A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele

To investigate the molecular mechanisms controlling the process of cold acclimation and to identify genes involved in plant freezing tolerance, mutations that impaired the cold acclimation capability of Arabidopsis thaliana (L.) Heynh. were screened for. A new mutation, frs1 (freezing sensitive 1), that reduced both the constitutive freezing tolerance as well as the freezing tolerance of Arabidopsis after cold acclimation was characterized. This mutation also produced a wilty phenotype and excessive water loss. Plants with the frs1 mutation recovered their wild-type phenotype, their capability to tolerate freezing temperatures and their capability to retain water after an exogenous abscisic acid (ABA) treatment. Measurements of ABA revealed that frs1 mutants were ABA deficient, and complementation tests indicated that frs1 mutation was a new allele of the ABA3 locus showing that a mutation in this locus leads to an impairment of freezing tolerance. These results constitute the first report showing that a mutation in ABA3 leads to an impairment of freezing tolerance, and not only strengthen the conclusion that ABA is required for full development of freezing tolerance in cold-acclimated plants, but also demonstrate that ABA mediates the constitutive freezing tolerance of Arabidopsis. Gene expression in frs1 mutants was altered in response to dehydration, suggesting that freezing tolerance in Arabidopsis depends on ABA-regulated proteins that allow plants to survive the challenges imposed by subzero temperatures, mainly freeze-induced cellular dehydration. Received: 16 December 1999 / Accepted: 31 March 2000     

Altered resource allocation during seed development in Arabidopsis caused by the abi3 mutation

The regulation of whole-plant resource allocation during seed development in Arabidopsis thaliana was investigated by examining growth rate and partitioning of ¹⁴CO₂ in wild-type plants and those carrying the abi3 mutation. Plants carrying the abi3 mutation partitioned more resources into seed development than the wild type. The extra resources were available as a result of delayed senescence of the cauline leaves in the mutant. After supply of ¹⁴CO₂ at later stages of reproductive development differences in patterns of ¹⁴C distribution between mutant and wild type were consistent with long-term changes in growth and allocation. The role of long-distance signals in the regulation of seed yield in Arabidopsis is discussed.     

Isolation of an Arabidopsis thaliana mutant in which accumulation of cucumber mosaic virus coat protein is delayed

Cucumber mosaic virus (CMV) is known to systemically infect Arabidopsis thaliana ecotype Columbia plants. In order to identify the host factors involved in the multiplication of CMV, we isolated an A. thaliana mutant in which the accumulation of the coat protein (CP) of CMV in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in the mutant plants was caused by a single, nuclear and recessive mutation designated cum1-1, which was located on chromosome IV. The cum1-1 mutation did not affect the multiplication of tobacco mosaic virus, turnip crinkle virus or turnip yellow mosaic virus, which belong to different taxonomic groups from CMV. Accumulation of CMV CP in the inoculated leaves of cum1-1 plants was also delayed either when CMV virion or CMV virion RNA was inoculated. On the other hand, when cum1-1 and the wild-type Col-0 protoplasts were inoculated with CMV virion RNA by electroporation, the accumulations of CMV-related RNAs and the coat protein were similar. These results suggest that the cum1-1 mutation did not affect the uncoating of CMV virion and subsequent replication in an initially infected cell but affected the spreading of CMV within an infected leaf, possibly the cell-to-cell movement of CMV in a virus-specific manner.     

A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana

The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.     

Characterization of green seed,an enhancer of abi3-1 in Arabidopsis that affects seed longevity

Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.     

The Arabidopsis peroxisome division mutant pdd2 is defective in the DYNAMIN-RELATED PROTEIN3A (DRP3A) gene

In plants, the division of peroxisomes is mediated by several classes of proteins, including PEROXIN11 (PEX11), FISSION1 (FIS1) and DYNAMIN-RELATED PROTEIN3 (DRP3). DRP3A and DRP3B are two homologous dynamin-related proteins playing overlapping roles in the division of both peroxisomes and mitochondria, with DRP3A performing a stronger function than DRP3B in peroxisomal fission. Here, we report the identification and characterization of the peroxisome division defective 2 (pdd2) mutant, which was later proven to be another drp3A allele. The pdd2 mutant generates a truncated DRP3A protein and exhibits pale green and retarded growth phenotypes. Intriguingly, this mutant displays much stronger peroxisome division deficiency in root cells than in leaf mesophyll cells. Our data suggest that the partial GTPase effector domain retained in pdd2 may have contributed to the distinct mutant phenotype of this mutant.Key words: peroxisome division, dynamin-related protein, arabidopsisIn eukaryotic cells, peroxisomes are surrounded by single membranes and house a variety of oxidative metabolic pathways such as lipid metabolism, detoxification and plant photorespiration.^1,2 To accomplish multiple tasks, the morphology, abundance and positioning of peroxisomes need to be highly regulated. Three families of proteins, whose homologs are present across different kingdoms, have been shown to be involved in peroxisome division in Arabidopsis. The PEX11 protein family is composed of five integral membrane proteins with primary roles in peroxisome elongation/tubulation, the initial step in peroxisome division.^3–5 Although the exact function of PEX11s has not been demonstrated, these proteins are believed to participate in peroxisome membrane modification.^6,7 The FIS1 family consists of two isoforms, which are C-terminal tail-anchored membrane proteins with rate limiting functions at the fission step.^8,9 DRP3A and DRP3B belong to a superfamily of dynamin-related proteins, which are large and self-assembling GTPases involved in the fission and fusion of membranes by acting as mechanochemical enzymes or signaling GTPases.¹⁰ The function of PEX11 seems to be exclusive to peroxisomes, whereas DRP3 and FIS1 are shared by the division machineries of both peroxisomes and mitochondria in Arabidopsis.^8,9,11–16 FIS1 proteins are believed to tether DRP proteins to the peroxisomal membrane,^17,18 but direct evidence has not been obtained from plants. DRP3A and DRP3B share 77% sequence identity at the protein level and are functionally redundant in regulating mitochondrial division; however, DRP3A''s role on the peroxisome seems stronger and cannot be substituted by DRP3B in peroxisome division.^8,13,15In a continuous effort to identify components of the plant peroxisome division apparatus from Arabidopsis, we performed genetic screens in a peroxisomal marker background expressing the YFP (yellow fluorescent protein)-PTS1 (peroxisome targeting signal 1, containing Ser-Lys-Leu) fusion protein. Mutants with defects in the morphology and abundance of fluorescently labeled peroxisomes are characterized. Following our analysis of the pdd1 mutant, which turned out to be a strong allele of DRP3A,⁸ we characterized the pdd2 mutant.In root cells of the pdd2 mutant, extremely elongated peroxisomes and a beads-on-a-string peroxisomal phenotype are frequently observed (Fig. 1A and B). These peroxisome phenotypes resemble those of pdd1 and other strong drp3A alleles previously reported.^8,15 However, the peroxisome phenotype seems to be less dramatic in leaf mesophyll cells. For instance, in addition to the decreased number of total peroxisomes, peroxisomes in leaf cells are only slightly elongated or exhibit a beads-on-a-string phenotype (Fig. 1C and D). Previously, we reported the phenotypes of three strong drp3A alleles, all of which contain a large number of peroxules, long and thin membrane extensions from the peroxisome,⁸ yet such peroxisomal structures are not observed in pdd2. On the other hand, pdd2 has a more severe growth phenotype than most drp3A alleles, as it is slow in growth and has pale green leaves (Fig. 1E). Genetic analysis showed that pdd2 segregates as a single recessive mutation (data not shown).Open in a separate windowFigure 1Phenotypic analyses of pdd2 and identification of the PDD2 gene. (A–D) Confocal micrographs of root and mesophyll cells in 3-week-old wild type and pdd2 mutant plants. Green signals show peroxisomes; red signals show chloroplasts. Scale bars = 20 µm. (E) Growth phenotype of 3-week-old mutants. (F) Map-based cloning of the PDD2 gene. Genetic distance from PDD2 is shown under each molecular marker. Positions for mutations in previously analyzed drp3A alleles and pdd2 are indicated in the gene schematic. drp3A-1 and drp3A-2 are T-DNA insertion mutants, whereas pdd1 is an EMS mutant containing a premature stop codon in exon 6. (G) A schematic of the DRP3A (PDD2) protein with functional domains indicated. The pdd2 allele encodes a truncated protein lacking part of the GED domain.The unique combination of peroxisomal and growth phenotypes of pdd2 prompted us to use map-based cloning to identify the PDD2 gene, with the hope to discover novel proteins in the peroxisome division machinery. A population of approximately 6,000 F₂ plants (pdd2 × Ler) was generated. After screening 755 F₂ mutants, the pdd2 mutation was mapped to the region between markers T10C21 and F4B14 on the long arm of chromosome 4 (Fig. 1F). Since this region contains DRP3A, we sequenced the entire DRP3A gene in pdd2 and identified a G→A transition at the junction of the 18^th exon and intron (Fig. 1F). Further analysis revealed that the point mutation at this junction caused mis-splicing of intron 18, introducing a stop codon in the GTPase effector domain GED near the C terminus (Fig. 1G).DRPs share with the classic dynamins an N-terminal GTPase domain, a middle domain (MD), and a regulatory motif named the GTPase effector domain (GED) (Fig. 1G). To date, a total of 26 drp3A mutant alleles carrying missense or nonsense mutations along the length of the DRP3A gene have been isolated.^8,15 The combined peroxisomal and growth phenotype of pdd2 and the nature of the mutation in this allele are unique among all the drp3A alleles, indicating that the partial GED domain retained in pdd2 may have created some novel function for this protein. Further analysis of the truncated protein may be necessary to test this prediction.     

A RING-H2 zinc-finger protein gene RIE1 is essential for seed development in Arabidopsis

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.     

A novel extinction screen in Arabidopsis thaliana identifies mutant plants defective in early microsporangial development

Few Arabidopsis mutants defective in early male or female germline development have been reported. A novel extinction screen has been devised which permits the identification of mutants deficient in the earliest stages of anther development. Using mutagenized plants carrying GUS reporter constructs driven by tapetal-specific promoters originally derived from Brassica genes, a wide spectrum of mutants have been identified in Arabidopsis, ranging from those defective in archesporial cell differentiation to others expressed later in development. Crosses between these lines and known anther development mutants have enabled the identification of lines carrying mutations in genes expressed during very early anther formation. Initial characterization reveals these early mutants fall into two classes, gne (GUS-negative) 1-like, and gne2-like. Members of the gne1 mutant class initiate all four layers of the anther wall and an appropriate number of sporogenous cells; however, as development proceeds the tapetal and middle-layer cells enlarge, eventually crushing the sporogenous cells. The gne2 class anthers are disrupted at an earlier stage, with the middle and tapetal layers failing to form, and an excess of sporogenous cells developing until the germline aborts late in meiosis II. Analysis of these mutants has already raised questions about the accuracy of current models of angiosperm anther development.