Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.     

The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1

Mitochondrial serine hydroxymethyltransferase (SHMT), combined with glycine decarboxylase, catalyzes an essential sequence of the photorespiratory C2 cycle, namely, the conversion of two molecules of glycine into one molecule each of CO2, NH4+, and serine. The Arabidopsis (Arabidopsis thaliana) mutant shm (now designated shm1-1) is defective in mitochondrial SHMT activity and displays a lethal photorespiratory phenotype when grown at ambient CO2, but is virtually unaffected at elevated CO2. The Arabidopsis genome harbors seven putative SHM genes, two of which (SHM1 and SHM2) feature predicted mitochondrial targeting signals. We have mapped shm1-1 to the position of the SHM1 gene (At4g37930). The mutation is due to a G --> A transition at the 5' splice site of intron 6 of SHM1, causing aberrant splicing and a premature termination of translation. A T-DNA insertion allele of SHM1, shm1-2, and the F1 progeny of a genetic cross between shm1-1 and shm1-2 displayed the same conditional lethal phenotype as shm1-1. Expression of wild-type SHM1 under the control of either the cauliflower mosaic virus 35S or the SHM1 promoter in shm1-1 abrogated the photorespiratory phenotype of the shm mutant, whereas overexpression of SHM2 or expression of SHM1 under the control of the SHM2 promoter did not rescue the mutant phenotype. Promoter-beta-glucuronidase analyses revealed that SHM1 is predominantly expressed in leaves, whereas SHM2 is mainly transcribed in the shoot apical meristem and roots. Our findings establish SHM1 as the defective gene in the Arabidopsis shm1-1 mutant.     

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.     

Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes

Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants. Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator. Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast. TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants. These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3. The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.     

Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development

The SWR1 complex (SWR1C) in yeast catalyzes the replacement of nucleosomal H2A with the H2AZ variant, which ensures full activation of underlying genes. We compared the phenotype of mutants in the homologs of SWR1C components in Arabidopsis thaliana. Mutations in Arabidopsis SWC6 (AtSWC6), SUPPRESSOR OF FRIGIDA 3 (SUF3) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), homologs of SWC6, ARP6 and SWR1, respectively, caused similar developmental defects, including leaf serration, weak apical dominance, flowers with extra petals and early flowering by reduction in expression of FLOWERING LOCUS C (FLC), a strong floral repressor. Chromatin immunoprecipitation assays showed that AtSWC6 and SUF3 bind to the proximal region of the FLC promoter, and protoplast transfection assays showed that AtSWC6 colocalizes with SUF3. Protein interaction analyses suggested the formation of a complex between PIE1, SUF3, AtSWC6 and AtSWC2. In addition, H2AZ, a substrate of SWR1C, interacts with both PIE1 and AtSWC2. Finally, knockdown of the H2AZ genes by RNA interference or artificial microRNA caused a phenotype similar to that of atswc6 or suf3. Our results strongly support the presence of an SWR1C-like complex in Arabidopsis that ensures proper development, including floral repression through full activation of FLC.     

Disruption of an Arabidopsis cytoplasmic ribosomal protein S13-homologous gene by transposon-mediated mutagenesis causes aberrant growth and development

We identified a Dissociation (Ds) transposon-inserted Arabidopsis mutant of a gene (AtRPS13A) homologous to cytoplasmic ribosomal protein (RP) S13. We named our mutant pointed first leaf (pfl) 2 because of its similar phenotype to the pfl1 mutant of the RPS18 gene. This mutant caused multiple phenotypic changes, including aberrant leaf and trichome morphology, retarded root growth, and late flowering. Microscopic analysis showed that the first leaf blade of pfl2 contained a significantly reduced number of palisade cells, which suggests that the mutant phenotype was caused by reduced cell division. However, no phenotypic changes were observed during reproductive growth. In Arabidopsis, the RPS13 protein was encoded by a small expressed gene family including AtRPS13A. A pfl1 pfl2 double mutant showed no additive effect. These results suggest that RPS13 functions in quantitative and pleiotropic ways during growth and development, and that mutations at different kinds of RP gene loci are accumulatable without serious growth defects because they belong to small gene families.     

Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.     

The C termini of Arabidopsis cryptochromes mediate a constitutive light response

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. Here we show that the signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype was not observed for mutants of cct1 corresponding to previously described cry1 alleles. We propose that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain.     

Characterization of plant phenotypes associated with loss-of-function of AtCNGC1, a plant cyclic nucleotide gated cation channel.

Of the 57 cation channel genes in the Arabidopsis genome, over a third encode cyclic nucleotide gated cation channels (CNGCs). CNGCs are ion channels regulated by cytosolic signaling molecules (cyclic nucleotides, calmodulin, and Ca(2+)), and which conduct Ca(2+) as well as K(+) and in some cases Na(+). Little is currently known about the role CNGCs may play in plant growth and development. Here, we examined the hypothesis that an Arabidopsis thaliana genotype containing a null mutation in one of the CGNC genes (AtCNGC1) would display cation uptake-related growth phenotype differences from wild type (WT) plants. We determined that AtCNGC1 protein is primarily expressed in the roots of Arabidopsis seedlings. Seedlings lacking this protein had slightly (6-22%) lower shoot Ca(2+) than WT plants. Primary roots of Atcngc1 mutant seedlings grew faster than roots of WT plants, and had larger angles of gravicurvature and less nitric oxide generation upon gravistimulation. We conclude that channels formed (at least in part) by AtCNGC1 contribute (along with other channels) to Ca(2+) uptake into plants, and that Ca(2+) uptake into roots through AtCNGC1 affects some aspects of growth in the primary root of Arabidopsis seedlings.     

AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues

Plants employ a specialized transport system composed of separate influx and efflux carriers to mobilize the plant hormone auxin between its site(s) of synthesis and action. Mutations within the permease-like AUX1 protein significantly reduce the rate of carrier-mediated auxin uptake within Arabidopsis roots, conferring an agravitropic phenotype. We are able to bypass the defect within auxin uptake and restore the gravitropic root phenotype of aux1 by growing mutant seedlings in the presence of the membrane-permeable synthetic auxin, 1-naphthaleneacetic acid. We illustrate that AUX1 expression overlaps that previously described for the auxin efflux carrier, AtPIN2, using transgenic lines expressing an AUX1 promoter::uidA (GUS) gene. Finally, we demonstrate that AUX1 regulates gravitropic curvature by acting in unison with the auxin efflux carrier to co-ordinate the localized redistribution of auxin within the Arabidopsis root apex. Our results provide the first example of a developmental role for the auxin influx carrier within higher plants and supply new insight into the molecular basis of gravitropic signalling.     

A gain-of-function phenotype conferred by over-expression of functional subunits of the COP9 signalosome in Arabidopsis

The COP9 signalosome is a conserved cellular regulator present in diverse organisms. To understand the structural and functional relationship of the COP9 signalosome with its subunits, we expressed in wild-type and mutant Arabidopsis backgrounds two orthologues of subunit 1, rice FUS6 (rFUS6) and human GPS1, and Arabidopsis subunit 8 (COP9). In Arabidopsis, rFUS6 can functionally replace Arabidopsis endogenous FUS6 to form the COP9 signalosome complex and rescue the null fus6-1 mutant phenotype. Moreover, light-grown rFUS6 over-expression seedlings displayed longer hypocotyls and reduced anthocyanin accumulation in comparison to wild-type seedlings, which is opposite to the fus6/cop11 mutant phenotype. The long-hypocotyl phenotype was also observed in transgenic seedlings over-expressing Arabidopsis COP9. This finding indicates that over-expression of a functional subunit 1 or subunit 8 of the COP9 signalosome confers a gain-of-function phenotype relative to the complex. Human GPS1, when expressed in the fus6-1 null mutant of Arabidopsis, can assemble into a chimeric COP9 signalosome at low efficiency, demonstrating the structural conservation of the complexes between human and Arabidopsis. This low-abundancy chimeric complex is insufficient to fully rescue the mutant but is able to attenuate the mutant severity.     

Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns

Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.     

Sulfurtransferases 1 and 2 play essential roles in embryo and seed development in Arabidopsis thaliana

Sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from a donor to a suitable acceptor molecule. The Arabidopsis thaliana genome encodes 20 putative STR proteins. The biological functions of most are unclear. We found that STR1 and STR2 play important roles in embryo/seed development. Mutation of STR1 alone resulted in a shrunken seed phenotype, although growth and development of vegetative and reproductive organs were not affected. The shrunken seed phenotype was associated with the delayed/arrested embryo development, in most cases, at the heart stage. The embryo defect of str1 mutant is not fully penetrant. Approximately 12.5% of embryos developed further and formed normal looking seeds. In severely shrunken seeds, no embryo could be identified after seed collection. Partially shrunken seeds that contained viable embryos could still germinate. However, cotyledons of the seedlings from such seeds were abnormal. An STR1-GUS fusion reporter revealed that the STR1 gene was universally expressed, with high levels of expression in specific tissues/organs including embryos. The incomplete penetrance of str1 embryo/seed phenotype is a result of functional STR2. Single str2 mutant had no phenotype. However, no str1(-/-)/str2(-/-) double mutant embryos were able to develop past the heart stage. Furthermore, STR2 is haplo-insufficient in str1 mutant background, and str1(-/-)/str2(+/-) embryos were 100% lethal. These data provide new insights into the biological functions of the ubiquitous sulfurtransferase in Arabidopsis embryogenesis and seed development.     

Two Arabidopsis cytochrome P450 monooxygenases, CYP714A1 and CYP714A2, function redundantly in plant development through gibberellin deactivation

The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA(12) slightly accumulated in the transgenic rice, and GA(19) highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops.     

EHD1 Functions in Endosomal Recycling and Confers Salt Tolerance

Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain.