Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

Temperature-controlled atmospheric-pressure plasma treatment induces protein uptake via clathrin-mediated endocytosis in tobacco cells.

Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N2 plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.

A collection of inducible transcription factor-glucocorticoid receptor fusion lines for functional analyses in Arabidopsis thaliana.

Arabidopsis possesses approximately 2000 transcription factors (TFs) in its genome. They play pivotal roles in various biological processes but analysis of their function has been hampered by the overlapping nature of their activities. To uncover clues to their function, we generated inducible TF lines using glucocorticoid receptor (GR) fusion techniques in Arabidopsis. These TF-GR lines each express one of 1255 TFs as a fusion with the GR gene. An average 14 lines of T2 transgenic TF-GR lines were generated for each TF to monitor their function. To evaluate these transcription lines, we induced the TF-GR lines of phytochrome-interacting factor 4, which controls photomorphogenesis, with synthetic glucocorticoid dexamethasone. These phytochrome-interacting factor 4-GR lines showed the phenotype described in a previous report. We performed screening of the other TF-GR lines for TFs involved in light signaling under blue and far-red light conditions and identified 13 novel TF candidates. Among these, we found two lines showing higher anthocyanin accumulation under light conditions and we examined the regulating genes. These results indicate that the TF-GR lines can be used to dissect functionally redundant genes in plants and demonstrate that the TF-GR line collection can be used as an effective tool for functional analysis of TFs.

Nerve root morphological and functional changes after degenerative cervical myelopathy surgery: preliminary study using ultrasound and electrophysiology.

STUDY DESIGN: A prospective observational study. OBJECTIVES: To depict morphological and functional changes in the cervical nerve roots before and after spinal cord decompression surgery for degenerative cervical myelopathy (DCM). SETTING: A general hospital in Japan. METHODS: Thirteen DCM patients who underwent posterior spinal cord decompression surgery, laminoplasty or laminectomy, were included in this study. The neural foramen shown on MRI and the cross-sectional area (CSA) of the nerve roots on ultrasound were used to evaluate the C5 and C6 nerve roots. The compound muscle action potentials (CMAPs) of deltoid and biceps muscle were also recorded. RESULTS: All patients showed sensorimotor functional improvement without the postoperative C5 palsy after surgery. Foraminal stenosis and preoperative CSA of the nerve root: C4/5 foramen and C5 nerve root, C5/6 foramen and C6 nerve root, had no significant correlation (P = 0.53 and 0.08). CSA of the C5 nerve root displayed no significant change before and after surgery (P = 0.2), however, that of the C6 nerve root reduced significantly after surgery (P = 0.038). The amplitude of the deltoid and biceps CMAPs displayed no significant change before and after surgery (P = 0.05 and 0.05). CONCLUSION: The C6 nerve root CSA change was observed after spinal cord decompression surgery with functional recovery. However, deltoid and biceps CMAPs amplitude showed no significant change. Independent CSA changes on ultrasound might be useful when conducting a functional evaluation of the postoperative nerve root. SPONSORSHIP: The Grant of Japan Orthopaedics and Traumatology Research Foundation No. 395.

Pyogenic Lumbar Facet Joint Infection with Foot Drop.

Pyogenic facet joint infection (PFJI) is a relatively rare spinal infection. Clinical suspicion of this condition is a key for diagnosis. We report a case of PFJI which required decompression surgery for severe neurological dysfunction. The patient was a 44-year-old woman who had a previous history of orthotic therapy for idiopathic scoliosis. The patient was admitted to our hospital with a history of two days of high fever and severe low back pain. There was no neurologic deficit, and blood tests revealed high levels of inflammatory markers. There was a slight amount of fluid that had collected at L4/5 facet joint in lumbar MRI. She was admitted for examination and treatment of fever of unknown origin and low back pain. Antibiotic treatment started the day after hospitalization since the first report of the blood culture taken upon admission tested positive to gram-positive cocci. As low back pain and fever persisted, an MRI was taken again on the fifth day of hospitalization. Repeated MRI showed fluid extension from the left facet joint to paravertebral muscles and epidural space. She was diagnosed with PFJI, and facet joint puncture was performed. At this time, it became clear that she had foot drop on the right, the contralateral side of the PFJI. She underwent irrigation, debridement, and partial laminectomy. Methicillin-sensitive Staphylococcus aureus (MSSA) was detected in blood cultures at the time of hospitalization, in the puncture fluid and tissue collected during surgery. The patient recovered completely from foot drop after the operation and a three-month course of antibiotics. As the imaging findings may be inadequate in the early stages of onset and PFJI potentially causes neurologic deficit such as foot drop, neurological findings need to be carefully observed even after hospitalization and one should reexamine the MRI if symptoms or clinical findings did not improve or were aggravated.

I-SceI Endonuclease-Mediated Plant Genome Editing by Protein Transport through a Bacterial Type III Secretion System.

Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.

Laser Doppler blood flowmeter as a useful instrument for the early detection of lower extremity peripheral arterial disease in hemodialysis patients: an observational study.

BACKGROUND: A simpler method for detecting atherosclerosis obliterans is required in the clinical setting. Laser Doppler flowmetry (LDF) is easy to perform and can accurately detect deterioration in skin perfusion. We performed LDF for hemodialysis patients to determine the correlations between blood flow in the lower limbs and peripheral arterial disease (PAD). METHODS: This retrospective study included 128 hemodialysis patients. Patients were categorized into the non-PAD group (n = 106) and PAD group (n = 22), 14 early stage PAD patients were included in the PAD group. We conducted LDF for the plantar area and dorsal area of the foot and examined skin perfusion pressure (SPP) during dialysis. RESULTS: SPP-Dorsal Area values were 82.1 ± 22.0 mmHg in the non-PAD, and 59.1 ± 20.3 mmHg in PAD group, respectively (p < 0.05). The LDF-Plantar blood flow (Qb) values were 32.7 ± 15.5 mL/min in non-PAD group and 21.5 ± 11.3 mL/min in PAD group (p < 0.001). A total of 21 non-PAD patients underwent LDF before and during dialysis. The LDF-Plantar-Qb values were 36.5 ± 17.6 mL/min before dialysis and 29.6 ± 17.7 mL/min after dialysis (p < 0.05). We adjusted SPP and LDF for PAD using logistic regression, SPP-Dorsal-Area and LDF-P were significantly correlated with PAD (p < 0.05). The receiver-operating characteristic curve analysis indicated cut-off values of 20.0 mL/min for LDF-Plantar-Qb during dialysis. CONCLUSION: LDF is a simple technique for sensitive detection of early-stage PAD. This assessment will help physicians identify early-stage PAD, including Fontaine stage II in clinical practice, thereby allowing prompt treatment.

Syntheses, Structures, and Antimicrobial Activities of Gold(I)- and Copper(I)- N-Heterocyclic Carbene (NHC) Complexes Derived from Basket-Shaped Dinuclear Ag(I)-NHC Complex.

Novel dinuclear gold(I)- and copper(I)- N-heterocyclic carbene (NHC, L-1a) complexes [M2(L-1a)](PF6)2 (M = AuI (Au-1·PF6) and M = CuI (Cu-1·PF6)) were synthesized by transmetalation of the dinuclear silver(I)-NHC complex [Ag2(L-1a)](PF6)2 (Ag-1·PF6) with [AuCl(Me2S)] or CuI in over 70% yield. These NHC complexes were characterized by CHN elemental analysis, Fourier transform infrared spectroscopy, thermogravimetry/differential thermal analysis, and solution (1H and 13C) NMR spectroscopy. X-ray crystallography revealed that Au-1·PF6 and Cu-1·PF6 are dinuclear molecules consisting of two linear intramolecular C-M-C bonds (M = AuI and CuI), one M···M interaction, and two metal atoms arranged in a T-shaped geometry; their molecular structures are very similar to the basket-shaped cage structure of the parent complex Ag-1·PF6. Because of the smaller ionic radius of copper(I), Cu-1·PF6 has the smallest upper space of the basket among the three complexes. Au-1·PF6 and Cu-1·PF6 were very stable in the solid state and in solution. They did not undergo nucleophilic reaction with thiols 2-mercaptoethanol and 2-benzimidazolethiol (Hbmt) at least for several hours, whereas Ag-1·PF6 did react, forming precipitates of silver(I) thiolate and the free ligand L-1a·PF6. The minimum inhibitory concentration values toward a panel of bacteria, yeasts, and molds were examined in a water-suspension system and a solution system containing dimethyl sulfoxide. The antimicrobial spectra of the three compounds were metal-dependent, and Au-1·PF6 showed the greatest activity toward Gram-positive bacteria.

Stress granule formation is induced by a threshold temperature rather than a temperature difference in Arabidopsis.

Stress granules, a type of cytoplasmic RNA granule in eukaryotic cells, are induced in response to various environmental stresses, including high temperature. However, how high temperatures induce the formation of these stress granules in plant cells is largely unknown. Here, we characterized the process of stress granule formation in Arabidopsis thaliana by combining live imaging and electron microscopy analysis. In seedlings grown at 22°C, stress granule formation was induced at temperatures above a critical threshold level of 34°C in the absence of transpiration. The threshold temperature was the same, regardless of whether the seedlings were grown at 22°C or 4°C. High-resolution live imaging microscopy revealed that stress granule formation is not correlated with the sizes of pre-existing RNA processing bodies (P-bodies) but that the two structures often associated rapidly. Immunoelectron microscopy revealed a previously unidentified characteristic of the fine structures of Arabidopsis stress granules and P-bodies: the lack of ribosomes and the presence of characteristic electron-dense globular and filamentous structures. These results provide new insights into the universal nature of stress granules in eukaryotic cells.

Direct protein introduction into plant cells using a multi-gas plasma jet.

Protein introduction into cells is more difficult in plants than in mammalian cells, although it was reported that protein introduction was successful in shoot apical meristem and leaves only together with a cell-penetrating peptide. In this study, we tried to introduce superfolder green fluorescent protein (sGFP)-fused to adenylate cyclase as a reporter protein without a cell-penetrating peptide into the cells of tobacco leaves by treatment with atmospheric non-thermal plasmas. For this purpose, CO2 or N2 plasma was generated using a multi-gas plasma jet. Confocal microscopy indicated that sGFP signals were observed inside of leaf cells after treatment with CO2 or N2 plasma without substantial damage. In addition, the amount of cyclic adenosine monophosphate (cAMP) formed by the catalytic enzyme adenylate cyclase, which requires cellular calmodulin for its activity, was significantly increased in leaves treated with CO2 or N2 plasma, also indicating the introduction of sGFP-fused adenylate cyclase into the cells. These results suggested that treatment with CO2 or N2 plasma could be a useful technique for protein introduction into plant tissues.

AtSKIP18 and AtSKIP31, F-box subunits of the SCF E3 ubiquitin ligase complex, mediate the degradation of 14-3-3 proteins in Arabidopsis.

14-3-3 proteins regulate numerous cellular processes through interaction with their target proteins in a phosphorylation dependent manner. Although proteins that are regulated by 14-3-3s have been studied, the regulatory mechanism of 14-3-3s is poorly understood. In the present study, F-box proteins, a component of Skp1-Cullin-F-box E3 ubiquitin ligase, were identified as 14-3-3 targets using yeast two-hybrid screening. Among them, AtSKIP18 and AtSKIP31, were shown to mediate the degradation of Arabidopsis 14-3-3s. Mutational analyses of AtSKIP18 and AtSKIP31 indicated that the phosphorylation of AtSKIPs is critical for interaction and degradation of 14-3-3s. The loss-of-function mutation in AtSKIP31 resulted in enhanced primary root growth under nitrogen deficient conditions. These findings suggest that AtSKIP31 regulates the primary root growth in nitrogen deficiency via degrading 14-3-3s.

Synthesis, characterization, and structure-activity relationship of the antimicrobial activities of dinuclear N-heterocyclic carbene (NHC)-silver(I) complexes.

The three dinuclear silver(I) complexes of N-heterocyclic carbene (NHC) ligands, [Ag2(L-1a)](PF6)2·2CH3CN (Ag-1a), [Ag2(L-2)2](PF6)2·H2O (Ag-2), and [Ag2(L-4)2](PF6)2·2CH3CN (Ag-4), were synthesized by reactions of Ag2O with the corresponding PF6- salt of the NHC precursors indicated as H4L-1(PF6)4, H2L-2(PF6)2, and H2L-4(PF6)2. H4L-1(PF6)4, which is the precursor of ligand L-1, was formed as mixed crystals of two geometric isomers, i.e., H4L-1a(PF6)4 (major) and H4L-1b(PF6)4 (minor), each of which was not isolated as single species. Ag2O reacted with the mixed isomers of H4L-1(PF6)4 to give a single, pure crystalline silver(I) complex Ag-1a with one isomer (L-1a) as the major product. The molecular structures of the precursors and their silver(I) complexes were determined by X-ray crystallography. A mononuclear NHC-silver(I) complex (Ag-3) was prepared by the reaction of Ag2O with the precursor HL-3Cl. The silver(I) complexes and NHC precursors prepared here were characterized by CHN elemental analysis, FTIR, Thermogravimetry/Differential thermal analysis, X-ray crystallography and solution (1H and 13C) NMR spectroscopy. Organometallic silver(I) complexes Ag-1a, Ag-2, and Ag-4 were dinuclear C-Ag-C bonding complexes, whereas Ag-3 was a mononuclear C-Ag-Cl bonding complex. These complexes are highly soluble in organic solvents such as acetone, acetonitrile, and dimethyl sulfoxide, and light-stable in the solid-state and in solution over one year. The antimicrobial activities of four silver(I) complexes and their NHC precursors against selected bacteria, yeasts, and molds in water-suspension systems were evaluated via the minimum inhibitory concentration; the activities were strongly dependent on the molecular structures of the dinuclear silver(I) complexes, which suggests a structure-activity relationship.

Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco.

The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.

Proteomic analysis of the 26S proteasome reveals its direct interaction with transit peptides of plastid protein precursors for their degradation.

The 26S proteasome is an ATP-dependent proteinase complex that is responsible for regulated proteolysis of polyubiquitinated proteins in eukaryotic cells. Here, we report novel 26S proteasome interacting proteins in Arabidopsis as revealed by LC-MS/MS analysis. We performed a two-step screening process that involved affinity purification of the 26S proteasome using Arabidopsis plants expressing a FLAG-tagged RPT2a subunit and partial purification of the 26S proteasome from cultured cells by glycerol density gradient centrifugation (GDG). Two plastid proteins, LTA2 and PDH E1α, which were commonly identified by both affinity purification and GDG, interacted with the 26S proteasome both in vitro and in vivo, and the transit peptides of LTA2 and PDH E1α were necessary for the interaction. Furthermore, the degradation of both LTA2 and PDH E1α was inhibited by MG132, a proteasome inhibitor. Similar to those two proteins, 26S proteasome subunits RPT2a/b and RPT5a interacted with the transit peptides of three other chloroplast proteins, which are known to be substrates of the ubiquitin-26S proteasome system. These results suggest that a direct interaction between the 26S proteasome and a transit peptide is important for the degradation of unimported plastid protein precursors to maintain cellular homeostasis.

Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis.

CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.

Cell wall proteomics of crops.

Cell wall proteins play key roles in cell structure and metabolism, cell enlargement, signal transduction, responses to environmental stress, and many other physiological events. Agricultural crops are often used for investigating stress tolerance because cultivars with differing degrees of tolerance are available. Abiotic and biotic stress factors markedly influence the geographical distribution and yields of many crop species. Crop cell wall proteomics is of particular importance for improving crop productivity, particularly under unfavorable environmental conditions. To better understand the mechanisms underlying stress response in crops, cell wall proteomic analyses are being increasingly utilized. In this review, the methods of purification and purity assays of cell wall protein fractions from crops are described, and the results of protein identification using gel-based and gel-free proteomic techniques are presented. Furthermore, protein composition of the cell walls of rice, wheat, maize, and soybean are compared, and the role of cell wall proteins in crops under flooding and drought stress is discussed. This review will be useful for clarifying the role of the cell wall of crops in response to environmental stresses.

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis.

Angiosperms have a unique sexual reproduction system called "double fertilization." One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.

A comprehensive analysis of interaction and localization of Arabidopsis SKP1-like (ASK) and F-box (FBX) proteins.

F-Box (FBX) proteins are encoded by a multigene family present in major lineages of eukaryotes. A number of FBX proteins are shown to be subunits of SCF complex, a type of E3 ligases composed of SKP1, CULLIN, FBX and RBX1 proteins. The Arabidopsis SKP-LIKE (ASK) proteins are also members of a family and some of them interact with FBX proteins directly. To clarify how FBX and ASK proteins combine, we carried out a large-scale interaction analysis between FBX and ASK proteins using yeast two-hybrid assay (Y2H) in Arabidopsis thaliana. FBX proteins randomly chosen from those proteins that interacted with more than one ASK protein were further analyzed for their subcellular localization and in vivo interaction with ASK proteins. Furthermore, the expression profiles of FBX and ASK genes were compared. This work reveals that FBX proteins had a preference for interacting with ASK proteins depending on the domains they contain such as the FBX-associated (FBA) domain, the Kelch domain and leucine rich repeat (LRR). In addition, it was found that a single FBX protein could form multiple SCF complexes by interacting with several ASK proteins in many cases. Furthermore, it was suggested that the variation of SCF complexes were especially abundant in tissues related to male gametophyte and seed development. More than half of the FBX proteins studied did not interact with any of the ASK proteins, implying the necessity for certain regulations for their interaction in vivo and/or distinct roles from subunits of the SCF complex.