Kelch Repeat Research Articles

Distinct roles of FKF1, Gigantea, and Zeitlupe proteins in the regulation of Constans stability in Arabidopsis photoperiodic flowering.

Many plants measure changes in day length to synchronize their flowering time with appropriate seasons for maximum reproductive success. In Arabidopsis, the day-length-dependent regulation of Constans (CO) protein stability is crucial to induce flowering locus T (FT) expression for flowering in long days. The flavin-binding, KELCH repeat, F-box1 (FKF1) protein binds to CO protein specifically in the long-day afternoon and stabilizes it, although the mechanism remains unknown. Here we demonstrated that the FKF1-interacting proteins Gigantea (GI) and Zeitlupe (ZTL) are involved in CO stability regulation. First, our immunoprecipitation-mass spectrometry analysis of FKF1 revealed that FKF1 forms an S-phase kinase-associated protein 1 (Skp1)/Cullin(CUL)/F-box complex through interactions with Arabidopsis Skp1-like 1 (ASK1), ASK2, and CUL1 proteins and mainly interacts with GI protein in vivo. GI interacts with CO directly and indirectly through FKF1. Unexpectedly, the gi mutation increases the CO protein levels in the morning in long days. This gi-dependent destabilization of CO protein was cancelled by the fkf1 mutation. These results suggest that there are other factors likely influenced by both gi and fkf1 mutations that also control CO stability. We found that ZTL, which interacts with GI and FKF1, may be one such factor. ZTL also interacts with CO in vivo. The CO protein profile in the ztl mutant resembles that in the gi mutant, indicating that ZTL activity also may be changed in the gi mutant. Our findings suggest the presence of balanced regulation among FKF1, GI, and ZTL on CO stability regulation for the precise control of flowering time.

Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf starch synthesis in different photoperiods.

Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5'-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated.

Evening expression of arabidopsis GIGANTEA is controlled by combinatorial interactions among evolutionarily conserved regulatory motifs.

Diurnal patterns of gene transcription are often conferred by complex interactions between circadian clock control and acute responses to environmental cues. Arabidopsis thaliana GIGANTEA (GI) contributes to photoperiodic flowering, circadian clock control, and photoreceptor signaling, and its transcription is regulated by the circadian clock and light. We used phylogenetic shadowing to identify three evolutionarily constrained regions (conserved regulatory modules [CRMs]) within the GI promoter and show that CRM2 is sufficient to confer a similar transcriptional pattern as the full-length promoter. Dissection of CRM2 showed that one subfragment (CRM2-A) contributes light inducibility, while another (CRM2-B) exhibits a diurnal response. Mutational analysis showed that three ABA RESPONSE ELEMENT LIKE (ABREL) motifs in CRM2-A and three EVENING ELEMENTs (EEs) in CRM2-B are essential in combination to confer a high amplitude diurnal pattern of expression. Genome-wide analysis identified characteristic spacing patterns of EEs and 71 A. thaliana promoters containing three EEs. Among these promoters, that of FLAVIN BINDING KELCH REPEAT F-BOX1 was analyzed in detail and shown to harbor a CRM functionally related to GI CRM2. Thus, combinatorial interactions among EEs and ABRELs confer diurnal patterns of transcription via an evolutionarily conserved module present in GI and other evening-expressed genes.

Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations.

BackgroundNemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site.MethodsWe produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT α- and β–tropomyosin, and for β-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro.ResultsWT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18).ConclusionsWe demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

Co-option of a photoperiodic growth-phase transition system during land plant evolution.

Photoperiodic control of the phase transition from vegetative to reproductive growth is critical for land plants. The GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) protein complex controls this process in angiosperms. However, little is known about how plants evolved this regulatory system. Here, we report that orthologues of GI and FKF1 are present in a basal plant, the liverwort Marchantia polymorpha, and describe the molecular interaction between their products. Knockout of either the GI or FKF1 orthologue completely abolishes the long-day-dependent growth-phase transition in M. polymorpha. Overexpression of either gene promotes growth-phase transition, even under short-day conditions. Introduction of the GI orthologue partially rescues the late-flowering phenotype of the Arabidopsis thaliana gi mutant. Our findings suggest that plants had already acquired the GI-FKF1 system to regulate growth-phase transition when they colonized land, and that this system was co-opted from gametophyte to sporophyte generation during evolution.

Diurnal expression of <i>CONSTANS-like</i> genes is independent of the function of cycling DOF factor (CDF)-like transcriptional repressors in <i>Physcomitrella patens</i>

The Dof-type transcriptional repressors CYCLING DOF FACTORS (CDFs) directly suppress the expression of CONSTANS (CO), which encodes a key regulator of photoperiodic gene expression, day-length perception and the floral transition in Arabidopsis thaliana. The genes encoding CDF-like (PpDof3 and PpDof4) and CO-like (PpCOL1-PpCOL3) proteins are present in the genome of the moss Physcomitrella patens, although P. patens lacks the genes encoding homologues of FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) and GIGANTEA (GI), which control the stability of CDFs in A. thaliana. In the current study, we investigated whether the functions of PpDof3 and PpDof4 are associated with the expression of PpCOL1-PpCOL3 in P. patens. We found that the diurnal expression patterns of PpDof3 and PpDof4 are similar to those of CDF genes and that like CDF1 from A. thaliana, PpDof3 and PpDof4 function as transcriptional repressors. However, targeted disruptions of PpDof3 and PpDof4 did not affect the expression of PpCOL1-PpCOL3, indicating that the expression of COLs is independent of the functions of PpDof3 and PpDof4 in P. patens.

Arabidopsis Kelch repeat F-box proteins regulate phenylpropanoid biosynthesis via controlling the turnover of phenylalanine ammonia-lyase.

Phenylalanine ammonia-lyase (PAL) catalyzes the first rate-limiting step in the phenylpropanoid pathway, which controls carbon flux to a variety of bioactive small-molecule aromatic compounds, and to lignin, the structural component of the cell wall. PAL is regulated at both the transcriptional and translational levels. Our knowledge about the transcriptional regulation of PAL is relatively comprehensive, but our knowledge of the molecular basis of the posttranslational regulation of PAL remains limited. Here, we demonstrate that the Arabidopsis thaliana Kelch repeat F-box (KFB) proteins KFB01, KFB20, and KFB50 physically interact with four PAL isozymes and mediate their proteolytic turnover via the ubiquitination-26S proteasome pathway. The KFB genes are differentially expressed in Arabidopsis tissues and respond to developmental and environmental cues. Up- or downregulation of their expression reciprocally affects the stability of the PAL enzymes, consequently altering the levels of phenylpropanoids. These data suggest that the KFB-mediated protein ubiquitination and degradation regulates the proteolysis of PALs, thus posttranslationally regulating phenylpropanoid metabolism. Characterizing the KFB-mediated proteolysis of PAL enzymes may inform future strategies for manipulating the synthesis of bioactive phenolics.

Identification and Molecular Characterization of FKF1 and GI Homologous Genes in Soybean

In Arabidopsis, FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) and GI (GIGANTEA) play important roles in flowering pathway through regulating daytime CO (CONSTANS) expression, and such a function is conserved across plants studied. But related reports are limited for soybean. In this study, we cloned FKF1 and GI homologs in soybean, and named as GmFKF1, GmFKF2, GmGI1, GmGI2, and GmGI3, respectively. GmGI1 had two alternative splicing forms, GmGI1α and GmGI1β. GmFKF1/2 transcripts were diurnally regulated, with a peak at zeitgeber time 12 (ZT12) in long days and at ZT10 in short days. The diurnal phases between GmGIs transcript levels greatly differed. GmGI2 expression was regulated by both the circadian clock and photoperiod. But the rhythmic phases of GmGI1 and GmGI3 expression levels were mainly conferred by long days. GmFKFs shared similar spatio-temporal expression profiles with GmGIs in all of the tissue/organs in different developmental stages in both LD and SD. Both GmFKF and GmGI proteins were targeted to the nucleus. Yeast two hybrid assays showed GmFKF1/GmFKF2 interacted with GmGI1/GmGI2/GmCDF1 (CYCLING DOF FACTOR CDF1 homolog in soybean); and the LOV (Light, Oxygen, or Voltage) domain in GmFKF1/GmFKF2 played an important role in these interactions. N-terminus of GmGI2 was sufficient to mediate its interaction with GmCDF1. Interestingly, N-terminus not full of GmGI3 interacted with GmFKF1/GmFKF2/GmCDF1. Ectopic over-expression of the GmFKF1 or GmFKF2 in Arabidopsis enhanced flowering in SD. Collectively, GmFKF and GmGI in soybean had conserved functional domains at DNA sequence level, but specific characters at function level with their homologs in other plants.

The F-Box Protein OsFBK12 Targets OsSAMS1 for Degradation and Affects Pleiotropic Phenotypes, Including Leaf Senescence, in Rice

Leaf senescence is related to the grain-filling rate and grain weight in cereals. Many components involved in senescence regulation at either the genetic or physiological level are known. However, less is known about molecular regulation mechanisms. Here, we report that OsFBK12 (an F-box protein containing a Kelch repeat motif) interacts with S-ADENOSYL-l-METHIONINE SYNTHETASE1 (SAMS1) to regulate leaf senescence and seed size as well as grain number in rice (Oryza sativa). Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays indicate that OsFBK12 interacts with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and with OsSAMS1. Biochemical and physiological data showed that OsFBK12 targets OsSAMS1 for degradation. OsFBK12-RNA interference lines and OsSAMS1 overexpression lines showed increased ethylene levels, while OsFBK12-OX lines and OsSAMS1-RNA interference plants exhibited decreased ethylene. Phenotypically, overexpression of OsFBK12 led to a delay in leaf senescence and germination and increased seed size, whereas knockdown lines of either OsFBK12 or OsSAMS1 promoted the senescence program. Our results suggest that OsFBK12 is involved in the 26S proteasome pathway by interacting with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and that it targets the substrate OsSAMS1 for degradation, triggering changes in ethylene levels for the regulation of leaf senescence and grain size. These data have potential applications in the molecular breeding of rice.

Identifying the Ubiquitin Ligase Complex That Regulates the NF1 Tumor Suppressor and Ras

The NF1 tumor suppressor protein neurofibromin is a negative regulator of Ras. Neurofibromin is dynamically regulated by the proteasome, and its degradation and reexpression are essential for maintaining appropriate levels of Ras-GTP. Like p53, NF1/neurofibromin can be inactivated in cancer by both mutations and excessive proteasomal destruction; however, little is known about the mechanisms that underlie this latter process. Here, we show that a Cullin 3 (Cul3)/kelch repeat and BTB domain-containing 7 complex controls both the regulated proteasomal degradation of neurofibromin and the pathogenic destabilization of neurofibromin in glioblastomas. Importantly, RNAi-mediated Cul3 ablation and a dominant-negative Cul3 directly stabilize neurofibromin, suppress Ras and extracellular signal-regulated kinase, and inhibit proliferation in an NF1-dependent manner. Moreover, in glioblastomas where neurofibromin is chronically destabilized, Cul3 inhibition restabilizes the protein and suppresses tumor development. Collectively, these studies show a previously unrecognized role for Cul3 in regulating Ras and provide a molecular framework that can be exploited to develop potential cancer therapies.

Plant F-box protein evolution is determined by lineage-specific timing of major gene family expansion waves.

F-box proteins (FBPs) represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in Arabidopsis thaliana, which carries a C-terminal F-box associated domain (FBA proteins) shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we de novo identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution.

Saltatory evolution of the ectodermal neural cortex gene family at the vertebrate origin.

The ectodermal neural cortex (ENC) gene family, whose members are implicated in neurogenesis, is part of the kelch repeat superfamily. To date, ENC genes have been identified only in osteichthyans, although other kelch repeat-containing genes are prevalent throughout bilaterians. The lack of elaborate molecular phylogenetic analysis with exhaustive taxon sampling has obscured the possible link of the establishment of this gene family with vertebrate novelties. In this study, we identified ENC homologs in diverse vertebrates by means of database mining and polymerase chain reaction screens. Our analysis revealed that the ENC3 ortholog was lost in the basal eutherian lineage through single-gene deletion and that the triplication between ENC1, -2, and -3 occurred early in vertebrate evolution. Including our original data on the catshark and the zebrafish, our comparison revealed high conservation of the pleiotropic expression pattern of ENC1 and shuffling of expression domains between ENC1, -2, and -3. Compared with many other gene families including developmental key regulators, the ENC gene family is unique in that conventional molecular phylogenetic inference could identify no obvious invertebrate ortholog. This suggests a composite nature of the vertebrate-specific gene repertoire, consisting not only of de novo genes introduced at the vertebrate origin but also of long-standing genes with no apparent invertebrate orthologs. Some of the latter, including the ENC gene family, may be too rapidly evolving to provide sufficient phylogenetic signals marking orthology to their invertebrate counterparts. Such gene families that experienced saltatory evolution likely remain to be explored and might also have contributed to phenotypic evolution of vertebrates.

Flowering time regulation: photoperiod- and temperature-sensing in leaves

Plants monitor changes in photoperiod and temperature to synchronize their flowering with seasonal changes to maximize fitness. In the Arabidopsis photoperiodic flowering pathway, the circadian clock-regulated components, such as FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 and CONSTANS, both of which have light-controlled functions, are crucial to induce the day-length specific expression of the FLOWERING LOCUS T (FT) gene in leaves. Recent advances indicate that FT transcriptional regulation is central for integrating the information derived from other important internal and external factors, such as developmental age, amount of gibberellic acid, and the ambient temperature. In this review, we describe how these factors interactively regulate the expression of FT, the main component of florigen, in leaves.

Interactions between Arabidopsis acyl-CoA-binding proteins and their protein partners

Protein-protein interactions are at the core of cellular interactomics and are essential for various biological functions. Since proteins commonly function as macromolecular complexes, it is important to identify their interacting partners to better understand their function and the significance in these interactions. The acyl-CoA-binding proteins (ACBPs) of eukaryotes show conservation in the presence of a lipid-binding acyl-CoA-binding domain. In Arabidopsis thaliana, four of six members from the AtACBP family possess ankyrin repeats (AtACBP1 and AtACBP2) or kelch motifs (AtACBP4 and AtACBP5), which can potentially mediate protein-protein interactions. Through yeast two-hybrid screens, a dozen putative protein partners interacting with AtACBPs have been isolated from an Arabidopsis cDNA library. Investigations in the past decade on the interaction between AtACBPs and their protein partners have revealed novel roles for AtACBPs, including functions in mediating oxidative stress responses, heavy metal tolerance and oxygen sensing. Recent progress and current questions on AtACBPs and their interactors are discussed in this review.

Update on the Kelch-like (KLHL) gene family

The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of β-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease.

Diverse Responses to Blue Light via LOV Photoreceptors

Plants utilize light not only as an energy source for photosynthesis, but also as an environmental cue to direct numerous developmental and physiological processes. For this purpose, plants have evolved two main types of photoreceptors: the phytochromes (Franklin and Quail 2010), which sense red/ far-red light, and the ultraviolet (UV)-A/blue light (BL)-sensing photoreceptors, which include cryptochrome (Liu et al. 2011), phototropin (Christie 2007) and other BL receptors described below. In addition, a third class of UV-B receptor, UVR8, was recently identified (Rizzini et al. 2011). The long history of research on BL receptors dates back to the initial observation made by the famous Charles Darwin, in which he found that plants were able to move towards light (Darwin and Darwin 1881). For a long time, the nature of the chromophore in BL receptors was much disputed as to whether it was a flavin or a carotenoid (Senger 1980). This issue was partially resolved through the identification of the first BL receptor, which corresponded to a flavin protein named cryptochrome (Ahmad and Cashmore 1993). Shortly after this discovery, the Briggs group demonstrated that the disrupted gene in Arabidopsis non-phototropic hypocotyl 1 (nph1) mutants encoded a BL receptor required for phototropic response (Huala et al. 1997, Christie et al. 1998). A homolog of NPH1, NON-PHOTOTROPIC HYPOCOTYL-LIKE 1 (NPL1), was soon identified (Kagawa et al. 2001); thereafter, NPH1 and NPL1 were renamed phototropin 1 (phot1) and phot2, respectively. Phototropins mediate diverse plant responses, such as chloroplast relocation movements (Kagawa et al. 2001, Sakai et al. 2001), stomatal opening (Kinoshita et al. 2001), early hypocotyl growth inhibition (Folta and Spaulding 2001), leaf flattening (Sakamoto and Briggs 2002), leaf positioning (Inoue et al. 2005), nuclear positioning (Iwabuchi et al. 2007, Tsuboi et al. 2007), sun tracking (Inoue et al. 2008b) and leaf photomorphogenesis (Kozuka et al. 2011), in which phot1 acts over a broad range of light intensities, whereas phot2 acts as a high-light sensor. These physiological responses contribute towards enhancing photosynthesis in the plant by increasing the absorption of light and CO2 (Takemiya et al. 2005). In addition to the two phototropins, a BL receptor family of proteins characteristically containing one light, oxygen or voltage (LOV) domain, an F-box and a Kelch repeat has been identified in Arabidopsis. These proteins include ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) and LOV KELCH PROTEIN 2 (LKP2), and are proposed to regulate the circadian clock and photoperiodic flowering by controlling BL-dependent protein degradation (Ito et al. 2012). A third type of LOV protein, aureochrome (AUREO), was identified in the stramenopile alga, Vaucheria frigida (Takahashi et al. 2007). AUREO has a basic region/leucine zipper (bZIP) domain as well as a LOV domain, and is thought to act as a BL-regulated transcription factor. Thus, the LOV BL receptor family in plants has divergent members who have a variety of physiological functions triggered by the photochemical reactions of FMN in the LOV domain.

Evolution of Three LOV Blue Light Receptor Families in Green Plants and Photosynthetic Stramenopiles: Phototropin, ZTL/FKF1/LKP2 and Aureochrome

Many organisms, including bacteria, fungi, animal, plants and algae, utilize blue light to adapt to a fluctuating light environment. Plants and algae, and photosynthetic stramenopiles in particular, require light energy for photosynthesis and have thus evolved a range of sophisticated light-sensing systems to utilize light information efficiently for growth, development and physiological responses. LOV (light, oxygen or voltage) domain photoreceptors are widely distributed among prokaryotic and eukaryotic organisms, and the number of specific LOV photoreceptors are increased in certain taxa. In this review, we summarize the molecular basis and physiological functions of three different families of LOV blue light receptors specific to green plants and photosynthetic stramenopiles: phototropin, ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LOV KELCH PROTEIN 2 (ZTL/FKF1/LKP2) and aureochrome.

A Kelch Motif‐Containing Serine/Threonine Protein Phosphatase Determines the Large Grain QTL Trait in Rice

A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F₂ population by crossing the large-grain japonica cultivar CW23 with Peiai 64 (PA64), an elite indica small-grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly-identified in this study. In particular, qGL3-1, a newly-identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine-mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution (C→A) at position 1092 in exon 10, resulting in replacement of Asp (D) in PA64 with Glu (E) in CW23 for the 364(th) amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near-isogenic line (NIL) for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection.

The Kelch Repeat Protein KLHDC10 Regulates Oxidative Stress-Induced ASK1 Activation by Suppressing PP5

Reactive oxygen species (ROS)-induced activation of Apoptosis signal-regulating kinase 1 (ASK1) plays crucial roles in oxidative stress-mediated cell death through the activation of the JNK and p38 MAPK pathways. However, the regulatory mechanism of ASK1 in the oxidative stress response remains to be elucidated. Here, we identified the kelch repeat protein, Slim, as an activator of ASK1 through a Drosophila misexpression screen. We also performed a proteomics screen and revealed that Kelch domain containing 10 (KLHDC10), a mammalian ortholog of Slim, interacted with Protein phosphatase 5 (PP5), which has been shown to inactivate ASK1 in response to ROS. KLHDC10 bound to the phosphatase domain of PP5 and suppressed its phosphatase activity. Moreover, KLHDC10 was required for H(2)O(2)-induced sustained activation of ASK1 and cell death in Neuro2A cells. These findings suggest that Slim/KLHDC10 is an activator of ASK1, contributing to oxidative stress-induced cell death through the suppression of PP5.

The P25 Pathogenicity Factor of Beet necrotic yellow vein virus Targets the Sugar Beet 26S Proteasome Involved in the Induction of a Hypersensitive Resistance Response via Interaction with an F-box Protein

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.