Plants sum and subtract stimuli over different timescales.

Mounting evidence suggests that plants engage complex computational processes to quantify and integrate sensory information over time, enabling remarkable adaptive growth strategies. However, quantitative understanding of these computational processes is limited. We report experiments probing the dependence of gravitropic responses of wheat coleoptiles on previous stimuli. First, building on a mathematical model that identifies this dependence as a form of memory, or a filter, we use experimental observations to reveal the mathematical principles of how coleoptiles integrate multiple stimuli over time. Next, we perform two-stimulus experiments, informed by model predictions, to reveal fundamental computational processes. We quantitatively show that coleoptiles respond not only to sums but also to differences between stimuli over different timescales, constituting evidence that plants can compare stimuli-crucial for search and regulation processes. These timescales also coincide with oscillations observed in gravitropic responses of wheat coleoptiles, suggesting shoots may combine memory and movement in order to enhance posture control and sensing capabilities.

A genetic framework for proximal secondary vein branching in the Arabidopsis thaliana embryo.

Over time, plants have evolved flexible self-organizing patterning mechanisms to adapt tissue functionality for continuous organ growth. An example of this process is the multicellular organization of cells into a vascular network in foliar organs. An important, yet poorly understood component of this process is secondary vein branching, a mechanism employed to extend vascular tissues throughout the cotyledon surface. Here, we uncover two distinct branching mechanisms during embryogenesis by analyzing the discontinuous vein network of the double mutant cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Similar to wild-type embryos, distal veins in cvp2 cvl1 embryos arise from the bifurcation of cell files contained in the midvein, whereas proximal branching is absent in this mutant. Restoration of this process can be achieved by increasing OCTOPUS dosage as well as by silencing RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) expression. Although RPK2-dependent rescue of cvp2 cvl1 is auxin- and CLE peptide-independent, distal branching involves polar auxin transport and follows a distinct regulatory mechanism. Our work defines a genetic network that confers plasticity to Arabidopsis embryos to spatially adapt vascular tissues to organ growth.

High temperature perception in leaves promotes vascular regeneration and graft formation in distant tissues.

Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing, but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were crucial for this effect, as blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures, and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospermum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism. This article has an associated 'The people behind the papers' interview.

Brassinosteroid regulates stomatal development in etiolated Arabidopsis cotyledons via transcription factors BZR1 and BES1.

Brassinosteroids (BRs) are phytohormones that regulate stomatal development. In this study, we report that BR represses stomatal development in etiolated Arabidopsis (Arabidopsis thaliana) cotyledons via transcription factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS SUPPRESSOR1 (BES1), which directly target MITOGEN-ACTIVATED PROTEIN KINASE KINASE 9 (MKK9) and FAMA, 2 important genes for stomatal development. BZR1/BES1 bind MKK9 and FAMA promoters in vitro and in vivo, and mutation of the BZR1/BES1 binding motif in MKK9/FAMA promoters abolishes their transcription regulation by BZR1/BES1 in plants. Expression of a constitutively active MKK9 (MKK9DD) suppressed overproduction of stomata induced by BR deficiency, while expression of a constitutively inactive MKK9 (MKK9KR) induced high-density stomata in bzr1-1D. In addition, bzr-h, a sextuple mutant of the BZR1 family of proteins, produced overabundant stomata, and the dominant bzr1-1D and bes1-D mutants effectively suppressed the stomata-overproducing phenotype of brassinosteroid insensitive 1-116 (bri1-116) and brassinosteroid insensitive 2-1 (bin2-1). In conclusion, our results revealed important roles of BZR1/BES1 in stomatal development, and their transcriptional regulation of MKK9 and FAMA expression may contribute to BR-regulated stomatal development in etiolated Arabidopsis cotyledons.

Electron tomography of prolamellar bodies and their transformation into grana thylakoids in cryofixed Arabidopsis cotyledons.

The para-crystalline structures of prolamellar bodies (PLBs) and light-induced etioplast-to-chloroplast transformation have been investigated via electron microscopy. However, such studies suffer from chemical fixation artifacts and limited volumes of 3D reconstruction. Here, we examined Arabidopsis thaliana cotyledon cells by electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed scanning transmission ET to image large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice-like carbon atoms in diamonds. Within 2 h after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets (pre-granal thylakoids), which folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids contained curved membranes in their tips, we examined the expression and localization of CURT1 (CURVATURE THYLAKOID1) proteins. CURT1A transcripts were most abundant in de-etiolating cotyledon samples, and CURT1A was concentrated at the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. In contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to cubic crystal growth in the dark.

Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum.

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral (x-y) pixel resolution of 10 nm and an axial (z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Developmental pleiotropy of SDP1 from seedling to mature stages in B. napus.

Rapeseed, an important oil crop, relies on robust seedling emergence for optimal yields. Seedling emergence in the field is vulnerable to various factors, among which inadequate self-supply of energy is crucial to limiting seedling growth in early stage. SUGAR-DEPENDENT1 (SDP1) initiates triacylglycerol (TAG) degradation, yet its detailed function has not been determined in B. napus. Here, we focused on the effects of plant growth during whole growth stages and energy mobilization during seedling establishment by mutation in BnSDP1. Protein sequence alignment and haplotypic analysis revealed the conservation of SDP1 among species, with a favorable haplotype enhancing oil content. Investigation of agronomic traits indicated bnsdp1 had a minor impact on vegetative growth and no obvious developmental defects when compared with wild type (WT) across growth stages. The seed oil content was improved by 2.0-2.37% in bnsdp1 lines, with slight reductions in silique length and seed number per silique. Furthermore, bnsdp1 resulted in lower seedling emergence, characterized by a shrunken hypocotyl and poor photosynthetic capacity in the early stages. Additionally, impaired seedling growth, especially in yellow seedlings, was not fully rescued in medium supplemented with exogenous sucrose. The limited lipid turnover in bnsdp1 was accompanied by induced amino acid degradation and PPDK-dependent gluconeogenesis pathway. Analysis of the metabolites in cotyledons revealed active amino acid metabolism and suppressed lipid degradation, consistent with the RNA-seq results. Finally, we proposed strategies for applying BnSDP1 in molecular breeding. Our study provides theoretical guidance for understanding trade-off between oil accumulation and seedling energy mobilization in B. napus.

Arabidopsis vascular complexity and connectivity controls PIN-FORMED1 dynamics and lateral vein patterning during embryogenesis.

Arabidopsis VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC) is a plant-specific transmembrane protein that controls the development of veins in cotyledons. Here, we show that the expression and localization of the auxin efflux carrier PIN-FORMED1 (PIN1) is altered in vcc developing cotyledons and that overexpression of PIN1-GFP partially rescues vascular defects of vcc in a dosage-dependent manner. Genetic analyses suggest that VCC and PINOID (PID), a kinase that regulates PIN1 polarity, are both required for PIN1-mediated control of vasculature development. VCC expression is upregulated by auxin, likely as part of a positive feedback loop for the progression of vascular development. VCC and PIN1 localized to the plasma membrane in pre-procambial cells but were actively redirected to vacuoles in procambial cells for degradation. In the vcc mutant, PIN1 failed to properly polarize in pre-procambial cells during the formation of basal strands, and instead, it was prematurely degraded in vacuoles. VCC plays a role in the localization and stability of PIN1, which is crucial for the transition of pre-procambial cells into procambial cells that are involved in the formation of basal lateral strands in embryonic cotyledons.

Glucose status within dark-grown etiolated cotyledons determines seedling de-etiolation upon light irradiation.

Exposure of dark-grown etiolated seedlings to light triggers the transition from skotomorphogenesis/etiolation to photomorphogenesis/de-etiolation. In the life cycle of plants, de-etiolation is essential for seedling development and plant survival. The mobilization of soluble sugars (glucose [Glc], sucrose, and fructose) derived from stored carbohydrates and lipids to target organs, including cotyledons, hypocotyls, and radicles, underpins de-etiolation. Therefore, dynamic carbohydrate biochemistry is a key feature of this phase transition. However, the molecular mechanisms coordinating carbohydrate status with the cellular machinery orchestrating de-etiolation remain largely opaque. Here, we show that the Glc sensor HEXOKINASE 1 (HXK1) interacts with GROWTH REGULATOR FACTOR5 (GRF5), a transcriptional activator and key plant growth regulator, in Arabidopsis (Arabidopsis thaliana). Subsequently, GRF5 directly binds to the promoter of phytochrome A (phyA), encoding a far-red light (FR) sensor/cotyledon greening inhibitor. We demonstrate that the status of Glc within dark-grown etiolated cotyledons determines the de-etiolation of seedlings when exposed to light irradiation by the HXK1-GRF5-phyA molecular module. Thus, following seed germination, accumulating Glc within dark-grown etiolated cotyledons stimulates a HXK1-dependent increase of GRF5 and an associated decrease of phyA, triggering the perception, amplification, and relay of HXK1-dependent Glc signaling, thereby facilitating the de-etiolation of seedlings following light irradiation. Our findings, therefore, establish how cotyledon carbohydrate signaling under subterranean darkness is sensed, amplified, and relayed, determining the phase transition from skotomorphogenesis to photomorphogenesis on exposure to light irradiation.

Identification of a novel and plant height-independent QTL for coleoptile length in barley and validation of its effect using near isogenic lines.

KEY MESSAGE: This study reported the identification and validation of novel QTL conferring coleoptile length in barley and predicted candidate genes underlying the largest effect QTL based on orthologous analysis and comparison of the whole genome assemblies for both parental genotypes of the mapping population. Coleoptile length (CL) is one of the most important agronomic traits in cereal crops due to its direct influence on the optimal depth for seed sowing which facilitates better seedling establishment. Varieties with longer coleoptiles are preferred in drought-prone areas where less moisture maintains at the top layer of the soil. Compared to wheat, genetic study on coleoptile length is limited in barley. Here, we reported a study on detecting the genomic regions associated with CL in barley by assessing a population consisting of 201 recombinant inbred lines. Four putative QTL conferring CL were consistently identified on chromosomes 1H, 5H, 6H, and 7H in each of the trials conducted. Of these QTL, the two located on chromosomes 5H and 6H (designated as Qcl.caf-5H and Qcl.caf-6H) are likely novel and Qcl.caf-5H showed the most significant effect explaining up to 30.9% of phenotypic variance with a LOD value of 15.1. To further validate the effect of this putative QTL, five pairs of near isogenic lines (NILs) were then developed and assessed. Analysis of the NILs showed an average difference of 21.0% in CL between the two isolines. Notably, none of the other assessed morphological characteristics showed consistent differences between the two isolines for each pair of the NILs. Candidate genes underlying the Qcl.caf-5H locus were also predicted by employing orthologous analysis and comparing the genome assemblies for both parental genotypes of the mapping population in the present study. Taken together, these findings expand our understanding on genetic basis of CL and will be indicative for further gene cloning and functional analysis underly this locus in barley.

Unveiling the mechanism of source-sink rebalancing in cucumber-pumpkin heterografts: the buffering roles of rootstock cotyledon.

Grafting onto pumpkin rootstock is widely applied in cucumber production to improve growth and yield, as well as to overcome soil-borne diseases and enhance resistance to abiotic stresses. In this study, we constructed the cucumber-pumpkin heterografts with the one-cotyledon grafting method, and examined the effects of heterografting on biomass allocation and sugar partitioning, with cucumber and pumpkin self-grafts used as control. Compared with cucumber self-grafts, heterografting onto pumpkin rootstock promoted photosynthesis in cucumber scion, and led to higher sucrose contents in the 1st true leaf (source) and newly emerged leaf (sink). Thereby, the scion part of heterografts accumulated more biomass than cucumber self-grafts. In contrast, when compared to pumpkin self-grafts, grafting with cucumber scion reduced root vigor and biomass but promoted cotyledon growth in pumpkin rootstock. The roots (sink) of heterografts contained less sucrose and hexoses, and showed reduced sucrose synthase (SuSy) and hexokinase (HXK) activities. However, the rootstock cotyledon (source) contained more sucrose and starch, and showed higher activities of HXK, cell-wall invertase (CWIN), and enzymes for starch synthesis and degradation. Furthermore, removal or shade of rootstock cotyledon led to reduced growth of root and scion. Silencing of CmoMEX1a gene in rootstock cotyledon inhibited maltose export and reduced root growth of heterografts. These results indicated that rootstock cotyledon, especially its starch content, played a buffering role in the growth regulation of cucumber-pumpkin heterografts. Taken together, our results provided a major contribution to our understanding of source-sink sugar partitioning and scion-rootstock growth balancing in cucumber-pumpkin heterografts.

Overexpression of R2R3-MYB IbMYB1a induces anthocyanin pigmentation in soybean cotyledon.

KEY MESSAGE: This is the first report showing anthocyanin accumulation in the soybean cotyledon via genetic transformation of a single gene. Soybean [Glycine max (L.) Merrill] contains valuable components, including anthocyanins. To enhance anthocyanin production in Korean soybean Kwangankong, we utilized the R2R3-type MYB gene (IbMYB1a), known for inducing anthocyanin pigmentation in Arabidopsis. This gene was incorporated into constructs using two promoters: the CaMV 35S promoter (P35S) and the ß-conglycinin promoter (Pß-con). Kwangankong was transformed using Agrobacterium, and the presence of IbMYB1a and Bar transgenes in T0 plants was confirmed through polymerase chain reaction (PCR), followed by gene expression validation. Visual inspection revealed that one P35S:IbMYB1a and three Pß-con:IbMYB1a lines displayed seed color change. Pß-con:IbMYB1a T1 seeds accumulated anthocyanins in cotyledon outer layers, whereas P35S:IbMYB1a and non-transgenic black soybean (Cheongja 5 and Seum) accumulated anthocyanins in the seed coat. During the germination and growth phase, T1 seedlings from Pß-con:IbMYB1a lines exhibited anthocyanin pigmentation in cotyledons for up to 1 month without growth aberrations. High-performance liquid chromatography confirmed cyanidin-3-O-glucoside as the major anthocyanin in the Pß-con:IbMYB1a line (#3). We analyzed the expression patterns of anthocyanin biosynthesis genes, chalcone synthase 7,8, chalcone isomerase 1A, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, dihydroflavanol reductase 1, dihydroflavanol reductase 2, anthocyanidin synthase 2, anthocyanidin synthase 3, and UDP glucose flavonoid 3-O-glucosyltransferase in transgenic and control Kwangankong and black soybean (Cheongja 5 and Seum) seeds using quantitative real-time PCR. We conclude that the induction of gene expression in transgenic plants in comparison with Kwangankong was attributable to IbMYB1a transformation. Notably, flavanone 3-hydroxylase, flavanone 3'-hydroxylase, and dihydroflavanol reductase 1 were abundantly expressed in black soybean seed coat, distinguishing them from transgenic cotyledons.

Role of sugars in the apical hook development of Arabidopsis etiolated seedlings.

KEY MESSAGE: The sugar supply in the medium affects the apical hook development of Arabidopsis etiolated seedlings. In addition, we provided the mechanism insights of this process. Dicotyledonous plants form an apical hook structure to shield their young cotyledons from mechanical damage as they emerge from the rough soil. Our findings indicate that sugar molecules, such as sucrose and glucose, are crucial for apical hook development. The presence of sucrose and glucose allows the apical hooks to be maintained for a longer period compared to those grown in sugar-free conditions, and this effect is dose-dependent. Key roles in apical hook development are played by several sugar metabolism pathways, including oxidative phosphorylation and glycolysis. RNA-seq data revealed an up-regulation of genes involved in starch and sucrose metabolism in plants grown in sugar-free conditions, while genes associated with phenylpropanoid metabolism were down-regulated. This study underscores the significant role of sugar metabolism in the apical hook development of etiolated Arabidopsis seedlings.

Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana.

KEY MESSAGE: The disruption of the SWL1 gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in Arabidopsis thaliana. And finally results in a dysfunction of chloroplast and plant growth. Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the SWL1 gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of swl1 lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the swl1 compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of swl1. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.

An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

Location and timing govern tripartite interactions of fungal phytopathogens and host in the stem canker species complex.

BACKGROUND: Leptosphaeria maculans "brassicae" (Lmb) and Leptosphaeria biglobosa "brassicae" (Lbb) make up a species complex involved in the stem canker (blackleg) disease of rapeseed (Brassica napus). They coinfect rapeseed together, from the early stage of infection on leaves to the final necrotic stage at the stem base, and both perform sexual crossings on plant residues. L. biglobosa is suggested to be a potential biocontrol agent against Lmb, but there has been no mechanistic investigation of the different types of interactions that may occur between the plant and the two fungal species. RESULTS: We investigated the bi- or tripartite interaction mechanisms by (i) confronting Lmb and Lbb in culture conditions or during cotyledon infection, with different timing and/or spore concentration regimes, (ii) performing RNA-Seq experiments in vitro or on the kinetics of infection of cotyledons infected by Lmb and/or Lbb to evaluate the transcriptomic activity and the plant response when both fungal species are inoculated together. Lbb infection of B. napus cotyledons was typical of a necrotrophic behavior, with a very early setup of one pathogenicity program and very limited colonization of tissues. This contrasted with the complex succession of pathogenicity programs of the hemibiotroph Lmb. During simultaneous co-infection by both species, Lmb was strongly impacted in its growth and transcriptomic dynamics both in vitro and in planta, while Lbb was unaffected by the presence of Lmb. However, the drastic inhibition of Lmb growth by Lbb was ineffective in the case of delayed inoculation with Lbb or a lower amount of spores of Lbb compared to Lmb. CONCLUSIONS: Our data suggest that Lmb growth inhibition by Lbb is the result of a combination of factors that may include competition for trophic resources, the generation by Lbb of an environment unsuitable for the lifecycle of Lmb or/and the effect on Lmb of plant defense responses induced by Lbb. It indicates that growth inhibition occurs in very specific conditions (i.e., co-inoculation at the same place of an equal amount of inoculum) that are unlikely to occur in the field where their coexistence does not prevent any species from completing their life cycle.

Improvement of Bioactive Polyphenol Accumulation in Callus of Salvia atropatana Bunge.

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line-HL, and root line-RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5-50.2 mg/g dry weight) and blue (51.4-53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

TCP15 interacts with GOLDEN2-LIKE 1 to control cotyledon opening in Arabidopsis.

After germination, exposure to light promotes the opening and expansion of the cotyledons and the development of the photosynthetic apparatus in a process called de-etiolation. This process is crucial for seedling establishment and photoautotrophic growth. TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors are important developmental regulators of plant responses to internal and external signals that are grouped into two main classes. In this study, we identified GOLDEN2-LIKE 1 (GLK1), a key transcriptional regulator of photomorphogenesis, as a protein partner of class I TCPs during light-induced cotyledon opening and expansion in Arabidopsis. The class I TCP TCP15 and GLK1 are mutually required for cotyledon opening and the induction of SAUR and EXPANSIN genes, involved in cell expansion. TCP15 also participates in the expression of photosynthesis-associated genes regulated by GLK1, like LHCB1.4 and LHCB2.2. Furthermore, GLK1 and TCP15 bind to the same promoter regions of different target genes containing either GLK or TCP binding motifs and binding of TCP15 is affected in a GLK1-deficient background, suggesting that a complex between TCP15 and GLK1 participates in the induction of these genes. We postulate that GLK1 helps to recruit TCP15 for the modulation of cell expansion genes in cotyledons and that the functional interaction between these transcription factors may serve to coordinate the expression of cell expansion genes with that of genes involved in the development of the photosynthetic apparatus.

Identification of novel regulators required for early development of vein pattern in the cotyledons by single-cell RNA-sequencing.

The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

DEFECTIVELY ORGANIZED TRIBUTARIES 5 is not required for leaf venation patterning in Arabidopsis thaliana.

The search for genetic regulators of leaf venation patterning started over 30 years ago, primarily focused on mutant screens in the eudicotyledon Arabidopsis thaliana. Developmental perturbations in either cotyledons or true leaves led to the identification of transcription factors required to elaborate the characteristic reticulated vein network. An ortholog of one of these, the C2H2 zinc finger protein DEFECTIVELY ORGANIZED TRIBUTARIES 5 (AtDOT5), was recently identified through transcriptomics as a candidate regulator of parallel venation in maize (Zea mays) leaves. To elucidate how AtDOT5 regulates vein patterning, we generated three independent loss-of-function mutations by gene editing in Arabidopsis. Surprisingly, none of them exhibited any obvious phenotypic perturbations. To reconcile our findings with earlier reports, we re-evaluated the original Atdot5-1 and Atdot5-2 alleles. By genome sequencing, we show that reported mutations at the Atdot5-1 locus are actually polymorphisms between Landsberg erecta and Columbia ecotypes, and that other mutations present in the background most likely cause the pleiotropic mutant phenotype observed. We further show that a T-DNA insertion in the Atdot5-2 locus has no impact on leaf venation patterns when segregated from other T-DNA insertions present in the original line. We thus conclude that AtDOT5 plays no role in leaf venation patterning in Arabidopsis.