Plant cell-cell communication plays a crucial role during growth and development. While plasmodesmata (PD) are well known in this communication, their biogenesis and regulation remain poorly understood. In this study, we demonstrate that xyloglucan endotransglucosylases (XETs), enzymes involved in transglycosylation of xyloglucan chains, could impact not only cellular growth but also PD development in the moss Physcomitrium patens (P. patens). Knockout mutant lines of two XET isoforms, named Ppxth5, Ppxth6, and Ppxth5,6dko, showed growth reductions in both protonemal and gametophytic stages. Subsequent analyses of these mutants indicated a decrease in PD density, abnormal clusters of xyloglucan and callose, and their colocalization, highlighting their potential involvement in PD formation. Further evaluation of these alterations using Dendra2 photoconvertible protein showed impaired intercellular communication, indicating reduced intercellular transport compared to WT. For PD structural changes, these mutants show higher ratios of incomplete simple PD and complex PD, highlighting the critical role of xyloglucan. Specifically, proper xyloglucan distribution, which is regulated by XET activities, is required for maintenance of PD density and structure, and for intercellular transport, possibly interacting with callose during cell plate formation.
Lateral bud outgrowth critically determines tomato plant architecture, canopy structure, photosynthate distribution, and fruit yield. SQUAMOSA promoter binding protein-like (SPL) transcription factors regulate multiple developmental processes; however, the role of SlSPL10 in controlling lateral bud outgrowth remains unknown. In this study, we used tomato cultivar Micro-Tom (wild-type, WT), SlSPL10 knockout mutants (slspl10#1 and slspl10#2), and overexpression lines (OE-SlSPL10#6 and OE-SlSPL10#7) to assess agronomic traits and phytohormone levels. The slspl10 mutants exhibited dwarfism, increased lateral bud number, and elongated buds, whereas OE-SlSPL10 lines suppressed bud outgrowth. Phytohormone and sucrose analyses revealed that slspl10 mutants displayed elevated levels of ZT, GA3, and sucrose, but reduced levels of IAA, ABA, and SA, whereas OE-SlSPL10 lines showed opposite trends. To dissect the underlying molecular mechanism, we demonstrated that SlSPL10 directly binds to GTAC motifs in the promoters of SlARF10a and SlABI3 using yeast one-hybrid (Y1H) assays, dual-luciferase reporter (DLR) assays, and chromatin immunoprecipitation-PCR (ChIP-PCR), thereby transactivating both target genes. Virus-induced gene silencing (VIGS) of SlARF10a or SlABI3 phenocopied the slspl10 mutants, showing increased lateral bud number and elongation. Hormone measurements in VIGS lines further indicated that silencing SlARF10a specifically reduced IAA content, whereas silencing SlABI3 specifically reduced ABA content; both lines also exhibited increased ZT, GA3, and sucrose, and decreased SA. Collectively, these results demonstrate that SlSPL10 directly targets SlARF10a and SlABI3 to modulate phytohormone and sucrose homeostasis, thereby precisely inhibiting lateral bud outgrowth in tomato.
Anther dehiscence is a developmentally programmed, mechanically executed event essential for sexual reproduction in flowering plants. Dehiscence must be synchronized with microspore and pollen maturation, coordinated with filament elongation to ensure correct anther positioning, and remain robust under fluctuating environmental conditions, particularly humidity, which strongly influences dehydration kinetics. At the same time, rapid, sufficiently wide opening requires that spatiotemporal developmental programs be converted into physical forces within a multilayered tissue. Yet despite extensive work on its genetic regulation and physical basis, these dimensions are often treated separately, leaving unresolved how developmental patterning is translated into controlled force generation in the anther wall. Here, focusing primarily on Arabidopsis, we integrate molecular patterning with biomechanics to explain (i) how lineage specification establishes the cellular architecture required for dehiscence; (ii) how hormonal and receptor-kinase signaling synchronize developmental timing with organ-level readiness; and (iii) how dehydration-driven mechanics are regulated and locally executed through epidermal transpiration, cell death, and cell-wall remodeling. We further contrast dehiscence with abscission to highlight a shared logic of spatially patterned reinforcement coupled with focal weakening, and examine how hydration dynamics, tissue mechanics, and geometry shape the timing and extent of opening. Together, these perspectives establish anther dehiscence as a model for understanding how developmental programs are translated into coordinated tissue mechanics and organ-level function.
Sex determination in Cannabis sativa L. has major implications for breeding, biotechnology, and crop management. In this species, sex is primarily governed by an XX/XY sex chromosome system, with genetic regulation constituting the principal level of control. Variation in sex expression, encompassing dioecious, monoecious, or hermaphroditic forms, may arise from interactions between sex chromosomes and autosomal factors X-to-autosome (X: A) chromosome balance. Secondary modulation of sex phenotype involves environmental and physiological influences, with phytohormonal regulation representing an important layer of sex plasticity. Epigenetic mechanisms may further contribute to the downstream fine-tuning of phenotypic sex expression. Importantly, sex expression in Cannabis can also be manipulated chemically. Several silver-based compounds, such as silver thiosulfate, are widely used to induce staminate flower formation in genetically female plants, whereas application of ethephon promotes female flower formation in genetically male individuals. Although both agents act through ethylene signalling pathway, their effects likely involve broader and more complex interactions within the molecular network, including cross-talk with other hormonal pathways. Within this context, transcriptional regulators associated with auxin signalling, including Aux/IAA - TOPLESS-RELATED co-repressors such as TPR1, may contribute to the activation of male developmental programmes by modulating hormonal response pathways. However, their precise role and position within the sex-regulatory hierarchy are yet to be clarified. The multi-layered regulation of sex determination in Cannabis sativa L. provides a valuable framework for investigating the molecular basis of this process through the integration of genetic, environmental, and hormonal factors. A more comprehensive understanding of these interacting regulatory layers may facilitate the development of strategies for early sex identification, improved control over flower sex conversion, and the optimisation of cannabinoid-rich female inflorescence production. Such advances would support both fundamental and applied approaches in Cannabis biotechnology and cultivation.
The ability to detect and respond to light signals is essential for a plant to maximise its fitness. These signals, including photoperiod, regulate key aspects of a plant's development, from the timing of seed germination through to the floral transition. How the photoperiod regulates hypocotyl elongation and the floral transition is now well understood at the molecular level. In contrast, how light and circadian signals are perceived and integrated in seeds is less clear. In this review, we highlight the latest insights into how light, photoperiod, and circadian signalling regulate development in seedlings and seeds. We also discuss how even though these two developmental stages employ many of the same components, re-wiring of the regulatory network that these components operate within enables the distinct developmental demands of germinating and seedling growth in responding to the environment cues. Finally, we highlight key knowledge gaps and future directions of research in how seedlings and seeds sense integrate light and circadian signals during development.
Grass Expression Atlas (GExA) is an interactive web-based resource for rapid exploration of gene expression across diverse tissues, developmental stages, and conditions in grass species. GExA integrates publicly available RNA sequencing (RNA-seq) datasets for four millets: pearl millet (Cenchrus americanus), foxtail millet (Setaria italica), proso millet (Panicum miliaceum), and finger millet (Eleusine coracana), and includes barley (Hordeum vulgare), sorghum (Sorghum bicolor) and rice (Oryza sativa) as reference species. Public RNA-seq datasets were reanalyzed using a unified workflow within each species to obtain read counts for each gene, which were then converted into consistently formatted expression matrices in transcripts per million (TPM). The current release comprises 4,753 samples from 457 BioProjects, including 987 pearl millet samples and 2,216 foxtail millet samples, and is provided through a user-friendly web interface. GExA is designed for scalable expansion to additional species via the pipeline used in this study. GExA is freely available athttps://www.gexa.anesc.u-tokyo.ac.jp/.
Sugars serve both as nutritional sources and signaling entities in plant root systems, engaging in crosstalk with phytohormones to modulate root morphogenesis and growth. Previous investigations demonstrated that heterologous expression of the Mirabilis jalapa mannanase gene (MirMAN) in Arabidopsis thaliana significantly promotes root development. Nevertheless, the mechanistic basis and associated signaling networks underpinning this phenomenon remain poorly elucidated. In this study, we found that MirMAN-mediated mannose could significantly promote plant root development, participate in root morphogenesis by promoting lateral root emergence and root elongation. Moreover, MirMAN-mediated mannose could increase the content of endogenous auxin, elevated the expression of auxin transport genes (AtLAX3/AtPIN2) and the response factor (AtARF7/19), lateral root development-related genes (AtLBD16/29), cell cycle-related gene (AtGATA23), sugar signaling-related genes (AtSnRK1) to promote lateral root development. Furthermore, the expression of AtMYB41 was significantly increased by MirMAN-mediated mannose. We further found that MYB41 directly bound to the promoter of AtDWF4 (a BR biosynthetic gene) and positively regulates the transcription of AtDWF4. Consistent with these findings, the myb41 mutants exhibited fewer lateral roots, reduced AtDWF4 expression, and lower brassinosteroid content. Collectively, These results provide new perspectives for understanding the mechanisms of endogenous plant sugar signaling, and provide new ideas for exploiting the plastic developmental capacity of plant roots.
Cannabis sativa is a multi-purpose crop with a wide range of industrial and medicinal end uses. Despite its long history of cultivation, it has not received the same breeding efforts as many other crops due to regulatory barriers. In particular, it bypassed the Green Revolution, which improved harvest indices of crops via developing semi-dwarf varieties by manipulating endogenous gibberellin signalling pathways. We chemically manipulated gibberellin signalling through the exogenous application of gibberellic acid and the gibberellin biosynthesis inhibitor paclobutrazol. We used multi-omic analysis to determine the effect of these applications on the proteome, transcriptome and secondary metabolite accumulation to assess the potential of gibberellin modulation for improved C. sativa end uses. Paclobutrazol treatments resulted in a semi-dwarf phenotype with an improved high harvest index and enhanced cannabinoid accumulation. Differentially expressed gene and weighted gene co-expression network analysis revealed the importance of source/sink dynamics for harvest index, with the increased expression of sucrose synthases and sugar transporters key to the phenotypic changes induced by gibberellin modulation. At the same time, the availability of hexanoate precursors via the degradation of long-chain fatty acids emerged as a likely constraint on cannabinoid accumulation. This study provides a basis for exploring the manipulation of gibberellin pathways in C. sativa varieties and their relevance to cannabinoid production.
Tetrapyrrole biosynthesis is an absolute essential metabolic pathway in plants that predominantly gives rise to chlorophyll, heme, and phytochromobilin. Dysregulation of tetrapyrrole biosynthesis severely impairs plant growth and development when heme and chlorophyll synthesis are not adjusted to the needs in the respective plant organ and when excessive accumulation of light-absorbing tetrapyrrole intermediates cause oxidative damage. However, how chlorophyll and heme synthesis are properly balanced during early development of greening seedlings remains largely elusive. In this study, we performed a suppressor screen on the photosensitive Arabidopsis thaliana pif1 pif3 (for phytochrome-interacting factors) double mutant, which exhibits excessive singlet oxygen (1O₂) accumulation, and identified four different point mutants of the GENOMES UNCOUPLED 3 (GUN3, also known as HY2) gene encoding phytochromobilin synthase. Further genetic analysis revealed that either mutation of GUN3 or also GUN2 (encoding heme oxygenase, also known as HY1/HO1), but not FC1 (encoding ferrochelatase), rescues the cell death phenotype of pif1 pif3, lowers 1O₂ levels, and suppresses 1O₂-responsive gene expression. Furthermore, gun2 and gun3 mutations lead to heme-mediated feedback inhibition of 5-aminolevulinic acid synthesis and consequently reduced protochlorophyllide accumulation in the pif1 pif3 background. Notably, GUN2 and GUN3 physically interact with GUN4, while their mutations markedly correspond with decreased content of GUN4 and GUN5, which are involved in Mg chelation at the beginning of chlorophyll biosynthesis. Our studies point to an important regulatory role of GUN2 and GUN3 for the mutual link of heme and chlorophyll synthesis.
Parasitic plants are a problem for world agriculture as their direct feeding on hosts can cause high yield losses. One approach to controlling parasitic plants would be to engineer host plants for resistance by expressing an effective defense in response to invasion by the parasite. While much attention has focused on identifying suitable resistance genes, little work has addressed the challenge of identifying promoters to drive specific expression of such genes. To meet this need, we set out to develop a parasite-inducible promoter. Starting with previously generated RNAseq data from Arabidopsis thaliana parasitized by Cuscuta campestris, we identified host genes induced by the parasite and then studied their regulatory regions to identify cis-regulatory elements (CREs). These elements, or transcription factor binding sites (TFBSs), were recombined into a set of minimum synthetic inducible promoters (MSIPs), fused to the β-glucuronidase (GUS) reporter gene, and stably transformed into A. thaliana. The MSIP lines were evaluated with and without Cuscuta, and GUS staining showed that most of the MSIPs were induced by Cuscuta parasitism, although they were also expressed in some non-parasitized tissues. The most parasite-specific MSIP contained stress-inducible TFBSs, and was also responsive to wounding, crushing and jasmonate treatment. Furthermore, A. thaliana parasitized by the root parasite Phelipanche aegyptiaca showed similar MSIP induction patterns in roots, suggesting that at least two types of parasitic plant trigger the same host transcriptional machinery. These MSIPs provide insights into host-parasite interactions and are potentially valuable for driving expression of novel parasite resistance genes.
Salt stress disrupts plant physiology and reprograms specialized metabolism. APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors integrate stress and hormone signals, but their roles in linking salt tolerance with phenylpropanoid metabolism in non-model alpine plants remain poorly understood. Here, we performed a genome-wide survey of AP2/ERF genes and functionally characterized a salt-responsive ERF in Herpetospermum pedunculosum. We identified 147 HpAP2/ERF genes, whose promoters were enriched in hormone- and stress-responsive cis-elements. Salt treatment induced widespread expression changes of HpAP2/ERF genes in roots, with HpERF144 among the most rapidly and strongly up-regulated members. Virus-induced silencing and ZYMV-mediated overexpression in H. pedunculosum, together with heterologous overexpression in Nicotiana tabacum, showed that HpERF144 positively regulates salt tolerance by modulating oxidative stress-related physiological responses. Moreover, HpERF144 silencing reduced, whereas overexpression increased, total lignin and total lignan accumulation in roots under salt stress. DAP-seq and dual-luciferase assays further indicated that HpERF144 associates with and activates selected phenylpropanoid- and cell wall-related promoters, including 4CL- and CCR-like targets. These findings identify HpERF144 as a link between salt stress signaling and phenylpropanoid-associated metabolic regulation in a lignan-rich medicinal plant.
Pseudotaxus chienii, an endangered relic conifer endemic to China, possesses significant medicinal potential, yet its phenolic acid biosynthesis and glycosylation mechanisms remain unelucidated. This study integrated metabolomics, mass spectrometry imaging (MSI), transcriptomics, and molecular biology to systematically characterize the phenolic acid pathway in P. chienii. Untargeted metabolomics identified distinct phenolic acid accumulation patterns between P. chienii and Taxus mairei. MALDI-2 MSI visualized ten phenolic acids, confirming leaves as the primary accumulation site. Genome-wide analysis revealed a complete phenolic acid biosynthesis pathway with key enzyme-encoding genes (4 PAL, 5 CCR, 2 F5H, etc.), while HQT was absent, indicating loss of chlorogenic acid synthesis. We identified 504 glycosyltransferase (GT) genes, with 48 leaf-specific ones (predominantly GT1 subfamily). Promoter and correlation analyses highlighted WRKY (PichiChr12G332970.1) and ERF (PichiChr3G086100.1) as core transcription factors (TFs). EMSA and dual-luciferase assays validated WRKY(PichiChr12G332970.1) directly activating three GT genes. This study clarifies the molecular basis and transcriptional regulation of phenolic acid glycosylation in P. chienii, providing a framework for exploiting its medicinal resources and guiding conservation-oriented breeding.
When ribosome biogenesis or function is perturbed, plant cells undergo ribosomal stress, leading to growth defects and developmental alterations. The plant ribosomal stress response has recently gained recognition, but its molecular mechanism remains elusive. Here, we characterized this response in Arabidopsis thaliana using ribosome biogenesis-impairing mutations (rid2 and rid3) and ribosome biogenesis/function-interfering drugs (5-fluorouracil and puromycin) as ribosomal stressors. These stressors repressed cell proliferation and increased ploidy levels indicative of endoreduplication. Under the ribosomal stress conditions, a subset of NAC transcription factor genes and CDK inhibitor genes were upregulated, while they tended to downregulate G2/M-specific genes. Overexpression of ANAC082, which encodes a NAC factor implicated in the ribosomal stress response pathway, phenocopied the ribosomal stress response in the cellular behavior and gene expression. These results together indicated that, predominantly mediated by ANAC082, ribosomal stress arrests the cell cycle at G2 and promotes endoreduplication. Comparison of gene expressions and genetic relations between the ribosomal and DNA damage stress responses, focusing on ANAC082 and the DNA damage stress response-mediating NAC factor SOG1, revealed that these stress response pathways are distinct and largely independent from each other, although they have similar outputs in the control of cell proliferation and endoreduplication.
A growing body of literature has illustrated the importance of understanding the circadian clock due to its connection to a wide array of molecular and physiological processes. Numerous methods have been developed in order to monitor the status of the circadian clock in living tissue; however, most methods are either costly or labor intensive, and often require precise conditions that lowers throughput and limits flexibility in the size, age, or type of plant to be assayed. Here, we present an affordable and adaptable methodology for assaying circadian period using the well-characterized circadian output of leaf movement. Employing fully automated time-lapse photography using Raspberry Pi cameras and predominantly automated image post-processing, this methodology minimizes manual input to expedite circadian analysis, thus improving throughput. Additionally, the top-down setup used in this method is appropriate for a wide range of sizes and ages of plants, allowing for an expansion of the scientific questions that can be assayed by this methodology.
