The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat

The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9_pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.     

Occurrence of steryl glycosides and acylated steryl glycosides in some marine algae

There is some controversy concerning the presence of steryl glycosides and acylated steryl glycosides in eucaryotic algae. These two classes of sterol compounds were investigated in species belonging to the three major groups of eucaryotic algae: green algae (Ulva gigantea, Cladophora rupestris), brown algae (Fucus vesiculosus, Ascophyllum nodosum), and red algae (Rhodymenia palmata, Porphyridium sp.). All these algae contain both steryl glycosides and acylated steryl glycosides. The sterol components of these compounds vary according to the alga but they are always the same as the free sterols of the alga in question. The most common sugar moiety is glucose. In the acylated steryl glycosides, the fatty acid is mainly palmitic acid. The percentage of these compounds (as a percentage of the total sterol content) is often low.     

Shading Influence on the Sterol Balance of Nicotiana tabacum L

Tobacco plants (Nicotiana tabacum L.) were grown in the field and the apex was removed at the 42-day stage. Shading screens were set up which produced 0, 26, 67, and 90% shade. Plants were grown an additional 25 days before leaves from top, middle, and bottom stalk positions were harvested. Each leaf group was analyzed for free sterol, steryl ester, steryl glycoside, and acylsteryl glycoside. The free sterol content was lowest in top leaves and highest in bottom leaves; however, the top leaves had more steryl ester than the bottom leaves. Leaf position had no effect on steryl glycosides and acylsteryl glycosides. Shading did not influence the level of any sterol class; but in general, shading increased stigmasterol and decreased sitosterol. This trend was observed for all sterol classes, and the free sterols showed the largest and most consistent change. The younger top leaves showed a greater response than the older bottom leaves, but bottom leaves always had more stigmasterol than sitosterol even without shade.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L

In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-¹⁴C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [¹⁴C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [¹⁴C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [¹⁴C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.     

Phytosterols in tobacco leaves at various stages of physiological maturity

Leaves of varying maturity from 84-day-old tobacco plants were harvested and analyzed for total sterol and their individual sterol components. The mature leaves had a significant higher sterol content than the immature leaves. Separation into free sterols, steryl esters, steryl glycosides, and acylated steryl glycosides showed that the free sterols accounted for most of the sterol increase, and stimgasterol was principally responsible for this increase.     

Atnoa1 mutant Arabidopsis plants induce compensation mechanisms to reduce the negative effects of the mutation

Alterations in temperature adaptation processes and changes in the content of stress-related compounds, polyamines and salicylic acid were evaluated in Atnoa1 (NO-associated 1) Arabidopsis mutant. The F_v/F_m chlorophyll-a fluorescence induction parameter and the actual quantum yield were significantly lower in the Atnoa1 mutant than in the wild-type. In the wild-type Col-0, the fastest increase in the non-photochemical quenching (NPQ) occurred in plants pre-treated at low temperature (4 °C), while the slowest was in those adapted to 30 °C. The NPQ showed not only a substantially increased level in the light-adapted state, but also more rapid light induction after the dark-adapted state in the Atnoa1 mutant than in the wild-type. The results of freezing tests indicated that both the wild-type and the mutant had better freezing tolerance after cold hardening, since no significant differences were found between the genotypes. The level of putrescine increased substantially, while that of spermine decreased by the end of the cold-hardening (4 °C, 4 d) period. The quantity of spermidine in Atnoa1 was significantly higher than in Col-0, at both control and cold-hardening temperatures. A similar trend was observed for spermine, but only under control conditions. The mutant plants showed substantially higher salicylic acid (SA) contents for both the free and bound forms. This difference was significant not only in the control, but also in the cold-hardened plants. These results suggest that there is a compensation mechanism in Atnoa1 mutant Arabidopsis plants to reduce the negative effects of the mutation. These adaptation processes include the stimulation of photoprotection and alterations in the SA and polyamine compositions.     

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.     

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.     

Mutations in UDP-Glucose:Sterol Glucosyltransferase in Arabidopsis Cause Transparent Testa Phenotype and Suberization Defect in Seeds

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis.Steryl glycosides (SGs) and acyl SGs (ASGs) are abundant constituents of the membranes of higher plants (Frasch and Grunwald, 1976; Warnecke and Heinz, 1994; Warnecke et al., 1997, 1999). SGs are synthesized by membrane-bound UDP-Glc:sterol glucosyltransferase (Hartmann-Bouillon and Benveniste, 1978; Ury et al., 1989; Warnecke et al., 1997), which catalyzes the glycosylation of the 3β-hydroxy group of sterols to produce a 3-β-d-glycoside. UGT80A2 has been found in the plasma membrane, Golgi vesicles, the endoplasmic reticulum membrane, and occasionally the tonoplast (Hartmann-Bouillon and Benveniste, 1978; Yoshida and Uemura, 1986; Ullmann et al., 1987; Warnecke et al., 1997). It has also been reported that a UDP-Glc-dependent glucosylceramide synthase from cotton (Gossypium hirsutum) is capable of synthesizing SG in plants (Hillig et al., 2003). All plant sterols can be glycosylated, given that sterol substrates are pathway end products (Δ⁵-sterols in Arabidopsis [Arabidopsis thaliana]) and not intermediates. The most commonly observed glycoside is Glc (Warnecke et al., 1997) but Xyl (Iribarren and Pomilio, 1985), Gal, and Man have been observed (Grunwald, 1978). Although rare in occurrence, SGs with di-, tri-, and tetraglucoside residues have also been reported (Kojima et al., 1989). SGs can be acylated, polyhydroxylated, or sulfated, but ASGs with fatty acids esterified to the primary alcohol group of the carbohydrate unit are the most common modifications (Lepage, 1964).SGs have been found as abundant membrane components in many species of plants, mosses, bacteria, fungi, and in some species of animals (Esders and Light, 1972; Mayberry and Smith, 1983; Murakami-Murofushi et al., 1987; Haque et al., 1996), yet relatively little is known about their biological functions. Because of the importance of sterols in membrane fluidity and permeability (Warnecke and Heinz, 1994; Warnecke et al., 1999; Schaller, 2003) and the phospholipid dependence of UDP-Glc:sterol glucosyltransferase (Bouvier-Nave et al., 1984), it has been postulated that SGs may have a role in adaptation to temperature stress (Palta et al., 1993). A difference in the proportion of glycosylated versus acylated sterols were reported in two different solanaceous species under the same cold acclimation experiment (Palta et al., 1993). In one species an increase in SG was correlated with a decrease in ASG. In contrast, the other species displayed no change in SG and ASG levels with cold acclimation conditioning. Hence, evidence for a role in temperature adaptation is lacking.Understanding the processes involved in SG production has additional human importance because SGs are highly bioactive food components and laboratory mice fed SGs faithfully lead to either amyotrophic lateral sclerosis or parkinsonism pathologies (Ly et al., 2007). Similarly, consumption of seeds of the cycad palm (Cycas micronesica), containing high SG levels, has been linked to an unusual human neurological disorder, amyotrophic lateral sclerosis-parkinsonism dementia complex, in studies of the people of Guam (Cruz-Aguado and Shaw, 2009). However, SG is a dominant moiety of all plant membranes and some of the most widely consumed plant products in the United States such as soybeans (Glycine max) have concentrations well within the dose range obtained by consumption of cycad seeds. Cholesterol glycoside is the SG most commonly identified in animal membranes and is exemplified by cases in snake epidermis cells (Abraham et al., 1987) and human fibroblast cells under heat shock (Kunimoto et al., 2000). Regarding a role for SGs in the membrane, in comparison to normal sterols, SG and ASG exchange more slowly between the monolayer halves of a bilayer, which could serve to regulate free sterol (FS) content and its distribution (Ullmann et al., 1987; Warnecke et al., 1999).A study of cellulose synthesis in herbicide-treated cotton fibers found that sitosterol β-glucoside (SSG) copurified with cellulose fragments (Peng et al., 2002), leading to speculation that SGs act as a primer for cellulose biosynthesis in higher plants (Peng et al., 2002). In support of the hypothesis, SSG biosynthesis was reported to be pharmacologically inhibited by the known cellulose biosynthesis inhibitor 2-6-dichlorobenzonitrile (DCB; Peng et al., 2002). However, in subsequent studies, DCB inhibition of cellulose synthesis was not reversed by the exogenous addition of SSG, and the effects of DCB on cellulose synthesis were so rapid that the turnover of SGs would need to be very fast to account for the effects of DCB on cellulose synthesis (DeBolt et al., 2007). Schrick et al. (2004) reported that sterol biosynthesis mutants fackel, hydra1, and sterol methyltransferase1/cephalopod have reduced levels of cellulose but a specific effect on SGs was not established. Hence, a role for SGs in plant growth and development remains speculative.Here we describe a genetic analysis of the biological roles of two isoforms of UDP-Glc:sterol glucosyltransferase, UGT80A2 and USGT80B1, that participate in the synthesis of SG in Arabidopsis. UDP-Glc-dependent glucosylceramide synthase may also be capable of synthesizing SG in plants (Hillig et al., 2003), but no analysis was performed herein. We show that mutations in one of these genes, UGT80B1, results in a lack of flavanoid accumulation in the seed coat and that it corresponds to transparent testa15 (tt15). Analysis of ugt80A2, ugt80B1, and a double mutant suggests that glycosylation of sterols by the UGT80A2 and UGT80B1 enzymes had no measurable consequence on cellulose levels in Arabidopsis seed, siliques, flowers, stems, trichomes, and leaves. Rather, we demonstrate that mutation of UGT80B1 principally alters embryonic development and seed suberin accumulation and cutin formation in the seed coat, leading to abnormal permeability and tetrazolium salt uptake.     

Conjugated and free sterols from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors and their effect on lipid metabolism and cholesterol transport in rat primary hepatocytes

Phytosterols have been widely studied for their cholesterol-lowering effect. Conjugated phytosterol forms have been found more active than free moieties. There are no reports about the sterol profile of black bean seed coats neither its effects on cholesterol metabolism. The aim of this research was to identify and quantify phytosterols from black bean seed coats and to determine their effects on cholesterol micellar solubility and on mRNA and key protein levels involved in lipid/cholesterol metabolism and cholesterol transport in primary rat hepatocytes. Free phytosterols, acylated steryl glycosides, and steryl glycosides were extracted from black bean seed coats. They were identified through HPLC–MS–TOF and quantified through HPLC equipped with UV–visible and evaporative light-scattering detectors. Free and conjugated phytosterols from the coats significantly increased the inhibitory effect of cholesterol micelle formation compared with stigmasterol, which was used as control (P < 0.05). In addition, phytosterols of black bean seed coat decreased lipogenesis by the downregulation of lipogenic proteins such as sterol regulatory element-binding protein 1 and fatty acid synthesis (FAS) in primary rat hepatocytes. Regarding β-oxidation, phytosterols upregulated the expression of carnitine palmitoyltransferase I and promoted the β-oxidation of long-chain fatty acids. Phytosterols inhibited cholesterol micellar solubility and reduced the activation of the liver X receptor, decreasing hepatic FAS and promoting hepatic β-oxidation of long-chain fatty acids.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

Exploiting Natural Variation of Secondary Metabolism Identifies a Gene Controlling the Glycosylation Diversity of Dihydroxybenzoic Acids in Arabidopsis thaliana

Plant secondary metabolism is an active research area because of the unique and important roles the specialized metabolites have in the interaction of plants with their biotic and abiotic environment, the diversity and complexity of the compounds and their importance to human medicine. Thousands of natural accessions of Arabidopsis thaliana characterized with increasing genomic precision are available, providing new opportunities to explore the biochemical and genetic mechanisms affecting variation in secondary metabolism within this model species. In this study, we focused on four aromatic metabolites that were differentially accumulated among 96 Arabidopsis natural accessions as revealed by leaf metabolic profiling. Using UV, mass spectrometry, and NMR data, we identified these four compounds as different dihydroxybenzoic acid (DHBA) glycosides, namely 2,5-dihydroxybenzoic acid (gentisic acid) 5-O-β-D-glucoside, 2,3-dihydroxybenzoic acid 3-O-β-D-glucoside, 2,5-dihydroxybenzoic acid 5-O-β-D-xyloside, and 2,3-dihydroxybenzoic acid 3-O-β-D-xyloside. Quantitative trait locus (QTL) mapping using recombinant inbred lines generated from C24 and Col-0 revealed a major-effect QTL controlling the relative proportion of xylosides vs. glucosides. Association mapping identified markers linked to a gene encoding a UDP glycosyltransferase gene. Analysis of Transfer DNA (T-DNA) knockout lines verified that this gene is required for DHBA xylosylation in planta and recombinant protein was able to xylosylate DHBA in vitro. This study demonstrates that exploiting natural variation of secondary metabolism is a powerful approach for gene function discovery.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Genetic Architecture of Natural Variation of Telomere Length in Arabidopsis thaliana

Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.