilization or distribution rather than defective Fe uptake. RNA-seq revealed extensive transcriptional reprogramming under Fe deficiency, with pronounced deregulation of genes involved in Fe homeostasis, redox processes and growth, particularly in the double mutant. Among these, SCOPOLETIN 8-HYDROXYLASE (S8H) emerged as a major target gene of MYB28. The expression of S8H was almost abolished in myb28 and myb28myb29 mutants, whereas expression of other coumarin biosynthetic genes remained largely unaffected. Promoter activation assays demonstrated that MYB28 activates the S8H promoter, and metabolic analyses showed accumulation of scopolin together with reduced fraxin levels in the mutants, consistent with impaired S8H activity. Collectively, our results identify MYB28 as a key regulator linking specialized metabolism to Fe homeostasis through control of coumarin biosynthesis, thereby expanding the biological functions of MYB28 and MYB29 transcription factors.

Flower opening is a complex developmental process involving coordinated changes in cell proliferation and cell expansion. Although several regulators of flower opening have been identified, how transcriptional programs are coordinated with the cellular and metabolic changes underlying petal expansion immediately before flower opening remains incompletely understood. Japanese morning glory (Ipomoea nil) is a suitable model for investigating these processes because its flowers open synchronously at a predictable time. This study aimed to identify transcriptional regulators involved in petal development and flower opening in Japanese morning glory. Temporal analyses of petal growth, sugar metabolism, and gene expression revealed that petal development was driven by both cell proliferation and cell expansion until approximately 48 h before flower opening, whereas cell expansion predominated thereafter. Weighted gene co-expression network analysis identified two genes encoding R2R3-MYB subgroup 19 transcription factors, InMYB21A and InMYB21B, as candidate regulators associated with petal development. CRISPR/Cas9-mediated knockout analysis revealed a prominent role for InMYB21B, whose loss markedly impaired petal cell expansion and prevented flower opening. InMYB21B knockout also impaired stamen and pistil development, resulting in male and female sterility. Starch degradation and glucose accumulation were impaired in InMYB21B knockout petals. Transcriptome analysis revealed delayed transcriptomic progression during petal development and reduced expression of genes associated with starch degradation, sucrose metabolism, cell wall remodeling, and water transport. These findings identify InMYB21B as a key regulator of petal cell expansion and flower opening in Japanese morning glory and show that loss of InMYB21B disrupts both metabolic and transcriptomic progression during late petal development.

Cajal bodies (CBs) are nuclear condensates known for their role in sn- and snoRNA maturation in animal, but their function in plant stress responses remains poorly understood. We compared wild-type (WT) Arabidopsis thaliana with the CB-lacking ncb-1 mutant during hypoxia and reoxygenation, focusing on nuclear retention of transcripts. In both genotypes, hypoxia caused nuclear accumulation of poly(A+) RNA and extensive reprogramming of gene expression. However, the transcriptional response, including induction of hypoxia-responsive genes, was considerably weaker in ncb-1, both during stress and recovery. In nuclear RNA, introns with low (intron retention index, IRX < 10%) and high (IRX > 50%) retention showed opposite, genotype-dependent dynamics. During hypoxia, efficiently spliced introns (IRX < 10%) accounted for over 70% of introns detected in nuclear RNA. Relative to normoxia, this proportion increased two- to threefold in WT but only moderately in the mutant. Together with poly(A+) RNA accumulation, this suggests that largely spliced transcripts, including those of genes involved in protein transport and ribosome biogenesis, are held in the nucleus during stress. Conversely, strongly retained introns, often terminal and located in genes related to hypoxia and other abiotic stresses, were most abundant in WT nuclear RNA under normoxia. Their proportion dropped sharply during hypoxia, and much less so in ncb-1. This indicate that WT plants maintain a nuclear reserve of incompletely spliced stress-related transcripts that are processed upon stress. The mutant also showed reduced interaction between U2 snRNA and U2B'', which leads to splicing defects. Our results indicate that CBs regulate which transcripts are held in the nucleus and how they are processed, contributing to plant tolerance of oxygen deprivation and recovery.

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana. Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

Changes in intracellular calcium ion (Ca{superscript 2}) concentrations generate characteristic signatures that are decoded by specialized Ca{superscript 2}-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca{superscript 2} at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca{superscript 2}-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca{superscript 2} sensor that couples Ca{superscript 2} perception to JA-mediated defense and abiotic stress responses in plants.

O_LIThe chloroplast CLP chaperone-protease is essential for chloroplast biogenesis. CLP substrate selection is aided by the N-recognin CLPS1 and CLPF adaptors. They interact with each other and the CLPC1 chaperone, but their specific functions are poorly understood. C_LIO_LIWe employed in vivo CLPC1 substrate-trapping in Arabidopsis by expressing a 35S:CLPC1-TRAP-STREPII transgene in wild-type (WT), clpf, clps1, and clpfclps1 to test the consequences of the loss of these adaptors on CLPC1-trapped proteins. Immunoblotting and protein half-life experiments were carried out for identified CLP substrates. C_LIO_LIExpression of the 35S:CLPC1-TRAP-STREPII in clps1cpf was embryo lethal. CLPF was trapped at a reduced level in clps1, supporting CLPS-CLPF interactions. Chloroplast 1O2 sensor EXECUTER1 (EX1) was trapped in WT and clps1 but not significantly in clpf. Steady-state protein accumulation of EX1 and its homolog EX2 increased 30-fold in the CLPC1-TRAP lines and clpr2-1, but not in clpf or clps1. In planta experiments showed that the half-life of EX1 is [~]3-fold longer in clpc1-1 than in WT, but EX1 half-life was unaffected in clpf. C_LIO_LIWe conclude that the CLP system plays a key role in EX1,2 homeostasis by keeping their intra-chloroplast concentrations low through continuous degradation, upstream of their 1O2 signaling function. C_LI

Flower colour is an important component of floral signaling, yet the genetic basis of naturally occurring colour variation remains poorly understood in many non-model plant groups. Here, we compare genome sequences of a normally pigmented and a largely unpigmented individual of Muscari commutatum, a species typically characterized by very dark violet flowers. Both assemblies were approximately 3.5 Gbp in size, with contig N50 values exceeding 130 Mbp and BUSCO completeness of about 99%. Most structural genes of the flavonoid biosynthesis and the corresponding transcriptional regulators were identified in both plants. Strikingly, no flavanone 3-hydroxylase (F3H) gene was detected in the genome sequence of the unpigmented plant, and additional manual searches provided no evidence for a bona fide F3H copy. Comparison of the two genome sequences revealed a lack of local synteny around the F3H locus of the anthocyanin-pigmented plant, suggesting that F3H might have been lost in the unpigmented plant in association with a genomic rearrangement.

Although the biosynthetic pathways of benzylisoquinoline alkaloids (BIAs) have been extensively investigated in several plant species, their transcriptional regulatory mechanisms remain only partially understood. Jasmonate (JA)-responsive group IX APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors (TFs) are well-known regulators of specialized plant metabolism, including the biosynthesis of various alkaloids. However, their specific roles in BIA biosynthesis remain largely elusive. Here, we isolated five novel group IX AP2/ERF TFs, designated Benzylisoquinoline alkaloid Jasmonate-responsive AP2/ERF (BJE1-5), from Coptis japonica. Phylogenetic analysis revealed that Benzylisoquinoline alkaloid Jasmonate-responsive AP2/ERF (BJE) proteins belong to subclades distinct from group IXa, which contains well-known AP2/ERF TFs involved in alkaloid biosynthesis. Transient expression analyses in C. japonica protoplasts demonstrated that certain BJEs, particularly CjBJE3 and CjBJE5, positively regulated BIA biosynthetic genes through a mutual regulatory network among BJE members. Moreover, CjBJE3 expression was regulated by CjbHLH1, a unique-type basic helix-loop-helix (bHLH) TF specific to BIA-producing plants. Furthermore, heterologous expression of CjBJE3 and CjBJE5 in cultured Eschscholzia californica cells significantly enhanced the overall BIA production, particularly by increasing end-product benzophenanthridine BIAs, highlighting several uncharacterized biosynthetic genes clustered in the genome. Our findings suggest that BIA-producing species have developed a specific regulatory network comprised of CjbHLH1 and BJE TFs, providing valuable clues for identifying novel biosynthetic enzymes.

Research ConductedTo elucidate the functions of glycosylceramides, we generated and characterized mutants deficient in multiple steps contributing to their assembly in the model moss Physcomitrium patens. We mutagenized SPHINGOLIPID {Delta}8-DESATURASE, whose products are preferentially incorporated into glycosylceramides, and a suite of higher-order mutants combining sphingolipid {Delta}4-desaturase and glycosyl ceramide synthase. MethodsWe used targeted lipidomics to describe the chemotypes of all mutants. We used quantitative phenotype analysis, transcriptomics, and phytohormone profiling to understand the effects of these chemotypes on development and physiology. Key ResultsThese mutants present a range of phenotypes that collectively indicate that in P. patens (1) glycosylceramide deficiency impairs development, largely due to imbalance in free ceramide homeostasis (2) the synthesis of glycosylceramides is dependent upon the presence of a specific free ceramide profile (3) the {Delta}4-, but not the {Delta}8-desaturation, is strictly required for glycosylceramide synthesis, (4) cell division and differentiation, but not cell expansion, are affected by sphingolipid imbalance, and (5) sphingolipid imbalance results in oxylipin accumulation. ConclusionCollectively, our results elucidate the assembly and functions of glycosylceramides in a model bryophyte, and highlight conserved and specialized aspects of sphingolipid metabolism among plants.

The root meristem is essential for stem cell maintenance and root development in plants. In Arabidopsis, Root meristem Growth Factor (RGF) peptides and their receptors regulate root meristem size through reactive oxygen species (ROS)-dependent signalling. RGF1-mediated ROS redistribution post-translationally stabilises the root meristem master regulator PLETHORA2 (PLT2). Although genomic studies suggest that RGF-receptor modules are evolutionarily conserved across land plants, their functional characterisation has remained largely limited to Arabidopsis. Here, we show that Oryza sativa RGF1-1 (OsRGF1-1) functions as a rice homologue of Arabidopsis RGF1 (AtRGF1). CRISPR/Cas9-generated Osrgf1-1 mutants exhibited shorter seminal roots, reduced root meristem size, and decreased superoxide (O2*-) accumulation. EdU staining further confirmed that cell proliferation activity was reduced in the Osrgf1-1 mutants. The Osrgf1-1 mutants were sensitive to low concentrations of chemically synthesised mature OsRGF1-1 peptide. This low dose of OsRGF1-1 peptide restored seminal root growth and O2*- accumulation in the Osrgf1-1 mutants but had no detectable effect on the wild type. Functional analyses using Arabidopsis rgfr receptor mutants further demonstrated that OsRGF1-1 is perceived through conserved RGF receptor machinery. Together, our findings provide the first functional evidence that the RGF1-receptor-ROS signalling module is evolutionarily conserved between dicots and monocots in the regulation of root meristem development.