Arabidopsis Thaliana Seeds Research Articles

Transcriptome and physiological analyses reveal the response of Arabidopsis thaliana to poly(aspartic acid)

Poly(aspartic acid) (PASP) is an environmentally friendly biopolymer used as a fertilizer synergist and known to increase agricultural yields. The mechanism of PASP enhancement has, however, not been established, although the general hypothesis is that the polymer functions to hold nutrients closer to the root zone. The objective of this study was to determine the physiological and molecular changes that occur when plants are exposed to PASP, with future directions leading to a proposed mode of action. A whole genome transcriptome study was conducted. Arabidopsis thaliana seeds were germinated and grown in sterile plates treated with 250 ppm PASP under continuous light. Total RNA was extracted from whole seedlings and sequenced. The results revealed 462 differentially expressed genes (DEGs), 245 of which were upregulated and 217 downregulated. Gene Ontology, KEGG and MAPman analyses revealed DEGs involved in photosynthesis with 11 light harvesting complexes upregulated (e.g. LHCB1.1, LHCB2.2, LHCA1, LHCB4.2, LHCB2.1, LHCA4, LHCB1.1, LHCB3, LHCA3); the peroxisome pathway had 6 DEGs (CAT1, KAT1 and XDH2) upregulated (CSD1, CSD2 and FSD2) downregulated, the phenylpropanoid biosynthesis pathway had 7 DEGs upregulated. Other key DEGs were associated with the amino acid (e.g. ASN1) and nitrogen metabolism pathways. Physiology assessment results showed significant differences between control and treated plants with a 33 % increase in leaf area, 25 % increase chlorophyll content (p ≤ 0.05) and a 4-fold increase in photosynthetic rate (p ≤ 0.001). This information helps to increase our understanding of the key genes and metabolic pathways associated with plant response to PASP.

Possible crosstalk between the Arabidopsis TSPO-related protein and the transcription factor WRINKLED1

This study uncovers a regulatory interplay between WRINKLED1 (WRI1), a master transcription factor for glycolysis and lipid biosynthesis, and Translocator Protein (TSPO) expression in Arabidopsis thaliana seeds. We identified potential WRI1-responsive elements upstream of AtTSPO through bioinformatics, suggesting WRI1's involvement in regulating TSPO expression. Our analyses showed a significant reduction in AtTSPO levels in wri1 mutant seeds compared to wild type, establishing a functional link between WRI1 and TSPO. This connection extends to the coordination of seed development and lipid metabolism, with both WRI1 and AtTSPO levels decreasing post-imbibition, indicating their roles in seed physiology. Further investigations into TSPO's impact on fatty acid synthesis revealed that TSPO misexpression alters WRI1's post-translational modifications and significantly enhances seed oil content. Additionally, we noted a decrease in key reserve proteins, including 12 S globulin and oleosin 1, in seeds with TSPO misexpression, suggesting a novel energy storage strategy in these lines. Our findings reveal a sophisticated network involving WRI1 and AtTSPO, highlighting their crucial contributions to seed development, lipid metabolism, and the modulation of energy storage mechanisms in Arabidopsis.

The tree peony DREB transcription factor PrDREB2D regulates seed α-linolenic acid accumulation.

α-Linolenic acid (ALA), an essential fatty acid (FA) for human health, serves as the precursor of 2 nutritional benefits, docosahexaenoic acid and eicosapentaenoic acid, and can only be obtained from plant foods. We previously found that phospholipid:diacylglycerol acyltransferase 2 (PrPDAT2) derived from ALA-rich tree peony (Paeonia rockii) can promote seed ALA accumulation. However, the regulatory mechanism underlying its promoting effect on ALA accumulation remains unknown. Here, we revealed a tree peony dehydration-responsive element binding transcription factor, PrDREB2D, as an upstream regulator of PrPDAT2, which is involved in regulating seed ALA accumulation. Our findings demonstrated that PrDREB2D serves as a nucleus-localized transcriptional activator that directly activates PrPDAT2 expression. PrDREB2D altered the FA composition in transient overexpression Nicotiana benthamiana leaves and stable transgenic Arabidopsis (Arabidopsis thaliana) seeds. Repressing PrDREB2D expression in P. rockii resulted in decreased PrPDAT2 expression and ALA accumulation. In addition, PrDREB2D strengthened its regulation of ALA accumulation by recruiting the cofactor ABA-response element binding factor PrABF2b. Collectively, the study findings provide insights into the mechanism of seed ALA accumulation and avenues for enhancing ALA yield via biotechnological manipulation.

Long-Term Imaging of Endoplasmic Reticulum Morphology in Embryos During Seed Germination.

Imaging plant embryos at the cellular level over time is technically challenging, since the embryo, once its protective seed coat is removed, must be kept viable and unstressed on a microscope slide for the duration of the experiment. Here we describe a procedure and suitable apparatus for the visualization, over several days, of changes in endoplasmic reticulum (ER) morphology associated with the process of germination in Arabidopsis thaliana seeds. Moreover, we also present a user-friendly image analysis tool, which enables subtle perturbations in the ER network to be measured.

Dormancy heterogeneity among Arabidopsis thaliana seeds is linked to individual seed size

Production of morphologically and physiologically variable seeds is an important strategy that helps plants to survive in unpredictable natural conditions. However, the model plant Arabidopsis thaliana and most agronomically essential crops produce visually homogenous seeds. Using automated phenotype analysis, we observed that small seeds in Arabidopsis tend to have higher primary and secondary dormancy levels than large seeds. Transcriptomic analysis revealed distinct gene expression profiles between large and small seeds. Large seeds have higher expression of translation-related genes implicated in germination competence. By contrast, small seeds have elevated expression of many positive regulators of dormancy, including a key regulator of this process, the DOG1 gene. Differences in DOG1 expression are associated with differential production of its alternative cleavage and polyadenylation isoforms; in small seeds, the proximal poly(A) site is selected, resulting in a short mRNA isoform. Furthermore, single-seed RNA sequencing analysis demonstrated that large seeds resemble DOG1 knockout mutant seeds. Finally, on the single-seed level, expression of genes affected by seed size is correlated with expression of genes that position seeds on the path toward germination. Our results demonstrate an unexpected link between seed size and dormancy phenotypes in a species that produces highly homogenous seed pools, suggesting that the correlation between seed morphology and physiology is more widespread than initially assumed.

Silver Nanoparticles Synthesized with Extracts of Leaves of Raphanus sativus L, Beta vulgaris L, and Ocimum basilicum and Its Application in Seed Disinfection

Silver nanoparticles (AgNPs) were produced by green synthesis using Raphanus sativus (RsAgNPs), Beta vulgaris (BvAgNPs), and Ocimum basilicum (ObAgNPs) leaf extracts as reducing agents. Plant phytochemical composition analysis indicated that they contain phenolic compounds that can participate in the synthesis reaction as flavonoids. Synthesized AgNPs presented maximal absorption peak at 430 nm (RsAgNP), 440 nm (BvAgNP), and 420 nm (ObAgNP) in ultraviolet-visible spectrophotometry analysis. Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis showed that RsAgNPs are 76 nm diameter spheres made of up to 54.1% silver, the BvAgNPs are 78 nm diameter spheres with 39.76% silver, and the ObAgNPs are cubes of 99 nm edges with 69.74% silver. The found Z potential values indicate that all the obtained AgNPs are stable in phosphate buffer. A disinfection of 100% and 90% was achieved for the in vitro culture of Arabidopsis thaliana and Psidium guajava (guava) seeds, respectively, with all AgNPs synthesized. Treatment with these AgNPs showed no negative effects on germination, and on the contrary, in guava, a higher germination percentage was observed when the seeds were exposed to RsAgNPs. Only at high concentration (10 mg/mL) of AgNPs, the growth of A. thaliana was decreased, while at low concentration (0.01 mg/mL) of ObAgNP and BvAgNPs, higher growth was showed, specifically 60 and 40% more than the control, respectively. All AgNPs showed antimicrobial activity against Escherichia coli and Klebsiella oxytoca which are bacteria of clinical interest, and against Agrobacterium tumefaciens, a bacterium used in the genetic transformation of plants.

Release of Arabidopsis seed dormancy by cold atmospheric plasma relies on cytoplasmic glass transition

When mature Arabidopsis thaliana seeds are dormant, their germination is prevented in apparently favorable conditions. This primary dormancy can be released during seed dry storage through a process called after-ripening whose duration can last several months. To reduce this delay, cold atmospheric plasmas (CAPs) can be used as sources of reactive oxygen species (ROS) capable of inducing heterogeneous chemical reactions. While CAP are known to stimulate the germination of various seed species, the relationship between CAP treatments and the amorphous solid state of dry seeds remains unexplored. Here, we demonstrate that seed dormancy can be alleviated using a cold plasma of ambient air and that this alleviation can be amplified for seeds with high water-content (typically 30%DW) or seeds heated at 60 °C during plasma treatment. Differential scanning micro-calorimetry shows that these characteristics control the glassy/rubbery state of the seed cytoplasm. This technique indicates also that a glass transition to the rubbery state strengthens the CAP effects to alleviate seed dormancy. We propose that lower cytoplasmic viscosity can promote the oxidative signaling induced by CAP which, in turn, improves the germination process.

BBX24 Increases Saline and Osmotic Tolerance through ABA Signaling in Arabidopsis Seeds.

Seed germination is a critical stage for survival during the life cycle of an individual plant. Genetic and environmental cues are integrated by individual seeds to determine germination, mainly achieved through regulation of the metabolism and signaling of gibberellins (GA) and abscisic acid (ABA), two phytohormones with antagonistic roles. Saline and drought conditions can arrest the germination of seeds and limit the seedling emergence and homogeneity of crops. This work aimed to study the function of BBX24, a B-Box transcription factor, in the control of germination of Arabidopsis thaliana seeds imbibed in saline and osmotic conditions. Seeds of mutant and reporter GUS lines of BBX24 were incubated at different doses of NaCl and polyethylene-glycol (PEG) solutions and with ABA, GA and their inhibitors to evaluate the rate of germination. We found that BBX24 promotes seed germination under moderated stresses. The expression of BBX24 is inhibited by NaCl and PEG. In addition, ABA suppresses BBX24-induced seed germination. Additional experiments suggest that BBX24 reduces ABA sensitivity, improving NaCl tolerance, and increases GA sensitivity in seeds imbibed in ABA. In addition, BBX24 inhibits the expression of ABI3 and ABI5 and genetically interacts upstream of HY5 and ABI5. This study demonstrates the relevance of BBX24 to induce drought and salinity tolerance in seed germination to ensure seedling emergence in sub-optimal environments.

Cystathionine γ-synthase expression in seeds alters metabolic and DNA methylation profiles in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) seeds expressing the feedback-insensitive form of cystathionine γ-synthase (AtD-CGS), the key gene of methionine (Met) synthesis, under the control of a seed-specific phaseolin promoter (SSE plants) show a significant increase in Met content. This elevation is accompanied by increased levels of other amino acids (AAs), sugars, total protein, and starch, which are important from a nutritional aspect. Here, we investigated the mechanism behind this phenomenon. Gas chromatography-mass spectrometry (GC-MS) analysis of SSE leaves, siliques, and seeds collected at 3 different developmental stages showed high levels of Met, AAs, and sugars compared to the control plants. A feeding experiment with isotope-labeled AAs showed an increased flux of AAs from nonseed tissues toward the developing seeds of SSE. Transcriptome analysis of leaves and seeds displayed changes in the status of methylation-related genes in SSE plants that were further validated by methylation-sensitive enzymes and colorimetric assay. These results suggest that SSE leaves have higher DNA methylation rates than control plants. This occurrence apparently led to accelerated senescence, together with enhanced monomer synthesis, which further resulted in increased transport of monomers from the leaves toward the seeds. The developing seeds of SSE plants, however, show reduced Met levels and methylation rates. The results provide insights into the role of Met in DNA methylation and gene expression and how Met affects the metabolic profile of the plant.

The tree peony nuclear factor Y transcription factor PrNF-YC2 promotes seed oil accumulation.

Seed oil not only provides energy for seed postgermination development but also provides essential nutrients and raw materials for human products. However, the transcriptional regulatory mechanism controlling seed oil accumulation remains largely unknown. Tree peony (Paeonia rockii) is an emerging woody oilseed crop in China that is known for its high-quality seed oil. Here, we revealed that a tree peony nuclear factor Y transcription factor, PrNF-YC2, is expressed predominantly in developing seeds and functions as an essential positive regulator of seed oil accumulation. PrNF-YC2 promoted oil accumulation in both transient ectopic overexpression Nicotiana benthamiana leaves and stable transgenic Arabidopsis thaliana seeds, globally upregulating the expression of genes involved in oil accumulation. In contrast, PrNF-YC2-silenced tree peony leaves using a virus-induced gene silencing system showed reduced oil content and expression of oil synthesis-related genes, including four master positive regulators contributing to oil accumulation, namely, LEAFY COTYLEDON1 (LEC1), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and WRINKLED1 (WRI1). We demonstrated that PrNF-YC2 directly activates PrLEC1 and PrABI3 alone and indirectly activates PrFUS3 and PrWRI1 by interacting with PrLEC1. Moreover, interaction with PrLEC1 also enhances the activation capacity of PrNF-YC2. The activation of these four master positive regulators by PrNF-YC2 triggered the upregulation of numerous oil synthesis-related genes, thus promoting oil accumulation. These findings provide new insights into the regulatory mechanism of seed oil accumulation and manipulation of PrNF-YC2 may be beneficial for enhancing oil yield in tree peony and other oilseed crops.

Mercury remediation potential of mercury-resistant strain Rheinheimera metallidurans sp. nov. isolated from a municipal waste dumping site

A novel mercury-resistant bacterium, designated strain DCL_24T, was isolated from the legacy waste at the Daddu Majra dumping site in Chandigarh, India. It showed resistance up to 300 µM of inorganic mercury (mercuric chloride). The isolate was found to be a Gram-negative, facultative anaerobic, motile, and rod-shaped bacterium that can grow at 4 – 30 °C (optimum 25 °C), pH 6.0 – 12.0 (optimum 7.0), and 0 – 4.0 % (w/v) NaCl (optimum 0.5 – 2.0 %). The 16 S rRNA gene-based phylogenetic analysis showed that DCL_ 24 T shared a 97.53 % similarity with itsºlosest type strain Rheinheimera muenzenbergensis E-49T. Insilico DNA-DNA hybridization and average nucleotide identity values were found to be 18.60 % and 73.77 %, respectively, between the genomes of DCL_24T and R. muenzenbergensis E-49T. The strain DCL_24T has 44.33 DNA G+C content (mol %). Based on the phenotypic, chemotaxonomic, and genotypic data, the strain DCL_24T represents a novel species within the genus Rheinheimera, for which the name Rheinheimera metallidurans sp. nov is proposed. The type strain is DCL_24T (MTCC13203T = NBRC115780T = JCM 35551 T). The isolate was found to volatilize and remove mercury efficiently, as demonstrated by X-ray film and dithizone-based colorimetric methods. Around 92 % of mercury removal was observed within 48 h. The mercury-resistant determinant mer operon consisting of merA, encoding the mercuric reductase enzyme, and transport and regulatory genes (merT, merP, merD, and merR) were found in the isolate. Relative expression analysis of merA at increasing concentrations of HgCl2 was confirmed by quantitative real-time PCR. These data indicate the merA-mediated reduction of toxic Hg2+ into a non-toxic volatile Hg0. The phytotoxicity assay performed using Arabidopsis thaliana seeds further demonstrated the mercury toxicity reduction potential of DCL_24T. The study shows that this novel isolate, DCL_24T, is an interesting candidate for mercury bioremediation. However, further studies are required to assess the bioremediation efficacy of the strain under the harsh environmental conditions prevailing in polluted sites.

Tree peony transcription factor PrWRI1 enhances seed oil accumulation

BackgroundWRINKLED1 (WRI1) encodes a transcription factor, belonging to the APETALA2 (AP2) family, and plays a key role in regulating plant oil biosynthesis. As a newly woody oil crop, tree peony (Paeonia rockii) was notable for the abundant unsaturated fatty acids in its seed oil. However, the role of WRI1 during the accumulation of P. rockii seeds oil remains largely unknown.ResultsIn this study, a new member of the WRI1 family was isolated from P. rockii and was named PrWRI1. The ORF of PrWRI1 consisted of 1269 nucleotides, encoding a putative protein of 422 amino acids, and was highly expressed in immature seeds. Subcellular localization analysis in onion inner epidermal cells showed that PrWRI1 was located at the nucleolus. Ectopic overexpression of PrWRI1 could significantly increase the total fatty acid content in Nicotiana benthamiana leaf tissue and even PUFAs in transgenic Arabidopsis thaliana seeds. Furthermore, the transcript levels of most genes related to fatty acids (FA) synthesis and triacylglycerol (TAG) assembly were also up-regulated in transgenic Arabidopsis seeds.ConclusionsTogether, PrWRI1 could push carbon flow to FA biosynthesis and further enhance the TAG amount in seeds with a high proportion of PUFAs.

Simultaneous determination of all aminobutyric acids by chiral derivatization and liquid chromatography-tandem mass spectrometry.

Aminobutyric acids include eight structural or stereoisomers that exhibit a wide range of biological activities. Recent evidence on some low abundant isomers have increased the demand for highly selective analysis of all the isomers; however, simultaneous separation of all the aminobutyric acid isomers has not been successful yet, except for a specialized method that uses multiple separation columns and a split of samples. In this study, we developed a new analytical method using chiral derivatization and liquid chromatography-tandem mass spectrometry to separate all the aminobutyric acid isomers in a single separation column. All the diastereomeric derivatives were resolved in a C18 column, and the derivatives showed characteristic fragmentation patterns in tandem mass spectrometry. By using the method, we analyzed the isomers in the Arabidopsis thaliana seeds and revealed the existence of three low abundant isomers, i.e., D-, L-β-aminoisobutyric acid, and D-β-aminobutyric acid. The proposed method uses a commercially available chiral derivatizing reagent and a broadly used column; therefore, it can be widely used in biological and food analyses.

Sucrose non-fermenting1-related protein kinase VcSnRK2.3 promotes anthocyanin biosynthesis in association with VcMYB1 in blueberry.

Sucrose non-fermenting1-related protein kinase-2 (SnRK2) is a plant-specific protein kinase family and an important component of the abscisic acid (ABA) signaling pathway. However, there is a lack of relevant studies in blueberry (Vaccinium corymbosum). In this study, we identified six SnRK2 family members (from VcSnRK2.1 to VcSnRK2.6) in blueberries for the first time. In addition, we found that VcSnRK2.3 expression was not only positively correlated with fruit ripening but was also induced by ABA signaling. Transient expression in blueberry fruits also proved that VcSnRK2.3 promoted anthocyanin accumulation and the expression of anthocyanin synthesis-related genes such as VcF3H, VcDFR, VcANS, and VcUFGT. Transgenic Arabidopsis thaliana seeds and seedlings overexpressing VcSnRK2.3 showed anthocyanin pigmentation. Yeast two-hybrid assays (Y2H) and Bimolecular fluorescence complementation assays (BiFC) demonstrated that VcSnRK2.3 could interact with the anthocyanin positive regulator VcMYB1. Finally, VcSnRK2.3 was able to enhance the binding of VcMYB1 to the VcDFR promoter. Via regulation transcription of anthocyanin biosynthesis genes, VcSnRK2.3 promoted anthocyanin accumulation in blueberry. The above results suggest that VcSnRK2.3 plays an important role in blueberry anthocyanin synthesis, is induced by ABA, and can interact with VcMYB1 to promote anthocyanin biosynthesis in blueberry.

A Novel Imaging Protocol for Investigating Arabidopsis thaliana Siliques and Seeds Using X-rays.

Understanding silique and seed morphology is essential to developmental biology. Arabidopsis thaliana is one of the best-studied plant models for understanding the genetic determinants of seed count and size. However, the small size of its seeds, and their encasement in a pod known as silique, makes investigating their numbers and morphology both time consuming and tedious. Researchers often report bulk seed weights as an indicator of average seed size, but this overlooks individual seed details. Removal of the seeds and subsequent image analysis is possible, but automated counts are often impossible due to seed pigmentation and shadowing. Traditional ways of analyzing seed count and size, without their removal from the silique, involve lengthy histological processing (24-48 h) and the use of toxic organic solvents. We developed a method that is non-invasive, requires minimal sample processing, and obtains data in a short period of time (1-2 h). This method uses a custom X-ray imaging system to visualize Arabidopsis siliques at different stages of their growth. We show that this process can be successfully used to analyze the overall topology of Arabidopsis siliques and seed size and count. This new method can be easily adapted for other plant models. Key features • No requirement for organic solvents for imaging siliques. • Easy image capture and rapid turnaround time for obtaining data. • Protocol may be easily adapted for other plant models.

Camelina cytosol-localized diacylglycerol acyltransferase 3 contributes to the accumulation of seed storage oils

In plants, triacylglycerol (TAG) is a form of seed storage oil that can provide a source of carbon and energy for seed germination and seedling development. Among three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) that synthesize TAG, DGAT3 is involved in TAG synthesis in the cytosol, however, its biological function is still unclear. In this study, three CsDGAT3 homoeologs ( CsDGAT3–1 , CsDGAT3–2 , and CsDGAT3–3 ) showed higher expression levels than CsDGAT1 in developing seeds of camelina ( Camelina sativa ). These were predominantly expressed in developing seeds, and the CsDGAT3 proteins were localized to the cytosol in the epidermal cells of Nicotiana benthamiana leaves. Seed weight and total seed fatty acid levels of transgenic Arabidopsis ( Arabidopsis thaliana ) seeds overexpressing CsDGAT3–2 or CsDGAT3–3 were approximately 13% and 11% higher, respectively, than those of nontransgenic seeds. In particular, the levels of C18:3 and C20:1 increased, while levels of C18:1 and C18:2 decreased. In CsDGAT3 -knockout camelina, as compared to wild type, the seed weights and total fatty acid levels were about 7% and 19% lower, and the C18:3 and C20:1 levels, specifically, were reduced. We detected no significant differences in total seed fatty acid levels or composition between the Arabidopsis dgat1 mutant and the dgat1 mutant overexpressing CsDGAT3–3 . Taken together, cytosol-localized DGAT3 contributes to the accumulation of seed storage oil and prefers the acylation of C18:3, which is the most abundant fatty acid in camelina seed oils. This finding can be applied to the development of oilseed plants with enhanced seed storage oils containing high levels of omega-3 fatty acids. • Camelina DGAT3 participates in TAG biosynthesis via the cytosolic DGAT-dependent pathway in seeds. • Camelina DGAT3 shows a preference for C18:3 and C20:1 as acyl-CoA substrates. • Cytosolic DGAT3-mediated TAG biosynthesis contributes to the accumulation of storage oils in seeds. • DGAT3 is a novel genetic resource to enhance seed oil contents with high omega-3 fatty acids levels.

Comparing the efficiency of six clearing methods in developing seeds of Arabidopsis thaliana

Key messageClearSee alpha and FAST9 were optimized for imaging Arabidopsis seeds up to the torpedo stages. The methods preserve the fluorescence of reporter proteins and seed shape, allowing phenotyping embryos in intact seeds.Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have already been reported in plants, whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds.

Enolase2 regulates seed fatty acid accumulation via mediating carbon partitioning in Arabidopsis thaliana.

In many higher plants, fatty acid (FA) biosynthesis is coordinately regulated at multiple levels by intricate regulatory networks. However, the factors and their regulatory mechanisms underlying seed oil accumulation are still limited. Here, we identified that loss of glycolytic metalloenzyme enolase2 (AtENO2) activity increased the contents of total FAs and salicylic acid (SA) but reduced the accumulation of flavonoids and mucilage by regulating the expression of key genes involved in their biosynthesis pathway in Arabidopsis thaliana seeds. AtENO2 physically interacts with the transcription factor AtTGA5, which may participate in the regulation of SA levels. Non-targeted metabolomics analysis of eno2- and WT also showed that the levels of three flavonoids, quercetin-3-galactoside, quercitrin, and epicatechin, were significantly decreased in eno2- , and the flavonoid biosynthesis pathway was also enriched in the KEGG analysis. Meanwhile, the mutation of AtENO2 delayed silique ripening, thereby prolonging silique photosynthesis time, allowing siliques to generate more photosynthesis products for FA biosynthesis. These results reveal a molecular mechanism by AtENO2 to regulate seed oil accumulation in A. thaliana, providing potential targets for improving crop seed oil quality.

Label-Free Quantitative Proteomics Reveal the Involvement of PRT6 in Arabidopsis thaliana Seed Responsiveness to Ethylene

In Arabidopsis thaliana, the breaking of seed dormancy in wild type (Col-0) by ethylene at 100 μL L−1 required at least 30 h application. A mutant of the proteolytic N-degron pathway, lacking the E3 ligase PROTEOLYSIS 6 (PRT6), was investigated for its role in ethylene-triggered changes in proteomes during seed germination. Label-free quantitative proteomics was carried out on dormant wild type Col-0 and prt6 seeds treated with (+) or without (−) ethylene. After 16 h, 1737 proteins were identified, but none was significantly different in protein levels in response to ethylene. After longer ethylene treatment (30 h), 2552 proteins were identified, and 619 Differentially Expressed Proteins (DEPs) had significant differences in protein abundances between ethylene treatments and genotypes. In Col, 587 DEPs were enriched for those involved in signal perception and transduction, reserve mobilization and new material generation, which potentially contributed to seed germination. DEPs up-regulated by ethylene in Col included S-adenosylmethionine synthase 1, methionine adenosyltransferase 3 and ACC oxidase involved in ethylene synthesis and of Pyrabactin Resistance1 acting as an ABA receptor, while DEPs down-regulated by ethylene in Col included aldehyde oxidase 4 involved in ABA synthesis. In contrast, in prt6 seeds, ethylene did not result in strong proteomic changes with only 30 DEPs. Taken together, the present work demonstrates that the proteolytic N-degron pathway is essential for ethylene-mediated reprogramming of seed proteomes during germination.

The nucleotide metabolome of germinating Arabidopsis thaliana seeds reveals a central role for thymidine phosphorylation in chloroplast development.

Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding of how thymidylates are formed in vivo. Analyzing a comprehensive collection of mutants and double mutants on the phenotypic and metabolic level, we report the effect of de novo thymidylate synthesis, salvage of thymidine, and conversion of cytidylates to thymidylates on thymidylate homeostasis during seed germination and seedling establishment in Arabidopsis (Arabidopsis thaliana). During germination, the salvage of thymidine in organelles contributes predominantly to the thymidylate pools and a mutant lacking organellar (mitochondrial and plastidic) thymidine kinase has severely altered deoxyribonucleotide levels, less chloroplast DNA, and chlorotic cotyledons. This phenotype is aggravated when mitochondrial thymidylate de novo synthesis is additionally compromised. We also discovered an organellar deoxyuridine-triphosphate pyrophosphatase and show that its main function is not thymidylate synthesis but probably the removal of noncanonical nucleotide triphosphates. Interestingly, cytosolic thymidylate synthesis can only compensate defective organellar thymidine salvage in seedlings but not during germination. This study provides a comprehensive insight into the nucleotide metabolome of germinating seeds and demonstrates the unique role of enzymes that seem redundant at first glance.