Arabidopsis Thaliana Seeds Research Articles

Arabidopsis thaliana L. Seeds are A novel source of somatic embryos

Somatic embryogenesis is a valuable tool for investigating the totipotency of plant cells. A simple and efficient protocol for inducing somatic embryogenesis from seeds of Arabidopsis thaliana were established. Surface sterilized seeds were placed on agar-solidified Murashige and Skoog (MS) medium free from growth regulators. Callus initiation began 7 days after seeds culture and became visible with the naked eye within 10-14 days. It was friable and yellowish white in color. Within 20 days, callus was transferred to Gamborg’s B5 medium containing 1.0 mgL-1 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 0.05 mgL−1 Kin for multiplication. The results indicated that somatic embryos had been recorded only in B5 medium supplemented with 0.4 mgL−1 TDZ (N-phenyl-N’-1,2, 3-thidiazol-5-ylurea) and it was the best one. Through our observation, different stages of somatic embryos have been found. The results revealed that the continuous transfer of small masses containing several embryos at different stages to the same induction medium subsequently formed a large cluster of shoots, which were rooted in MS medium free from growth regulators and MS hormone-free medium with 0.2activated charcoal. The percentages of rooting were 63% and 51% respectively. This study proved that Arabidopsis thaliana seeds are a novel source for somatic embryos.

Advantages and Limitations of Surface Analysis Techniques on Plasma-Treated Arabidopsis thaliana Seeds

Surface characterization of plasma-treated seeds has made significant progress over the last decade. Most papers in the literature use scanning electron microscopy (SEM) and contact angle goniometry to investigate surface modifications. However, very few papers address the chemical modifications to the seed coat after plasma treatment. Here, a summary of the methods used to analyze plasma-treated seeds is presented, such as SEM, contact angle goniometry, energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The results obtained on Arabidopsis thaliana Col-0 seeds and the limitations of these techniques are discussed. An experiment was designed in order to compare the relative advantages and limitations of these surface analysis techniques by investigating the separate effects of plasma, heat, and ozone on A. thaliana seeds.

Application of expansion microscopy on developing Arabidopsis seeds.

Expansion microscopy (ExM) improves image resolution of specimens without requirements of sophisticated techniques or equipment. Probes or proteins are anchored onto an acrylamide gel matrix which is then expanded with osmotic pressure. As the physical distance between two signal points increases, previously confounded signals can be resolved while their relative spatial locations are retained. ExM has been successfully applied to several animal tissues, but its application to plant tissues was only recently demonstrated. Here we provide a detailed ExM protocol for plant tissues using fluorescent immunostaining of developing Arabidopsis thaliana (Arabidopsis) seeds as an example. This modified ExM protocol enables expansion of ovule/seed samples, and preserves the majority of fluorescent protein signals in the expanded samples. The fluorescent immunostaining observed using this protocol demonstrates the feasibility of detecting cellular events and subcellular structures in expanded plant samples. This ExM protocol variant for plants can serve as a guideline for applying ExM to various plant tissues and help increase the resolution of corresponding microscopy based studies.

14C-Tracing of Lipid Metabolism.

Lipids are produced through a dynamic metabolic network involving branch points, cycles, reversible reactions, parallel reactions in different subcellular compartments, and distinct pools of the same lipid class involved in different parts of the network. For example, diacylglycerol (DAG) is a biosynthetic and catabolic intermediate of many different lipid classes. Triacylglycerol can be synthesized from DAG assembled de novo, or from DAG produced by catabolism of membrane lipids, most commonly phosphatidylcholine. Quantification of lipids provides a snapshot of the lipid abundance at the time they were extracted from the given tissue. However, quantification alone does not provide information on the path of carbon flux through the metabolic network to synthesize each lipid. Understanding lipid metabolic flux requires tracing lipid metabolism with isotopically labeled substrates over time in living tissue. [14C]acetate and [14C]glycerol are commonly utilized substrates to measure the flux of nascent fatty acids and glycerol backbones through the lipid metabolic network in vivo. When combined with mutant or transgenic plants, tracing of lipid metabolism can provide information on the molecular control of lipid metabolic flux. This chapter provides a method for tracing in vivo lipid metabolism in developing Arabidopsis thaliana seeds, including analysis of 14C labeled lipid classes and fatty acid regiochemistry through both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) approaches.

Transport of UDP-rhamnose by URGT2, URGT4, and URGT6 modulates rhamnogalacturonan-I length.

Rhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage (SM) of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of SM polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in Rhamnogalacturonan-I (RG-I) elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage (AM). Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains.

ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

Sequestering miR165/166 enhances seed germination in Arabidopsis thaliana under normal condition and ABA treatment

Seed germination is a dynamic process involving imbibition, increased metabolic activity and protrusion of a tiny plantlet rupturing the seed coat. Many genes, phytohormones like ABA and GA have been implicated in germination of Arabidopsis thaliana seeds. Although many microRNAs (miRNAs) have been shown to be differentially expressed during seed germination process, their role remains mostly unaddressed. Here we address the role of developmentally important miR165/166 in the process of seed germination. We demonstrate that the seeds of transgenic A. thaliana having target mimic-miR165/166 (eTM-miR165/166), where miR165/166 is sponged, show better germination efficiency. The seeds of this line also maintain better germination even under ABA treatment, which is a negative regulator of seed germination. Thus, our results suggest that, sequestering miR165/166 activity enhances seed germination efficiency under normal and ABA-stress condition.

High-efficiency procedure to characterize, segment, and quantify complex multicellularity in raw micrographs in plants

BackgroundThe increasing number of novel approaches for large-scale, multi-dimensional imaging of cells has created an unprecedented opportunity to analyze plant morphogenesis. However, complex image processing, including identifying specific cells and quantitating parameters, and high running cost of some image analysis softwares remains challenging. Therefore, it is essential to develop an efficient method for identifying plant complex multicellularity in raw micrographs in plants.ResultsHere, we developed a high-efficiency procedure to characterize, segment, and quantify plant multicellularity in various raw images using the open-source software packages ImageJ and SR-Tesseler. This procedure allows for the rapid, accurate, automatic quantification of cell patterns and organization at different scales, from large tissues down to the cellular level. We validated our method using different images captured from Arabidopsis thaliana roots and seeds and Populus tremula stems, including fluorescently labeled images, Micro-CT scans, and dyed sections. Finally, we determined the area, centroid coordinate, perimeter, and Feret’s diameter of the cells and harvested the cell distribution patterns from Voronoï diagrams by setting the threshold at localization density, mean distance, or area.ConclusionsThis procedure can be used to determine the character and organization of multicellular plant tissues at high efficiency, including precise parameter identification and polygon-based segmentation of plant cells.

Effects of intentionally treated water and seeds on the growth of Arabidopsis thaliana

Objective. A previously reported experiment indicated that Arabidopsis thaliana seeds with cryptochrome mutation His-CRY2 showed more robust photomorphogenic growth when hydrated with intentionally treated water as compared to untreated water. The present study attempted to replicate that outcome, adding a condition where the seeds were also intentionally treated. Arabidopsis seeds were used because they contain a photosensitive flavoprotein called cryptochrome (CRY). CRY has been proposed as a possible “transducer” of intention in living systems because it is thought to have quantum biological properties, and as such, it might potentially be sensitive to quantum observer effects. DesignThree Buddhist monks directed their attention toward commercially bottled water and Arabidopsis seeds while holding the intention to improve the growth of the plant. As a control condition, no attention was directed at water or seeds from the same sources. Under double-blinded conditions, treated and untreated seeds were placed in an incubator, hydrated with treated or untreated water, and exposed to either continuous blue light or blue plus far-red light. The seed germination process was repeated three times, each time using new seeds. A 2 × 2 × 2 ANOVA, with water, seeds, and light as factors, was used to analyze the results. Results. Treated water was associated with enhanced photomorphogenic growth, as reflected by a shorter hypocotyl length (p = 0.04) and greater amounts of chlorophyll (p = 0.0005) and anthocyanin (p = 2 × 10−6). Treated seeds resulted in greater amounts of chlorophyll (p = 0.04), but also a longer hypocotyl (p = 0.0004) and less anthocyanin (p = 0.01). Plants exposed to blue plus far-red light were constantly more robust than plants grown under blue light, regardless of the type of water or seed (p < 10−10). Conclusion. Intentionally treated water improved the growth of the His-CRY2 variant of Arabidopsis, confirming results of an earlier experiment. Enhanced growth associated with exposure to blue plus far-red light also confirmed to known effects. A more complex relationship was observed with treated seeds. Further research is required to understand the latter outcome, as it may provide clues about the underlying mechanisms of intentional influences.

Sequence analysis of Ricinus communis small heat-shock protein (sHSP) subfamily and its role in abiotic stress responses

Small heat shock proteins (sHSPs) possess major roles in plant defense mechanisms towards abiotic stresses. sHSPs act as molecular chaperones providing the necessary tools to sustain cellular homeostasis under adverse conditions. sHSP genes display specific expression signatures, which depend on tissue-specificity, developmental stage and the nature of the abiotic stress. Despite the fact that Ricinus communis is an important oilseed crop with large socioeconomic impact on small family farmers in semi-arid regions worldwide, the characterization of RcsHSP genes and their possible contribution to plant survival under harsh environmental conditions has not been addressed. Hence, this study aimed at characterizing the R. communis sHSP subfamily, through phylogeny, gene structure, duplication, and expression profile analysis, as well as by characterizing Arabidopsis thaliana seeds overexpressing RcsHSP genes. We identified 41 RcsHSP genes with the α-crystallin domain and compatible molecular weight (<43 kDa). The RcsHSP subfamily showed different homology levels with sHSP genes from other plant species, suggesting the occurrence of specific gene expansion and loss. The RcsHSP subfamily was classified according to the cellular locations of the genes, which included cytosolic, chloroplastic, mitochondrial, and endoplasmic reticulum groups. Ten putative motifs were found among RcsHSP genes, but only motifs 4, 6 and 8 were sHSP protein domains. The RcsHSP subfamily showed 19 genes produced by tandem duplication events, which might have been crucial for RcsHSP diversification and acquisition of tolerance in R. communis. Gene expression analysis showed that the RcsHSP subfamily possesses different regulatory mechanisms in response to various abiotic stresses. Additionally, overexpression of RcsHSP genes in A. thaliana was followed by enhanced SOD activity and higher content of osmoprotectants, which ultimately led to enhanced seed germination under a variety of abiotic stresses. Our results may contribute to breeding programs aiming at developing high tolerant R. communis plants, providing economic and social support for farmers in semiarid areas worldwide.

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeoleiVT8.

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.

Long-Chain Hydrocarbons (C21, C24, and C31) Released by Bacillus sp. MH778713 Break Dormancy of Mesquite Seeds Subjected to Chromium Stress

Volatile organic compounds (VOCs) produced by rhizobacteria have been proven to stimulate plant growth during germination and seedling stages. However, the modulating effect of bacterial volatiles on the germination of seeds subjected to heavy metal stress is scarcely studied. In this work, the ability of volatiles released by Bacillus sp. MH778713 to induce seed dormancy breakage in Prosopis laevigata and Arabidopsis thaliana seeds were examined. The minimal inhibitory concentration of chromium (Cr) VI that prevents seed germination of P. laevigata and A. thaliana on water-Cr-agar plates was 2500 and 100 mg L–1, respectively. Remarkably, partitioned Petri-dish co-cultivation of Bacillus sp. MH778713 and plant seeds under Cr-stress showed the beneficial effect of volatiles emitted by Bacillus sp. MH778713, helping plant seeds to overcome Cr-stress. Among the metabolites emitted by Bacillus sp. MH778713, octadecane, heneicosane, 2,4-di-tert-butylphenol, hexadecane, eicosane, octacosane, and tetratriacontane were the most abundant. To confirm that these long-chain compounds produced by Bacillus sp. MH778713 could be responsible for the seed dormancy breakage, high pure organic compounds (2,4-di-tert-butylphenol, heneicosane, hentriacontane, and tetracosane) were used directly in germination assays of P. laevigata and A. thaliana seeds instead of volatiles emitted by Bacillus sp. MH778713. All organic compounds allowed Prosopis and Arabidopsis seeds to overcome Cr-toxicity and germinate. The results of this study provide new insight into the role of long-chain bacterial compounds produced by Bacillus sp. MH778713 as triggers of seed abiotic stress tolerance, surmounting chromium stress and stimulating seedling development.

A simple, non-toxic method for separating seeds based on density, and its application in isolating Arabidopsis thaliana seed oil mutants.

PremiseSeed oil is an economically important trait in Brassica oilseed crops. A novel method was developed to isolate Arabidopsis thaliana seeds with altered oil content.Methods and ResultsIn A. thaliana, seed oil content is correlated with seed density, with high‐oil seeds being less dense than wild type and tending to float in solution, and low‐oil seeds being denser and tending to sink. In contrast to previous methods, which used toxic chemicals and density gradient centrifugation, different concentrations of calcium chloride (CaCl2) were employed to separate seeds without the need for centrifugation. The method was validated using known seed oil mutants, and 120,822 T‐DNA mutagenized A. thaliana lines were then screened for novel seed density phenotypes.ConclusionsA number of candidate mutants, as well as new alleles of two genes known to influence seed oil biosynthesis, were successfully isolated.

Impact of sunflower (Helianthus annuus L.) plastidial lipoyl synthases genes expression in glycerolipids composition of transgenic Arabidopsis plants

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM.

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

Glucosinolate Content in Dormant and Germinating Arabidopsis thaliana Seeds Is Affected by Non-Functional Alleles of Classical Myrosinase and Nitrile-Specifier Protein Genes.

While the defensive function of glucosinolates is well established, their possible role as a nutrient reservoir is poorly understood and glucosinolate turnover pathways have not been elucidated. Previous research showed that glucosinolate content in germinating seeds of Arabidopsis thaliana Columbia-0 (Col-0) increases within the first two to four days on culture medium and then decreases below the level at day 0. In this study we used previously characterized T-DNA mutants to investigate if enzymes known to be involved in glucosinolate breakdown upon tissue damage affect the time course of glucosinolate content in germinating seeds. Besides dormant seeds, we analyzed seeds subjected to stratification in water for up to 72 h or germination on plates for up to ten days. Although seeds of tgg1 tgg2 (deficient in above-ground classical myrosinases) had higher glucosinolate levels than Col-0, the changes during germination were not different to those in seeds of Col-0. This demonstrates that TGG1/TGG2 are not responsible for the decline in glucosinolate content upon germination and suggests the involvement of other enzymes. Expression data extracted from publically available databases show a number of β-glucosidases of the BGLU18–BGLU33 clade to be expressed at specific time points of seed maturation and germination identifying them as good candidates for a role in glucosinolate turnover. Although nitrile-specifier proteins (NSPs) act downstream of myrosinases upon glucosinolate breakdown in tissue homogenates, mutants deficient in either seed-expressed NSP2 or seedling-expressed NSP1 were affected in glucosinolate content in seeds and during stratification or germination when compared to Col-0 indicating a direct role in turnover. The mutant lines nsp1-1, nsp2-1 and nsp2-2 had significantly higher glucosinolate levels in dry seeds than Col-0. After 24 h of stratification in water, nsp2-2 seeds contained 2.3 fold higher levels of glucosinolate than Col-0 seeds. This might indicate downregulation of hydrolytic enzymes when nitrile formation following glucosinolate hydrolysis is impaired. The time course of total glucosinolate content during ten days of germination depended on functional NSP1. Based on the present data, we propose a number of experiments that might aid in establishing the pathway(s) of glucosinolate turnover in germinating A. thaliana seeds.

Arabidopsis thaliana SEED DORMANCY 4-LIKE regulates dormancy and germination by mediating the gibberellin pathway.

The molecular mechanisms underlying seed dormancy and germination are not fully understood. Here, we show that Arabidopsis thaliana SEED DORMANCY 4-LIKE (AtSdr4L) is a novel specific regulator of dormancy and germination. AtSdr4L encodes a protein with an unknown biochemical function that is localized in the nucleus and is expressed specifically in seeds. Loss of function of AtSdr4L results in increased seed dormancy. The germination of freshly harvested seeds of the Atsdr4l mutant is insensitive to gibberellin (GA). After-ripened mutant seeds are hypersensitive to the GA biosynthesis-inhibitor paclobutrazol but show unaltered sensitivity to abscisic acid. Several GA biosynthesis genes and GA-regulated cell wall remodeling genes are down-regulated in the mutant in both dormant and after-ripened seeds. These results suggest that the Atsdr4l mutation causes both decreased GA biosynthesis and reduced responses. In addition, a genetic analysis indicated that AtSdr4L is epistatic to DELAY OF GERMINATION1 (DOG1) for dormancy and acts upstream of RGA-LIKE 2 (RGL2) in the GA pathway. We propose that AtSdr4L regulates seed dormancy and germination by mediating both the DOG1 and GA pathways.

Natural Variation Reveals a Key Role for Rhamnogalacturonan I in Seed Outer Mucilage and Underlying Genes.

On imbibition, Arabidopsis (Arabidopsis thaliana) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis.

Lipidomic Analysis of Plastidial Octanoyltransferase Mutants of Arabidopsis thaliana.

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2, and NADH. For the proper functioning of this complex, lipoic acid is required to be bound to the dihydrolipoamide S-acetyltransferase E2 subunit of PDH. Octanoyltransferase (LIP2; EC 2.3.1.181) and lipoyl synthase (LIP1; EC 2.8.1.8) are the enzymes involved in the biosynthesis of this essential cofactor. In Arabidopsis plastids, an essential lipoyl synthase (AtLIP1p) and two redundant octanoyltransferases (AtLIP2p1 and AtLIP2p2) have been described. In the present study, the lipidomic characterization of Arabidopsis octanoyltransferase mutants reveals new insight into the lipoylation functions within plastid metabolism. Lipids and fatty acids from mature seeds and seedlings from Atlip2p1 and Atlip2p2 mutants were analysed by gas chromatography (GC) and liquid chromatography–electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS2), the analysis revealed changes in fatty acid profiles that showed similar patterns in both mutant seeds and seedlings and in the lipid species containing those fatty acids. Although both mutants showed similar tendencies, the lack of the AtLIP2p2 isoform produced a more acute variation in its lipids profile. These changes in fatty acid composition and the increase in their content per seed point to the interference of octanoyltransferases in the fatty acid synthesis flux in Arabidopsis thaliana seeds.

Alternative Splicing Regulation During Light-Induced Germination of Arabidopsis thaliana Seeds.

Seed dormancy and germination are relevant processes for a successful seedling establishment in the field. Light is one of the most important environmental factors involved in the relief of dormancy to promote seed germination. In Arabidopsis thaliana seeds, phytochrome photoreceptors tightly regulate gene expression at different levels. The contribution of alternative splicing (AS) regulation in the photocontrol of seed germination is still unknown. The aim of this work is to study gene expression modulated by light during germination of A. thaliana seeds, with focus on AS changes. Hence, we evaluated transcriptome-wide changes in stratified seeds irradiated with a pulse of red (Rp) or far-red (FRp) by RNA sequencing (RNA-seq). Our results show that the Rp changes the expression of ∼20% of the transcriptome and modifies the AS pattern of 226 genes associated with mRNA processing, RNA splicing, and mRNA metabolic processes. We further confirmed these effects for some of the affected AS events. Interestingly, the reverse transcriptase–polymerase chain reaction (RT–PCR) analyses show that the Rp modulates the AS of splicing-related factors (At-SR30, At-RS31a, At-RS31, and At-U2AF65A), a light-signaling component (At-PIF6), and a dormancy-related gene (At-DRM1). Furthermore, while the phytochrome B (phyB) is responsible for the AS pattern changes of At-U2AF65A and At-PIF6, the regulation of the other AS events is independent of this photoreceptor. We conclude that (i) Rp triggers AS changes in some splicing factors, light-signaling components, and dormancy/germination regulators; (ii) phyB modulates only some of these AS events; and (iii) AS events are regulated by R and FR light, but this regulation is not directly associated with the intensity of germination response. These data will help in boosting research in the splicing field and our understanding about the role of this mechanism during the photocontrol of seed germination.