FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.

Winter-annual ecotypes of Arabidopsis are relatively late flowering, unless the flowering of these ecotypes is promoted by exposure to cold (vernalization). This vernalization-suppressible, late-flowering phenotype results from the presence of dominant, late-flowering alleles at two loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). In this study, we report that flc null mutations result in early flowering, demonstrating that the role of active FLC alleles is to repress flowering. FLC was isolated by positional cloning and found to encode a novel MADS domain protein. The levels of FLC mRNA are regulated positively by FRI and negatively by LUMINIDEPENDENS. FLC is also negatively regulated by vernalization. Overexpression of FLC from a heterologous promoter is sufficient to delay flowering in the absence of an active FRI allele. We propose that the level of FLC activity acts through a rheostat-like mechanism to control flowering time in Arabidopsis and that modulation of FLC expression is a component of the vernalization response.     

Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark‐grown plants. While both COP1 and the four SPA proteins contain coiled‐coil and WD‐repeat domains, SPA proteins differ from COP1 in carrying an N‐terminal kinase‐like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N‐terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N‐terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N‐terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase‐like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase‐like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase‐like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N‐terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N‐terminal domain. Together, these results uncover an important, but complex role of the SPA1 N‐terminus in the suppression of photomorphogenesis.     

EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis

We have characterized Arabidopsis esd1 mutations, which cause early flowering independently of photoperiod, moderate increase of hypocotyl length, shortened inflorescence internodes, and altered leaf and flower development. Phenotypic analyses of double mutants with mutations at different loci of the flowering inductive pathways suggest that esd1 abolishes the FLC-mediated late flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. We found that ESD1 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of esd1 in a flc-3 null genetic background and the downregulation of other members of the FLC-like/MAF gene family in esd1 mutants suggest that flowering inhibition mediated by ESD1 occurs through both FLC-and FLC-like gene-dependent pathways. The ESD1 locus was identified through a map-based cloning approach. ESD1 encodes ARP6, a homolog of the actin-related protein family that shares moderate sequence homology with conventional actins. Using chromatin immunoprecipitation (ChIP) experiments, we have determined that ARP6 is required for both histone acetylation and methylation of the FLC chromatin in Arabidopsis.     

SHOOT MERISTEMLESS trafficking controls axillary meristem formation,meristem size and organ boundaries in Arabidopsis

The shoot stem cell niche, contained within the shoot apical meristem (SAM) is maintained in Arabidopsis by the homeodomain protein SHOOT MERISTEMLESS (STM). STM is a mobile protein that traffics cell‐to‐cell, presumably through plasmodesmata. In maize, the STM homolog KNOTTED1 shows clear differences between mRNA and protein localization domains in the SAM. However, the STM mRNA and protein localization domains are not obviously different in Arabidopsis, and the functional relevance of STM mobility is unknown. Using a non‐mobile version of STM (2xNLS‐YFP‐STM), we show that STM mobility is required to suppress axillary meristem formation during embryogenesis, to maintain meristem size, and to precisely specify organ boundaries throughout development. STM and organ boundary genes CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 regulate each other during embryogenesis to establish the embryonic SAM and to specify cotyledon boundaries, and STM controls CUC expression post‐embryonically at organ boundary domains. We show that organ boundary specification by correct spatial expression of CUC genes requires STM mobility in the meristem. Our data suggest that STM mobility is critical for its normal function in shoot stem cell control.     

Analysis of the molecular basis of flowering time variation in Arabidopsis accessions

Allelic variation at the FRI (FRIGIDA) and FLC (FLOWERING LOCUS C) loci are major determinants of flowering time in Arabidopsis accessions. Dominant alleles of FRI confer a vernalization requirement causing plants to overwinter vegetatively. Many early flowering accessions carry loss-of-function fri alleles containing one of two deletions. However, some accessions categorized as early flowering types do not carry these deletion alleles. We have analyzed the molecular basis of earliness in five of these accessions: Cvi, Shakhdara, Wil-2, Kondara, and Kz-9. The Cvi FRI allele carries a number of nucleotide differences, one of which causes an in-frame stop codon in the first exon. The other four accessions contain nucleotide differences that only result in amino acid substitutions. Preliminary genetic analysis was consistent with Cvi carrying a nonfunctional FRI allele; Wil-2 carrying either a defective FRI or a dominant suppressor of FRI function; and Shakhdara, Kondara, and Kz-9 carrying a functional FRI allele with earliness being caused by allelic variation at other loci including FLC. Allelic variation at FLC was also investigated in a range of accessions. A novel nonautonomous Mutator-like transposon was found in the weak FLC allele in Landsberg erecta, positioned in the first intron, a region required for normal FLC regulation. This transposon was not present in FLC alleles of most other accessions including Shakhdara, Kondara, or Kz-9. Thus, variation in Arabidopsis flowering time has arisen through the generation of nonfunctional or weak FRI and FLC alleles.     

Arabidopsis OTU1, a linkage‐specific deubiquitinase,is required for endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is part of the ER protein quality‐control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N‐terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N‐terminal region. These observations collectively define AtOTU1 as an OTU domain‐containing deubiquitinase involved in Arabidopsis ERAD.     

FRIGIDA and related proteins have a conserved central domain and family specific N- and C- terminal regions that are functionally important

Arabidopsis accessions are either winter-annuals, which require cold winter temperatures for spring flowering, or rapid-cycling summer annuals. Typically, winter annual accessions have functional FRIGIDA (FRI) and FRIGIDA-LIKE 1 (FRL1) proteins that promote high expression of FLOWERING LOCUS C (FLC), which prevents flowering until after winter. In contrast, many rapid-cycling accessions have low FLC levels because FRI is inactive. Using biochemical, functional and bioinformatic approaches, we show that FRI and FRL1 contain a stable, central domain that is conserved across the FRI superfamily. This core domain is monomeric in solution and primarily α-helical. We analysed the ability of several FRI deletion constructs to function in Arabidopsis plants. Our findings suggest that the C-terminus, which is predicted to be disordered, is required for FRI to promote FLC expression and may mediate protein:protein interactions. The contribution of the FRI N-terminus appears to be limited, as constructs missing these residues retained significant activity when expressed at high levels. The important N- and C-terminal regions differ between members of the FRI superfamily and sequence analysis identified five FRI families with distinct expression patterns in Arabidopsis, suggesting the families have separate biological roles.     

The transmembrane domain of N –acetylglucosaminyltransferase I is the key determinant for its Golgi subcompartmentation

Golgi‐resident type–II membrane proteins are asymmetrically distributed across the Golgi stack. The intrinsic features of the protein that determine its subcompartment‐specific concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of Nicotiana benthamiana N–acetylglucosaminyltransferase I (GnTI) for its cis/medial‐Golgi localization and for protein–protein interaction in the Golgi. The individual GnTI protein domains were replaced with those from the well‐known trans‐Golgi enzyme α2,6–sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co‐localization analysis and N–glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial‐Golgi localization. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub‐Golgi localization was not able to complement the GnTI‐dependent glycosylation defect. Our results suggest that sequence‐specific features in the transmembrane domain of GnTI account for its steady‐state distribution in the cis/medial‐Golgi in plants, which is a prerequisite for efficient N–glycan processing in vivo.     

Role of FRIGIDA and FLOWERING LOCUS C in determining variation in flowering time of Arabidopsis

Arabidopsis (Arabidopsis thaliana) accessions provide an excellent resource to dissect the molecular basis of adaptation. We have selected 192 Arabidopsis accessions collected to represent worldwide and local variation and analyzed two adaptively important traits, flowering time and vernalization response. There was huge variation in the flowering habit of the different accessions, with no simple relationship to latitude of collection site and considerable diversity occurring within local regions. We explored the contribution to this variation from the two genes FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), previously shown to be important determinants in natural variation of flowering time. A correlation of FLC expression with flowering time and vernalization was observed, but it was not as strong as anticipated due to many late-flowering/vernalization-requiring accessions being associated with low FLC expression and early-flowering accessions with high FLC expression. Sequence analysis of FRI revealed which accessions were likely to carry functional alleles, and, from comparison of flowering time with allelic type, we estimate that approximately 70% of flowering time variation can be accounted for by allelic variation of FRI. The maintenance and propagation of 20 independent nonfunctional FRI haplotypes suggest that the loss-of-function mutations can confer a strong selective advantage. Accessions with a common FRI haplotype were, in some cases, associated with very different FLC levels and wide variation in flowering time, suggesting additional variation at FLC itself or other genes regulating FLC. These data reveal how useful these Arabidopsis accessions will be in dissecting the complex molecular variation that has led to the adaptive phenotypic variation in flowering time.     

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

The E3 ubiquitin ligase SP1‐like 1 plays a positive role in peroxisome biogenesis in Arabidopsis

Peroxisomes are dynamic organelles crucial for a variety of metabolic processes during the development of eukaryotic organisms, and are functionally linked to other subcellular organelles, such as mitochondria and chloroplasts. Peroxisomal matrix proteins are imported by peroxins (PEX proteins), yet the modulation of peroxin functions is poorly understood. We previously reported that, besides its known function in chloroplast protein import, the Arabidopsis E3 ubiquitin ligase SP1 (suppressor of ppi1 locus1) also targets to peroxisomes and mitochondria, and promotes the destabilization of the peroxisomal receptor–cargo docking complex components PEX13 and PEX14. Here we present evidence that in Arabidopsis, SP1's closest homolog SP1‐like 1 (SPL1) plays an opposite role to SP1 in peroxisomes. In contrast to sp1, loss‐of‐function of SPL1 led to reduced peroxisomal β‐oxidation activity, and enhanced the physiological and growth defects of pex14 and pex13 mutants. Transient co‐expression of SPL1 and SP1 promoted each other's destabilization. SPL1 reduced the ability of SP1 to induce PEX13 turnover, and it is the N‐terminus of SP1 and SPL1 that determines whether the protein is able to promote PEX13 turnover. Finally, SPL1 showed prevalent targeting to mitochondria, but rather weak and partial localization to peroxisomes. Our data suggest that these two members of the same E3 protein family utilize distinct mechanisms to modulate peroxisome biogenesis, where SPL1 reduces the function of SP1. Plants and possibly other higher eukaryotes may employ this small family of E3 enzymes to differentially modulate the dynamics of several organelles essential to energy metabolism via the ubiquitin‐proteasome system.     

Plasticity genes and plasticity costs: a new approach using an Arabidopsis recombinant inbred population

Earlier flowering is triggered by vernalization in some but not all Arabidopsis ecotypes, often reflecting allelic variation at the FRIGIDA (FRI) locus. Using a recombinant inbred (RI) population polymorphic at FRI, we examined fitness consequences of variation for plasticity. Flowering and fitness were scored for 68 RI genotypes following full and partial vernalization treatments. Within-environment and mixed-model anovas estimated variance components for a genotype effect and a G x E term, respectively. Selection analyses examined whether delayed bolting increases fitness; a plasticity costs analysis asked whether increased plasticity lowers fitness. We also explored whether trait QTL had environment-specific effects, colocated in the immediate vicinity of FRI, or overlapped with fitness QTL. Selection may favor fri alleles and constitutive early flowering, especially in conditions that only partially vernalize plants. Plasticity costs, detected only after partial vernalization and only marginally significant, were nonetheless consistent with FRI-FLC function. We discuss how information about QTL with environment-specific effects, fitness QTL, and knowledge about plasticity genes can improve interpretation of selection or plasticity cost analyses.