Towards the genetic architecture of seed lipid biosynthesis and accumulation in Arabidopsis thaliana

We report the quantitative genetic analysis of seed oil quality and quantity in six Arabidopsis thaliana recombinant inbred populations, in which the parent accessions were from diverse geographical origins, and were selected on the basis of variation for seed oil content and lipid composition. Although most of the biochemical steps involved in lipid biosynthesis are known and the key genes have been identified, the regulation of the processes that results in the final oil composition and total amount is not understood. By using physically anchored markers it was possible to compare results across populations. A total of 219 quantitative trait loci (QTLs) were identified, of which 81 were significant at P<0.001. Some of these colocalise with QTLs identified previously, but many novel QTLs were also identified. The results highlight the importance of studying traits in multiple populations, which will lead to a better understanding of the contribution that natural variation makes to the genetic architecture of a phenotype.     

Age-dependent loss of seed viability is associated with increased lipid oxidation and hydrolysis

The accumulation of reactive oxygen species has been associated with a loss of seed viability. Therefore, we have investigated the germination ability of a range of seed stocks, including two wheat collections and one barley collection that had been dry-aged for 5–40 years. Metabolite profiling analysis revealed that the accumulation of glycerol was negatively correlated with the ability to germinate in all seed sets. Furthermore, lipid degradation products such as glycerol phosphates and galactose were accumulated in some seed sets. A quantitative analysis of nonoxidized and oxidized lipids was performed in the wheat seed set that showed the greatest variation in germination. This analysis revealed that the levels of fully acylated and nonoxidized storage lipids like triacylglycerols and structural lipids like phospho- and galactolipids were decreasing. Moreover, the abundance of oxidized variants and hydrolysed products such as mono-/diacylglycerols, lysophospholipids, and fatty acids accumulated as viability decreased. The proportional formation of oxidized and nonoxidized fatty acids provides evidence for an enzymatic hydrolysis of specifically oxidized lipids in dry seeds. The results link reactive oxygen species with lipid oxidation, structural damage, and death in long-term aged seeds.     

华山松籽油中多不饱和脂肪酸的分离研究

华山松籽油中富含不饱和脂肪酸,尤其是亚油酸。采用尿素包合法对华山松籽油中的多不饱和脂肪酸进行分离。一次尿素包合后气相色谱(GC)分析华山松籽油亚油酸从63％增加到93％,油酸由26％减少到6％,不饱和脂肪总量提高到99．9％。尿素包合法能有效地分离华山松籽油中的多不饱和脂肪酸,其较理想的包合条件为：乙醇作溶剂,脂肪酸、尿素的比为1;1．5,包合温度为5℃。     

The effects of graded levels of concentrate supplementation on colour and lipid stability of beef from pasture finished late-maturing bulls

Finishing late-maturing bulls on grass may alter the antioxidant/prooxidant balance leading to beef with higher susceptibility to lipid oxidation and a lower colour stability compared to bulls finished on cereal concentrates. In this context, lipid oxidation and colour stability of beef from late-maturing bulls finished on pasture, with or without concentrate supplements, or indoors on concentrate was assessed. Charolais or Limousin sired bulls (n = 48) were assigned to four production systems: (1) pasture only (P), (2) pasture plus 25% dietary DM intake as barley-based concentrate (PC25), (3) pasture plus 50% dietary DM intake as barley-based concentrate (PC50) or (4) a barley-based concentrate ration (C). Following slaughter and postmortem ageing, M. Longissimus thoracis et lumborum was subjected to simulated retail display (4°C, 1000 lux for 12 h out of 24 h) for 3, 7, 10 and 14 days in modified atmosphere packs (O₂ : CO₂; 80 : 20). Lipid oxidation was determined using the 2-thiobarbituric acid-reactive substances assay; α-tocopherol was determined by HPLC; fatty acid methyl esters were determined using Gas Chromatography. Using a randomised complete block design, treatment means were compared by either ANOVA or repeated measures ANOVA using the MIXED procedure of SAS. Total polyunsaturated fatty acid (PUFA) concentrations were not affected by treatment, n-3 PUFAs were higher (P < 0.001) and the ratio of n-6 to n-3 PUFAs was lower (P < 0.001) in muscle from P, PC25 and PC50 compared to C. α-Tocopherol concentration was higher in muscle from P compared to PC50 and C bulls (P = 0.001) and decreased (P < 0.001) in all samples by day 14. Lipid oxidation was higher in muscle from C compared to P bulls on day 10 and day 14 of storage (P < 0.01). Finishing on pasture without supplementation did not affect beef colour stability and led to lower lipid oxidation, possibly due to the higher α-tocopherol concentration compared to concentrate finished beef.     

Arabidopsis thaliana seed germination and early seedling growth are inhibited by monoethanolamine salts of para-halogenated benzoic acids

Abstract

In spite of the simplicity of its molecules, the complex effects of benzoic acids on the regulation of plant growth are an increasingly attractive field of research to chemists and biologists. Halide substituted benzoic acids offer an excellent opportunity to explore the effect of electron withdrawing substituents (fluoro-, chloro-, bromo- and iodo-) on the response of plant growth stage. Under normal physiological conditions, benzoic acids are ionized molecules that exhibit low solubility in water. Monoethanolamine, a natural alkanolamine, was used to generate salts of monoethanolamine of halogenated para-substituted benzoic acids, new compounds with biological activity. This study reports on the biological effects of these substances at different concentrations on Arabidopsis thaliana seed germination and early seedling growth. Seed germination at 22°C, in a vertical position, under a photoperiod of 16 h light and 8 h darkness, was variable depending on the concentration of the compounds applied. Final germination percentages were similar for all treatments and control at 0.05 mM and 0.1 mM (exception p-Br BA and p-I MEASPBA). No germination occurred when seeds were treated with more than 0.5 mM. The results also revealed that the primary root length and the number of secondary roots are reduced in a concentration-dependent manner and also in relation to increasing atomic size of the substituents (F < Cl < Br < I). It is concluded that uptake rates of benzoic acid anions by roots decrease with a decrease in hydrophilic character of the anion and with an increase in molecular size.     

Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

Different effects of phospholipase Dζ2 and non‐specific phospholipase C4 on lipid remodeling and root hair growth in Arabidopsis response to phosphate deficiency

Phosphate (Pi) deficiency in soils is a major limiting factor for plant growth. In response to Pi deprivation, one prominent metabolic adaptation in plants is the decrease in membrane phospholipids that consume approximately one‐third cellular Pi. The level of two phospholipid‐hydrolyzing enzymes, phospholipase Dζ2 (PLDζ2) and non‐specific phospholipase C4 (NPC4), is highly induced in Pi‐deprived Arabidopsis. To determine the role of PLDζ2 and NPC4 in plant growth under Pi limitation, Arabidopsis plants deficient in both PLDζ2 and NPC4 (npc4pldζ2) were generated and characterized. Lipid remodeling in leaves and roots was analyzed at three different durations of Pi deficiency. NPC4 affected lipid changes mainly in roots at an early stage of Pi deprivation, whereas PLDζ2 exhibited a more overt effect on lipid remodeling in leaves at a later stage of Pi deprivation. Pi deficiency‐induced galactolipid increase and phospholipid decrease were impeded in pldζ2 and npc4pldζ2 plants. In addition, seedlings of npc4pldζ2 had the same root hair density as pldζ2 but shorter root hair length than pldζ2 in response to Pi deficiency. The loss of NPC4 decreased root hair length but had no effect on root hair density. These data suggest that PLDζ2 and NPC4 mediate the Pi deprivation‐induced lipid remodeling in a tissue‐ and time‐specific manner. PLDζ2 and NPC4 have distinctively different roles in root hair growth and development in response to Pi deprivation; PLDζ2 negatively modulates root hair density and length, whereas NPC4 promotes root hair elongation.     

Emergence timing and fitness consequences of variation in seed oil composition in Arabidopsis thaliana

Early seedling emergence can increase plant fitness under competition. Seed oil composition (the types and relative amounts of fatty acids in the oils) may play an important role in determining emergence timing and early growth rate in oilseeds. Saturated fatty acids provide more energy per carbon atom than unsaturated fatty acids but have substantially higher melting points (when chain length is held constant). This characteristic forms the basis of an adaptive hypothesis that lower melting point seeds (lower proportion of saturated fatty acids) should be favored under colder germination temperatures due to earlier germination and faster growth before photosynthesis, while at warmer germination temperatures, seeds with a higher amount of energy (higher proportion of saturated fatty acids) should be favored. To assess the effects of seed oil melting point on timing of seedling emergence and fitness, high‐ and low‐melting point lines from a recombinant inbred cross of Arabidopsis thaliana were competed in a fully factorial experiment at warm and cold temperatures with two different density treatments. Emergence timing between these lines was not significantly different at either temperature, which aligned with warm temperature predictions, but not cold temperature predictions. Under all conditions, plants competing against high‐melting point lines had lower fitness relative to those against low‐melting point lines, which matched expectations for undifferentiated emergence times.     

Localization and regulation of phospholipase D2 by ARF6

Phospholipase D (PLD) and ADP-ribosylation factor 6 (ARF6) have been implicated in vesicular trafficking and rearrangement of the actin cytoskeleton. We have explored the co-localization of rat PLD1b and rat PLD2 with wild type and mutant forms of ARF6 in HeLa cells and studied their activation by ARF6 and the role of the actin cytoskeleton. GFP-tagged PLD1 had a similar pattern to multivesicular and late endosomes and the trans-Golgi apparatus, but not to other organelles. When wild type or dominant negative ARF6 and PLD1 or PLD2 were co-expressed, they had a similar localization in cytosolic particles and at the cell periphery. In contrast, dominant active ARF6 caused cell shrinkage and had a similar localization with PLD1 and PLD2 in dense structures, containing the trans-Golgi apparatus and actin. Disruption of the actin cytoskeleton with cytochalasin D did not induce the formation of these structures. To determine, if ARF6 selectively activated PLD1 or PLD2, wild type and mutant forms of the ARF isoform were transfected together with PLD1 or PLD2. Wild type ARF6 did not affect either PLD isozyme, but dominant active ARF6 selectively activated PLD2 and dominant negative ARF6 selectively inhibited PLD2. In contrast, dominant active ARF1 or Rac1 stimulated both PLD isozymes but the ARF1 effect on PLD2 was very small. Cytochalasin D did not affect the activation of PLD by phorbol ester. The localizations of PLD and ARF6 were also analyzed by fractionation after methyl-beta-cyclodextrin extraction to deplete cholesterol. The results showed that all PLD isoforms and ARF6 mutants existed in the membrane fraction, but only wild type ARF6 was dependent on the presence of cholesterol. These experiments showed that wild type ARF6 had a similar location with PLD isoforms on cell staining, but it did not colocalize with PLD isoforms in fractionation experiments. It is proposed that activated ARF6 translocates to the cholesterol independent microdomain and then activates PLD2 there. It is further concluded that PLD2 is selectively activated by ARF6 in vivo and that disruption of the actin cytoskeleton does not affect this activation.     

A comparative study of seed morphology in relation to desiccation tolerance and other physiological responses in 71 Eastern Australian rainforest species

Seed characteristics were measured in 71 Eastern Australian rainforest species representing 30 families. Sensitivity to desiccation to low moisture contents (< 10%) occurred in 42% of species. We estimate, based on findings from 100 species from this present study and previously published reports, that 49% of Eastern Australian rainforest species have non‐orthodox seeds. Germination level and time to 50% germination were not significantly different between desiccation sensitive (DS) and desiccation tolerant (DT) seeds. The estimation of seed desiccation sensitivity based on predictors is an important tool underpinning ex situ conservation efforts. Seed characteristics differed significantly between DS and DT seeds; that is, DS seeds had: (i) larger fruits (19 949 mg vs 8322 mg); (ii) larger seeds (1663 mg vs 202 mg); (iii) higher seed moisture contents (49.7% vs 35.5% fresh weight); (iv) lower oil content (7.3% vs 24.8% yield); and (v) less investment in seed coats (0.19 vs 0.48 seed coat ratio). Only 25% of DS seeded species had oily seeds compared with 87% of DT seeded species. Most green embryos were DS. Seed coat ratio was the best predictor of seed DS (80% correctly predicted). Seed moisture content at maturity was also related to germination time. Mean seed size was correlated (?0.657, P = 0.01) with mean seed oil content in 46 species. Further research on seed storage physiology of possible oily and/or DS seeded species is crucial to ensure future long‐term security of this biodiversity, particularly for species currently threatened in situ and/or of socioeconomic importance in Eastern Australian rainforests.     

Non‐specific phospholipase C5 and diacylglycerol promote lateral root development under mild salt stress in Arabidopsis

Developing a robust root system is crucial to plant survival and competition for soil resources. Here we report that the non‐specific phospholipase C5 (NPC5) and its derived lipid mediator diacylglycerol (DAG) mediate lateral root (LR) development during salt stress in Arabidopsis thaliana. T‐DNA knockout mutant npc5‐1 produced few to no LR under mild NaCl stress, whereas overexpression of NPC5 increased LR number. Roots of npc5‐1 contained a lower level of DAG than wild type, whereas NPC5 overexpressor exhibited an increase in DAG level. Application of DAG, but not phosphatidic acid, fully restored LR growth of npc5‐1 to that of wild type under NaCl stress. NPC5 expression was significantly induced in Arabidopsis seedlings treated with NaCl. Npc5‐1 was less responsive to auxin‐mediated root growth than the wild type. These results indicate that NPC5 mediates LR development in response to salt stress and suggest that DAG functions as a lipid mediator in the stress signalling.     

The diversity of algal phospholipase D homologs revealed by biocomputational analysis

Phospholipase D (PLD) participates in the formation of phosphatidic acid, a precursor in glycerolipid biosynthesis and a second messenger. PLDs are part of a superfamily of proteins that hydrolyze phosphodiesters and share a catalytic motif, HxKxxxxD, and hence a mechanism of action. Although HKD‐PLDs have been thoroughly characterized in plants, animals and bacteria, very little is known about these enzymes in algae. To fill this gap in knowledge, we performed a biocomputational analysis by means of HMMER iterative profiling, using most eukaryotic algae genomes available. Phylogenetic analysis revealed that algae exhibit very few eukaryotic‐type PLDs but possess, instead, many bacteria‐like PLDs. Among algae eukaryotic‐type PLDs, we identified C2‐PLDs and PXPH‐like PLDs. In addition, the dinoflagellate Alexandrium tamarense features several proteins phylogenetically related to oomycete PLDs. Our phylogenetic analysis also showed that algae bacteria‐like PLDs (proteins with putative PLD activity) fall into five clades, three of which are novel lineages in eukaryotes, composed almost entirely of algae. Specifically, Clade II is almost exclusive to diatoms, whereas Clade I and IV are mainly represented by proteins from prasinophytes. The other two clades are composed of mitochondrial PLDs (Clade V or Mito‐PLDs), previously found in mammals, and a subfamily of potentially secreted proteins (Clade III or SP‐PLDs), which includes a homolog formerly characterized in rice. In addition, our phylogenetic analysis shows that algae have non‐PLD members within the bacteria‐like HKD superfamily with putative cardiolipin synthase and phosphatidylserine/phosphatidylglycerophosphate synthase activities. Altogether, our results show that eukaryotic algae possess a moderate number of PLDs that belong to very diverse phylogenetic groups.     

Ensuring seed quality in ecological restoration: native seed cleaning and testing

Seeds are a critical and limited resource for restoring biodiversity and ecological function to degraded and fragmented ecosystems. Cleaning and quality testing are two key steps in the native seed supply chain. Optimizing the practices used in these steps can ensure seed quality. Post‐collection handling of seeds can have a profound impact on their viability, longevity in storage, and establishment potential. The first section of this article describes seed cleaning, outlines key considerations, and details traditional and novel approaches. Despite the growth of the native seed industry and the need for seed quality standards, existing equipment and standards largely target agricultural, horticultural, and commercial forestry species. Native plant species typically have complex seed traits, making it difficult to directly transfer existing cleaning and quality standards to these species. Furthermore, in ecological restoration projects, where diversity is valued over uniformity crop standards can be unsuitable. We provide an overview and recommendations for seed quality testing (sampling, purity, viability, germinability, vigor), identity reporting, and seed transfer as well as highlight the need to implement internationally recognized standards for certification for native seeds. Novel and improved cleaning and testing methods are needed for native species from a range of ecosystems to meet the challenges and goals of the United Nations Decade on Ecosystem Restoration. The guidelines outlined in this article along with others in the Special Issue of Restoration Ecology “Standards for Native Seeds in Ecological Restoration” can serve as a foundation for this critical work.     

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-Plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.     

地菍果实成熟过程中品质及种子油脂肪酸组分的变化

对地菍果实不同成熟期品质的研究表明,成熟时的地菍果实,维生素C、总糖含量达最高值,酸度则降至最低。此期风味最好,软硬适宜,色泽甚佳,为地菍鲜食的最佳采收期。对不同成熟期的种子油脂肪酸分析表明,种子油以不饱和脂肪酸为主,尤其是亚油酸含量很高,达84%;果实成熟过程中脂肪酸各级分的含量变化趋势不明显。