The nuclease FAN1 is involved in DNA crosslink repair in Arabidopsis thaliana independently of the nuclease MUS81

Fanconi anemia is a severe genetic disorder. Mutations in one of several genes lead to defects in DNA crosslink (CL) repair in human cells. An essential step in CL repair is the activation of the pathway by the monoubiquitination of the heterodimer FANCD2/FANCI, which recruits the nuclease FAN1 to the CL site. Surprisingly, FAN1 function is not conserved between different eukaryotes. No FAN1 homolog is present in Drosophila and Saccharomyces cerevisiae. The FAN1 homolog in Schizosaccharomyces pombe is involved in CL repair; a homolog is present in Xenopus but is not involved in CL repair. Here we show that a FAN1 homolog is present in plants and it is involved in CL repair in Arabidopsis thaliana. Both the virus-type replication-repair nuclease and the ubiquitin-binding ubiquitin-binding zinc finger domains are essential for this function. FAN1 likely acts upstream of two sub-pathways of CL repair. These pathways are defined by the Bloom syndrome homolog RECQ4A and the ATPase RAD5A, which is involved in error-free post-replicative repair. Mutations in both FAN1 and the endonuclease MUS81 resulted in greater sensitivity against CLs than in the respective single mutants. These results indicate that the two nucleases define two independent pathways of CL repair in plants.     

A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes

The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.     

Arabidopsis FAB1/PIKfyve Proteins Are Essential for Development of Viable Pollen

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P₂ is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P₂ in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P₂ production being central to cellular responses to changes in osmotic conditions.Phosphoinositides make up a minor fraction of total membrane lipids in all eukaryotic organisms. Their production is spatially restricted to the cytoplasmic leaflet of specific organellar membranes and temporally regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Three of the five hydoxyl groups of PtdIns can be phosphorylated, either singly or combinatorially, to produce seven different phosphoinositides. These different phosphoinositides can recruit and/or activate proteins with specific phosphoinositide-binding domains and have been implicated in the regulation of many important cellular functions, including membrane trafficking, cell growth, and cytoskeleton remodeling (Di Paolo and De Camilli, 2006).In animal cells, phosphorylation at the 3 position of PtdIns and its phosphorylated derivatives can be carried out by three different classes of PtdIns 3-kinase (classes I–III; Cantley, 2002). Plants and yeast only have class III PtdIns 3-kinases that are orthologs of the Saccharomyces cerevisiae protein Vps34p (Mueller-Roeber and Pical, 2002). Vps34p orthologs are thought to use PtdIns as their sole lipid substrate and produce PtdIns 3-phosphate (PtdIns3P). PtdIns3P is involved in endosomal/lysosomal protein sorting in eukaryotic cells in addition to cellular signaling events (Backer, 2008).In plants, PtdIns3P is essential for normal growth and development. Arabidopsis (Arabidopsis thaliana) plants carrying a VPS34 antisense construct have severe developmental defects (Welters et al., 1994). Furthermore, using pharmacological inhibitors of PtdIns3P production and analysis of transgenic plants defective in downstream signaling from PtdIns3P, it has been shown that this lipid has a role to play in many diverse physiological processes, such as root hair growth (Lee et al., 2008a). The phenotypes observed in studies of PtdIns3P function in plants are consistent with a role in endosomal and vacuolar trafficking in plants (Kim et al., 2001; Lee et al., 2008a), as in other eukaryotes. Recently, an attempt to generate vps34 homozygous mutant plant lines was unsuccessful due to failure of the mutant vps34 allele to transmit through the male germ line (Lee et al., 2008b).Importantly, PtdIns3P is the precursor to another phosphoinositide, PtdIns 3,5-bisphosphate [PtdIns(3,5)P₂], which also has vital roles in endosomal trafficking in eukaryotes (Dove et al., 2009). Thus, it is possible that some of the effects in plants attributed to PtdIns3P in previous studies may actually be due to an inability of cells to produce PtdIns(3,5)P₂. PtdIns(3,5)P₂ is produced by the PtdIns3P 5-kinases PIKfyve and Fab1p in animal and yeast cells, respectively. PIKfyve/Fab1p proteins have an N-terminal FYVE domain necessary for binding to PtdIns3P-containing membranes, a central Cpn60_TCP1 (for HSP chaperonin T complex 1) homology domain, and a C-terminal kinase domain. In Arabidopsis, there are a number of genes encoding putative Fab1p homologs, but only two of them, FAB1A (At4g33240) and FAB1B (At3g14270), encode proteins having FYVE domains at their N termini (Mueller-Roeber and Pical, 2002). It is likely that these proteins are PtdIns3P 5-kinases in Arabidopsis. Despite the importance of PtdIns(3,5)P₂ in yeast and animals, very little is known about its function in plants. However, it has been shown that hyperosmotic stress can induce the rapid synthesis of PtdIns(3,5)P₂ in cell suspension cultures from a number of plant species (Meijer and Munnik, 2003) and in pollen tubes from tobacco (Nicotiana tabacum; Zonia and Munnik, 2004). This production is consistent with a requirement for PtdIns(3,5)P₂ in vacuolar membrane reorganization, as water moves from the vacuole to the cytosol upon cells being placed under hyperosmotic stress. In animal cells, defective PtdIns(3,5)P₂ production leads to cytoplasmic vacuolation of endosome-derived membranes (Ikonomov et al., 2001; Jefferies et al., 2008). It seems that there is a general requirement in all eukaryotes for PtdIns(3,5)P₂ production in endomembrane remodeling. This remodeling could be mediated by proteins that bind to PtdIns(3,5)P₂. A number of candidates have been identified, including yeast Svp1p (Dove et al., 2004), its mammalian homolog WIP149 (Jeffries et al., 2004), CHMP3 (Whitley et al., 2003), and Ent3p (Friant et al., 2003).In this study, we aimed to further investigate the role of PtdIns(3,5)P₂ in plant physiology and the function of PIKfyve/Fab1p orthologs in Arabidopsis by generating mutant plant lines homozygous for T-DNA insertions in both FAB1A and FAB1B. We failed to generate double homozygous fab1a/fab1b knockout plants but observed subtle phenotypes in both fab1a and fab1b single homozygous plants. The data show that pollen with a fab1a/fab1b genotype has an abnormal vacuolar phenotype and does not contribute to the next generation. Our data are consistent with the hypothesis that the male gametophytic defect observed in vps34 mutant pollen (Lee et al., 2008b) is due to an inability of this pollen to generate PtdIns(3,5)P₂ and is not a direct result of the lack of PtdIns3P.     

Lipoate-Protein Ligase and Octanoyltransferase Are Essential for Protein Lipoylation in Mitochondria of Arabidopsis

Prosthetic lipoyl groups are required for the function of several essential multienzyme complexes, such as pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), and the glycine cleavage system (glycine decarboxylase [GDC]). How these proteins are lipoylated has been extensively studied in prokaryotes and yeast (Saccharomyces cerevisiae), but little is known for plants. We earlier reported that mitochondrial fatty acid synthesis by ketoacyl-acyl carrier protein synthase is not vital for protein lipoylation in Arabidopsis (Arabidopsis thaliana) and does not play a significant role in roots. Here, we identify Arabidopsis lipoate-protein ligase (AtLPLA) as an essential mitochondrial enzyme that uses octanoyl-nucleoside monophosphate and possibly other donor substrates for the octanoylation of mitochondrial PDH-E2 and GDC H-protein; it shows no reactivity with bacterial and possibly plant KGDH-E2. The octanoate-activating enzyme is unknown, but we assume that it uses octanoyl moieties provided by mitochondrial β-oxidation. AtLPLA is essential for the octanoylation of PDH-E2, whereas GDC H-protein can optionally also be octanoylated by octanoyltransferase (LIP2) using octanoyl chains provided by mitochondrial ketoacyl-acyl carrier protein synthase to meet the high lipoate requirement of leaf mesophyll mitochondria. Similar to protein lipoylation in yeast, LIP2 likely also transfers octanoyl groups attached to the H-protein to KGDH-E2 but not to PDH-E2, which is exclusively octanoylated by LPLA. We suggest that LPLA and LIP2 together provide a basal protein lipoylation network to plants that is similar to that in other eukaryotes.Lipoic acid (LA; 6,8-dithiooctanoic acid) prosthetic groups are essential for the catalytic activity of four important multienzyme complexes in plants and other organisms: pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), branched-chain α-ketoacid dehydrogenase (BCDH), and the Gly cleavage system (glycine decarboxylase [GDC]; Perham, 2000; Douce et al., 2001; Mooney et al., 2002). In all these multienzyme complexes, LA is covalently attached to the ε-amino group of a particular lysyl residue of the respective protein subunit. Lipoylated E2 subunits of PDH, KGDH, and BCDH are dihydrolipoyl acyltransferases that interact with E1 and E3 subunits to pass acyl intermediates to CoA (Mooney et al., 2002). By contrast, the lipoylated H-protein of GDC acts as a cosubstrate of three other GDC proteins and has no enzymatic activity itself (Douce et al., 2001). In the course of their respective reaction cycles, LA becomes reduced to dihydrolipoic acid. Most of these enzymes are confined to the mitochondrion. As the only exception, PDH is also present in plastids, where it provides acetyl-CoA for fatty acid biosynthesis (Ohlrogge et al., 1979; Lernmark and Gardeström, 1994; Lin et al., 2003).Mitochondria and plastids each have their own route of de novo LA synthesis, both of which start with the synthesis of protein-bound octanoyl chains (Shimakata and Stumpf, 1982; Ohlrogge and Browse, 1995; Wada et al., 1997; Gueguen et al., 2000; Yasuno et al., 2004). These octanoyl moieties are passed on by organelle-specific octanoyltransferases (Wada et al., 2001a, 2001b) to the respective target apoproteins where lipoyl synthase (LIP1) inserts two sulfur atoms to finally produce functional lipoyl groups (Yasuno and Wada, 1998, 2002; Zhao et al., 2003). A similar pathway has been identified in mammalian mitochondria (Morikawa et al., 2001; Witkowski et al., 2007). In quantitative terms, leaf mesophyll mitochondria have an extraordinarily high requirement for lipoate, because they contain very large amounts of GDC to catalyze the photorespiratory Gly-to-Ser conversion (Bauwe et al., 2010). For this reason, leaf mesophyll mitochondria are the major site of LA synthesis in plants (Wada et al., 1997).It was thought that the octanoyl chains provided by mitochondrial β-ketoacyl-acyl carrier protein synthase (mtKAS) represent the solitary source for protein lipoylation in plant mitochondria (Yasuno et al., 2004). As we reported earlier, however, leaves of mtKAS-deficient knockout mutants show considerable lipoylation of mitochondrial PDH-E2 and KGDH-E2 subunits and some residual lipoylation of GDC H-protein; roots are not at all impaired. Accordingly, the phenotype of such mutants can be fully cured in the low-photorespiratory condition of elevated CO₂ (Ewald et al., 2007). These observations indicated that plant mitochondria, in addition to the mtKAS-LIP2-LIP1 route of protein lipoylation, can resort to an alternative pathway. This would not be uncommon. In Escherichia coli, for example, a salvage pathway utilizes free octanoate or LA in an ATP-dependent two-step reaction catalyzed by the bifunctional enzyme lipoate-protein ligase A (LPLA; Morris et al., 1995). Archaea (Christensen and Cronan, 2009; Posner et al., 2009) and vertebrates (Tsunoda and Yasunobu, 1967) require two separate enzymes to first activate octanoate or LA to lipoyl-nucleoside monophosphate (NMP) and then, in a second step, to convey the activated lipoyl group to the respective target proteins. The lipoate-activating enzyme (LAE) of mammals was identified as a refunctioned medium-chain acyl-CoA synthetase that utilizes GTP to produce lipoyl-GMP (Fujiwara et al., 2001). LIP3 from yeast (Saccharomyces cerevisiae) can use octanoyl-CoA to octanoylate apoE2 proteins (Hermes and Cronan, 2013), whereas octanoyl groups from fatty acid biosynthesis are first attached to H-protein and then passed on to apoE2 proteins (Schonauer et al., 2009).The physiological significance of lipoyl-protein ligases in plants is not exactly known. Such enzymes do not operate in plastids (Ewald et al., 2014) but could be present in mitochondria. A single-gene-encoded LPLA with predicted mitochondrial localization has been identified in rice (Oryza sativa; Kang et al., 2007). Complementation studies with the lipoylation-deficient E. coli mutant TM137 (Morris et al., 1995) suggested that OsLPLA belongs to the bifunctional type of LPLAs. We report the identification of the homologous enzyme in Arabidopsis (Arabidopsis thaliana), provide evidence for its mitochondrial location, and show that Arabidopsis LPLA requires a separate enzyme for octanoate/lipoate activation. We also examine the interplay between LPLA, LIP2, and the mtKAS route of protein lipoylation and suggest a model for protein lipoylation in plant mitochondria.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

TEN1 Is Essential for CDC13-Mediated Telomere Capping

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.GENOME stability is critically dependent upon functional telomeres. DNA ends that lack telomeres, or that have dysfunctional telomeres, are metabolized by DNA repair processes; without an appropriate repair template, such chromosome ends can be resected or joined inappropriately with other chromosome ends. Thus, genomic integrity can be significantly compromised by telomere dysfunction, particularly in proliferating cells where cycles of instability may ensue due to creation of dicentric chromosomes (Bailey and Murnane 2006). Protein complexes that bind to the duplex and single-stranded telomere repeats are key for stabilizing the chromosome ends (de Lange 2005). In proliferating cells, this job is complicated not only because the terminal chromatin must be opened during the process of chromosome replication, but also because additional processes that metabolize DNA ends are active. For example, while nonhomologous end joining processes are preferentially used in repair of DNA double-strand breaks in G1, homologous recombination is preferentially used for this repair in S and G2 (Ferreira and Cooper 2004; Zierhut and Diffley 2008). Given these complexities, it is not surprising that our molecular understanding of how telomere proteins protect chromosomes ends is incomplete.Budding yeast has been useful for dissecting how cells correctly metabolize their chromosome ends. In Saccharomyces cerevisiae, the terminal DNA comprises approximately 300 bp of TG_1-3/C_1-3A sequences, ending with a short single-stranded overhang of the G-rich repeats. This 3′ overhang is ∼12–14 nucleotides, although during the late S/G2 phase of the cell cycle, it becomes longer, >30 nucleotides in length (Wellinger et al. 1993b; Dionne and Wellinger 1996; Larrivee et al. 2004). Central among factors that prevent inappropriate telomere degradation in S. cerevisiae is Cdc13, a protein that binds to single-stranded telomere G-rich repeats (Garvik et al. 1995; Lin and Zakian 1996; Nugent et al. 1996). Reducing Cdc13 function through either the cdc13-1 temperature sensitive (ts) allele or the cdc13-td conditional null (degron) allele results in telomere C-strand loss, with degradation continuing into the subtelomeric chromosomal regions (Garvik et al. 1995; Vodenicharov and Wellinger 2006). Correspondingly, homologous recombination at chromosome termini increases in cdc13-1 strains (Carson and Hartwell 1985; Garvik et al. 1995). The loss of Cdc13 unmasks the telomeres, provoking activation of the DNA damage checkpoint (Weinert and Hartwell 1993; Garvik et al. 1995). This protective role of Cdc13 is most likely its essential function.A thorough, mechanistic understanding of how Cdc13 mediates chromosome end protection is hampered in part because the activities responsible for the loss of the telomere C strand are not fully known. At normal telomeres, the Mre11-Rad50-Xrs2 complex has a role regulating resection required for telomere addition, whereas the Exo1 nuclease, Rad9 and Rad24 checkpoint proteins each influence the resection process at uncapped telomeres (Lydall and Weinert 1995; Maringele and Lydall 2002; Larrivee et al. 2004; Zubko et al. 2004). The 5′-to-3′ resection of both normal and uncapped telomeres is regulated by the activity of Cdk1, the yeast cyclin-dependent kinase (Frank et al. 2006; Vodenicharov and Wellinger 2006). Similar to the activities that promote 5′-to-3′ degradation of DNA ends at double-strand breaks (Aylon et al. 2004; Ira et al. 2004), the activities that lead to telomere resection are active in late S and G2 cell cycle phases (Wellinger et al. 1993a, 1996; Marcand et al. 2000; Vodenicharov and Wellinger 2006). Interestingly, Cdc13 is required to prevent degradation at telomeres only in proliferating cells and not when cells are blocked in stationary phase (Vodenicharov and Wellinger 2006). Additional factors, such as the S. cerevisiae Rap1 protein, prevent chromosome fusions by nonhomologous recombination during the G1 phase of the cell cycle (Pardo and Marcand 2005; Marcand et al. 2008).At least two additional proteins, Stn1 and Ten1, aid the capping role of Cdc13. Like CDC13, both STN1 and TEN1 are essential, and loss of their function leads to excessive single-stranded telomeric DNA (Grandin et al. 1997, 2001; Petreaca et al. 2007). STN1 was originally identified as a high copy suppressor of cdc13-1 temperature sensitivity (Grandin et al. 1997), and TEN1 was similarly isolated as a dosage suppressor of stn1-13 (Grandin et al. 2001). Combining either the cdc13-1 allele with stn1 mutations or the ten1-31 allele with stn1-13 is lethal (Grandin et al. 2001; Petreaca et al. 2007). The essential nature of these genes makes it difficult to clearly differentiate whether these genes operate in the same, or in parallel pathways to protect telomeres. A compelling argument that Cdc13, Stn1, and Ten1 likely function in a common pathway is that, in addition to these genetic interactions, Stn1 and Ten1 proteins interact with one another both in vivo and in vitro (Grandin et al. 2001; Gao et al. 2007), and each associates with Cdc13 in the yeast two-hybrid assay (Grandin et al. 1997, 2001; Petreaca et al. 2007). From these data, Cdc13, Stn1, and Ten1 are suggested to function as a single complex that mediates chromosome end protection in S. cerevisiae. Such a complex would share some similarities with the single-stranded DNA binding complex RPA (Gao et al. 2007). Whether these proteins normally operate exclusively as a heterotrimeric complex is still not entirely clear. Stn1 and Ten1 can make contributions to capping that are independent of Cdc13, as shown in experiments where overproducing the Stn1 essential domain with Ten1 replaced the essential function of Cdc13 (Petreaca et al. 2006). In addition, while the Schizosaccharomyces pombe Stn1 and Ten1 homologs are critical for telomere protection, they do not interact with Pot1, the single-stranded telomere binding protein that is also critical for telomere capping (Martin et al. 2007).The role of Ten1 in maintaining both telomere integrity and length homeostasis is not understood. It has been assumed that Stn1 and Ten1 play the same role as Cdc13 in maintaining telomere integrity, namely, preventing inappropriate terminal resection. However, whether this is in fact the case is not entirely clear. For one, disrupting the DNA replication machinery can give rise to an excess of terminal single-stranded DNA, although in this case, the ssDNA accumulation is attributed to a failure to synthesize the lagging DNA strand rather than removing a block to telomere resection (Diede and Gottschling 1999; Adams Martin et al. 2000). Although both Cdc13 and Stn1 are thought to act as capping proteins, each can interact with Polα subunits (Qi et al. 2003; Grossi et al. 2004; Petreaca et al. 2006), making it important to evaluate Ten1 function more carefully. Our goal here was to compare how Cdc13 and Ten1 promote chromosome end protection, first by testing whether Ten1 acts to prevent telomere resection from activities comparable to those that degrade telomeres in cdc13-1, and second by determining the impact of ten1 dysfunction upon Cdc13. The cdc13-1 allele has been extremely useful in analyzing the CDC13 essential function; TEN1 analysis has been hindered by a lack of equivalent genetic reagents. Here we have created a collection of ten1-ts alleles useful for probing the essential role of TEN1. Analysis of these alleles, which show constitutive telomere elongation, reveals that Ten1 promotes telomere capping with a similar cell cycle dependency as Cdc13, protecting ends during the period in which mitotic forms of Cdk1 are active. Critically, by showing that single-stranded DNA is generated in ten1-ts strains under conditions where semi-conservative replication is complete, we conclude that Ten1 truly can function as a capping protein. Moreover, the ten1-ts strains fail to restrain degradation of chromosome ends and induce formation of Rad52 repair foci, despite the association of wild-type Cdc13 with telomeres, indicating not only that Cdc13 binds telomeres independent of Ten1 function, but also that Cdc13 telomere localization is not sufficient for end protection. Finally, although the ten1-ts capping-deficient phenotypes parallel cdc13-1, only the ten1-ts strains are highly sensitive to impaired POL1 function, leaving open the possibility that TEN1 function additionally impacts terminal replication.     

DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development

Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.     

MUS81 generates a subset of MLH1-MLH3-independent crossovers in mammalian meiosis

Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81-/- animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.     

Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance in Arabidopsis

Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway.     

Drosophila FANCM Helicase Prevents Spontaneous Mitotic Crossovers Generated by the MUS81 and SLX1 Nucleases

Several helicases function during repair of double-strand breaks and handling of blocked or stalled replication forks to promote pathways that prevent formation of crossovers. Among these are the Bloom syndrome helicase BLM and the Fanconi anemia group M (FANCM) helicase. To better understand functions of these helicases, we compared phenotypes of Drosophila melanogaster Blm and Fancm mutants. As previously reported for BLM, FANCM has roles in responding to several types of DNA damage in preventing mitotic and meiotic crossovers and in promoting the synthesis-dependent strand annealing pathway for repair of a double-strand gap. In most assays, the phenotype of Fancm mutants is less severe than that of Blm mutants, and the phenotype of Blm Fancm double mutants is more severe than either single mutant, indicating both overlapping and unique functions. It is thought that mitotic crossovers arise when structure-selective nucleases cleave DNA intermediates that would normally be unwound or disassembled by these helicases. When BLM is absent, three nucleases believed to function as Holliday junction resolvases—MUS81-MMS4, MUS312-SLX1, and GEN—become essential. In contrast, no single resolvase is essential in mutants lacking FANCM, although simultaneous loss of GEN and either of the others is lethal in Fancm mutants. Since Fancm mutants can tolerate loss of a single resolvase, we were able to show that spontaneous mitotic crossovers that occur when FANCM is missing are dependent on MUS312 and either MUS81 or SLX1.     

FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.The plant vacuole is the largest organelle in a plant cell in which proteins, metabolites, and ions can be stored or sequestered. The vacuole is essential for plant development and growth and is directly or indirectly involved in various biotic and abiotic stress responses (Zhang et al., 2014). The vacuole is also the central organelle for degradation of endocytic and autophagic protein substrates through the activity of vacuolar proteases. In both degradation pathways, substrates are transported to the vacuole by intracellular membrane trafficking. In endocytic degradation, plasma membrane-localized proteins are targeted to the vacuole for degradation by endosomes (Reyes et al., 2011). This process is important, among others, to control the abundance of plasma membrane receptors and thus downstream signaling events. Autophagic degradation is mainly involved in nutrient recycling. During this process, cytosolic proteins and organelles are either selectively or nonselectively transported by double membrane autophagosomes to the vacuole to be degraded (Liu and Bassham, 2012). Vacuolar transport defines an intracellular transport pathway by which de novo synthesized proteins or metabolic compounds are carried to the vacuole by vesicle transport (Drakakaki and Dandekar, 2013).In yeast (Saccharomyces cerevisiae), forward genetic screens aimed at finding mutants with defective vacuolar transport or vacuolar morphology have identified more than 30 VACUOLAR PROTEIN SORTING (VPS) and VACUOLAR MORPHOLOGY (VAM) genes (Banta et al., 1988; Raymond et al., 1992; Wada and Anraku, 1992). Closer analyses have shown that many of these mutants have defects both in protein sorting and in vacuole biogenesis, suggesting a close link between these processes. vps and vam mutants were classified into six mutant classes according to their phenotypes. The strategic success of these screens has been confirmed when later studies revealed that many of the genes categorized in the same mutant class were coding for subunits of the same protein complexes. Among them were complexes important for membrane transport and fusion events, such as the endosomal sorting complex required for transport (ESCRT)-I to ESCRT-III (Henne et al., 2011) or the homotypic fusion and vacuole protein sorting (HOPS) complex (Balderhaar and Ungermann, 2013).Sequence homologs of most yeast VPS genes can be found in the Arabidopsis (Arabidopsis thaliana) genome (Sanderfoot and Raikhel, 2003; Bassham et al., 2008), and some of them were reported to be involved in intracellular trafficking as well as vacuole biogenesis. For example, the Arabidopsis vacuoleless (vcl)/vps16 mutant is embryo lethal and lacks lytic vacuoles (Rojo et al., 2001). VPS16 is a subunit of the HOPS complex, suggesting that membrane fusion events mediated by VCL/VPS16 are also important for plant vacuole biogenesis. Several other Arabidopsis vps mutants were also shown to have altered vacuole morphology at the mature embryo stage (Shimada et al., 2006; Sanmartín et al., 2007; Ebine et al., 2008, 2014; Yamazaki et al., 2008; Zouhar et al., 2009; Shahriari et al., 2010), showing that there is a conserved mechanism regulating vacuolar transport and vacuole biogenesis. However, in contrast to yeast, in which mutants without vacuole or severe biogenesis defects are viable, plant vacuoles seem to be essential for plant development.We have previously shown that defects in the deubiquitinating enzyme (DUB) ASSOCIATED MOLECULE WITH THE Src homology-3 DOMAIN OF STAM3 (AMSH3) also lead to a severe vacuole biogenesis defect (Isono et al., 2010). AMSH homologs do not exist in budding yeast but are conserved in animals and plants. Our previous studies have shown that AMSH3 can directly interact with ESCRT-III subunits (Katsiarimpa et al., 2013). ESCRT-III is a multiprotein complex that is essential for multivesicular body (MVB) sorting (Winter and Hauser, 2006) and hence for plant growth and development (Haas et al., 2007; Spitzer et al., 2009; Katsiarimpa et al., 2011; Cai et al., 2014). AMSH proteins regulate intracellular trafficking events, including endocytic degradation, vacuolar transport, and autophagic degradation through its interaction with ESCRT-III (Isono et al., 2010; Katsiarimpa et al., 2011, 2013, 2014). Prior to our characterization of the amsh3 mutant, AMSH proteins had not been implicated in vacuole biogenesis. Thus, we reasoned that there might be additional, yet unidentified, factors important for regulating vacuole biogenesis in plants. Further, we reasoned that other mutants with a defect in vacuole biogenesis, analogous to amsh3, might also exhibit seedling lethality.Thus, with the goal to identify and characterize these factors, we carried out a two-step mutant screen. We first selected seedling lethal mutants from an ethyl methansulfonate (EMS)-mutagenized population and then examined the vacuole morphology in these mutants. The isolated mutants were designated vacuolar fusion defective (vfd). vfd1 is affected in the expression of a functional Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing FYVE1 protein. FYVE1 was originally identified in silico as one of 16 FYVE domain-containing proteins in Arabidopsis with no apparent homologs in yeast and mammals (van Leeuwen et al., 2004). FYVE domains bind phosphatidylinositol 3-P, a phospholipid that is a major constituent of endosomal membranes. Hence, FYVE domain-containing proteins are implicated in intracellular trafficking (van Leeuwen et al., 2004; Wywial and Singh, 2010). In a previous work, we have shown that a null mutant of FYVE1, fyve1-1, is defective in IRON-REGULATED TRANSPORTER1 (IRT1) polarization and that FYVE1 is essential for plant growth and development (Barberon et al., 2014). A very recent publication describing the same mutant has shown that FYVE1/FYVE domain protein required for endosomal sorting1 (FREE1) is also important for the early and late endosomal trafficking events (Gao et al., 2014). In this study, we show that FYVE1 is also regulating ubiquitin-dependent membrane protein degradation, vacuolar transport, autophagy, and vacuole biogenesis. Altogether, our results point toward FYVE1 being a key component of the intracellular trafficking machinery in plants.     

The Copper Transporter RAN1 Is Essential for Biogenesis of Ethylene Receptors in Arabidopsis

Plants utilize ethylene as a hormone to regulate multiple developmental processes and to coordinate responses to biotic and abiotic stress. In Arabidopsis thaliana, a small family of five receptor proteins typified by ETR1 mediates ethylene perception. Our previous work suggested that copper ions likely play a role in ethylene binding. An independent study indicated that the ran1 mutants, which display ethylene-like responses to the ethylene antagonist trans-cyclooctene, have mutations in the RAN1 copper-transporting P-type ATPase, once again linking copper ions to the ethylene-response pathway. The results presented herein indicate that genetically engineered Saccharomyces cerevisiae expressing ETR1 but lacking the RAN1 homolog Ccc2p (Δccc2) lacks ethylene-binding activity. Ethylene-binding activity was restored when copper ions were added to the Δccc2 mutants, showing that it is the delivery of copper that is important. Additionally, transformation of the Δccc2 mutant yeast with RAN1 rescued ethylene-binding activity. Analysis of plants carrying loss-of-function mutations in ran1 showed that they lacked ethylene-binding activity, whereas seedlings carrying weak alleles of ran1 had normal ethylene-binding activity but were hypersensitive to copper-chelating agents. Altogether, the results show an essential role for RAN1 in the biogenesis of the ethylene receptors and copper homeostasis in Arabidopsis seedlings. Furthermore, the results indicate cross-talk between the ethylene-response pathway and copper homeostasis in Arabidopsis seedling development.     

Expression and Stability of Arabidopsis CDC6 Are Associated with Endoreplication

Studies on the CDC6 protein, which is crucial to the control of DNA replication in yeast and animal cells, are lacking in plants. We have isolated an Arabidopsis cDNA encoding the AtCDC6 protein and studied its possible connection to the occurrence of developmentally regulated endoreplication cycles. The AtCDC6 gene is expressed maximally in early S-phase, and its promoter contains an E2F consensus site that mediates the binding of a plant E2F/DP complex. Transgenic plants carrying an AtCDC6 promoter-beta-glucuronidase fusion revealed that it is active in proliferating cells and, interestingly, in endoreplicating cells. In particular, the extra endoreplication cycle that occurs in dark-grown hypocotyl cells is associated with upregulation of the AtCDC6 gene. This was corroborated using ctr1 Arabidopsis mutants altered in their endoreplication pattern. The ectopic expression of AtCDC6 in transgenic plants induced endoreplication and produced a change in the somatic ploidy level. AtCDC6 was degraded in a ubiquitin- and proteosome-dependent manner by extracts from proliferating cells, but it was degraded poorly by extracts from dark-grown hypocotyl endoreplicating cells. Our results indicate that endoreplication is associated with expression of the AtCDC6 gene and, most likely, the stability of its product; it also apparently requires activation of the retinoblastoma/E2F/DP pathway. These conclusions may apply to endoreplicating cells in other tissues of the plant and to endoreplicating cells in other eukaryotes.     

MUS81 Generates a Subset of MLH1-MLH3–Independent Crossovers in Mammalian Meiosis

Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81^−/− animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.     

NBS1在DNA断裂损伤反应和维持端粒稳定中的作用

NBS1作为MRE11/RAD50/NBS1复合物的组分之一,是细胞应答DNA损伤的一个关键蛋白质,在DNA双链断裂修复和维持基因组稳定中发挥重要的作用。端粒是染色体末端由DNA重复序列和蛋白质构成的复合体,其独特结构与DNA双链断裂非常相似。最近几年的研究发现NBS1与端粒也有着十分密切的联系。综述了NBS1在DNA损伤反应中的作用,并探讨NBS1参与维持端粒稳定中的分子机制。