Arabidopsis Seeds Research Articles

EgmiR5179 from the mesocarp of oil palm (Elaeis guineensis Jacq.) regulates oil accumulation by targeting NAD transporter 1

Oil palm (Elaeis guineensis Jacq.) is characterized by the accumulation of palm oil in the fruit mesocarp. In this paper, conserved microRNA, named EgmiR5179, excavated from a previous deep sequencing data, was observed to vary considerably during the maturation process of the oil palm mesocarp. A genome-wide screening for predicting target genes of EgmiR5179 was performed, and the function was verified by genetic transformation and metabolic analysis. Ectopic over-expression of EgmiR5179 in Arabidopsis indicated that EgmiR5179 positively regulated oil and linolenic acid accumulation in transgenic plant seeds. Further studies revealed that the NAD transporter 1 (NDT1) gene, which encodes a transporter of NAD+ partly modulated the NAD+ and NADH content by importing NAD+ into chloroplasts from the cytosol, was the downstream target of EgmiR5179 in oil palm and Arabidopsis. Therefore, EgmiR5179 regulates metabolite biosynthesis by down-regulating its target gene NDT1, leading to a reduction of NAD+ and NADH content, and the ratio of NAD+/NADH. The reduction of NAD+ content and NAD+/NADH ratio enhanced the triglyceride assembly and resulted in increases in oil content and decreases in starch content in seeds of transgenic Arabidopsis. The functions and effects of EgmiR5179 on the regulation of metabolism in oil palm mesocarp were demonstrated and their potential applications to oil palm metabolism engineering were discussed.

Family quarrels in seeds and rapid adaptive evolution in Arabidopsis

Evolutionary conflict can drive rapid adaptive evolution, sometimes called an arms race, because each party needs to respond continually to the adaptations of the other. Evidence for such arms races can sometimes be seen in morphology, in behavior, or in the genes underlying sexual interactions of host-pathogen interactions, but is rarely predicted a priori. Kin selection theory predicts that conflicts of interest should usually be reduced but not eliminated among genetic relatives, but there is little evidence as to whether conflict within families can drive rapid adaptation. Here we test multiple predictions about how conflict over the amount of resources an offspring receives from its parent would drive rapid molecular evolution in seed tissues of the flowering plant Arabidopsis As predicted, there is more adaptive evolution in genes expressed in Arabidopsis seeds than in other specialized organs, more in endosperms and maternal tissues than in embryos, and more in the specific subtissues involved in nutrient transfer. In the absence of credible alternative hypotheses, these results suggest that kin selection and conflict are important in plants, that the conflict includes not just the mother and offspring but also the triploid endosperm, and that, despite the conflict-reducing role of kinship, family members can engage in slow but steady tortoise-like arms races.

Plant growth-promoting activity of beta-propeller protein YxaL secreted from Bacillus velezensis strain GH1-13.

YxaL is conserved within the Bacillus subtilis species complex associated with plants and soil. The mature YxaL protein contains a repeated beta-propeller domain, but the subcellular location and function of YxaL has not been determined. The gene encoding the mature YxaL protein was PCR amplified from genomic DNA of B. velezensis strain GH1-13 and used for recombinant protein production. A rabbit polyclonal antibody against the purified YxaL was generated and used for western blotting to determine the constitutive expression and secretion of YxaL. During normal culture growth of strain GH1-13, levels of the constitutively secreted YxaL were slowly rising to 100 μg L-1, and degraded with a half-life of 1.6 h in the culture medium. When the effects of YxaL on plant seed germination and seedling growth were examined, it was shown that seed treatment of Arabidopsis thaliana and rice (Oryza sativa L.) with purified YxaL at the optimal concentration of 1 mg L-1 was effective at improving the root growth of plants. Seedlings from the treated Arabidopsis seeds markedly increased transcription of a 1-aminocyclopropane-1-carboxylate synthetase marker gene (ACS11) but reduced expression of auxin- and abscisic acid-responsive marker genes (IAA1, GH3.3, and ABF4), especially when provided with exogenous auxin. Horticulture experiments showed that pepper (Capsicum annuum) seeds treated with 1 mg L-1 YxaL in a soaking solution increased shoot growth and improved tolerance to drought stress. We hypothesize that YxaL secreted from plant growth-promoting Bacillus cells has a significant impact on plant roots, with the potential to improve plant growth and stress tolerance.

Identification of cotton MOTHER OF FT AND TFL1 homologs, GhMFT1 and GhMFT2, involved in seed germination.

Plant phosphatidylethanolamine-binding protein (PEBP) is comprised of three clades: FLOWERING LOCUS T (FT), TERMINAL FLOWER1 (TFL1) and MOTHER OF FT AND TFL1 (MFT). FT/TFL1-like clades regulate identities of the determinate and indeterminate meristems, and ultimately affect flowering time and plant architecture. MFT is generally considered to be the ancestor of FT/TFL1, but its function is not well understood. Here, two MFT homoeologous gene pairs in Gossypium hirsutum, GhMFT1-A/D and GhMFT2-A/D, were identified by genome-wide identification of MFT-like genes. Detailed expression analysis revealed that GhMFT1 and GhMFT2 homoeologous genes were predominately expressed in ovules, and their expression increased remarkably during ovule development but decreased quickly during seed germination. Expressions of GhMFT1 and GhMFT2 homoeologous genes in germinating seeds were upregulated in response to abscisic acid (ABA), and their expressions also responded to gibberellin (GA). In addition, ectopic overexpression of GhMFT1 and GhMFT2 in Arabidopsis inhibited seed germination at the early stage. Gene transcription analysis showed that ABA metabolism genes ABA-INSENSITIVE3 (ABI3) and ABI5, GA signal transduction pathway genes REPRESSOR OF ga1-3 (RGA) and RGA-LIKE2 (RGL2) were all upregulated in the 35S:GhMFT1 and 35S:GhMFT2 transgenic Arabidopsis seeds. GhMFT1 and GhMFT2 localize in the cytoplasm and nucleus, and both interact with a cotton bZIP transcription factor GhFD, suggesting that both of GhMFT1, 2 have similar intracellular regulation mechanisms. Taken together, the results suggest that GhMFT1 and GhMFT2 may act redundantly and differentially in the regulation of seed germination.

Overexpression of two plastidial fatty acid desaturase genes from tree peony affects the ratio of α-linolenic acid to linoleic acid in Arabidopsis seeds

ABSTRACTTree peony (Paeonia section Moutan DC.) has recently been recognised as an important woody oil plant of high economic value, with its seeds containing high levels of α-linolenic acids (ALA, C18:3). Here, we identified two novel FA desaturase genes from Paeonia. rockii, designated PrFAD6 and PrFAD7. Sequence, structure, and phylogenetic analyses revealed that they share high similarity with ω-6 and ω-3 plastidial FA desaturases. qRT-PCR showed that PrFAD6 and PrFAD7 were expressed in all studied tissues, particularly high in stamen, pistil, seed and leaf. Heterologous expression of PrFAD6 in Arabidopsis resulted in a 2.3–2.9% reduction in the ratio of ALA to linoleic acid (LA, C18:2), whilst overexpression of PrFAD7 increased the ALA/LA ratio by 7.0–9.9%. Moreover, the transgenic Arabidopsis plants overexpressing PrFAD6 and PrFAD7 exhibited varied abundances of a few FA components, particularly increased C18:2 and C18:3 levels, respectively. These results support the biological role of PrFAD6 and PrFAD7 in the biosynthesis of ALA and LA in tree peony seeds. Our study is the first attempt to verify the functions of plastidial FA desaturases in tree peony and will facilitate future research and industrial utilisation of tree peony as an important oil plant.

Powerdress as the novel regulator enhances Arabidopsis seeds germination tolerance to high temperature stress by histone modification of SOM locus

Seeds germination or dormancy is strictly controlled by endogenous phytohormone signal and environment cues. High temperature (HT) suppresses seeds germination or triggers seeds dormancy but underlying mechanism by which HT mediates seeds germination thermoinhibition needs more investigating. SOM is reported as the critical factor negatively controls light-irradiation seeds germination by altering Abscisic acid (ABA) and gibberellin acid (GA) biosynthesis. Here we found that HT accelerates SOM expressing through ABA signal transduction component ABI3, both of abi3 and som mutants seeds show high germination rate under HT in contrast to wild type seeds. Using ABI3 as the bait, we identified the epigenetic factor Powerdress (PWR) as the ABI3 interaction protein. Genetic and physiological analysis showed that PWR negatively control the expressing of SOM, and overexpressing PWR enhanced, while pwr mutant reduced, seeds germination thermotolerance. Without HT stress, PWR accelerated the histone H3 deacetylation level and H2A.Z deposition at SOM locus, and thus suppressed ABI3-dependent SOM transcription for seeds germination, HT stress block PWR transcriptional level, thus attenuated the inhibition effect of PWR on SOM expressing, resulting into seeds germination thermoinhibition. Thus our finding propose a new function of PWR in controlling seeds germination under HT through histone acetylation modification and H2A.Z deposition.

Isolation and characterization of a novel seed-specific promoter from peanut (Arachis hypogaea L.).

Peanut, whose seeds are ideal bioreactors for the production of recombinant proteins and/or nutrient metabolites, is one of the most important crop species worldwide. As important molecular tools, seed-specific promoters (SSPs) can direct the expression of foreign proteins specifically in seeds to avoid constitutive expression that can damage plants. However, few SSPs have been identified from this species. In this study, we isolated a novel SSP (we named it AHSSP2) from peanut. Several cis-acting elements commonly found in SSPs, including 3 copies of RYREPEAT elements, were dispersed throughout the 1970-bp sequence of AHSSP2. The sequence was then substituted in place of the 35S promoter sequence in a pBI121 plasmid, which was subsequently transformed into Arabidopsis. Beta-glucuronidase (GUS) staining showed that AHSSP2 can drive GUS gene expression in the mature seeds of transgenic Arabidopsis, excluding within the testa. The cotyledons and hypocotyls of the germinating seeds of transgenic Arabidopsis seedlings also exhibited GUS activity, even after the seedlings became adult plants. No GUS activity was detected in nontransformed Arabidopsis at any stage. These results strongly suggested that AHSSP2 could drive the expression of foreign genes in a seed-specific manner. This study enriched SSP resources, and the results showed that AHSSP2 could be potentially utilized in peanut and other crop species to improve seed quality, such as modifications to seed oil content.

Dynamic hydrolase labelling as a marker for seed quality in Arabidopsis seeds.

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Arabidopsis thaliana Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality.

ScDREB10, an A-5c type of DREB Gene of the Desert Moss Syntrichia caninervis, Confers Osmotic and Salt Tolerances to Arabidopsis.

Drought and salinity are major factors limiting crop productivity worldwide. DREB (dehydration-responsive element-binding) transcription factors play important roles in plant stress response and have been identified in a wide variety of plants. Studies on DREB are focused on the A-1 (DREB1) and A-2 (DREB2) groups. Studies on A-5 group DREBs, which represent a large proportion of the DREB subfamily, is limited. In this study, we characterized and analyzed the stress tolerance function of ScDREB10, an A-5c type DREB gene from the desert moss Syntrichia caninervis. Transactivation assay in yeast showed that ScDREB10 had transactivation activity. Transient expression assay revealed that ScDREB10 was distributed both in the nucleus and cytosol of tobacco leaf epidermal cells. Overexpression of ScDREB10 significantly increased the germination percentage of Arabidopsis seeds under osmotic and salt stresses, and improved the osmotic and salt stress tolerances of Arabidopsis at the seedling stage and is associated with the expression of downstream stress-related genes and improved reactive oxygen species (ROS) scavenging ability. Our study provides insight into the molecular mechanism of stress tolerance of A-5 type DREB proteins, as well as providing a promising candidate gene for crop salt and drought stress breeding.

Expression of a novel PSK-encoding gene from soybean improves seed growth and yield in transgenic plants.

Expression of GmPSKγ1 , a novel PSK-encoding gene from soybean, increases seed size and yield in transgenic plants by promoting cell expansion. Phytosulfokine-α (PSK-α), a sulfated pentapeptide hormone with the sequence YIYTQ, plays important roles in many aspects of plant growth and development. In this study, we identified a pair of putative precursor genes in soybean, GmPSKγ1 and -2, encoding a PSK-like peptide: PSK-γ. Similar to PSK-α in amino acid composition, the sequence of PSK-γ is YVYTQ, and the tyrosines undergo sulfonylation. Treatment of Arabidopsis seedlings with synthetic sulfated PSK-γ significantly enhanced root elongation, indicating that PSK-γ might be a functional analog of PSK-α. Expression pattern analysis revealed that the two GmPSKγ genes, especially GmPSKγ1, are primarily expressed in developing soybean seeds. Heterologous expression of GmPSKγ1 under the control of a seed-specific promoter markedly increased seed size and weight in Arabidopsis, and this promoting effect of PSK-γ on seed growth was further confirmed in transgenic tobacco constitutively expressing GmPSKγ1. Cytological analysis of transgenic Arabidopsis seeds revealed that PSK-γ promotes seed growth by inducing embryo cell expansion. In addition, expression analysis of downstream candidate genes suggested that PSK-γ signaling might regulate cell wall loosening to promote cell expansion in Arabidopsis seeds. Overall, our results shed light on the mechanism by which PSK-γ promotes seed growth, paving the way for the use of this new peptide for biotechnological improvement of crop seed/grain size and yield.

Transgenic Arabidopsis plants expressing CsBCATs affect seed germination under abiotic stress conditions

We investigated the responses of transgenic Arabidopsis plants that express cucumber branched-chain aminotransferases (CsBCATs) under abiotic or biotic stresses. The expression of CsBCAT3 and CsBCAT7 was dramatically increased by various abiotic treatments. The germination of CsBCAT3- or CsBCAT7-expressing Arabidopsis seeds was increased under salt and dehydration treatments, which was correlated with the increased or decreased expression levels of the germination-responsive genes. Although salicylic acid and jasmonic acid increased CsBCAT2 or CsBCAT7 expression, transgenic Arabidopsis plants that express CsBCAT2 or CsBCAT7 did not show the disease resistance to bacteria or fungus. Our results indicate that the CsBCATs affect seed germination of plants under abiotic stresses.

Analysis of Monosaccharides from Arabidopsis Seed Mucilage and Whole Seeds Using HPAEC-PAD

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Phytochrome Regulation of Seed Germination.

Seed germination assays consist of counting the number of germinated seeds, defined as seeds in which the radicle has ruptured the endosperm and emerged from the seed coat. In Arabidopsis seed germination assays, Arabidopsis seeds are surface-sterilized, plated on agar plates containing test compounds, and incubated at specific temperatures under specific light conditions, after which the germinated seeds are counted, either with the naked eye or under a microscope. This chapter describes step-by-step protocols for Arabidopsis seed germination assays under phytochrome-dependent conditions.

Regulation of Seed Germination: The Involvement of Multiple Forces Exerted via Gibberellic Acid Signaling

Regulation of Seed Germination: The Involvement of Multiple Forces Exerted via Gibberellic Acid Signaling

Interaction of DGAT1 and PDAT1 to enhance TAG assembly in Arabidopsis

ABSTRACTSeeds contain a large quantity of oils, which are mainly constituted of triacylglycerol (TAG), a fundamental source of carbon and energy. TAG biosynthesis is catalyzed by a series of multiple enzymes. In particular, two key enzymes catalyzing the last acylation step for TAG production, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), are rate-limiting enzymes that determine TAG accumulation in Arabidopsis seeds. We recently showed that the two enzymes are transcriptionally coordinated by the R2R3-type MYB96 transcription factor to promote TAG assembly during seed maturation in Arabidopsis. Here, we further found that DGAT1 and PDAT1 physically associate, possibly to enhance the efficiency of TAG production. Overall, our findings suggest that TAG biosynthesis is intricately regulated at multiple levels, and these molecular strategies can potentially be used for metabolic engineering in plants.

Regulation of Root-Knot Nematode Behavior by Seed-Coat Mucilage-Derived Attractants

Seed exudates influence the behavior of soil organisms, but how this occurs remains unclear, particularly for multicellular animals. Here we show that compounds associated with Arabidopsis seed-coat mucilage regulate the behavior of soil-borne animals, specifically root-knot nematodes (RKNs). Infective RKN J2 larvae actively travel toward Arabidopsis seeds through chemotaxis. Analysis of Arabidopsis mucilage mutants demonstrated that the attraction of RKNs to Arabidopsis seeds requires the synthesis and extrusion of seed-coat mucilage. Extracted mucilage alone is not sufficient to attract RKNs, but seed-surface carbohydrates and proteins are required for this process. These findings suggest that the RKN chemoattractant is synthesized de novo upon mucilage extrusion but may be highly unstable. RKNs attracted by this mucilage-dependent mechanism can infect the emerging seedling. However, the attraction signal from seedling roots likely acts independently of the seed-coat signal and may mask the attraction to seed-coat mucilage after germination. Multiple RKN species are attracted by Arabidopsis seeds, suggesting that this mechanism is conserved in RKNs. These findings indicate that seed exudate can regulate the behavior of multicellular animals and highlight the potential roles of seed-coat mucilage in biotic interactions with soil microorganisms.

Cloning and molecular characterization of three lysophosphatidic acid acyltransferases expressed in flax seeds

In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.

Revisiting the Role of Ethylene and N-End Rule Pathway on Chilling-Induced Dormancy Release in Arabidopsis Seeds.

Dormant Arabidopsis (Arabidopsis thaliana) seeds do not germinate easily at temperatures higher than 10–15 °C. Using mutants affected in ethylene signaling (etr1, ein2 and ein4) and in the N-end-rule pathway of the proteolysis (prt6 and ate1-ate2) we have investigated the effects of cold and ethylene on dormancy alleviation. Ethylene (10–100 ppm) and 2–4 days chilling (4 °C) strongly stimulate the germination of wild type (Col-0) seeds at 25 °C. Two to four days of chilling promote the germination at 25 °C of all the mutants suggesting that release of dormancy by cold did not require ethylene and did not require the N-end-rule pathway. One mutant (etr1) that did not respond to ethylene did not respond to GA3 either. Mutants affected in the N-end rule (prt6 and ate1-ate2) did not respond to ethylene indicating that also this pathway is required for dormancy alleviation by ethylene; they germinated after chilling and in the presence of GA3. Cold can activate the ethylene signaling pathway since it induced an accumulation of ETR1, EINI4, and EIN2 transcripts, the expression of which was not affected by ethylene and GA3. Both cold followed by 10 h at 25 °C and ethylene downregulated the expression of PRT6, ATE1, ATE2, and of ABI5 involved in ABA signaling as compared to dormant seeds incubated at 25 °C. In opposite, the expression of RGA, GAI, and RGL2 encoding three DELLAs was induced at 4 °C but downregulated in the presence of ethylene.

Use of ionic liquid for X-ray micro-CT specimen preparation of imbibed seeds.

X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

Effects of fungal volatile organic compounds on Arabidopsis thaliana growth and gene expression

Abstract Many microorganisms produce volatile organic compounds (VOCs) with biological effects on plants. In this study, Arabidopsis seeds or 14-day-old vegetative plants were exposed to 0.5 μg/l of chemical standards of 26 VOCs previously identified from the biocontrol fungus Trichoderma. Seven compounds (1-decene, 2-heptylfuran, 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1- butanol, 2-heptanone, and 1-octen-3-ol) were further tested at the physiological concentration (10 ng/l) and 3-methyl-1-butanol, 1-decene, and 2-heptylfuran induced significant increases in fresh weight and total chlorophyll content. Plants exposed to 1-decene had the greatest increase in plant fresh shoot weight (38.9%) and chlorophyll content (67.8%). An RNA sequencing analysis was performed on plants treated with vapors of 1-decene. The expression of 123 genes was differentially affected, encompassing genes involved in cell wall modification, auxin induction, stress, and defense responses, with several major classes of stress-related genes showing down-regulation. To our knowledge, this is the first report of the effect of a plant growth promoting VOC on gene expression in Arabidopsis thaliana. As the role of fungal VOCs in biocontrol moves from correlative studies to more hypothesis driven approaches, our findings can guide both basic and applied studies in agricultural research.