Network Analysis Identifies ELF3 as a QTL for the Shade Avoidance Response in Arabidopsis

Quantitative Trait Loci (QTL) analyses in immortal populations are a powerful method for exploring the genetic mechanisms that control interactions of organisms with their environment. However, QTL analyses frequently do not culminate in the identification of a causal gene due to the large chromosomal regions often underlying QTLs. A reasonable approach to inform the process of causal gene identification is to incorporate additional genome-wide information, which is becoming increasingly accessible. In this work, we perform QTL analysis of the shade avoidance response in the Bayreuth-0 (Bay-0, CS954) x Shahdara (Sha, CS929) recombinant inbred line population of Arabidopsis. We take advantage of the complex pleiotropic nature of this trait to perform network analysis using co-expression, eQTL and functional classification from publicly available datasets to help us find good candidate genes for our strongest QTL, SAR2. This novel network analysis detected EARLY FLOWERING 3 (ELF3; AT2G25930) as the most likely candidate gene affecting the shade avoidance response in our population. Further genetic and transgenic experiments confirmed ELF3 as the causative gene for SAR2. The Bay-0 and Sha alleles of ELF3 differentially regulate developmental time and circadian clock period length in Arabidopsis, and the extent of this regulation is dependent on the light environment. This is the first time that ELF3 has been implicated in the shade avoidance response and that different natural alleles of this gene are shown to have phenotypic effects. In summary, we show that development of networks to inform candidate gene identification for QTLs is a promising technique that can significantly accelerate the process of QTL cloning.     

Inhibition of saturated very-long-chain fatty acid biosynthesis by mefluidide and perfluidone,selective inhibitors of 3-ketoacyl-CoA synthases

The trifluoromethanesulphonanilides mefluidide and perfluidone are used in agriculture as plant growth regulators and herbicides. Despite the fact that mefluidide and perfluidone have been investigated experimentally for decades, their mode of action is still unknown. In this study, we used a cascade approach of different methods to clarify the mode of action and target site of mefluidide and perfluidone. Physiological profiling using an array of biotests and metabolic profiling in treated plants of Lemna paucicostata suggested a common mode of action in very-long-chain fatty acid (VLCFA) synthesis similar to the known 3-ketoacyl-CoA synthase (KCS) inhibitor metazachlor. Detailed analysis of fatty acid composition in Lemna plants showed a decrease of saturated VLCFAs after treatment with mefluidide and perfluidone. To study compound effects on enzyme level, recombinant KCSs from Arabidopsis thaliana were expressed in Saccharomyces cerevisiae. Enzyme activities of seven KCS proteins from 17 tested were characterized by their fatty acid substrate and product spectrum. For the KCS CER6, the VLCFA product spectrum in vivo, which consists of tetracosanoic acid, hexacosanoic acid and octacosanoic acid, is reported here for the first time. Similar to metazachlor, mefluidide and perfluidone were able to inhibit KCS1, CER6 and CER60 enzyme activities in vivo. FAE1 and KCS2 were inhibited by mefluidide only slightly, whereas metazachlor and perfluidone were strong inhibitors of these enzymes with IC₅₀ values in μM range. This suggests that KCS enzymes in VLCFA synthesis are the primary herbicide target of mefluidide and perfluidone.     

Quantitative trait loci controlling root growth and architecture in Arabidopsis thaliana confirmed by heterogeneous inbred family

Arabidopsis thaliana provides a scientifically attractive and simple model for studying root growth and architecture and, subsequently, for discovering new genes involved in the control of these characters in plants. We have used the natural variation available in Arabidopsis accessions and mapped quantitative trait loci (QTLs) for primary root length (PRL), lateral root number (LRN) and density (LRD) and for total length of the lateral root system (LRL) in the Bay-0 × Shahdara population. Total phenotypic variation was very large, and despite the importance of the environmental component we were able to map 13 QTLs and one epistatic interaction between QTLs. Our results highlight the biological relevance and genetic control of lateral root density in this material. We were also able to show that variation in the extent of the lateral root system depends mainly on the growth of the existing lateral roots rather than in a change in LRN. Factors controlling lateral root growth seemed to have no major effect on primary root growth. Moreover, Shahdara QTL alleles always increased the length of the lateral roots, which may be taken as an adaptation to its very dry natural environment in Tadjikistan. A QTL for PRL was confirmed using a type of near-isogenic line called a heterogeneous inbred family (HIF), and this QTL is a candidate for further fine-mapping and cloning.     

Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa

Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production.     

拟南芥低温黑暗发芽基因的鉴定及QTL定位

低温发芽是一个重要农艺性状,提高作物的耐寒性并确定控制低温发芽的基因是一个全球性的重大育种课题。通过对300多份拟南芥种子进行发芽试验,鉴定了低温黑暗下发芽的自然变异,并比较了两种生态型Bay-0和Shahdara低温黑暗下的发芽特性,这种自然变异被用来鉴定低温黑暗中发芽基因的遗传位点;利用这两个生态型杂交产生的重组近交系,结合分子标记和QTL方法,检测到种子在低温黑暗下发芽至少受6个QTL所控制,其中3个为主效位点,它们所解释的自然变异为61%,利用异源近亲代(HIF)验证了这3个主要QTL,为低温黑暗发芽基因的精细定位和基因克隆等研究奠定了基础。     

Bay-0 x Shahdara recombinant inbred line population: a powerful tool for the genetic dissection of complex traits in Arabidopsis

Natural genetic variation in Arabidopsis is considerable, but has not yet been used extensively as a source of variants to identify new genes of interest. From the cross between two genetically distant ecotypes, Bay-0 and Shahdara, we generated a Recombinant Inbred Line (RIL) population dedicated to Quantitative Trait Locus (QTL) mapping. A set of 38 physically anchored microsatellite markers was created to construct a robust genetic map from the 420 F6 lines. These markers, evenly distributed throughout the five chromosomes, revealed a remarkable equilibrium in the segregation of parental alleles in the genome. As a model character, we have analysed the genetic basis of variation in flowering time in two different environments. The simultaneous mapping of both large- and small-effect QTLs responsible for this variation explained 90% of the total genotypic variance. Two of the detected QTLs colocalize very precisely with FRIGIDA and FLOWERING LOCUS C genes; we provide information on the polymorphism of genes confirming this hypothesis. Another QTL maps in a region where no QTL had been found previously for this trait. This confirms the accuracy of QTL detection using the Bay-0 x Shahdara RIL population, which constitutes the largest in size available so far in Arabidopsis. As an alternative to mutant analysis, this population represents a powerful tool which is currently being used to undertake the genetic dissection of complex metabolic pathways.     

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis Epidermis Induces Cell Death and Suggests a Critical Role for Phospholipase A2 in This Process

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific β-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.     

Quantitative trait loci analysis of primary cell wall composition in Arabidopsis

Quantitative trait loci (QTL) analysis was used to identify genes underlying natural variation in primary cell wall composition in Arabidopsis (Arabidopsis thaliana). The cell walls of dark-grown seedlings of a Bay-0 x Shahdara recombinant inbred line population were analyzed using three miniaturized global cell wall fingerprinting techniques: monosaccharide composition analysis by gas chromatography, xyloglucan oligosaccharide mass profiling, and whole-wall Fourier-transform infrared microspectroscopy. Heritable variation and transgression were observed for the arabinose-rhamnose ratio, xyloglucan side-chain composition (including O-acetylation levels), and absorbance for a subset of Fourier-transform infrared wavenumbers. In total, 33 QTL, corresponding to at least 11 different loci controlling dark-grown hypocotyl length, pectin composition, and levels of xyloglucan fucosylation and O-acetylation, were identified. One major QTL, accounting for 51% of the variation in the arabinose-rhamnose ratio, affected the number of arabinan side chains presumably attached to the pectic polysaccharide rhamnogalacturonan I, paving the way to positional cloning of the first gene underlying natural variation in pectin structure. Several QTL were found to be colocalized, which may have implications for the regulation of xyloglucan metabolism. These results demonstrate the feasibility of combining fingerprinting techniques, natural variation, and quantitative genetics to gain original insight into the molecular mechanisms underlying the structure and metabolism of cell wall polysaccharides.     

Functional identification of ELO-like genes involved in very long chain fatty acid synthesis in Arabidopsis thaliana

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.     

Natural variation for carbohydrate content in Arabidopsis. Interaction with complex traits dissected by quantitative genetics

Besides being a metabolic fuel, carbohydrates play important roles in plant growth and development, in stress responses, and as signal molecules. We exploited natural variation in Arabidopsis (Arabidopsis thaliana) to decipher the genetic architecture determining carbohydrate content. A quantitative trait locus (QTL) approach in the Bay-0 x Shahdara progeny grown in two contrasting nitrogen environments led to the identification of 39 QTLs for starch, glucose, fructose, and sucrose contents representing at least 14 distinct polymorphic loci. A major QTL for fructose content (FR3.4) and a QTL for starch content (ST3.4) were confirmed in heterogeneous inbred families. Several genes associated with carbon (C) metabolism colocalize with the identified QTL. QTLs for senescence-related traits, and for flowering time, water status, and nitrogen-related traits, previously detected with the same genetic material, colocalize with C-related QTLs. These colocalizations reflect the complex interactions of C metabolism with other physiological processes. QTL fine-mapping and cloning could thus lead soon to the identification of genes potentially involved in the control of different connected physiological processes.     

Natural Variation at the FRD3 MATE Transporter Locus Reveals Cross-Talk between Fe Homeostasis and Zn Tolerance in Arabidopsis thaliana

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator.     

Quantitative trait loci analysis of water and anion contents in interaction with nitrogen availability in Arabidopsis thaliana

In plants, water and anion parameters are linked, for example through the integration of nutritional signaling and the response to diverse stress. In this work, Arabidopsis thaliana is used as a model system to dissect the genetic variation of these parameters by quantitative trait loci (QTL) mapping in the 415 recombinant inbred lines of the Bay-0 x Shahdara population. Water, nitrate, chloride, and phosphate contents were measured at the vegetative stage in the shoots of plants grown in controlled conditions. Two contrasting nitrogen (N) conditions were studied, one leading to the complete depletion of the nitrate pool in the plants. Most of the observed genetic variation was identified as QTL, with medium but also large phenotypic contributions. QTL colocalization provides a genetic basis for the correlation between water and nitrate contents in nonlimiting N conditions and water and chloride contents in limiting N conditions. The 34 new QTL described here represent at least 19 loci polymorphic between Bay-0 and Shahdara; some may correspond to known genes from water/anion transport systems, while others clearly identify new genes controlling or interacting with water/anion absorption and accumulation. Interestingly, flowering-time genes probably play a role in the regulation of water content in our conditions.     

Quantitative trait loci analysis of nitrogen use efficiency in Arabidopsis

Improving plant nitrogen (N) use efficiency or controlling soil N requires a better knowledge of the regulation of plant N metabolism. This could be achieved using Arabidopsis as a model genetic system, taking advantage of the natural variation available among ecotypes. Here, we describe an extensive study of N metabolism variation in the Bay-0 x Shahdara recombinant inbred line population, using quantitative trait locus (QTL) mapping. We mapped QTL for traits such as shoot growth, total N, nitrate, and free-amino acid contents, measured in two contrasting N environments (contrasting nitrate availability in the soil), in controlled conditions. Genetic variation and transgression were observed for all traits, and most of the genetic variation was identified through QTL and QTL x QTL epistatic interactions. The 48 significant QTL represent at least 18 loci that are polymorphic between parents; some may correspond to known genes from the N metabolic pathway, but others represent new genes controlling or interacting with N physiology. The correlations between traits are dissected through QTL colocalizations: The identification of the individual factors contributing to the regulation of different traits sheds new light on the relations among these characters. We also point out that the regulation of our traits is mostly specific to the N environment (N availability). Finally, we describe four interesting loci at which positional cloning is feasible.     

The VLCFA elongase gene family in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: phylogenetic analysis, 3D modelling and expression profiling

As precursors of wax compounds, very long chain fatty acids participate in the limitation of non-stomatal water loss and the prevention of pathogen attacks. They also serve as energy storage in seeds and as membrane building blocks. Their biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes (KCS, KCR, HCD and ECR). Twenty-one 3-ketoacyl-CoA synthase (KCS) genes have been identified in Arabidopsis thaliana genome. In this paper we present an overview of the acyl-CoA elongase genes in Arabidopsis focusing on the entire KCS family. We show that the KCS family is made up of 8 distinct subclasses, according to their phylogeny, duplication history, genomic organization, protein topology and 3D modelling. The analysis of the subcellular localization in tobacco cells of the different subunits of the acyl-CoA elongase shows that all these proteins are localized in the endoplasmic reticulum demonstrating that VLCFA production occurs in this compartment. The expression patterns in Arabidopsis of the acyl-CoA elongase genes suggest several levels of regulations at the tissular or organ level but also under stress conditions suggesting a complex organization of this multigenic family.     

Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis

In the absence of cell migration, plant architecture is largely determined by the direction and extent of cell expansion during development. In this report, we show that very-long-chain fatty acid (VLCFA) synthesis plays an essential role in cell expansion. The Arabidopsis thaliana eceriferum10 (cer10) mutants exhibit severe morphological abnormalities and reduced size of aerial organs. These mutants are disrupted in the At3g55360 gene, previously identified as a gene coding for enoyl-CoA reductase (ECR), an enzyme required for VLCFA synthesis. The absence of ECR activity results in a reduction of cuticular wax load and affects VLCFA composition of seed triacylglycerols and sphingolipids, demonstrating in planta that ECR is involved in all VLCFA elongation reactions in Arabidopsis. Epidermal and seed-specific silencing of ECR activity resulted in a reduction of cuticular wax load and the VLCFA content of seed triacylglycerols, respectively, with no effects on plant morphogenesis, suggesting that the developmental phenotypes arise from abnormal sphingolipid composition. Cellular analysis revealed aberrant endocytic membrane traffic and defective cell expansion underlying the morphological defects of cer10 mutants.     

AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana

Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long‐chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast‐2‐hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co‐overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild‐type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.     

Light-related loci controlling seed germination in Ler x Cvi and Bay-0 x Sha recombinant inbred-line populations of Arabidopsis thaliana

Background and Aims

Dormancy is a complex trait finely regulated by hormones and environmental factors. The phytochromes that sense red:far-red (R:FR) are the sole photoreceptors involved in the termination of dormancy and the induction of germination by light. The aims of this study were to identify and characterize loci controlling this process in seeds of Arabidopsis thaliana.

Methods

Recombinant inbred lines (RILs) derived from Landsberg erecta and Cape Verde Islands (Ler × Cvi), and Bayreuth and Shahdara (Bay-0 × Sha) were used to map loci related to light effects in seeds previously exposed to chilling and after-ripening periods.

Key Results

Substantial genetic variation was found between accessions of A. thaliana in the induction of germination by light. Twelve loci were identified under R, FR or darkness, some of which were novel loci: DOG8, DOG9, DOG13, DOG14 and DOG15 detected in the Ler × Cvi RIL population; and DOG10, DOG11 and DOG12 mapped in the Bay-0 × Sha RIL population. Furthermore, independent loci were mapped for the induction of germination by low fluence (DOG-LF1 and DOG-LF2) and very low fluence of light (DOG-VLF1) in the Ler × Cvi RIL population. Several loci were confirmed and characterized after different after-ripening and chilling treatments through near-isogenic lines (NILs) and heterogeneous inbred families (HIFs).

Conclusions

The results show that one group of loci act in a wide range of environmental scenarios, whereas a smaller group of loci are relevant only under a narrower set of conditions when the influence of the most-prevalent loci is reduced as a consequence of changes in the physiological status of the seeds. In addition, the identification of specific loci controlling the action modes of the phytochromes improves our understanding of the two independent signalling pathways that promote germination in response to light.Key words: Arabidopsis thaliana, dormancy, germination, phytochromes, very-low-fluence response (VLFR), low-fluence response (LFR), natural genetic variation, quantitative trait loci (QTL), recombinant inbred line (RIL), near-isogenic line (NIL) heterogeneous inbred family (HIF)