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.

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.

NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

Cooperative transcriptional regulation by ATAF1 and HY5 promotes light-induced cotyledon opening in Arabidopsis thaliana.

The opening of the embryonic leaves (cotyledons) as seedlings emerge from the dark soil into the light is crucial to ensure the survival of the plant. Seedlings that sprout in the dark elongate rapidly to reach light but keep their cotyledons closed. During de-etiolation, the transition from dark to light growth, elongation slows and the cotyledons open. Here, we report that the transcription factor ACTIVATING FACTOR1 (ATAF1) participates in de-etiolation and facilitates light-induced cotyledon opening. The transition from dark to light rapidly induced ATAF1 expression and ATAF1 accumulation in cotyledons. Seedlings lacking or overexpressing ATAF1 exhibited reduced or enhanced cotyledon opening, respectively, and transcriptomic analysis indicated that ATAF1 repressed the expression of genes associated with growth and cotyledon closure. The activation of the photoreceptor phytochrome A (phyA) by far-red light induced its association with the ATAF1 promoter and stimulation of ATAF1 expression. The transcription factor ELONGATED HYPOCOTYL5 (HY5), which is also activated in response far-red light, cooperated with phyA to induce ATAF1 expression. ATAF1 and HY5 interacted with one another and cooperatively repressed the expression of growth-promoting and cotyledon closure genes. Together, our study reveals a mechanism through which far-red light promotes cotyledon opening.

Pea protein extraction method impacts the protein (micro)structural organisation and in vitro digestion kinetics.

There is increasing interest in including pulse proteins into food products due to their nutrient-rich and sustainable character. However, little is known regarding the consequences of different extraction approaches on the pulse protein structure and the subsequent protein (micro)structural organization and protein digestion kinetics. Therefore, three green pea protein extracts were created: (i) cooking followed by cotyledon cell isolation, (ii) alkaline extraction followed by isoelectric precipitation, or (iii) salt extraction, and compared to the original pea flour as well as to sodium caseinate. The results showed that encapsulated, denatured protein inside pea cotyledon cells presented the (s)lowest digestion, while accessible and more native protein (e.g., pea flour, pea protein salt extract) presented much faster and higher digestion. Moreover, the alkali extracted pea protein was denatured to some extent, significantly lowering in vitro digestion kinetics. In the second part, three different in vitro approaches were applied to digest the salt extracted pea protein. Semi-dynamic gastric digestion approaches simulate in vivo conditions more closely which especially impacted the rate of digestion.

OsEIL1 and OsEIL2, two master regulators of rice ethylene signaling, promote the expression of ROS scavenging genes to facilitate coleoptile elongation and seedling emergence from soil.

Successful emergence from the soil is a prerequisite for survival of germinating seeds in their natural environment. In rice, coleoptile elongation facilitates seedling emergence and establishment, and ethylene plays an important role in this process. However, the underlying regulatory mechanism remains largely unclear. Here, we report that ethylene promotes cell elongation and inhibits cell expansion in rice coleoptiles, resulting in longer and thinner coleoptiles that facilitate seedlings emergence from the soil. Transcriptome analysis showed that genes related to reactive oxygen species (ROS) generation are upregulated and genes involved in ROS scavenging are downregulated in the coleoptiles of ethylene-signaling mutants. Further investigations showed that soil coverage promotes accumulation of ETHYLENE INSENSITIVE 3-LIKE 1 (OsEIL1) and OsEIL2 in the upper region of the coleoptile, and both OsEIL1 and OsEIL2 can bind directly to the promoters of the GDP-mannose pyrophosphorylase (VTC1) gene OsVTC1-3 and the peroxidase (PRX) genes OsPRX37, OsPRX81, OsPRX82, and OsPRX88 to activate their expression. This leads to increased ascorbic acid content, greater peroxidase activity, and decreased ROS accumulation in the upper region of the coleoptile. Disruption of ROS accumulation promotes coleoptile growth and seedling emergence from soil. These findings deepen our understanding of the roles of ethylene and ROS in controlling coleoptile growth, and this information can be used by breeders to produce rice varieties suitable for direct seeding.

Exogenous Serotonin (5-HT) Promotes Mesocotyl and Coleoptile Elongation in Maize Seedlings under Deep-Seeding Stress through Enhancing Auxin Accumulation and Inhibiting Lignin Formation.

Serotonin (5-HT), an indoleamine compound, has been known to mediate many physiological responses of plants under environmental stress. The deep-seeding (≥20 cm) of maize seeds is an important cultivation strategy to ensure seedling emergence and survival under drought stress. However, the role of 5-HT in maize deep-seeding tolerance remains unexplored. Understanding the mechanisms and evaluating the optimal concentration of 5-HT in alleviating deep-seeding stress could benefit maize production. In this study, two maize inbred lines were treated with or without 5-HT at both sowing depths of 20 cm and 3 cm, respectively. The effects of different concentrations of 5-HT on the growth phenotypes, physiological metabolism, and gene expression of two maize inbred lines were examined at the sowing depths of 20 cm and 3 cm. Compared to the normal seedling depth of 3 cm, the elongation of the mesocotyl (average elongation 3.70 cm) and coleoptile (average elongation 0.58 cm), secretion of indole-3-acetic acid (IAA; average increased 3.73 and 0.63 ng g-1 FW), and hydrogen peroxide (H2O2; average increased 1.95 and 0.63 µM g-1 FW) in the mesocotyl and coleoptile were increased under 20 cm stress, with a concomitant decrease in lignin synthesis (average decreased 0.48 and 0.53 A280 g-1). Under 20 cm deep-seeding stress, the addition of 5-HT activated the expression of multiple genes of IAA biosynthesis and signal transduction, including Zm00001d049601, Zm00001d039346, Zm00001d026530, and Zm00001d049659, and it also stimulated IAA production in both the mesocotyl and coleoptile of maize seedlings. On the contrary, 5-HT suppressed the expression of genes for lignin biosynthesis (Zm00001d016471, Zm00001d005998, Zm00001d032152, and Zm00001d053554) and retarded the accumulation of H2O2 and lignin, resulting in the elongation of the mesocotyl and coleoptile of maize seedlings. A comprehensive evaluation analysis showed that the optimum concentration of 5-HT in relieving deep-seeding stress was 2.5 mg/L for both inbred lines, and 5-HT therefore could improve the seedling emergence rate and alleviate deep-seeding stress in maize seedlings. These findings could provide a novel strategy for improving maize deep-seeding tolerance, thus enhancing yield potential under drought and water stress.

Spatial Lipidomics Reveals Lipid Changes in the Cotyledon and Plumule of Mung Bean Seeds during Germination.

Seed germination is a vital process in plant development involving dynamic biochemical transformations such as lipid metabolism. However, the spatial distribution and dynamic changes of lipids in different seed compartments during germination are poorly understood. In this study, we employed liquid chromatography/mass spectrometry (LC/MS)-based lipidomics and MALDI mass spectrometry imaging (MSI) to investigate lipid changes occurring in the cotyledon and plumule of mung bean seeds during germination. Lipidomic data revealed that the germination process reduced the levels of many glycerolipids (e.g., triglyceride) and phosphatidylglycerols (e.g., phosphatidylcholine) while increased the levels of lysophospholipids (e.g., lysophosphatidylcholine) in both the cotyledon and plumule. Sphingolipids (e.g., sphingomyelin) displayed altered levels solely in the plumule. Sterol levels increased in the cotyledon but decreased in the plumule. Further imaging results revealed that MALDI-MSI could serve as a supplement and validate LC-MS data. These findings enhance our understanding of the metabolic processes underlying seedling development, with potential implications for crop improvement and seed quality control.

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.

Smooth Elongation of Pavement Cells Induced by RIC1 Overexpression Leads to Marginal Protrusions of the Cotyledon in Arabidopsis thaliana.

The interdigitated pavement cell shape is suggested to be mechanically rational at both the cellular and tissue levels, but the biological significance of the cell shape is not fully understood. In this study, we explored the potential importance of the jigsaw puzzle-like cell shape for cotyledon morphogenesis in Arabidopsis. We used a transgenic line overexpressing a Rho-like GTPase-interacting protein, ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN 1 (RIC1), which causes simple elongation of pavement cells. Computer-assisted microscopic analyses, including virtual reality observation, revealed that RIC1 overexpression resulted in abnormal cotyledon shapes with marginal protrusions, suggesting that the abnormal organ shape might be explained by changes in the pavement cell shape. Microscopic, biochemical and mechanical observations indicated that the pavement cell deformation might be due to reduction in the cell wall cellulose content with alteration of cortical microtubule organization. To examine our hypothesis that simple elongation of pavement cells leads to an abnormal shape with marginal protrusion of the cotyledon, we developed a mathematical model that examines the impact of planar cell growth geometry on the morphogenesis of the organ that is an assemblage of the cells. Computer simulations supported experimental observations that elongated pavement cells resulted in an irregular cotyledon shape, suggesting that marginal protrusions were due to local growth variation possibly caused by stochastic bias in the direction of cell elongation cannot be explained only by polarity-based cell elongation, but that an organ-level regulatory mechanism is required.

In vitro germination and reserve mobilization of Vriesea friburgensis Mez.

Studies on the germination and establishment of plants are key pieces to understanding the reproductive success of plants. This work aimed to describe in vitro germination and reserve mobilization in the bromeliad Vriesea friburgensis through morphological, histochemical, and biochemical analysis. The conditions used in this study for the in vitro germination are adequate. From the third day of in vitro inoculation, a uniform germination of 98% was obtained, exhibiting a high physiological quality of the seeds and a high potential to produce seedlings (94%). There is early reserve mobilization, which began in the imbibition phase. The accumulated reserves in the endosperm cytoplasm are degraded by hydrolytic enzymes provided by the aleurone layer. It is possible that compounds in the cell walls of the endosperm contribute to a lesser extent in mobilization. Additionally, it was observed that starch accumulation in the cotyledon increases when the seedling has formed. Results from this study provide insights for future studies on ecology, seed technology, and conservation in this species. This study contributes to the limited knowledge of the dynamics of reserves during germination and seedling establishment in Bromeliaceae. To the best of our knowledge, this is the first study with this approach in the genus Vriesea.

Salt adopted in soaking solution controls the yield and starch digestion kinetics of intact pulse cotyledon cells.

Intact cellular powders have gained attention as a functional ingredient due to their lower glycemic response and potential benefits in colon. The isolation of intact cells in the laboratory and pilot plant settings is mainly achieved through thermal treatment with or without the use of limited salts. However, the effects of salt type and concentration on cell porosity, and their impact on the enzymic hydrolysis of encapsulated macro-nutrients such as starch, have been overlooked. In this study, different salt-soaking solutions were used to isolate intact cotyledon cells from white kidney beans. The use of Na2CO3 and Na3PO4 soaking treatments, with high pH (11.5-12.7) and high amount of Na ion (0.1, 0.5 M), greatly improved the yield of cellular powder (49.6-55.5 %), due to the solubilization of pectin through ß-elimination and ion exchange. Intact cell walls serve as a physical barrier, significantly reducing the susceptibility of cell to amylolysis when compared to white kidney bean flour and starch counterparts. However, the solubilization of pectin may facilitate enzyme access into the cells by enlarging cell wall permeability. These findings provide new insights into the processing optimization to improve the yield and nutritional value of intact pulse cotyledon cells as a functional food ingredient.

Wild soybean salt tolerance metabolic model: Assessment of storage protein mobilization in cotyledons and C/N balance in the hypocotyl/root axis.

Salt stress has become one of the main factors limiting crop yield in recent years. The post-germinative growth is most sensitive to salt stress in soybean. In this study, cultivated and wild soybeans were used for an integrated metabonomics and transcriptomics analysis to determine whether wild soybean can resist salt stress by maintaining the mobilization of stored substances in cotyledons and the balance of carbon and nitrogen in the hypocotyl/root axis (HRA). Compared with wild soybean, the growth of cultivated soybean was significantly inhibited during the post-germinative growth period under salt stress. Integrating analysis found that the breakdown products of proteins, such as glutamate, glutamic acid, aspartic acid, and asparagine, increased significantly in wild soybean cotyledons. Asparagine synthase and fumarate hydratase genes and genes encoding HSP20 family proteins were specifically upregulated. In wild soybean HRA, levels of glutamic acid, aspartic acid, asparagine, citric acid, and succinic acid increased significantly, and the glutamate decarboxylase gene and the gene encoding carbonic anhydrase in nitrogen metabolism were significantly upregulated. The metabolic model indicated that wild soybean enhanced the decomposition of stored proteins and the transport of amino acids to the HRA in cotyledons and the GABA shunt to maintain carbon and nitrogen balance in the HRA to resist salt stress. This study provided a theoretical basis for cultivating salt-tolerant soybean varieties and opened opportunities for the development of sustainable agricultural practices.

Phenotypically abnormal cotyledonary Vitis vinifera embryos differ in anatomy, endogenous hormone levels and transcriptome profiles.

In many perennial fruit species, including grapevine (Vitis vinifera L.), the highly complex process of somatic embryogenesis (SE) can result in the formation of a deformed embryo, although the underlying reasons are still poorly understood. Here, V. vinifera cv. 'Chardonnay' cotyledonary embryos with distinct morphologies were used to address this issue. Normal cotyledonary embryos (NCEs) and elongated cotyledonary embryos (ECEs) were observed to have better-developed vasculature and shoot meristems than the vitrified cotyledonary embryos (VCEs) and fused cotyledonary embryos (FCEs), but ECEs were less developed. We determined that the morphological differences in these phenotypically abnormal embryos were likely associated with endogenous hormone levels, since concentrations of the phytohormones indoleacetic acid (IAA) and abscisic acid (ABA) in NCEs were higher than in the other three types. Comparative transcriptome analysis revealed large differences in gene expression of the hormone signaling pathways in normal and abnormal cotyledonary embryos. Weighted gene co-expression network analysis of the different cotyledonary types allowed the identification of co-regulated gene modules associated with SE, suggesting a role for ERF family genes and other transcription factors (TFs) in regulating morphology. Moreover, an analysis of morphology-specific gene expression indicated that the activation of a specific protein kinase, small heat shock proteins (sHSPs) and certain TFs was closely associated with the formation of normal cotyledonary embryos. Our comparative analyses provide insights into the gene networks regulating somatic cotyledon development and open new avenues for research into plant regeneration and functional genomic studies of malformed embryos.

Machine learning and feature analysis of the cortical microtubule organization of Arabidopsis cotyledon pavement cells.

The measurement of cytoskeletal features can provide valuable insights into cell biology. In recent years, digital image analysis of cytoskeletal features has become an important research tool for quantitative evaluation of cytoskeleton organization. In this study, we examined the utility of a supervised machine learning approach with digital image analysis to distinguish different cellular organizational patterns. We focused on the jigsaw puzzle-shaped pavement cells of Arabidopsis thaliana. Measurements of three features of cortical microtubules in these cells (parallelness, density, and the coefficient of variation of the intensity distribution of fluorescently labeled cytoskeletons [as an indicator of microtubule bundling]) were obtained from microscopic images. A random forest machine learning model was then used with these images to differentiate mutant and wild type, and Taxol-treated and control cells. Using these three metrics, we were able to distinguish wild type from bpp125 triple mutant cells, with approximately 80% accuracy; classification accuracy was 88% for control and Taxol-treated cells. Different features contributed most to the classification, namely, coefficient of variation for the wild-type/mutant cells and parallelness for the Taxol-treated/control cells. The random forest method used enabled quantitative evaluation of the contribution of features to the classification, and partial dependence plots showed the relationships between metric values and classification accuracy. While further improvements to the method are needed, our small-scale analysis shows the potential for this approach in large-scale screening analyses.

Ethylene-mediated regulation of coleoptile elongation in rice seedlings.

Rice is an important food crop in the world and the study of its growth and plasticity has a profound influence on sustainable development. Ethylene modulates multiple agronomic traits of rice as well as abiotic and biotic stresses during its lifecycle. It has diverse roles, depending on the organs, developmental stages and environmental conditions. Compared to Arabidopsis (Arabidopsis thaliana), rice ethylene signalling pathway has its own unique features due to its special semiaquatic living environment and distinct plant structure. Ethylene signalling and responses are part of an intricate network in crosstalk with internal and external factors. This review will summarize the current progress in the mechanisms of ethylene-regulated coleoptile growth in rice, with a special focus on ethylene signaling and interaction with other hormones. Insights into these molecular mechanisms may shed light on ethylene biology and should be beneficial for the genetic improvement of rice and other crops.

Response of sugar metabolism in the cotyledons and roots of Ricinus communis subjected to salt stress.

Ricinus communis is an important oilseed crop worldwide and is also considered one of the best potential plants for salt-affected soil improvement in northeast China. However, little is known about photosynthesis and carbohydrate metabolism in this plant, nor the distribution of carbohydrates in cotyledons and roots under salinity stress. In the present study, seedling growth, gas exchange parameters (PN , E, gs and Ci ), carbohydrate (fructose, sucrose, glucose, soluble sugar and starch) metabolism and related enzymes and genes were measured in Ricinus plants. Under salt stress, PN of cotyledons decreased significantly (P < 0.05), resulting in weak photosynthetic capacity. Furthermore, salt stress increased sucrose and glucose content in cotyledons, but decreased soluble sugar and starch content. However, sucrose increased and starch decreased in roots. This may be correlated with the increasing sugar metabolism under salinity, including notable changes in sugar-related enzyme activities (SPS, SuSy, α-amylase and ß-amylase) and gene expression of RcINV, RcSUS, RcAmY, RcBAM and RcGBE1. The results suggest that salinity reduces photosynthesis of cotyledons, alters carbohydrate allocation between cotyledons and roots and also promotes starch utilization in cotyledons and starch biosynthesis in roots, leading to a functional imbalance between cotyledons and roots. Together, these findings provide insights into the crucial role of sugar metabolism in improving salt-tolerance of Ricinus during the early seedling growth stage.

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.

A vicilin-like protein extracted from Clitoria fairchildiana cotyledons was toxic to Callosobruchus maculatus (Coleoptera: Chrysomelidae).

Callosobruchus maculatus is the main pest cowpea (Vigna unguiculata). Given its relevance as an insect pest, studies have focused in finding toxic compounds which could prevent its predatory action towards the seeds. Clitoria fairchildiana is a native Amazon species, whose seeds are refractory to insect predation. This characteristic was the basis of our interest in evaluating the toxicity of its seed proteins to C. maculatus larvae. Seed proteins were fractioned, according to their solubility, to albumins (F1), globulins (F2), kaphyrins (F3), glutelins (F4), linked kaphyrins (F5) and cross-linked glutelins (F6). The fractionated proteins were quantified, analysed by tricine-SDS-PAGE and inserted into the diet of this insect pest in order to evaluate their insecticidal potential. The most toxic fraction to C. maculatus, the propanol soluble F3, was submitted to molecular exclusion chromatography and all of the peaks obtained, F3P1, F3P2, F3P3, caused a reduction of larval mass, especially F3P1, seen as a major ~12 kDa electrophoretic band. This protein was identified as a vicilin-like protein by mass spectrometry and BLAST analysis. The alignment of the Cfvic (C. fairchildiana vicilin) peptides with a V. unguiculata vicilin sequence, revealed that Cfvic has at least five peptides (ALLTLVNPDGR, AILTLVNPDGR, NFLAGGKDNV, ISDINSAMDR, NFLAGEK) which lined up with two chitin binding sites (ChBS). This finding was corroborated by chitin affinity chromatography and molecular docking of chitin-binding domains for N-Acetyl-D-glucosamine and by the reduction of Cfvic chitin affinity after chemical modification of its Lys residues. In conclusion, Cfvic is a 12 kDa vicilin-like protein, highly toxic to C. maculatus, acting as an insect toxin through its ability to bind to chitin structures present in the insect midgut.

ELONGATED HYPOCOTYL 5-mediated suppression of melatonin biosynthesis is alleviated by darkness and promotes cotyledon opening.

Melatonin (N-acetyl-5-methoxytryptamine) biosynthesis in plants is induced by darkness and high-intensity light; however, the underlying transcriptional mechanisms and upstream signalling pathways are unknown. We identified a dark-induced and highly expressed melatonin synthetase in Arabidopsis thaliana, AtSNAT6, encoding serotonin N-acetyltransferase. We assessed melatonin content and AtSNAT6 expression in mutants lacking key regulators of light/dark signalling. AtCOP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and AtHY5 (ELONGATED HYPOCOTYL 5), which control light/dark transition and photomorphogenesis, promoted and suppressed melatonin biosynthesis, respectively. Using EMSA and ChIP-qPCR analysis, we showed that AtHY5 inhibits AtSNAT6 expression directly. An analysis of melatonin content in snat6 hy5 double mutant and AtHY5+AtSNAT6-overexpressing plants confirmed the regulatory function of AtHY5 and AtSNAT6 in melatonin biosynthesis. Exogenous melatonin further inhibited cotyledon opening in hy5 mutant and AtSNAT6-overexpressing seedlings, but partially reversed the promotion of cotyledon opening in AtHY5-overexpressing seedlings and snat6. Additionally, CRISPR/Cas9-mediated mutation of AtSNAT6 increased cotyledon opening in hy5 mutant, and overexpression of AtSNAT6 decreased cotyledon opening in AtHY5-overexpressing seedlings via changing melatonin biosynthesis, confirming that AtHY5 decreased melatonin-mediated inhibition of cotyledon opening. Our data provide new insights into the regulation of melatonin biosynthesis and its function in cotyledon opening.