SPA Proteins Research Articles

Genome-Wide Characterization of Tomato FAD Gene Family and Expression Analysis under Abiotic Stresses.

The fatty acid desaturase (FAD) gene family plays a crucial regulatory role in the resistance process of plant biomembranes. To understand the role of FADs in tomato growth and development, this study identified and analyzed the tomato FAD gene family based on bioinformatics analysis methods. In this study, 26 SlFADs were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that the SlFAD gene family was divided into six branches, and the exon-intron composition and conserved motifs of SlFADs clustered in the same branch were quite conservative. Several hormone and stress response elements in the SlFAD promoter suggest that the expression of SlFAD members is subject to complex regulation; the construction of a tomato FAD protein interaction network found that SlFAD proteins have apparent synergistic effects with SPA and GPAT proteins. qRT-PCR verification results show that SlFAD participates in the expression of tomato root, stem, and leaf tissues; SlFAD8 is mainly highly expressed in leaves; SlFAD9 plays a vital role in response to salt stress; and SlFAB5 regulates all stages of fruit development under the action of exogenous hormones. In summary, this study provides a basis for a systematic understanding of the SlFAD gene family. It provides a theoretical basis for in-depth research on the functional characteristics of tomato SlFAD genes.

SP-A and SP-D surfactant proteins and conventional risk factors for chronic non-infectious human diseases

Surfactant proteins SP-A and SP-D, which belong to the family of collagen-containing type C lectins, are used as diagnostic and prognostic markers for many acute and chronic respiratory diseases. The aim of the study is to assess the impact of conventional risk factors for chronic non-infectious diseases on SP-A and SP-D protein levels by means of systemic and structural analysis on the basis of relevant publications from international databases and official WHO reports. This analytical review concludes that widespread expression of SP-A and SP-D is documented in numerous studies, and, although the lungs remain the main site of synthesis of surfactant proteins, one can expect its significant impact on the immune and inflammatory response in many organs and tissues. The authors note that there are several known extrapulmonary effects of these proteins. However, many mechanisms of additional cellular effects of SP-A and SP-D outside the bronchopulmonary system still remain unstudied, which indicates the prospects for further research in this area.

Regulation of Surfactant Protein Gene Expression by Aspergillus fumigatus in NCl-H441 Cells.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

Co-action of COP1, SPA and cryptochrome in light signal transduction and photomorphogenesis of the moss Physcomitrium patens.

The Arabidopsis COP1/SPA ubiquitin ligase suppresses photomorphogenesis in darkness. In the light, photoreceptors inactivate COP1/SPA to allow a light response. While SPA genes are specific to the green lineage, COP1 also exists in humans. This raises the question of when in evolution plant COP1 acquired the need for SPA accessory proteins. We addressed this question by generating Physcomitrium Ppcop1 mutants and comparing their visible and molecular phenotypes with those of Physcomitrium Ppspa mutants. The phenotype of Ppcop1 nonuple mutants resembles that of Ppspa mutants. Most importantly, both mutants produce green chloroplasts in complete darkness. They also exhibit dwarfed gametophores, disturbed branching of protonemata and absent gravitropism. RNA-sequencing analysis indicates that both mutants undergo weak constitutive light signaling in darkness. PpCOP1 and PpSPA proteins form a complex and they interact via their WD repeat domains with the VP motif of the cryptochrome CCE domain in a blue light-dependent manner. This resembles the interaction of Arabidopsis SPA proteins with Arabidopsis CRY1, and is different from that with Arabidopsis CRY2. Taken together, the data indicate that PpCOP1 and PpSPA act together to regulate growth and development of Physcomitrium. However, in contrast to their Arabidopsis orthologs, PpCOP1 and PpSPA proteins execute only partial suppression of light signaling in darkness. Hence, additional repressors may exist that contribute to the repression of a light response in dark-exposed Physcomitrium.

The role of surfactant proteins SP-A and SP-D in viral infection: a focus on COVID-19

An immune response to invasion of viral pathogens is an integral part of maintaining the physiological functioning of the bronchopulmonary system and effective gas exchange. Collagen-containing C-type lectins (lung collectins) are some of the key proteins in the identification of viral particles. They have image-recognizing receptors that identify pathogen-associated molecular patterns, particularly viral glycoproteins. The surfactant proteins SP-A and SP-D, which are composed of trimerized units, belong to pulmonary collectins and oligomerize into higher-order structures. These proteins play an essential role in recognition and elimination of microbial pathogens (viruses, bacteria, fungi, parasites, nanoparticles, allergens) through a variety of mechanisms. Taking into account the burden of the novel coronavirus infection caused by the SARS-CoV-2 virus, it is important to consider the role of the surfactant proteins SP-A and SP-D in the pathogenesis of the immune response to viral invasion. Currently, there are data on the direct relationship between surfactant proteins and viruses belonging to the Coronaviridae family. The SP-A and SP-D proteins modulate inflammatory responses and cytokine synthesis, but prevent an excessive inflammatory response (cytokine storm). There is also an assumption that SARSCoV-2 directly suppresses and alters the production of surfactant proteins. Thus, the key pathogenetic role of the surfactant proteins SP-A and SP-D in the response to the viral pathogen SARS-CoV-2 is evident. Today, this is a promising area of translational medicine, which will contribute to a profound understanding of the pathogenesis of coronavirus infection for assessing the diagnostic and prognostic potentials of the surfactant proteins SP-A and SP-D in COVID-19. Additionally, it will help evaluate the therapeutic potential of recombinant fragments of human SP-A and SP-D.

INDOMETHACIN CAN PROTECT PLACENTAL INFLAMMATION AND DELAY PRETERM BIRTH IN THE LPS-INDUCED PRETERM DELIVERY MODEL

The major cause of prematurity is preterm birth (PTB), associated with intrauterine inflammation. Defects in the Notch pathway harm placentation, and there is evidence between Notch activation and the inflammatory environment. In the action of PTB, surfactant A (SP-A) may have a pro-inflammatory or anti-inflammatory effect, and increased synthesis of prostaglandins illustrates their crucial roles in gestational tissues at parturition. Altogether, the potential of SP-A and prostaglandin inhibitors to prevent PTB through the placenta is worth exploring. This study evaluates the preventive effect of SP-A and Indomethacin (IND) treatment on placental inflammation in the LPS-induced PTB model. Forty-eight female CD-1 mice were distributed to pregnant control (PC), Sham, PBS, IND (2 mg/kg; intraperitoneally), LPS (25μg/100μl; intrauterine), LPS+IND, SP-A block (SP-A B; 20ug/100μl; intrauterine) groups. The injections were performed on day 14.5 of pregnancy. Placentae were removed on day 15.5 of pregnancy, and immunohistochemical analyzes were performed. Differences in staining intensities between the groups for Cox-1, Notch-1 (N1), Dll-1, Jagged-2 (Jag-2), SP-A, Tlr-2, and Tlr-4 proteins were compared using ANOVA and Sidak’s Multiple Comparison test. P values <0.05 were considered statistically significant. PTB rates were; 100%, 66% (in this group, delivery delayed for about 5 hours), and 50% in LPS, LPS+IND, SP-A B groups, respectively. LPS application caused damage to fetal and maternal vascular structures in the placenta, especially in the labyrinth zone (LZ). Placental volume decreased, and lymphocyte infiltration was observed. The morphological distinction between the compartments was unclear. N1 expression increased in both the junctional zone (JZ) and LZ. Cox-1 expression in the LZ decreased significantly (p<0.05), while the expression of N1, Dll- 1, and Jag-2 increased significantly (p<0.05). Tlr-2 and Tlr-4 expression increased significantly in LZ and JZ, respectively. In the LPS+IND group, the LZ morphology was similar to the control, and placenta zone boundaries were distinguishable. In the LPS+IND group, N1, Jag-2, and Tlr-4 expression decreased significantly (p<0.05). In the SP-A B group, Cox-1 expression increased significantly (p<0.05). In the PTB model, Notch signaling, SP-A, and prostaglandin-associated signaling are disturbed in the maternal-fetal exchange site, the LZ and hormonal production site, the JZ of the placentae. While SP-A modulates the LPS-induced inflammatory response related to PTB, IND can prevent PTB via decreasing inflammation in the LZ.

Functional comparison of the WD-repeat domains of SPA1 and COP1 in suppression of photomorphogenesis.

The Arabidopsis COP1/SPA complex acts as a cullin4-based E3 ubiquitin ligase to suppress photomorphogenesis in darkness. It is a tetrameric complex of two COP1 and two SPA proteins. Both COP1 and SPA are essential for the activity of this complex, and they both contain a C-terminal WD-repeat domain responsible for substrate recruitment and binding of DDB1. Here, we used a WD domain swap-approach to address the cooperativity of COP1 and SPA proteins. We found that expression of a chimeric COP1 carrying the WD-repeat domain of SPA1 mostly complemented the cop1-4-mutant phenotype in darkness, indicating that the WD repeat of SPA1 can replace the WD repeat of COP1. In the light, SPA1-WD partially substituted for COP1-WD. In contrast, expression of a chimeric SPA1 protein carrying the WD repeat of COP1 did not rescue the spa-mutant phenotype. Together, our findings demonstrate that a SPA1-type WD repeat is essential for COP1/SPA activity, while a COP1-type WD is in part dispensible. Moreover, a complex with four SPA1-WDs is more active than a complex with only two SPA1-WDs. A homology model of SPA1-WD based on the crystal structure of COP1-WD uncovered two insertions and several amino acid substitutions at the predicted substrate-binding pocket of SPA1-WD.

Illuminating the COP1/SPA Ubiquitin Ligase: Fresh Insights Into Its Structure and Functions During Plant Photomorphogenesis.

CONSTITUTIVE PHOTOMORPHOGENIC 1 functions as an E3 ubiquitin ligase in plants and animals. Discovered originally in Arabidopsis thaliana, COP1 acts in a complex with SPA proteins as a central repressor of light-mediated responses in plants. By ubiquitinating and promoting the degradation of several substrates, COP1/SPA regulates many aspects of plant growth, development and metabolism. In contrast to plants, human COP1 acts as a crucial regulator of tumorigenesis. In this review, we discuss the recent important findings in COP1/SPA research including a brief comparison between COP1 activity in plants and humans.

Association of SP-A and SP-D Surfactant Proteins with the Severity of CommunityAcquired Pneumonia

Relevance. In current clinical practice, there is a need for research to find new diagnostic tests for the purpose of determining the patients with the highest risk of death from pneumonia. Surfactant proteins SP-A and SP-D play a key role in the pathogenesis of the response to microbial invasion of lung tissue, which participate in a cascade of reactions of both innate and adaptive immunity, and therefore proteins SP-A and SP-D may be considered as markers of the severity of community-acquired pneumonia (CAP).Aim of study. To evaluate the associations of surfactant proteins SP-A and SP-D in blood plasma with the severity of CAP.Material and methods. The study included 247 patients admitted to the therapeutic department. The group of patients with CAP (n=188) was divided into groups of severe (n=103) and non-severe (n=85) pneumonia. The comparison group (n=59) consisted of patients without acute and chronic diseases of the bronchi and lungs. The mean age (years, Me, 25th; 75th percentile) of patients was 55 (47; 68), 55 (47; 70), and 61 (37; 63) years, respectively. All patients underwent clinical, functional, diagnostic and laboratory studies (including determination of the content of SP-A and SP-D proteins by enzyme immunoassay).Results. In the group of patients with severe pneumonia unlike mild pneumonia, and group of comparison higher levels of proteins SP-A and SP-D were observed. Correlation analysis described below revealed statistically significant connection: protein SP-D — direct relation with leukocyte levels (r=0.320, p&lt;0.0001), erythrocyte sedimentation rate (r=0.331, p&lt;0.0001), inverse relation with blood oxygen saturation (r=-0.407, p&lt;0.0001), for SP-A protein — direct relation with body temperature (r=0.355, p&lt;0.0001), erythrocyte sedimentation rate (r=0.369, p&lt;0.0001) in the blood C-reactive protein (r=0.446, p&lt;0.0001), SP-D (r=0.357, p&lt;0.0001), and also relation with the duration of clinical symptoms (r=0.528, p&lt;0.0001) and blood oxygen saturation (r=-0.401, p&lt;0.0001). When conducting ROCanalysis for the surfactant protein SP-A, the area under the ROC- curve was 0.70, the optimal sensitivity for severe pneumonia was 68%, the specificity was 69% at the SP-A level in blood plasma equal to 42.9 ng/ml. When performing ROC analysis for the surfactant protein SP-D, the area under the ROC curve was 0.64 for severe pneumonia, the optimal sensitivity was 62%, and the specificity was 62% at the SP-D content in blood plasma equal to 319.2 ng/ml.Conclusion. According to the results of this study, the SP-A and SP-D proteins are associated with clinical and laboratory signs that reflect the severity of CAP. Thus, SP-A and SP-D are new laboratory markers of CAP severity.

Exoproteomic profiling uncovers critical determinants for virulence of livestock-associated and human-originated Staphylococcus aureus ST398 strains

ABSTRACT Staphylococcus aureus: with the sequence type (ST) 398 was previously associated with livestock carriage. However, in recent years livestock-independent S. aureus ST398 has emerged, representing a potential health risk for humans especially in nosocomial settings. Judged by whole-genome sequencing analyses, the livestock- and human originated strains belong to two different S. aureus ST398 clades but, to date, it was not known to what extent these clades differ in terms of actual virulence. Therefore, the objective of this study was to profile the exoproteomes of 30 representative S. aureus ST398 strains by mass spectrometry, to assess clade-specific differences in virulence factor secretion, and to correlate the identified proteins and their relative abundance to the strains’ actual virulence. Although the human-originated strains are more heterogeneous at the genome level, our observations show that they are more homogeneous in terms of virulence factor production than the livestock-associated strains. To assess differences in virulence, infection models based on larvae of the wax moth Galleria mellonella and the human HeLa cell line were applied. Correlation of the exoproteome data to larval killing and toxicity toward HeLa cells uncovered critical roles of the staphylococcal Sbi, SpA, SCIN and CHIPS proteins in virulence. These findings were validated by showing that sbi or spa mutant bacteria are attenuated in G. mellonella and that the purified SCIN and CHIPS proteins are toxic for HeLa cells. Altogether, we show that exoproteome profiling allows the identification of critical determinants for virulence of livestock-associated and human-originated S. aureus ST398 strains.

Effects of smoking on the level of sp-a and sp-d surfactant proteins in the blood of patients without bronchopulmonary diseases

Every year, about six million people die from tobacco use. Respiratory epithelium is the first line of defense against exogenous invasion, in particular, harmful inhaled particles, pathogens and allergens. However, the epithelium of the respiratory tract is also a regulator of immunological and inflammatory reactions through secretion of inflammation and immune cell recruitment mediators. An important component of the pulmonary immune system is the surfactant, and, in particular, its proteins SP-A and SP-D, synthesized mainly by type II pneumocytes. Aim . To assess the levels of surfactant proteins SP-A and SP-D in the blood of smoking patients without bronchopulmonary diseases. Materials and мethods . The study included 59 patients admitted to the department of internal medicine with hypertension. The general group was divided into subgroups: non-smoking patients ( n = 31) and healthy smokers ( n = 28). All patients underwent clinical, functional, diagnostic and laboratory tests. The content of surfactant proteins SP-A and SP-D in the blood was determined by enzyme immunoassay. Results. The subgroups did not differ in sex, age, height, body weight, blood pressure, heart rate, respiratory rate, and the distribution of comorbidities. The subgroups differed in the platelet level; in other main parameters of complete blood count and blood biochemistry no differences were revealed. It was found that the blood levels of surfactant proteins SP-A and SP-D in the subgroup of healthy smokers were significantly higher in comparison with the subgroup of non-smoking patients. The correlation analysis revealed a direct relationship between surfactant proteins SP-A and SP-D and smoking ( R = 0.360, p = 0.006, R = 0.274, p = 0.037), a negative correlation between SP-D protein and age ( R = –0.315, p = 0.016), and a direct relationship between SP-A protein and diastolic blood pressure ( R = 0.271, p = 0.039). In the non-smoking subgroup, a negative correlation between SP-D and age ( R = –0.438, p = 0.016) and between SP-D and systolic blood pressure ( R = –0.433, p = 0.017) was identified. Conclusion. The direct relationship between higher levels of the surfactant proteins SP-A and SP-D and smoking in the group of healthy smokers is justified (inflammatory changes, structural abnormalities in the lung parenchyma under the influence of cigarette smoke). The SP-D protein is more significant in comparison with the SP-A protein in vascular wall remodeling, lung tissue matrix, oxidative lung tissue damage, and apoptosis, which explains its negative correlation with age and systolic blood pressure.

A COP1-PIF-HEC regulatory module fine-tunes photomorphogenesis in Arabidopsis.

Light responses mediated by the photoreceptors play crucial roles in regulating different aspects of plant growth and development. An E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)1/SUPPRESSOR OF PHYA (SPA), one of the central repressors of photomorphogenesis, is critical for maintaining skotomorphogenesis. It targets several positive regulators of photomorphogenesis for degradation in darkness. Recently, we revealed that basic helix-loop-helix factors, HECATEs (HECs), function as positive regulators of photomorphogenesis by directly interacting and antagonizing the activity of another group of repressors called PHYTOCHROME-INTERACTING FACTORs (PIFs). It was also shown that HECs are partially degraded in the dark through the ubiquitin/26S proteasome pathway. However, the underlying mechanism of HEC degradation in the dark is still unclear. Here, we show that HECs also interact with both COP1 and SPA proteins in darkness, and that HEC2 is directly targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway. Moreover, COP1-mediated polyubiquitylation and degradation of HEC2 are enhanced by PIF1. Therefore, the ubiquitylation and subsequent degradation of HECs are significantly reduced in both cop1 and pif mutants. Consistent with this, the hec mutants partially suppress photomorphogenic phenotypes of both cop1 and pifQ mutants. Collectively, our work reveals that the COP1/SPA-mediated ubiquitylation and degradation of HECs contributes to the coordination of skoto/photomorphogenic development in plants.

Genomic evidence reveals SPA-regulated developmental and metabolic pathways in dark-grown Arabidopsis seedlings.

Photomorphogenesis is repressed in the dark mainly by an E3 ubiquitin ligase complex comprising CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and four homologous proteins called SUPPRESSOR OF PHYA-105 (SPA1-SPA4) in Arabidopsis. This complex induces the ubiquitination and subsequent degradation of positively acting transcription factors (TFs; e.g. ELONGATED HYPOCOTYL (HY5), LONG HYPOCOTYL IN FAR-RED 1 (HFR1), PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) and others] in the dark to repress photomorphogenesis. Genomic evidence showed a large number of genes regulated by COP1 in the dark, of which many are direct targets of HY5. However, the genomic basis for the constitute photomorphogenic phenotype of spaQ remains unknown. Here, we show that >7200 genes are differentially expressed in the spaQ background compared to wild-type in the dark. Comparison of the RNA sequencing (RNA-Seq) data between cop1 and spaQ revealed a large overlapping set of genes regulated by the COP1-SPA complex. In addition, many of the genes coordinately regulated by the COP1-SPA complex are also regulated by HY5 directly and indirectly. Taken together, our data reveal that SPA proteins repress photomorphogenesis by controlling gene expression in concert with COP1, likely through regulating the abundance of downstream TFs in light signaling pathways. Moreover, SPA proteins may function both in a COP1-dependent and -independent manner in regulating many biological processes and developmental pathways in Arabidopsis.

A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) is a highly conserved E3 ubiquitin ligase from plants to animals and acts as a central repressor of photomorphogenesis in plants. SUPPRESSOR OF PHYA-105 1 family members (SPA1-SPA4) directly interact with COP1 and enhance COP1 activity. Despite the presence of a kinase domain at the N-terminus, no COP1-independent role of SPA proteins has been reported. Here we show that SPA1 acts as a serine/threonine kinase and directly phosphorylates PIF1 in vitro and in vivo. SPAs are necessary for the light-induced phosphorylation, ubiquitination and subsequent degradation of PIF1. Moreover, the red/far-red light photoreceptor phyB interacts with SPA1 through its C-terminus and enhances the recruitment of PIF1 for phosphorylation. These data provide a mechanistic view on how the COP1-SPA complexes serve as an example of a cognate kinase-E3 ligase complex that selectively triggers rapid phosphorylation and removal of its substrates, and how phyB modulates this process to promote photomorphogenesis.

Characterization of spa mutants in the moss Physcomitrella provides evidence for functional divergence of SPA genes during the evolution of land plants.

The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage. To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb. Light-grown PpspaAB double mutants exhibit smaller gametophores than the wild-type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light-regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark-grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb. The data suggest that COP1/SPA-mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness.

Dynamic regulation of PIF5 by COP1–SPA complex to optimize photomorphogenesis in Arabidopsis

Light signal provides the spatial and temporal information for plants to adapt to the prevailing environmental conditions. Alterations in light quality and quantity can trigger robust changes in global gene expression. In Arabidopsis thaliana, two groups of key factors regulating those changes in gene expression are CONSTITUTIVE PHOTOMORPHOGENESIS/DEETIOLATED/FUSCA (COP/DET/FUS) and a subset of basic helix-loop-helix transcription factors called PHYTOCHROME-INTERACTING FACTORS (PIFs). Recently, rapid progress has been made in characterizing the E3 ubiquitin ligases for the light-induced degradation of PIF1, PIF3 and PIF4; however, the E3 ligase(s) for PIF5 remains unknown. Here, we show that the CUL4COP 1- SPA complex is necessary for the red light-induced degradation of PIF5. Furthermore, COP1 and SPA proteins stabilize PIF5 in the dark, but promote the ubiquitination and degradation of PIF5 in response to red light through the 26S proteasome pathway. Genetic analysis illustrates that overexpression of PIF5 can partially suppress both cop1-4 and spaQ seedling de-etiolation phenotypes under dark and red-light conditions. In addition, the PIF5 protein level cycles under both diurnal and constant light conditions, which is also defective in the cop1-4 and spaQ backgrounds. Both cop1-4 and spaQ show defects in diurnal growth pattern. Overexpression of PIF5 partially restores growth defects in cop1-4 and spaQ under diurnal conditions, suggesting that the COP1-SPA complex plays an essential role in photoperiodic hypocotyl growth, partly through regulating the PIF5 level. Taken together, our data illustrate how the CUL4COP 1- SPA E3 ligase dynamically controls the PIF5 level to regulate plant development.

The blue light-induced interaction of cryptochrome 1 with COP1 requires SPA proteins during Arabidopsis light signaling.

Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that—in turn—COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex.

SPA Proteins Affect the Subcellular Localization of COP1 in the COP1/SPA Ubiquitin Ligase Complex during Photomorphogenesis.

The Arabidopsis (Arabidopsis thaliana) COP1/SPA ubiquitin ligase is a central repressor that suppresses light signaling in darkness by targeting positive regulators of the light response, mainly transcription factors, for degradation. Light inactivates COP1/SPA, in part by excluding COP1 from the nucleus. SPA proteins are essential cofactors of COP1, but their exact role in the COP1/SPA complex is thus far unknown. To unravel a potential role of SPA proteins in COP1 nucleocytoplasmic partitioning, we monitored the subcellular localization of COP1 in a spa1234 quadruple mutant (spaQn). We analyzed a YFP-COP1-expressing transgenic line and endogenous COP1 after subcellular fractionation. In dark-grown seedlings, both YFP-COP1 and endogenous COP1 accumulated in the nucleus in the absence and presence of SPA proteins, indicating that SPA proteins are not required for nuclear localization of COP1 in darkness. In contrast, in white light-grown seedlings, spaQn mutants failed to relocalize COP1 from the nucleus to the cytoplasm. Hence, SPA proteins are necessary for the light-controlled change in COP1 subcellular localization. We conclude that SPA proteins have a dual role: (1) they are required for light-responsiveness of COP1 subcellular localization, and (2) they promote COP1 activity in darkness in a fashion that is independent of the nuclear import/nuclear retention of COP1.

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 invitro and invivo, 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.

SPA proteins: SPAnning the gap between visible light and gene expression.

In this review we focus on the role of SPA proteins in light signalling and discuss different aspects, including molecular mechanisms, specificity, and evolution. The ability of plants to perceive and respond to their environment is key to their survival under ever-changing conditions. The abiotic factor light is of particular importance for plants. Light provides plants energy for carbon fixation through photosynthesis, but also is a source of information for the adaptation of growth and development to the environment. Cryptochromes and phytochromes are major photoreceptors involved in control of developmental decisions in response to light cues, including seed germination, seedling de-etiolation, and induction of flowering. The SPA protein family acts in complex with the E3 ubiquitin ligase COP1 to target positive regulators of light responses for degradation by the 26S proteasome to suppress photomorphogenic development in darkness. Light-activated cryptochromes and phytochromes both repress the function of COP1, allowing accumulation of positive photomorphogenic factors in light. In this review, we highlight the role of the SPA proteins in this process and discuss recent advances in understanding how SPAs link light-activation of photoreceptors and downstream signaling.