The Plant Journal

Whether Photosystem I (PSI) core subunits accumulate prior to light exposure in developing angiosperm seedlings remains unresolved, with conflicting reports across species. Here, we investigated the presence and membrane colocalization of the PSI core subunit PsaA in etioplasts of dark-grown angiosperms representing dicot and monocot species. Immunoblotting showed that PsaA accumulates in etioplasts of all three dicot species examined (pea, Arabidopsis, and runner bean), whereas in the monocot oat it was detected only after prolonged etiolation, at substantially lower levels and with an anomalously high apparent molecular weight. Blue-native PAGE analysis reveals that a fraction of PsaA co-migrates with LPOR, PsaB, FNR, and chlorophyll synthase, suggesting co-localization within a shared membrane microdomain rather than stable complex formation. The thylakoid insertase Alb3 was more abundant in dicot etioplasts, consistent with a potential role in the early integration of PsaA into the membrane. Upon illumination, pea reached PSI functionality faster than oat, with P700 oxidation detectable 30 min earlier, linking the dark accumulation of PsaA to an accelerated photosynthetic onset. These findings demonstrate light-independent accumulation of a PSI core subunit in a species-dependent manner and point to early steps in PSI biogenesis that precede full photosynthetic complex assembly. Highlight Contrary to prevailing models, a Photosystem I core subunit PsaA accumulates in dark-grown angiosperm seedlings before light exposure, revealing light-independent early steps in photosynthetic complex biogenesis.

The red/far-red sensing photoreceptor phytochrome B (phyB) governs multifaceted plant development and responses to light and temperature stimuli. PhyB photoconversion between red-absorbing, inactive Pr and far red-absorbing, active Pfr states, imparted by its covalently bound bilin chromophore, enables rapid switching and plasticity of phyB signaling activities. The phyBY276H variant (YHB) is photochemically inert but adopts a constitutively active Pfr-like structure regardless of light conditions, which becomes a versatile model to dissect phyB signaling mechanisms. Here, we conducted a large-scale EMS mutagenesis screen on YHB-expressing transgenic lines, mining intragenic suppressor mutations that would unveil critical residues for phyB structure-function relationships. Comparative analyses of 26 nonsense variants suggested modular organization of phyB overall structure and dispensability of the C-terminal HKRD domain for phyB signaling. Amongst fourteen novel and nine known loss-of-function missense variants identified herein, G284E was of particular interest for its fully suppressed constitutive activity in darkness and its restored photochemistry and light responsiveness. The G284E mutation was further tested to also nullify another constitutively active phyBY303V allele by eliminating chromophore attachment. P309L was the sole variant identified which fully suppressed YHB in both dark and light conditions. C402Y profoundly elicited YHB protein instability. Three variants G118R, C402Y and G538D markedly reduced chromophorylation levels of YHB. Although the chromophore binding site variant C357Y was a strong loss-of-function allele, it retained residual signaling activity with respect to PIF3 protein turnover in dark-grown seedlings, presumably due to its ability to noncovalently bind chromophore. Two tandem prolines (P799, P800) proved critical to YHB structural integrity/stability as well as signaling activity. In summary, these diverse variants shed new insights into multiple levels by which the YHB (and thereby phyB) signaling is initiated, tuned, and disseminated.

Biolistic transformation is a versatile tool in plant science, yet high equipment costs and tissue damage from high-pressure gas remain significant barriers. Building on our previously developed "TSGMAC", a low-cost, helium-free biolistic system, we report three major advancements to enhance its throughput, delivery quality, and quantitative capability. First, a "guide barrel" assembled from commercial DIY fittings was developed; it effectively eliminates physical tissue damage and ensures uniform particle distribution, even in soft tissues like bok choy (Brassica rapa subsp. chinensis). Second, a rapid gene expression platform using PCR products was characterized. Results demonstrate that linear DNA constructs are efficiently circularized via non-homologous end joining (NHEJ) in plant cells, and protein expression is robust regardless of the relative positions of the promoter, coding sequence, and terminator. This system bypasses time-consuming cloning. Third, a cost-effective, highly sensitive dual-luciferase assay system utilizing teal Luc (teLuc) and inexpensive firefly luciferase (FLuc) inhibitors was established. This integrated workflow enables rapid, quantitative molecular biology using supermarket-obtained materials and standard PCR reagents. Our findings provide a practical foundation for plant scientists, synergistically accelerating gene functional analysis and genetic tool development.

Plant architecture is a crucial component of maize productivity. Tailoring architectural component traits like leaf area and angle can increase productivity by promoting deeper light penetration into the canopy and better resource utilization. Novel genetic variants can increase the rate of gain for optimized plant architecture. Here, we map a moderate-effect mutation denoted reduced leaf area1 (rdla1) to the RAGGED5 (RGD5) locus and characterize it as a transposon insertion allele. Mutant leaf area reductions were most extreme in mid-upper canopy positions. Photosynthetic gas exchange rates were not significantly impacted in rdla1 relative to wild-type, indicating that mutant leaf structure, but not function, is altered. Functional annotations of RDLA1 were supported by metabolite profiles suggesting a role in cuticular wax biosynthesis. Introgression of the rdla1 allele into 27 commercially relevant genetic backgrounds identified differences in effect size across genotypes, revealing modifier effects that could serve as targets for modulating plant architecture.

Improving regeneration of ribulose-1,5-bisphosphate (RUBP) is a promising approach to improve photosynthesis and plant growth. In addition to transgenic overexpression of target genes, it could be possible to directly overexpress endogenous target genes, through transcriptional enhancements. As shown by the recent discovery of a short sequence motif, that resembles the known octopine synthase (ocs) enhancer, transcriptional enhancement is achievable by relatively short endogenous sequences. In this study, we query the genome of several model and crop plant genomes for the presence of short enhancer motifs. We find hits across all genomes including some in promoter regions of genes. By using derivatives of these motifs in a transient fluorescence assay, we show that several of these are capable of inducing target gene expression in different promoter contexts. A motif scan of the created constructs, for the presence of known transcription factor binding sites, shows that the insertion of these motifs has created binding sites for different TGA-, NAC- and bZIP-transcription factors. Taken together our study shows the feasibility of finding enhancer sequences in the genomes of different plants. With advancement in gene-editing technologies, like prime editing, using such endogenous enhancer sequences, could allow for precise cisgenic promoter engineering of target genes.

Transcriptional regulation is one of the fundamental approaches for young plants to cope with environmental fluctuations and maintain active development. The transposable element (TE) subclass long terminal-repeat retrotransposons (LTR-RTs) can act as additional regulators for genes through enhancer and promoter activity, but their promoters, transcription initiation, and contributions during maize development remain uncharacterized. Here, we developed IsoClassifier to resolve the transcription start site (TSS) and RNA isoforms of LTR-RTs based on long-read transcriptomics, delineating LTR U3 regions as the native promoter and enhancer of LTR-RTs. We reveal conserved motifs associated with core promoter activity in transcribed LTR-RTs that are highly comparable to gene promoters. Further, we found that LTR-RT transcription in maize was dominated by spliced, long non-coding RNA. Finally, a genome-wide coexpression analysis revealed that LTR-RTs are transcribed as hub-like elements in coexpression networks, suggesting important roles in gene regulation. We conclude that LTR-RTs have similar promoter compositions to gene promoters and likely share similar transcription regulation programs.

Local adaptation of a species involves the selection of adaptive alleles that confer a fitness advantage in their local environment. Inversions prevent recombination between the standard and inverted heterozygous hybrids. Inversions can play a crucial role in local adaptation by locking together a set of co-adapted alleles, acting as supergenes. Inv4m is a 13 Mb inversion in maize prevalent in highland maize and highland wild relatives from Mexico. Maize from the highlands of the Trans-Mexican volcanic belt has been shown to be well-adapted to volcanic, acidic soils with low phosphorus availability. Inv4m carries several genes involved in P acquisition and utilization. We therefore tested the hypothesis that Inv4m contributes to maize adaptation to these environments through enhanced phosphorus acquisition or utilization. Alternatively, Inv4m possible adaptive value may operate through constitutive developmental effects independent of nutrient stress responses. To test this hypothesis, we introgressed a highland maize variety from the highlands of Michoacan, Mexico, carrying Inv4m into the temperate line B73 and developed Near-Introgression Lines (NILs) carrying Inv4m. We then grew NILs carrying the inversion and controls without it in soils with different phosphorus levels and evaluated the fitness effects of the inversion, as well as changes in gene expression using RNA-Seq. Our results show that P starvation elicits highly conserved transcriptomic, lipidomic, and ionomic responses, independently of the Inv4m inversion genotype. Therefore, phosphorus deficiency does not seem to be driving the adaptive value of Inv4m. Additionally, we observed a phosphorus modulated transcriptional gradient from the collar leaf downward, characterized by a decrease in the expression of photosynthesis genes and an increase in the expression of senescence-associated genes, corresponding to the positional onset and initial stages of sequential leaf senescence. Although the magnitude of the phosphorus response increased with leaf age, we did not observe significant interactions with Inv4m. Our multi-omics analysis of the maize phosphorus starvation response identified and characterized two coordinately regulated molecular programs, light harvesting shutdown and accelerated senescence, whose deployment depends on leaf developmental stage, with older leaves below the collar integrating nutrient limitation into the natural progression toward senescence.

The genus Rhynchospora Vahl (beak-sedges) comprises approximately 381 accepted species with a worldwide distribution, all of which possess holocentric chromosomes, where centromeric activity is distributed almost along the entire chromosome. Despite the recent advances, the mechanisms governing the dynamics of meiotic recombination in holocentric plants remain poorly understood. Here, we developed haplotype-specific oligo-FISH probes for chromosomes 1, 2, and 3 based on a haplotype-phased genome assembly of Rhynchospora breviuscula (n = 5), enabling homolog-specific chromosome painting. Each probe set was labelled with a distinct fluorophore and hybridised in situ to metaphase chromosomes of the reference plant and seven F1 individuals derived from self-crossed reference plants. This approach allowed the unambiguous discrimination of homologous haplotypes and the indirect visualisation of crossover (CO) events in recombined chromosomes. We observed that recombination events were predominantly located in terminal chromosomal regions, consistent across individuals. These results corroborate previous findings from single-cell recombination mapping and provide independent cytological validation of the recombination landscape in this species. Our study establishes haplotype-specific chromosome painting as a robust tool for high-resolution mapping of meiotic recombination in holocentric plants across generations. Furthermore, these probes provided a foundation for future investigations into inverted meiosis, a mechanism characterized by an alternative pattern of chromosome segregation in holocentric species.

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.

Propagation mode strongly influences crop establishment, yet its impact on whole-plant transport strategies remains poorly understood. Here, we examined whether seed- and set-derived plants deploy contrasting aquaporin networks associated with different physiological behaviours in onion (Allium cepa L.). We combined genome-wide gene-family characterisation with transcriptomic, biochemical, and physiological analyses. Forty-eight aquaporin genes were identified and classified into four subfamilies (15 PIPs, 19 TIPs, 8 NIPs, and 6 SIPs), with evidence of lineage-specific expansion in the PIP1 and TIP2. Expression analyses revealed clear propagation-dependent patterns. Set-derived plants displayed higher expression of AcPIP1.1, several PIP2 isoforms and most TIP2 members in both roots and leaves, consistent with enhanced water and CO2 transport, higher stomatal conductance, transpiration, and photosynthetic rates. In contrast, seed-derived plants showed increased expression of specific PIPs, and several NIPs (AcNIP1.1, AcNIP3.1, AcNIP5.1, AcNIP5.2, and AcNIP2.1) associated with solute and H2O2 transport, coinciding with higher boron and hydrogen peroxide levels. These findings indicate that propagation origin is associated with alternative aquaporin-mediated transport strategies: set-derived plants favour a high-flux strategy that supports rapid growth, whereas seed-derived plants prioritize tighter internal regulation through solute redistribution and redox homeostasis. Our results provide a molecular-physiological framework linking propagation origin with resource-use strategies in onion. HighlightPropagation origin reprograms aquaporin expression in onion, generating contrasting hydraulic, metabolic and growth strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/728701v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@c3a3a6org.highwire.dtl.DTLVardef@2d0674org.highwire.dtl.DTLVardef@2d2cborg.highwire.dtl.DTLVardef@15d2c34_HPS_FORMAT_FIGEXP M_FIG C_FIG

Grain legumes such as pea (Pisum sativum L.) accumulate large amounts of seed storage proteins without nitrogen fertilization due to their symbiosis with nitrogen-fixing bacteria, making them a key source of plant-based proteins. Seed growth and the accumulation of seed storage proteins are tightly regulated by complex gene networks; however, the mechanisms governing these processes in pea remain poorly understood. In this study, we generated a comprehensive seed expression atlas covering six developmental stages in pea (cv Cameor), including the key transition stage from embryogenesis to early seed filling, providing a detailed temporal resolution of transcriptional dynamics throughout seed development in this species. Co-expression network analysis highlighted several candidate transcription factors potentially involved in the transition towards seed filling. Among them, we characterized the seed-specific NF-YB transcription factor PsLEC1-like (PsL1L), the major LEC1-type factor expressed during early pea seed development. Functional analyses using TILLING mutants demonstrated that loss of PsL1L function reduces seed size and seed nitrogen content and impairs early embryo growth from the end of embryogenesis. Finally, we show that the expression of the B3-domain transcription factor PsFUS3, but not that of PsLEC2 or PsABI3, is reduced in the loss-of-function l1l mutant, suggesting that PsL1L acts upstream of PsFUS3 to control seed size.

Lipids are the predominant building blocks of plant membranes and are essential for plant growth and development. They are crucial for survival during times of stress as lipids are involved in multiple signaling pathways, and their relative abundances can change in response to environmental factors. To better characterize the lipid composition of the vital food crop maize, we generated a comprehensive glycerolipid atlas using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. We surveyed the lipid profiles of three different maize genotypes: B73, a temperate inbred; CML312, a subtropical inbred; and Palomero Toluqueno, an open-pollinated variety from the Mexican highlands. We collected leaf samples from 4 developmental stages and 6 leaves. From one growth stage, we also sampled along with three leaf zones: base, center, and tip. The genotype and leaf number were the major drivers of lipid differences. Phosphatidylcholine, lysophosphatidylcholine, and triacylglycerol genotypic differences were particularly high. We generated an eFP browser to be integrated into the maize genome browser, as well as a separate web interface to easily browse and compare lipid levels across tissues and genotypes, available at https://rrellan.shinyapps.io/Zea-Lip/. SIGNIFICANCE STATEMENTThis work creates a spatial map of lipids in maize leaves across four growth stages for three genotypes: a lowland, a sub-tropical, and a highland. The resources generated here will directly benefit both the maize and lipid communities by creating a large dataset that can be used to generate new hypotheses in understanding lipid metabolism and environmental responses in maize.

Hydathodes are specialized leaf tissues that mediate guttation, the release of liquid water droplets from vascular plants. They consist of xylem endings, water pores, and the epithem. The epithem comprises small cells whose cell types and functional properties remain poorly understood. While molecular genetic information on hydathodes has accumulated--particularly in the model plant Arabidopsis thaliana through recent RNA-seq analyses--high-resolution anatomical insights into their spatial organization remain limited. In this study, we aimed to characterize the specialized anatomy of Arabidopsis hydathodes using complementary imaging approaches. A fluorescent dye taken up from roots stained hydathodes, consistent with a connection between hydathodes and the vasculature. Microfocus-X-ray computed tomography revealed that the hydathodes protrude from the abaxial leaf surface and are more densely packed internally than surrounding tissues. Light microscopy showed that the epithem was heterogeneous, with elongated cells proximally and rounded cells distally. We found that the epithem was surrounded by cells (hereafter referred to as "boundary cells") with plastids that resembled those of mesophyll cells. Transmission electron microscopy showed that the organelles in epithem cells were immature, and the apoplast contained electron-dense material. Finally, using transgenic plants expressing a secreted fluorescent marker protein, we detected GFP in guttation droplets, indicating that the droplets can contain apoplastic material. Together, these findings reveal that Arabidopsis hydathodes possess a dense and spatially heterogeneous epithem associated with the release of apoplastic fluid.

O_LIShoot branching is critical not only in breeding for yield but also for ornamentals architecture. In the ornamental plant poinsettia (Euphorbia pulcherrima), shoot branching has traditionally been induced by phytoplasma (Candidatus Phytoplasma pruni) inoculation. This study aimed to identify regulatory genes that could be leveraged in future breeding-by-genetic engineering efforts to develop phytoplasma-free, branching poinsettia plants. C_LIO_LITo elucidate mechanisms of phytoplasma-induced shoot branching, we performed RNA-sequencing and assembled an axillary bud-specific transcriptome for expression analyses in phytoplasma-infected and -free poinsettia. Phenotyping and RNA-sequencing were also conducted on Arabidopsis mutants and wild-type lines to investigate the transcriptional regulatory effects of candidate genes. C_LIO_LIThe transcription factors EpMAF3 and EpbZIP67 were highly de-regulated in phytoplasma-infected poinsettia. We also found a two-fold increase in primary-stem branching levels of the Arabidopsis maf3 and bzip67 mutants, suggesting the two transcription factors as potential shoot branching suppressors. AtTcp1, a CYC-clade TCP transcription factor, was up-regulated (78x) in leaves of the maf3 mutants. Analyzing previously reported protein-level interactions for the differentially expressed genes (e.g., AtClamt, AtGh3.9/3.15, AtSaur32/36, AtAbi3, AtGamt2, AtTcp3, and AtDwf4) in bzip67 mutants shed light on other shoot branching regulators such as TCPs, PINs, ABIs, DWARF14, and BES1, highlighting two regulatory sub-networks including membrane transport and hormonal signaling. C_LIO_LIThe results open the way to rational engineering of shoot branching in poinsettia by targeted mutagenesis of MAF3 and bZIP67. In that way, the tedious and viral-infection prone process of phytoplasma inoculation can be avoided and poinsettia plants would have more homogenous branching. C_LI One-sentence summaryPhytoplasma infection in poinsettia induces bud-specific repression of the transcription factors EpMaf3 and EpbZip67, consistent with the enhanced stem branching observed in Arabidopsis maf3 and bzip67 mutant lines.

O_LIPlants continuously adjust photosynthesis to balance growth and photoprotection under changing environmental conditions. Environmental fluctuations frequently impose a mismatch between energy production and CO2 assimilation. How photochemical reactions are regulated to maintain performance under these conditions remains a central question in plant biology. C_LIO_LIWe previously developed transplastomic tobacco (Fd1-OE plants) overexpressing ferredoxin (Fd) displaying enhanced photoprotection and growth penalties with a variegated leaf phenotype under greenhouse conditions. Here, we investigate how these plants respond to different growth irradiances using physiological, ultrastructural, and photosynthetic analyses, including PAM, gas exchange, and P700 absorbance measurements, and dynamic-light assays. C_LIO_LIFd1-OE plants progressively recovered growth, leaf phenotype and photosynthetic performance as growth irradiance increased, reaching near WT performance at 1400 mol m-{superscript 2} s-{superscript 1}. This enhanced adaptation to "high-light" was associated with a larger fraction of open PSII reaction centers and enhanced NPQ. Dynamic-light analyses further revealed faster plastoquinone (PQ) turnover, a more oxidized PQ pool and enhanced electron withdrawal downstream of PSI. C_LIO_LIOur results indicate that Fd overexpression redefines the balance between photochemistry and photoprotection. This adjustment shifts adaptation toward higher irradiance and enhances photosynthetic performance under changing light environments. Electron partitioning downstream of PSI emerges as a promising target to improve photosynthetic resilience. C_LI One sentence summaryOverexpression of Fd1 in tobacco plants adjusts photosynthesis/photoprotection trade off to enhance high-light adaptation

The loss of photosynthesis triggers extreme plastid genome (ptDNA) decay, including complete genome loss. Of the multiple transitions to heterotrophy among angiosperms, the ptDNA status remains poorly defined in lineages such as the endophytic Mitrastemonaceae (Ericales). Adopting a panplastome perspective, we characterized genomic variation across Mitrastemon yamamotoi individuals, assembling two complete circular ptDNAs and re-evaluating all available genomic resources for the genus. Our results reveal a highly minimized ptDNA (18-26 kb) with extreme AT content (>77%) and loss of the typical quadripartite architecture. Despite the absence of the stabilizing inverted repeats, the Mitrastemon panplastome exhibits remarkable structural stability and collinearity among individuals. The reduced suite of 26 genes, which includes accD, infA, clpP, ycf1, ycf2, and the essential tetrapyrrole precursor trnE-UUC, exhibit elevated substitution rates. Evolutionary rate analyses (dN/dS) demonstrate that the core ribosomal suite remains under strong purifying selection ({omega}<1), confirming the organelles functional status. Furthermore, transcriptomic analysis identified a nearly complete set of nuclear-encoded DNA-RRR genes, with the notable exception of the MUTS2 surveillance system. The convergent loss of these homologs in Mitrastemon and another holoparasitic lineage may be linked to the shared structural instability and mutational bias. Our findings demonstrate that despite extreme genome compaction, accelerated substitution rates, and severe AT-bias, the Mitrastemon panplastome remains quite stable, providing a definitive genomic framework for understanding plastid evolution within the endoparasitic Mitrastemonaceae.

The Nucleotide-Binding Domain Leucine-Rich Repeat (NLR) gene family is a central component of plant immune systems, yet its diversity and evolutionary dynamics remain poorly characterized in long-lived tree species. Here, we performed a genome-wide analysis of the NLR gene family in Quercus suber (cork oak) using InterNLR, a new annotation tool, and explored their expression regulation in response to biotic and abiotic stresses. A total of 918 NLR and NLR-like genes were identified, encompassing both canonical and non-canonical members. Phylogenetic analyses based on the NB-ARC domain highlighted the distinct evolutionary trajectory of RNL proteins, which function as helper NLRs and show evidence of clade-specific gene duplication. Transcriptomic analyses revealed pronounced tissue-specific expression patterns, with RNLs exhibiting significantly elevated expression in xylem, suggesting a specialized role in this tissue. Under drought stress, seven NLR genes were differentially expressed and shared orthology with known abiotic stress-responsive genes. Notably, a CNL gene (LOC111996439) responded to both biotic and abiotic stresses, indicating a potential role as an integrative regulator of early defence responses, while an ADR1 orthologue (LOC112022539) suggests molecular crosstalk between stress signaling pathways. Population genetic analyses further revealed signatures of both positive and balancing selection acting on NLR genes. Together, these results provide new insights into the evolution, expression, and functional diversification of NLRs in cork oak. This work advances our understanding of immune gene architecture in an ecologically and economically important forest tree species.

Understanding plant growth dynamics requires imaging across day-and-night cycles to quantify growth, movement and development in the aerial plant body and to capture the rhythmic nature of these processes. This requires imaging in light during the day and in darkness at night without perturbing plant physiology. Nighttime imaging has typically depended on infrared (IR) illumination, producing monochrome datasets that require specialised hardware and separate analysis pipelines when combined with daytime RGB imaging. Here, we evaluated very low-intensity green (dimG) illumination from standard LEDs as a practical alternative for colour-consistent nighttime imaging and assessed its physiological impact in Arabidopsis thaliana and Lactuca sativa (lettuce). We show that high resolution colour images can be obtained under dimG using low- cost cameras, with sufficient consistency between full-spectrum and dimG images to allow direct comparison and unified image analysis. We show that very low-fluence green light (<0.5 mol m-2 s-1) does not sustain circadian oscillations of gene activity under continuous exposure and does not perturb rhythms when applied during the dark phase of diel cycles. DimG imaging enabled accurate detection of diel leaf movement profiles in Arabidopsis circadian mutants, revealing genotype-specific phase differences under varying photoperiods. In lettuce, dimG pulses and continuous dimG enabled accurate quantification of diel leaf movement without affecting growth, stomatal opening, electron transport rate or chlorophyll content. Motion profiles under continuous dimG mirrored those under darkness. Our findings establish dim green illumination as a cost-effective solution for night-time imaging, simplifying phenotyping workflows with minimal impact on physiology.

Starch synthesis in wheat endosperm involves the initiation of large A-type starch granules during early grain development, followed by small B-type granules in later grain development. It is established that MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1) plays an important role in granule initiation in Arabidopsis chloroplasts, but how it influences A- and B-type initiations in wheat amyloplasts is not known. We discovered that due to a gene duplication in cereals, wheat contains two MFP1 paralogs, MFP1.1 and MFP1.2, which are both expressed in the developing endosperm. We generated a series of durum wheat mutants defective in all homoeologs of either MFP1.1 or MFP1.2, or both. While starch granule size distributions and granule morphology of mfp1.1 and mfp1.2 mutants were identical to those of the wild-type, the mfp1.1 mfp1.2 mutants had fewer, but larger B-type granules - suggesting that the two paralogs play redundant roles in B-type granule initiation. Consistent with this, both paralogs interacted with B-GRANULE CONTENT 1 (BGC1), a key protein required for proper B-type granule initiation in wheat, and both paralogs could partially complement defects in starch initiation in the Arabidopsis mfp1 mutant. Our work demonstrates that MFP1 is required for establishing correct starch granule number in non-photosynthetic amyloplasts, but its role in wheat is limited to B-type granule initiation. One-sentence summaryWheat has two MFP1 paralogs that interact with the granule initiation protein, BGC1 and influence B-type granule initiation in non-photosynthetic amyloplasts of endosperm.

In Asteraceae species, the capitulum is a compact inflorescence, featuring a characteristic reproductive structure. Despite the identification of a few key regulatory factors, the transcriptome-level information on the developing capitulum remains limited. Here, we applied single-cell and spatial transcriptome sequencing to investigate the developing Gerbera hybridas capitulum during floret differentiation. We obtained a transcriptomics atlas encompassing different stages of the Gerbera capitulum and analyzed the cellular and spatial dynamics of gene expression. Using marker gene expression and GO enrichment of cluster-specific DEGs, we annotated putative cell types and described changes in gene expression across sampled stages, potentially associated with ongoing developmental processes. We detected activity of previously undescribed MADS-box genes and defined their spatial expression patterns. Notably, the MADS-box gene GAGL12 was found to be enriched in the putative capitulum phloem cells. The GAGL12 protein was shown in yeast two-hybrid assays to interact with several other MADS-domain proteins with hypothesized functions in vasculature development, and further detailed in silico analyses supported a candidate role in the development of capitulum vasculature. Altogether, we provide integrative and dynamic transcriptomic insight into capitulum and floret development and lay a basis for future functional studies of the control and development of this intriguing reproductive structure.