Complementation Tests Research Articles

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization.

Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

WAX INDUCER 1 Regulates β-Diketone Biosynthesis by Mediating Expression of the Cer-cqu Gene Cluster in Barley.

Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with β-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the β-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

Recessive embryonic lethal mutations uncovered in heterozygous condition in silkworm semiconsomic strains

In this study, we found two embryonic lethal mutations, t04 lethal (l-t04) and m04 lethal (l-m04), in semiconsomic strains T04 and M04, respectively. In these semiconsomic strains, the entire diploid genome, except for one chromosome 4 of the wild silkworm Bombyx mandarina, is substituted with chromosomes of the domesticated silkworm B. mori, and l-t04 and l-m04 mutations are located on B. mandarina-derived chromosome 4. To clarify the cause of the lethalities and the genes responsible for these mutations, positional cloning and CRISPR/Cas9 mediated knockout screening were performed. Finally, genetic complementation tests identified the mutations responsible for the l-t04 and l-m04 as the Bombyx homolog of imaginal discs arrested (Bmida) and TATA box binding protein-associated factor 5 (BmTaf5), respectively. Lethal stages of each knockout mutant indicated the importance of these genes in B. mori late embryogenesis. The lethal mutations responsible for l-t04 and l-m04 were not found in parental strains or wild B. mandarina collected from 39 distinct locations in Japan, indicating that both mutations were independently introduced during or after the development of the semiconsomic strains. We conclude that the recessive embryonic lethality in the T04 and M04 strains is due to deleterious mutations produced in B. mandarina-derived chromosome 4.

The trans-zeatin-type side-chain modification of cytokinins controls rice growth.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

OsABC1K3, encodes an ABC1-like kinase, involved PGR5-dependent cyclic electron transport in rice

Light is an essential environmental factor for the growth and development of plants, but excessive light will damage the photosynthetic system and seriously affect crop yield. ABC1K is a class of atypical protein kinases commonly found in prokaryotes and eukaryotes. Chloroplast ABC1K can participate in the plant response to light signals, but its regulatory network in rice is not fully determined. In this study, a zebra leaf mutant osabc1k3 was isolated and characterized from the japonica rice Shennong9816 after EMS mutagenesis. At the tillering stage, chlorophyll was unevenly distributed, the contents of H2O2, O2- and MDA were increased in the leaves of abc1k3, and apoptosis was detected by TUNEL analysis. Fine mapping and DNA sequencing revealed a single base substitution on OsABC1K3, leading to premature termination of translation. Complementation tests further demonstrated that the mutation in osabc1k3 was responsible for the zebra leaf phenotype. OsABC1K3 belongs to the ABC1K gene family and encodes an activity of bc1 complex (ABC1) kinase 3 protein. OsABC1K3 can interact with FdC2 and PGR5 in vitro and in vivo. Compared with WT, osabc1k3 exhibited weakened photosynthesis. RLC analysis revealed that osabc1k3 showed decreased adaptability to strong light. OsABC1K3, a member of the ABC1K family, encodes an ABC1-like kinase 3 protein. OsABC1K3 can interact with FdC2 and PGR5, and is critical for adaptation to supersaturated light intensity in rice.

Cloning and Characterisation of a novel phosphate transporter gene EcPT1 from Eichhornia crassipes

ABSTRACT Eichhornia crassipes (Mart.) Solms can absorb nitrogen and phosphorus efficiently from water. Cloning the phosphate transporter 1 from E. crassipes (EcPHT1) and studying its characteristics can help to explain the molecular mechanism of absorption and transportation of inorganic phosphate (Pi) in E. crassipes and develop new genetic resources for phosphate transport to aid genetic breeding of crops with increased phosphate use efficiency. In this paper, we have used RACE method to clone the EcPT1 with 1572 bps which encoded a polypeptide of 523 amino acids and contained 11 transmembrane regions which was the typical structure of plant PHT1. The phylogenetic analysis also showed EcPT1 was closely related to other plant PHT1s. Quantitative real-time RT-PCR (qRT-PCR) showed that the EcPT1 expression in roots was up-regulated under low phosphorus stress . Moreover the EcPT1 expression in leaves was suppressed after E. crassipes was transferred to sufficient Pi from deficient Pi. Yeast functional complementation tests showed that the EcPT1 could complement the phosphate transport function of the Pi deficient yeast PHO84 in 175 μM Pi and make it grow best in pH = 5 or 6. All these results indicated that EcPT1 might be a phosphate transporter gene. Its expression was different in different tissues.

Identification of Thermo-Sensitive Chloroplast Development Gene TSCD5 Required for Rice Chloroplast Development under High Temperature

Chloroplasts are critical organelles for photosynthesis and play significant roles in plant growth and metabolism. High temperature is one of abiotic stresses affecting the growth and development of plants, involving chlorophyll biosynthesis and chloroplast development. It is well known that the methyl erythritol 4-phosphate (MEP) pathway is vital to photosynthesis and plant growth, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the enzyme that catalyze the first step of the MEP pathway. Although DXS has been widely studied in microbes and plants, no DXS gene has been identified in rice. Here, a novel thermo-sensitive chlorophyll-deficient 5 (tscd5) mutant was isolated in rice with decreased chlorophyll contents, impaired chloroplasts, and albino leaves at high temperature (35 °C). Fine mapping and DNA sequencing of TSCD5 found a missense mutation (G to A) in the sixth exon of LOC_Os05g33840 in tscd5. The TSCD5 gene encodes a 1-deoxy-D-xylulose-5-phosphate synthase 1 (OsDXS1) localized in chloroplast. Complementation tests and overexpression assay demonstrated that the mutation in LOC_Os05g33840 caused the tscd5 phenotype. qRT-PCR of TSCD5 showed it was constitutively expressed in all tissues, and its transcript amounts were reduced in tscd5 under high temperature. Here, TSCD5 is verified to be crucial to chloroplast development under high temperature in rice, which may facilitate the elucidation of the molecular mechanisms which underlie acclimation to high temperature stress in plants.

Complement as a Biomarker for Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a disease of immune complex deposition; therefore, complement plays a vital role in the pathogenesis of SLE. In general, complement levels in blood and complement deposition in histological tests are used for the management of SLE. Thus, the evaluation of complement status can be useful in the diagnosis of SLE, assessment of disease activity, and prediction of treatment response and prognosis. In addition, novel complement biomarkers, such as split products and cell-bound complement activation products, are considered to be more sensitive than traditional complement markers, such as serum C3 and C4 levels and total complement activity (CH50), which become more widely used. In this review, we report the complement testing in the management of SLE over the last decade and summarize their utility.

IlAP2, an AP2/ERF Superfamily Gene, Mediates Cadmium Tolerance by Interacting with IlMT2a in Iris lactea var. chinensis

Cadmium (Cd) stress has a major impact on ecosystems, so it is important to find suitable Cd-tolerant plants while elucidating the responsible molecular mechanism for phytoremediation to manage Cd soil contamination. Iris lactea var. chinensis is an ornamental perennial groundcover plant with strong tolerance to Cd. Previous studies found that IlAP2, an AP2/ERF superfamily gene, may be an interacting partner of the metallothionein gene IlMT2a, which plays a key role in Cd tolerance. To study the role of IlAP2 in regulating Cd tolerance in I. lactea, we analyzed its regulation function and mechanism based on a yeast two-hybrid assay, a bimolecular fluorescence complementation test, quantitative real-time PCR, transgenics and transcriptome sequencing. The results showed that IlAP2 interacts with IlMT2a and may cooperate with other transcription factors to regulate genes involved in signal transduction and plant hormones, leading to reduced Cd toxicity by hindering Cd transport. These findings provide insights into the mechanism of IlAP2-mediated stress responses to Cd and important gene resources for improving plant stress tolerance in phytoremediation.

Functional Analysis of the P-Type ATPases Apt2-4 from Cryptococcus neoformans by Heterologous Expression in Saccharomyces cerevisiae.

Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen Cryptococcus neoformans contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient S. cerevisiae strain dnf1Δdnf2Δdrs2Δ, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the C. neoformans Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and -choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of dnf1Δdnf2Δdrs2Δ, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a β-subunit. These results identify C. neoformans Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions.

LG5, a Novel Allele of EUI1, Regulates Grain Size and Flag Leaf Angle in Rice.

Grain size and flag leaf angle are two important traits that determining grain yield in rice. However, the mechanisms regulating these two traits remain largely unknown. In this study, a rice long grain 5 (lg5) mutant with a large flag leaf angle was identified, and map-based cloning revealed that a single base substitution followed by a 2 bp insertion in the LOC_Os05g40384 gene resulted in larger grains, a larger flag leaf angle, and higher plant height than the wild type. Sequence analysis revealed that lg5 is a novel allele of elongated uppermost internode-1 (EUI1), which encodes a cytochrome P450 protein. Functional complementation and overexpression tests showed that LG5 can rescue the bigger grain size and larger flag leaf angle in the Xiushui11 (XS) background. Knockdown of the LG5 transcription level by RNA interference resulted in elevated grain size and flag leaf angle in the Nipponbare (NIP) background. Morphological and cellular analyses suggested that LG5 regulated grain size and flag leaf angle by promoting cell expansion and cell proliferation. Our results provided new insight into the functions of EUI1 in rice, especially in regulating grain size and flag leaf angle, indicating a potential target for the improvement of rice breeding.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

New Fructokinase, OsFRK3, Regulates Starch Accumulation and Grain Filling in Rice.

Plant fructokinase (FRK) guarantees the growth and development of higher plants by participating in carbohydrate metabolism. In this study, a new fructokinase, OsFRK3, was identified using bioinformatics analysis, enzyme assay, bacterial growth assay, and yeast complementation test. Then, we created OsFRK3 knockout transgenic lines (osfrk3-1 and osfrk3-2) by the CRISPR/Cas9 technology. We found that the 1000-grain weight decreased notably (approximately -3.6% and -6.1%, respectively) in osfrk3-1 and osfrk3-2. Evidently decreased grain width, grain thickness, and endosperm filling rate were detected in the osfrk3 mutants (osfrk3-1 and osfrk3-2) compared with those of the WT. In addition, the content of seed total starch was significantly decreased by 3.42 and 4.80% in osfrk3 lines, compared with that in the WT. The level of maltose was significantly reduced in the mutants, while that of sucrose and fructose was obviously increased in the mutants. The transcript levels of OsGBSS1, OsBEIIb, OsPFP1β, and OsAGPL1 were significantly decreased in the osfrk3 mutants. These results suggest that OsFRK3 may positively regulate the accumulation of starch through influencing the sugar metabolism.

Functional analysis of the dehydratase domains of the PUFA synthase from Emiliania huxleyi in Escherichia coli and Arabidopsis thaliana

BackgroundPolyunsaturated fatty acid (PUFA) synthase is a multi-domain mega-enzyme that effectively synthesizes a series of PUFAs in marine microorganisms. The dehydratase (DH) domain of a PUFA synthase plays a crucial role in double bond positioning in fatty acids. Sequencing results of the coccolithophore Emiliania huxleyi (E. huxleyi, Eh) indicated that this species contains a PUFA synthase with multiple DH domains. Therefore, the current study, sought to define the functions of these DH domains (EhDHs), by cloning and overexpressing the genes encoding FabA-like EhDHs in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana).ResultsA complementation test showed that the two FabA-like DH domains could restore DH function in a temperature-sensitive (Ts) mutant. Meanwhile, overexpression of FabA-like EhDH1 and EhDH2 domains increased the production of unsaturated fatty acids (UFAs) in recombinant E. coli by 43.5–32.9%, respectively. Site-directed mutagenesis analysis confirmed the authenticity of active-site residues in these domains. Moreover, the expression of tandem EhDH1-DH2 in A. thaliana altered the fatty acids content, seed weight, and germination rate.ConclusionsThe two FabA-like DH domains in the E. huxleyi PUFA synthase function as 3-hydroxyacyl-acyl carrier protein dehydratase in E. coli. The expression of these domains in E. coli and A. thaliana can alter the fatty acid profile in E. coli and increase the seed lipid content and germination rate in A. thaliana. Hence, introduction of DH domains controlling the dehydration process of fatty acid biosynthesis in plants might offer a new strategy to increase oil production in oilseed plants.

Long term follow-up of complement parameters to improve the management of acquired angioedema due to C1-inhibitor deficiency

Acquired angioedema due to C1-inhibitor deficiency (C1–INH-AAE) is a rare disease that can be diagnosed via complement testing. It often accompanies lymphoproliferative underlying diseases. Our study aimed to examine if there is a connection between complement parameters and the clinical symptoms of C1–INH-AAE, and, in case of a known underlying disease, its activity. The other question is how a connection, if proven, could help in the development of the therapeutic strategy of C1–INH-AAE patients.In the past 30 years, out of the 3938 patients sent to the Angioedema Center with angioedema symptoms, we have diagnosed C1–INH-AAE in 19 cases. An underlying disease was diagnosed in 15 patients. Most often lymphoma (6/19 patients) and monoclonal gammopathy of undetermined significance (6/19 patients) were found.Angioedema specific long-term prophylaxis did not result in an improvement in neither the frequency of the attacks nor in the complement parameters.A connection has been found between the presence and activity of any underlying disease, the frequency of the angioedema attacks and the decreased level of proteins of the complement system. Decreasing complement parameters warn about the appearance or the worsening of the underlying disease. The treatment of the underlying disease brings improvement in the complement parameters.Rituximab treatment reduced the number of attacks or completely made them disappear, and we experienced positive changes in complement parameters. Complement parameters supported the long-term efficacy of rituximab treatment for C1–INH-AAE.The change in complement parameters predict the relapse of the underlying disease, and it is a good indicator for the prediction of angioedematous attacks. In C1–INH-AAE, it is essential to examine the patients for underlying diseases, and to regularly follow up the patient's complement parameters.

Grape CIPK18 acts as a positive regulator of CBF cold signaling pathway by modulating ROS homeostasis

Cold temperatures trigger a diverged Ca 2+ decoding paradigm that includes the proteins CBL (Calcineurin B-like proteins) and CIPK (CBL-interacting protein kinases). Here, putative CBL and CIPK genes were surveyed in cold-tolerant wild Amur grape ( Vitis amurensis ), designated as VaCBLs and VaCIPKs , respectively. The transcripts of 8 VaCBLs and 19 VaCIPKs encoded in this genome were confirmed, among which VaCIPK18 physically interacted with all 8 VaCBLs through the C-terminal regulatory domain (NAF), indicating that it may serve as a node regulator. VaCIPK18 was ubiquitously expressed in various tissues and was highly induced by cold, drought, salt, and ABA . The NAF domain of VaCIPK18 was required for functional homodimerization in yeast as well as in planta . Complementation tests confirmed that VaCIPK18 rescued the cold sensitivity of ArabidopsisAtcipk3 mutant, in which the expressions of CBFs were suppressed and is sensitive to cold and freezing stresses. In addition, overexpression of VaCIPK18 enhanced the cold tolerance by upregulating the C-repeat binding transcription factor (CBF) transcriptional pathway under normal or cold conditions, and reducing reactive oxygen species production. Thus, our study demonstrates that VaCIPK18 positively regulates cold signaling at least partly through the direct transcriptional control of cold-regulated CBFs and modulation of ROS homeostasis . Moreover, VaCIPK18 was found to interact with VaMYB4a in vivo and in vitro . Our findings provide evidence that VaCIPK18 functions as a key node in the CBL-CIPK network and may interact with VaMYB4a to contribute to cold response in grape plants. • An Amur grape VaCIPK18 could function as a node regulator of CBL-CIPK network. • VaCIPK18 modulates cold tolerance by directly controlling CBFs and ROS homeostasis. • 'VaCIPK18-VaMYB4a' module may regulate cold tolerance.

The Role of PnTCP2 in the Lobed Leaf Formation of Phoebe neurantha var. lobophylla.

A lobed leaf is a common trait in plants, but it is very rare in Lauraceae plants, including species of Phoebe. In the study of germplasm resources of Phoebe neurantha, we found lobed leaf variant seedlings, and the variation could be inherited stably. Studying the lobed leaf mechanism of P. neurantha var. lobophylla can offer insight into the leaf development mechanism of woody plants. RNA-seq and small RNA-seq analysis results showed that a total of 8091 differentially expressed genes (DEGs) and 16 differentially expressed miRNAs were identified in P. neurantha var. lobophylla. Considering previous research results, a leaf margin morphological development related miRNA, pne-miRNA319a, was primary identified as a candidate miRNA. Target gene prediction showed that a total of 2070 genes were predicted to be the target genes of differentially expressed miRNAs. GO enrichment analysis of differentially expressed target genes suggested that PnTCP2 is related to lobed leaf formation. The TRV-VIGS gene silencing of PnTCP2 led to lobed leaves in P. neurantha seedlings. The downregulation of PnTCP2 led to lobed leaves. The yeast two-hybrid test and bimolecular fluorescence complementation test confirmed that the PnTCP2 protein interacted with the PnLBD41 protein. Based on the expression analysis of gene-silenced leaves and RNA-seq and small RNA-seq analysis results, pne- miRNA319a and PnLBD41 might also play important roles in this process. In conclusion, PnTCP2 plays an important and vital role in the formation of the lobed leaves of P. neurantha var. lobophylla.

Gw2.1, a new allele of GW2, improves grain weight and grain yield in rice

Grain weight is an important characteristic of grain shape and a key contributing factor to the grain yield in rice. Here, we report that gw2.1, a new allele of the Grain Width and Weight 2 (GW2) gene, regulates grain size and grain weight. A single nucleotide substitution in the coding sequence (CDS) of gw2.1 resulted in the change of glutamate to lysine (E128K) in GW2.1 protein. Complementation tests and GW2 overexpression experiments demonstrated that the missense mutation in gw2.1 was responsible for the phenotype of enlarged grain size in the mutant line jf42. The large grain trait of the near-isogenic line NIL-gw2.1 was found to result from increased cell proliferation during flower development. Meanwhile, NIL-gw2.1 was shown to increase grain yield without compromising the grain quality. The GW2 protein was localized to the cell nucleus and membrane, and interacted with CHB705, a subunit of the chromatin remodeling complex. Finally, the F1 hybrids from crosses of NIL-gw2.1 with 7 cytoplasmic male-sterile lines exhibited large grains and desirable grain appearance. Thus, gw2.1 is a promising allele that could be applied to improve grain yield and grain appearance in rice. Availability of data and materialsThe datasets generated and/or analyzed in the study are available from the corresponding author on reasonable request.

A mutation in SlCHLH encoding a magnesium chelatase H subunit is involved in the formation of yellow stigma in tomato (Solanum lycopersicum L.)

Chlorophylls are ubiquitous pigments responsible for the green color in plants. Changes in the chlorophyll content have a significant impact on photosynthesis, plant growth and development. In this study, we used a yellow stigma mutant (ys) generated from a green stigma tomato WT by using ethylmethylsulfone (EMS)-induced mutagenesis. Compared with WT, the stigma of ys shows low chlorophyll content and impaired chloroplast ultrastructure. Through map-based cloning, the ys gene is localized to a 100 kb region on chromosome 4 between dCAPS596 and dCAPS606. Gene expression analysis and nonsynonymous SNP determination identified the Solyc04g015750, as the potential candidate gene, which encodes a magnesium chelatase H subunit (CHLH). In ys mutant, a single base C to T substitution in the SlCHLH gene results in the conversion of Serine into Leucine (Ser92Leu) at the N-terminal region. The functional complementation test shows that the SlCHLH from WT can rescue the green stigma phenotype of ys. In contrast, knockdown of SlCHLH in green stigma tomato AC, observed the yellow stigma phenotype at the stigma development stage. Overexpression of the mutant gene Slys in green stigma tomato AC results in the light green stigma. These results indicate that the mutation of the N-terminal S92 to Leu in SlCHLH is the main reason for the formation of the yellow stigma phenotype. Characterization of the ys mutant enriches the current knowledge of the tomato chlorophyll mutant library and provides a novel and effective tool for understanding the function of CHLH in tomato.

Evaluating the clinical utility of measuring levels of factor H and the related proteins

After years of disappointing clinical results, the tide has finally changed and complement targeted-therapies have become a validated and accepted treatment option for several diseases. These accomplishments have revitalized the field and brought renewed attention to the prospects that complement therapeutics can offer. Streamlining diagnostics and therapeutics is imperative in this new era of clinical use of complement therapeutics. However, the incredible success in therapeutics has not been accompanied by the development of novel standardized tools for complement testing. Complement biomarkers can assist in the risk assessment and diagnosis of diseases as well as the prediction of disease progression and treatment response. Recently, a group of complement proteins has been suggested to be highly relevant in various complement-associated disorders, namely the human factor H (FH) protein family. This family of closely related proteins consists of FH, FH-like protein 1, and five factor H-related proteins, and they have been linked to eye, kidney, infectious, vascular, and autoimmune diseases as well as cancer. The goal of this review is to provide a comprehensive overview of the available data on circulating levels of FH and its related proteins in different pathologies. In addition, we examined the current literature to determine the clinical utility of measuring levels of the FH protein family in health and disease. Finally, we discuss future steps that are needed to make their clinical translation a reality.