Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism

The Arabidopsis acyl-CoA oxidase (ACX) family comprises isozymes with distinct fatty acid chain-length specificities that together catalyse the first step of peroxisomal fatty acid beta-oxidation. We have isolated and characterized T-DNA insertion mutants in the medium to long-chain (ACX1) and long-chain (ACX2) acyl-CoA oxidases, and show that the corresponding endogenous activities are decreased in the mutants. Lipid catabolism during germination and early post-germinative growth was unaltered in the acx1-1 mutant, but slightly delayed in the acx2-1 mutant, with 3-day-old acx2-1 seedlings accumulating long-chain acyl-CoAs. In acx1-1 and acx2-1, seedling growth and establishment in the absence of an exogenous supply of sucrose was unaffected. Seedlings of the double mutant acx1-1 acx2-1 were unable to catabolize seed storage lipid, and accumulated long-chain acyl-CoAs. The acx1-1 acx2-1 seedlings were also unable to establish photosynthetic competency in the absence of an exogenous carbon supply, a phenotype that is shared with a number of other Arabidopsis mutants disrupted in storage lipid breakdown. Germination frequency of the double mutant was significantly reduced compared with wild-type seeds. This was unaffected by the addition of exogenous sucrose, but was improved by dormancy-breaking treatments such as cold stratification and after-ripening. We show that the acx1-1, acx2-1 and acx1-2 acx2-1 double mutants and the ketoacyl-CoA thiolase-2 (kat2) mutant exhibit a sucrose-independent germination phenotype comparable with that reported for comatose (cts-2), a mutant in a peroxisomal ABC transporter which exhibits enhanced dormancy. This demonstrates an additional role beyond that of carbon provision for the beta-oxidation pathway during germination or in dormant seeds.     

The gluconeogenic enzyme phosphoenolpyruvate carboxykinase in Arabidopsis is essential for seedling establishment

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate in the gluconeogenic production of sugars from storage oil in germinating oilseeds. Here, we present the results of analysis on PEPCK antisense Arabidopsis plants with a range of enzyme activities from 20% to 80% of wild-type levels. There is a direct correlation between enzyme activity and seedling establishment during early post-germinative growth, thus demonstrating the absolute requirement of PEPCK and gluconeogenesis in this process. Soluble sugar levels in the 35S-PCK1 antisense seedlings are reduced and seedling establishment can be rescued with an exogenous supply of sucrose. We observed an increase in the respiration of acetyl coenzyme A units released from fatty acid beta-oxidation and a corresponding decrease in the production of sugars with decreasing enzyme activity in 2-d-old antisense seedlings. The 35S-PCK1 antisense lines have a more extreme phenotype when compared with Arabidopsis mutants disrupted in the glyoxylate cycle. We conclude that the 35S-PCK1 antisense seedlings are compromised in the ability to use both storage lipid and storage protein through gluconeogenesis to produce soluble sugars.     

AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that β-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.     

Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans

A site-directed mutagenesis of the GFA1 gene encoding Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase afforded its GFA1S208A version. A product of the modified gene, lacking the putative phosphorylation site for protein kinase A (PKA), exhibited all the basic properties identical to those of the wild-type enzyme but was no longer a substrate for PKA. Comparison of the C. albicans Deltagfa1/GFA1 and Deltagfa1/GFA1S208A cells, grown under conditions stimulating yeast-to-mycelia transformation, revealed that the latter demonstrated lower GlcN-6-P synthase specific activity, decreased chitin content and formed much fewer mycelial forms. All these findings, as well as the observed effects of specific inhibitors of protein kinases, suggest that a loss of the possibility of GlcN-6-P synthase phosphorylation by PKA strongly reduces but not completely eliminates the germinative response of C. albicans cells.     

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.     

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.     

The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal beta-oxidation is essential for seedling establishment

The multifunctional protein (MFP) of peroxisomal beta-oxidation catalyses four separate reactions, two of which (2-trans enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase) are core activities required for the catabolism of all fatty acids. We have isolated and characterized five Arabidopsis thaliana mutants in the MFP2 gene that is expressed predominantly in germinating seeds. Seedlings of mfp2 require an exogenous supply of sucrose for seedling establishment to occur. Analysis of mfp2-1 seedlings revealed that seed storage lipid was catabolized more slowly, long-chain acyl-CoA substrates accumulated and there was an increase in peroxisome size. Despite a reduction in the rate of beta-oxidation, mfp2 seedlings are not resistant to the herbicide 2,4-dichlorophenoxybutyric acid, which is catabolized to the auxin 2,4-dichlorophenoxyacetic acid by beta-oxidation. Acyl-CoA feeding experiments show that the MFP2 2-trans enoyl-CoA hydratase only exhibits activity against long chain (C18:0) substrates, whereas the MFP2 L-3-hydroxyacyl-CoA dehydrogenase is active on C6:0, C12:0 and C18:0 substrates. A mutation in the abnormal inflorescence meristem gene AIM1, the only homologue of MFP2, results in an abnormal inflorescence meristem phenotype in mature plants (Richmond and Bleecker, Plant Cell 11, 1999, 1911) demonstrating that the role of these genes is very different. The mfp2-1 aim1double mutant aborted during the early stages of embryo development showing that these two proteins share a common function that is essential for this key stage in the life cycle.     

Spermine is not essential for survival of Arabidopsis

Spermine is the final product of the polyamine biosynthetic pathway and is ubiquitously present in most organisms. The genome of Arabidopsis thaliana has two genes encoding spermine synthase: ACAULIS5 (ACL5), whose loss-of-function mutants show a severe defect in stem elongation, and SPMS. In order to elucidate the function of spermine in plants, we isolated a T-DNA insertion mutant of the SPMS gene. Free and conjugated spermine levels in the mutant, designated spms-1, were significantly decreased compared with those in the wild-type, but no obvious morphological phenotype was observed in spms-1 plants. We further confirmed that acl5-1 spms-1 double mutants contained no spermine. Surprisingly, acl5-1 spms-1 was fully as viable as the wild-type and showed no phenotype except for the reduced stem growth due to acl5-1. These results indicate that spermine is not essential for survival of Arabidopsis, at least under normal growth conditions.     

Thellungilla halophila is more adaptive to salinity than Arabidopsis thaliana at stages of seed germination and seedling establishment

Thellungiella halophila is a salt tolerant relative of Arabidopsis thaliana with high genetic and morphological similarity. In the present study, effects of salinity on germination and seedling growth of T. halophila and A. thaliana were compared. The present results showed that the salinity inhibited seed germination in both species. Unexpectedly, percentages of seed germination in A. thaliana were higher than T. halophila in a range of 0?C200?mM NaCl. Seeds of both species could not germinate when the concentration of NaCl was over 200?mM. However, when compared with A. thaliana, seeds of T. halophila did not suffer ion toxicity, as evidenced by the higher final germination rate after ungerminated seeds pretreated with NaCl were transferred to distilled water. Seedlings of T. halophila were more salt tolerant than those of A. thaliana, e.g., seedlings of T. halophila had better plant growth (root length, fresh and dry mass), higher chlorophyll content, less MDA content and higher proline content and K⁺/Na⁺ ratio under salinity. These results indicate that T. halophila is more salt tolerant than A. thaliana during both seed germination and seedling stages and explain why A. thaliana is excluded from saline locations and T. halophila can survive in saline soils.     

Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana

In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.     

Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

Clathrin is spindle-associated but not essential for mitosis

Background

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

Methodology/Principal Findings

Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

Conclusions/Significance

We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.     

The GCR2 gene family is not required for ABA control of seed germination and early seedling development in Arabidopsis

Background

The plant hormone abscisic acid (ABA) regulates diverse processes of plant growth and development. It has recently been proposed that GCR2 functions as a G-protein-coupled receptor (GPCR) for ABA. However, the structural relationships and functionality of GCR2 have been challenged by several independent studies. A central question in this controversy is whether gcr2 mutants are insensitive to ABA, because gcr2 mutants were shown to display reduced sensitivity to ABA under one experimental condition (e.g. 22°C, continuous white light with 150 µmol m^-2 s⁻¹) but were shown to display wild-type sensitivity under another slightly different condition (e.g. 23°C, 14/10 hr photoperiod with 120 µmol m⁻² s⁻¹). It has been hypothesized that gcr2 appears only weakly insensitive to ABA because two other GCR2-like genes in Arabidopsis, GCL1 and GCL2, compensate for the loss of function of GCR2.

Principal Findings

In order to test this hypothesis, we isolated a putative loss-of-function allele of GCL2, and then generated all possible combinations of mutations in each member of the GCR2 gene family. We found that all double mutants, including gcr2 gcl1, gcr2 gcl2, gcl1 gcl2, as well as the gcr2 gcl1 gcl2 triple mutant displayed wild-type sensitivity to ABA in seed germination and early seedling development assays, demonstrating that the GCR2 gene family is not required for ABA responses in these processes.

Conclusion

These results provide compelling genetic evidence that GCR2 is unlikely to act as a receptor for ABA in the context of either seed germination or early seedling development.     

An Arabidopsis mutant impaired in coenzyme A biosynthesis is sugar dependent for seedling establishment

Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4'-phosphopantothenoyl-cysteine decarboxilase that generates 4'-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in beta-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.     

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose-response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.     

The Saccharomyces cerevisiae RAD54 gene is important but not essential for natural homothallic mating-type switching

Homothallic Saccharomyces cerevisiae strains switch their mating-type in a specific gene conversion event induced by a DNA double strand break made by the HO endonuclease. The RAD52 group genes control recombinational repair of DNA double strand breaks, and we examined their role in native homothallic mating-type switching. Surprisingly, we found that the Rad54 protein was important but not essential for mating-type switching under natural conditions. As an upper limit, we estimate that 29% of the rad54 spore clones can successfully switch their mating-type. The RAD55 and RAD57 gene products were even less important, but their presence increased the efficiency of the process. In contrast, the RAD51 and RAD52 genes are essential for homothallic mating-type switching. We propose that mating-type switching in RAD54 mutants occurs stochastically with a low probability, possibly reflecting different states of chromosomal structure.     

Seed mass and seedling dimensions in relation to seedling establishment

Several experiments have shown that seedlings from larger-seeded species are better able to survive various hazards during establishment. Previous work has suggested a general mechanism might underpin this outcome. Larger-seeded species might tend to mobilize their metabolic resources over a longer period into the autotrophically functioning structures of the seedling. Consequently relatively more resources would remain uncommitted at any given time during the early period of the seedling’s growth, and available to support respiration during carbon deficit. An important aspect of this larger-seed-later-commitment mechanism would be that at a given time, larger-seeded species would hold more resources uncommitted not just absolutely, but relative to the functional seedling structures that needed to be supported. Here we quantify, across a wide range of phanerocotylar species, an allometric pattern that supports the generality of a larger-seed-later-commitment mechanism as an explanation for superior performance by larger-seeded species in face of the hazards of seedling establishment. Larger-seeded species allocate relatively less to cotyledon area, reflecting the initial functional size of the seedling, and relatively more to dry mass per unit area of cotyledon, reflecting stored metabolic reserves. The shift in relative allocation is progressive, rather than seedlings falling into discrete morphological types. The allometry is similar whether considered as correlated evolutionary divergences (phylogenetically independent contrasts) or as correlation across present-day species. Received: 7 April 1999 / Accepted: 29 March 2000