Mutant Phenotype Research Articles

The RING-type E3 ligase RIE1 sustains leaf longevity by specifically targeting AtACS7 to fine-tune ethylene production in Arabidopsis

Ethylene is widely recognized as a positive regulator of leaf senescence. However, how plants coordinate the biosynthesis of ethylene to meet the requirements of senescence progression has not been determined. The rate-limiting enzyme in the ethylene biosynthesis pathway is ACC synthase. AtACS7 was previously considered one of the major contributors to the synthesis of "senescence ethylene" in Arabidopsis. However, the "brake signal" that fine-tunes the expression of AtACS7 to ensure optimal ethylene production during leaf development has yet to be identified. In the present study, the RING-H2 zinc-finger protein RIE1 was found to specifically interact with and ubiquitinate AtACS7, among all functional ACSs in Arabidopsis, to promote its degradation. Overexpression of RIE1 markedly decreased ethylene biosynthesis and delayed leaf senescence, whereas loss of function of RIE1 significantly increased ethylene emission and accelerated leaf senescence. The ethylene-related phenotypes of RIE1 overexpressing or knockout mutants were effectively rescued by the ethylene precursor ACC or the competitive inhibitor of ACS, respectively. In particular, AtACS7-induced precocious leaf senescence was strongly enhanced by the loss of RIE1 but was significantly attenuated by the overexpression of RIE1. The specific regions of interaction between AtACS7 and RIE1, as well as the major ubiquitination sites of AtACS7, were further investigated. All results demonstrated that RIE1 functions as an important modulator of ethylene biosynthesis during leaf development by specifically targeting AtACS7 for degradation, thereby enabling plants to produce the optimal levels of ethylene needed.

Overexpression of vacuolar H+-pyrophosphatase from a recretohalophyte Reaumuria trigyna enhances vegetative growth and salt tolerance in transgenic Arabidopsis thaliana

Reaumuria trigyna, a wild and endangered salt-secreting small shrub, is distributed in arid and semi-arid areas of Inner Mongolia, China. An H+-pyrophosphatase gene (RtVP1) was isolated from R. trigyna according to transcriptomic data, which encoded a plasma membrane and tonoplast-localized protein. RtVP1 was quickly upregulated by NaCl and exogenous abscisic acid treatment and rescued the sucrose deficiency sensitive phenotype of the AtVP1 mutant (avp1). Transgenic Arabidopsis overexpressing RtVP1 exhibited a higher leaf area, plant height, fresh weight, root length, and soluble carbohydrate accumulation compared to the wild type (WT) under normal conditions. RtVP1 overexpression increased the seed germination rate and decreased the reduction rate of fresh weight, root length, and chlorophyll content in transgenic plants under salt stress. Catalase enzyme activity, proline content, relative water content, and soluble sugar content were significantly increased in transgenic Arabidopsis under salt stresses, but the malondialdehyde content was dramatically decreased. More K+ and less Na+ were accumulated in transgenic Arabidopsis leaves, resulting in a relatively lower Na+/K+ ratio. In transgenic Arabidopsis roots, K+ was unchanged, but Na+ and the Na+/K+ ratios were reduced compared to those in WT. More Na+ and K+ were accumulated in the intracellular of transgenic yeast, and the Na+/K+ ratio was significantly reduced compared to the control. These results showed that R. trigyna RtVP1 promotes the vegetative growth of plants, mainly by regulating carbohydrate metabolism, and confers salt tolerance in transgenic Arabidopsis by maintaining Na+/K+ homeostasis and enhancing the antioxidant and osmotic regulatory capacity. These results indicated that RtVP1 can serve as an important candidate gene for genetic improvement of crop yield and salt tolerance.

TRX h2-PP2AC2 module serves as a convergence node for aluminum stress and leaf senescence signals, regulating cell death via ABA-mediated ROS pathway.

ROS/redox signaling plays an important role in the regulation of signal transduction and acclimation pathways activated by multiple abiotic stresses and leaf senescence. However, the regulatory events that produce ROS under different stimuli are far from clear. Here, we report the elucidation of the molecular mechanism of an h type thioredoxin, AhTRX h2, positively regulates Al sensitivity and leaf senescence by promoting ROS. AhTRX h2 transcript levels increased greatly during both natural senescence and Al stress condition in peanut. Ectopic expression of AhTRX h2 in Arabidopsis conferred Al sensitivity as well as premature leaf senescence, manifested by multiple indices, including inhibiting root elongation, severe cell death, and accelerated expression of MC1 and CEX17. AhTRX h2 exhibited similar functions to AtTRX h2, as AhTRX h2 was able to restore the phenotypes of the AtTRX h2 defective mutant (trxh2-4) which showed Al tolerant and late senescence phenotypes. The knock down of AhTRX h2 markedly suppressed Al- and senescence-induced cell death in peanut. AhTRX h2 could recruit catalytic subunit of protein phosphatase 2A (PP2AC2) to form a stable complex. The interaction between AhTRX h2 and AtPP2AC2, as well as AhPP2AC2 and AtTRX h2 was also proved. Overexpression of AhPP2AC2 significantly enhanced Al sensitivity and leaf senescence in Arabidopsis. Protein stability assay revealed that AhTRX h2 was more stable during aging or aluminum stress. Moreover, PP2AC2 could greatly enhance the stability of AhTRX h2 in vivo. Consistent with these observations, overexpression of AhPP2AC2 effectively enhanced AhTRX h2-induced Al sensitivity and precocious leaf senescence. AhTRX h2 and AhPP2AC2 required ABA and ROS in response to cell death under Al stress and senescence, and it was evidence to suggest that ABA acted upstream of ROS in this process. Together, AhTRX h2 and AhPP2AC2 constitute a stable complex that promotes the accumulation of ABA and ROS, effectively regulate cell death. These findings suggest that TRX h2-PP2AC2-mediated pathway may be a widespread mechanism in regulating Al stress and leaf senescence.

Glucocerebrosidase deficiency leads to neuropathology via cellular immune activation.

Mutations in GBA (glucosylceramidase beta), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the strongest genetic risk factor for the neurodegenerative disorders Parkinson's disease (PD) and Lewy body dementia. Recent work has suggested that neuroinflammation may be an important factor in the risk conferred by GBA mutations. We therefore systematically tested the contributions of immune-related genes to neuropathology in a Drosophila model of GCase deficiency. We identified target immune factors via RNA-Seq and proteomics on heads from GCase-deficient flies, which revealed both increased abundance of humoral factors and increased macrophage activation. We then manipulated the identified immune factors and measured their effect on head protein aggregates, a hallmark of neurodegenerative disease. Genetic ablation of humoral (secreted) immune factors did not suppress the development of protein aggregation. By contrast, re-expressing Gba1b in activated macrophages suppressed head protein aggregation in Gba1b mutants and rescued their lifespan and behavioral deficits. Moreover, reducing the GCase substrate glucosylceramide in activated macrophages also ameliorated Gba1b mutant phenotypes. Taken together, our findings show that glucosylceramide accumulation due to GCase deficiency leads to macrophage activation, which in turn promotes the development of neuropathology.

The DeoR-like pleiotropic regulator SCO1897 controls specialised metabolism, sporulation, spore germination, and phosphorus accumulation in Streptomyces coelicolor

Streptomycetes are bacteria of significant biotechnological interest due to their production of bioactive specialised metabolites used in medicine and agriculture. In these bacteria, specialised metabolism and morphological differentiation are linked and typically repressed under high phosphate conditions. This study characterises a DeoR-like transcriptional regulator, SCO1897, in Streptomyces coelicolor, whose expression increases during sporulation. Disruption of sco1897 results in reduced biosynthesis of specialised metabolites, delayed sporulation, higher spore phosphate content, and impaired germination. Transcriptomic analysis revealed 1420 genes differentially expressed in the sco1897 mutant compared to the S. coelicolor wild-type strain. The sco1897 gene is located upstream and transcribed in the same direction as six genes, including sco1898-1900 encoding sub-units of an ABC transporter annotated as involved in carbohydrate transport. SCO1897 negatively regulates its own expression, that of the sco1898-1900 ABC transporter, and sco4142, encoding the PstS phosphate-binding protein. The overexpression of sco1898-1900 in the S. coelicolor wild-type strain leads to a significant increase in intracellular spore phosphate levels, similar to those observed in the sco1897 mutant. These findings suggest a complex regulatory network involving the sco1897-sco1900 region. Hypotheses are proposed to explain the various phenotypes of the sco1897 mutant and the complex regulation of the genes of the sco1897-sco1900 region.

The dwarf & pale leaf mutation reduces chloroplast numbers, resulting in sugar depletion that inhibits leaf growth of maize seedlings

Plant growth is ultimately driven by cell division and expansion, but how these processes are regulated to mediate a wide range of genotypic variation in organ size is still poorly understood. To address this, we screened an EMS maize mutant population to identify a new EMS maize dwarf mutant with small, pale-yellow leaves (dpl). The mutation was mapped to a region of 11.58 Mb at the 3’ end of chromosome 7. We identified Zm00001d022394 as a potential causal gene for the dpl phenotype, encoding a pentatricopeptide repeat-containing (PPR) family protein involved in chloroplast gene expression and function, explaining the pale color of dpl. Mature dpl leaves are thinner and shorter due to a reduced number of cells of approximately normal length. The chloroplasts of dpl are reduced in size and number, correlating with a decreased chlorophyll content, however chloroplast ultrastructure was not affected. Consistent with the reduced chlorophyll content photosynthetic rate of dpl were reduced by 50 % and a 30 reduction of Fv/Fm suggests photoinhibition. As a consequence, soluble and insoluble sugar levels are severely reduced throughout the leaf growth zone. At the cell level reduced cell division rates and size of the division zone, explain the reduced leaf elongation rate (LER). The growth of dpl leaves can be restored by supplying growing leaves with sucrose through their cut tips, which also restores sucrose levels in the division zone of maize leaf, demonstrating that limited sugar availability explains the reduced growth phenotype. Inversely, we phenocopied the mutant growth phenotype by inhibiting photosynthetic electron transport in wild type plants with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Our study of dpl provides a functional link between inhibition of photosynthesis, soluble sugar flux to the leaf growth zone, the regulation of cell division and whole leaf growth.

Blind But Alive - Congenital Loss of atoh7 Disrupts the Visual System of Adult Zebrafish.

Vision is the predominant sense in most animal species. Loss of vision can be caused by a multitude of factors resulting in anatomic as well as behavioral changes. In mice and zebrafish, atoh7 mutants are completely blind as they fail to generate retinal ganglion cells (RGCs) during development. In contrast to mice, raising blind zebrafish to adulthood is challenging and this important model is currently missing in the field. Here, we report the phenotype of homozygous mutant adult zebrafish atoh7 mutants that have been raised using adjusted feeding and holding conditions. The phenotype of adult mutants was characterized using classical histology and immunohistochemistry as well as optical coherence tomography. In addition, the optokinetic response was characterized. Adult atoh7 mutants display dark body pigmentation and significantly reduced body length. They fail to form RGCs, the resulting nerve fiber layer as well as the optic nerve, and consequently behave completely blindly. In contrast, increased amounts of other retinal neurons and Müller glia are formed. In addition, the optic tectum is anatomically reduced in size, presumably due to the missing retinal input. Taken together, we provide a comprehensive characterization of a completely blind adult zebrafish mutant with focus on retinal and tectal morphology, as a useful model for glaucoma and optic nerve aplasia.

16S rRNA methyltransferase KsgA contributes to oxidative stress and antibiotic resistance in Pseudomonas aeruginosa

Ribosomal RNA (rRNA) modifications are involved in multiple biological processes. KsgA is a 16S rRNA adenine dimethyltransferase that methylates at the adenines 1518 and 1519 (A1518/1519) positions, which are located near the ribosome decoding center. These methylations are conserved and important for ribosome biogenesis and protein translation. In this study, we demonstrated the absence of A1518/1519 methylation in the 16S rRNA of a Pseudomonas aeruginosa ksgA mutant. Biolog phenotypic microarrays were used to screen the phenotypes of the ksgA mutant against various antimicrobial agents. The loss of ksgA led to increased sensitivity to menadione, a superoxide generator, which was, at least in part, attributed to decreased in a superoxide dismutase (SOD) activity. Interestingly, the decrease in SOD activity in the ksgA mutant was linked to a decrease in the SodM protein levels, but not the sodM mRNA levels. Furthermore, the ksgA mutant strain exhibited sensitivity to hygromycin B and tylosin antibiotics. The tylosin-sensitive phenotype was correlated with decreased transcriptional levels of tufA, tufB, and tsf, which encode elongation factors. Additionally, the ksgA mutant showed resistance to kasugamycin. Collectively, these findings highlight the role of KsgA in oxidative stress responses and antibiotic sensitivity in P. aeruginosa.

Myosin-binding protein 13 mediates primary seed dormancy via abscisic acid biosynthesis and signaling in Arabidopsis.

Dormancy is an essential characteristic that enables seeds to survive in unfavorable conditions while germinating when conditions are favorable. Myosin-binding proteins (MyoBs) assist in the movement of organelles along actin microfilaments by attaching to both organelles and myosins. In contrast to studies on yeast and metazoans, research on plant MyoBs is still in its early stages and primarily focuses on tip-growing cells. In this study, we found that Arabidopsis MyoB13 is highly expressed in dry mature seeds. The myob13 mutant, created using CRISPR/Cas9, exhibits a preharvest sprouting phenotype, which can be mitigated by after-ripening treatment, indicating that MyoB13 plays a positive role in primary seed dormancy. Furthermore, we show that MyoB13 negatively regulates ABA biosynthesis and signaling pathways. Notably, the expression of MyoB13 orthologs from maize and soybean can completely restore the phenotype of the Arabidopsis myob13 mutant, suggesting that the function of MyoB13 in ABA-induced seed dormancy is evolutionarily conserved. Therefore, the functional characterization of MyoB13 offers an additional genetic resource to help prevent vivipary in crop species.

C. elegans epicuticlins define specific compartments in the apical extracellular matrix and function in wound repair.

The apical extracellular matrix (aECM) of external epithelia often contains lipid-rich outer layers that contribute to permeability barrier function. The external aECM of nematodes is known as the cuticle and contains an external lipid-rich layer - the epicuticle. Epicuticlins are a family of tandem repeat cuticle proteins of unknown function. Here, we analyze the localization and function of the three C. elegans epicuticlins (EPIC proteins). EPIC-1 and EPIC-2 localize to the surface of the cuticle near the outer lipid layer, as well as to interfacial cuticles and adult-specific struts. EPIC-3 is expressed in dauer larvae and localizes to interfacial aECM in the buccal cavity. Skin wounding in the adult induces epic-3 expression, and EPIC proteins localize to wound sites. Null mutants lacking EPIC proteins are viable with reduced permeability barrier function and normal epicuticle lipid mobility. Loss of function in EPIC genes modifies the skin blistering phenotypes of Bli mutants and reduces survival after skin wounding. Our results suggest EPIC proteins define specific cortical compartments of the aECM and promote wound repair.

Maize DLR1/NHX7 Is Required for Root Development Under Potassium Deficiency.

Root System Architecture (RSA) is a crucial plant trait that governs a plant's ability to absorb water and nutrients. In this study, we describe a mutant with nutrient-dependent defects in root development, affecting both the primary root and lateral roots (LRs). This mutant, identified through a screen for defects in LR development, has been designated dlr1-1. The dlr1-1 mutant exhibits impaired LR emergence rather than defects in the LR primordium (LRP) formation, particularly under potassium (K+)-deprivation conditions. This impairment likely stems from inhibited cell proliferation caused by the dlr1-1 mutation. K+ deprivation specifically leads to the accumulation of salicylic acid (SA) in the dlr1-1 mutant, consistent with the upregulation of SA biosynthesis genes. Moreover, exogenous application of SA to wild-type plants (B73) mimics the dlr1-1 phenotype. Conversely, treatment of the dlr1-1 mutant with 2-aminoindane-2-phosphonic acid, an SA biosynthesis inhibitor, partially restores LR emergence, indicating that elevated SA levels may be responsible for the mutant's developmental defects. MutMap analysis and allelism tests confirmed that the phenotypes of the dlr1-1 mutant results from the loss of the Na+/H+ antiporter, ZmNHX7. Additionally, the application of NaCl exacerbates the dlr1-1 mutant phenotype, suggesting that the root defects in dlr1-1 mutant depend on ion homoeostasis. In conclusion, our findings demonstrate that maize DLR1/NHX7 is essential for root development under potassium deprivation.

Strain variation in Candida albicans glycolytic gene regulation.

Central carbon metabolism is vital for the proliferation of Candida albicans, a fungus that is prominent as a commensal and pathogen. Glycolytic genes are activated by overlapping activities of the transcription factors Tye7 and Gal4, as shown by studies in the SC5314 genetic background. However, regulatory relationships can vary among C. albicans isolates. Here, we analyzed Tye7- and Gal4-related phenotypes in five diverse clinical isolates of C. albicans. We tested growth properties and gene expression impact through Nanostring profiling and, for the two strains SC5314 and P87, RNA sequencing. Our results lead to three main conclusions. First, the functional redundancy of Tye7 and Gal4 for glycolytic gene activation is preserved among all strains tested. Second, at the gene expression level, strain P87 is an outlier with regard to tye7Δ/Δ impact, and strain SC5314 is an outlier with regard to gal4Δ/Δ impact. Third, while Gal4 is well known to be dispensable for induction of the GAL1, GAL7, and GAL10 galactose-specific metabolic genes, we find that gal4Δ/Δ mutants of several strains have a mild galactose fermentation defect, as assayed by growth on galactose with the respiration inhibitor antimycin A. Our findings indicate that even a central metabolic regulatory network is subject to strain variation and illustrates an unexpected genotype-phenotype relationship.The fungal commensal and pathogen Candida albicans rely upon metabolic flexibility to colonize and infect host niches. Central carbon metabolism is governed by two regulators, Tye7 and Gal4, as defined in the reference strain SC5314. Here, we have explored the impact of Tye7 and Gal4 on carbon utilization and gene expression across five diverse C. albicans clinical isolates. Novel aspects of this study are the finding that even a central metabolic regulatory network is subject to strain variation and the observation of an unexpected mutant phenotype.

Functions of the Sinorhizobium meliloti LsrB Substrate-Binding Domain in Oxidized Glutathione Resistance, Alfalfa Nodulation Symbiosis, and Growth.

To successfully colonize legume root nodules, rhizobia must effectively evade host-generated reactive oxygen species (ROS). LsrB, a redox regulator from Sinorhizobium meliloti, is essential for symbiosis with alfalfa (Medicago sativa). The three cysteine residues in LsrB's substrate domain play distinct roles in activating downstream redox genes. The study found that LsrB's substrate-binding domain, dependent on the cysteine residue Cys146, is involved in oxidized glutathione (GSSG) resistance and alfalfa nodulation symbiosis. LsrB homologues from other rhizobia, with Cys172/Cys238 or Cys146, enhance GSSG resistance and complement lsrB mutant's symbiotic nodulation. Substituting amino acids in Azorhizobium caulinodans LsrB with Cys restores lsrB mutant phenotypes. The lsrB deletion mutant shows increased sensitivity to NCR247, suggesting an interaction with host plant-derived NCRs in alfalfa nodules. Our findings reveal that the key cysteine residue in the LsrB's substrate domain is vital for rhizobium-legume symbiosis.

Genome-wide analysis of glutathione S-transferase genes in four Prunus species and the function of PmGSTF2, activated by PmMYBa1, in regulating anthocyanin accumulation in Prunus mume

Glutathione S-transferases (GSTs) are proteases with multiple physiological functions and play an important role in plant responses to abiotic stresses. Nevertheless, there is a paucity of systematic research on GST genes in Prunus genus. Here, 330 GST genes in four Prunus species were identified for the first time and classified into eight subgroups based on protein sequence and conserved structure, among which Tau subfamily genes had the largest number. The amino acid lengths of GST-encoded proteins in the four species ranged from 66 to 1152 aa, most of which were soluble proteins and located in the cytoplasm and chloroplasts. The GST family was propelled by tandem duplications, yet robust purifying selection constrained its divergence. Conserved motif and domain analysis revealed that the majority of PmGSTs exhibited a highly conserved GST-N structure. The expression pattern of PmGSTs exhibited tissue specificity and spatiotemporal specificity. qRT-PCR validated the transcriptome results and 11 genes were differentially expressed in varieties with different flower and stem colors. In addition, we discovered an anthocyanin-related gene PmGSTF2, which can effectively restore the anthocyanin and proanthocyanidin deficiency-related phenotypes of the Arabidopsis tt19 mutant. Recombinant PmGSTF2 enhanced the water solubility of cyanidin and cyanidin-3-O-glucoside in vitro. Moreover, PmMYBa1 could directly bind to the promoter of PmGSTF2 and activate its expression. The findings revealed that GSTs were preserved in Prunus species and that PmGSTF2 was critical in regulating anthocyanin accumulation.

CLAVATA3 INSENSITIVE RECEPTOR KINASEs regulate lateral root initiation and spacing in Arabidopsis.

The root system architecture is very critical for plants to adapt to ever-changing environmental stimulations and is largely affected by lateral roots (LRs). Therefore, how plants regulate LR initiation and spacing is a key point for root system development. Previous studies have shown that RECEPTOR-LIKE KINASE 7 (RLK7) and its ligand TARGET OF LBD SIXTEEN 2 (TOLS2) control the initiation and spacing of LRs. However, the molecular mechanism underlying the perception and transduction of the TOLS2 signal by RLK7 remains to be elucidated. In this study, we explored whether CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs) are critical signaling components during Arabidopsis (Arabidopsis thaliana) LR development by investigating phenotypes of cik mutants and examining interactions between CIKs and members of the RLK7-mediated signaling pathway. Our results showed that high-order cik mutants generated more LRs because of more LR initiation and defective LR spacing. The cik mutants showed reduced sensitivity to applied TOLS2 peptides. TOLS2 application enhanced the interactions between CIKs and RLK7 and the RLK7-dependent phosphorylation of CIKs. In addition, overexpression of transcription factor PUCHI and constitutive activation of MITOGEN-ACTIVATED PROTEIN KINASE KINASE 4 (MKK4) and MKK5 partially rescued the spacing defects of LRs in cik and rlk7-3 mutants. Moreover, we discovered that auxin maximum in pericycle cells altered subcellular localization of CIKs to determine lateral root founder cells. These findings revealed that CIKs and RLK7 function together to perceive the TOLS2 signal and regulate LR initiation and spacing through the MKK4/5-MPK3/6-PUCHI cascade.

The functional roles of zebrafish HoxA- and HoxD-related clusters in the pectoral fin development

The paralogs 9–13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa−/−;hoxab−/−;hoxda−/− larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

Full-Length Transcriptome Analysis and Characterization of DFRs Involved in the Formation of Anthocyanin in Allium wallichii

Allium wallichii is famous for its reddish-purple flowers, which can be utilized as cut flowers and garden landscaping. Flower color is mainly determined by flavonoids, betalains, carotenoids, as well as other pigments. However, there is no research on the color formation mechanism in A. wallichii, which restricts its genetic improvement and development of superior varieties. The flower of A. wallichii was collected for full-length transcriptome sequencing and metabolome analysis using PacBio SMART and UPLC-MS, respectively. A total of 45 anthocyanins were detected in its flower, and 75,778 transcripts of 107,208 non-redundant transcripts were annotated. Then, two AwDFRs were cloned and characterized using bioinformatics tools. Enzyme activity assays revealed that both AwDFR1 and AwDFR2 possessed DFR activity in vitro that only accepted DHQ and DHM as substrates, except for DHK. Finally, physiological results showed that AwDFR1 and AwDFR2 could restore the lacking phenotypes of Arabidopsis tt3 mutant and increase the content of anthoycanin in tobacco petals. The anthocyanins and transcriptome in A. wallichii were firstly reported, and AwDFR1 and AwDFR2 are key enzymes participating in the biosynthesis of anthocyanins. This research provides important guidance for future key gene mining, color improvement, and horticultural breeding in A. wallichii.

Assessing the Role of AtGRP7 Arginine 141, a Target of Dimethylation by PRMT5, in Flowering Time Control and Stress Response.

PROTEIN ARGININE METHYLTRANSFERASES (PRMTs) catalyze arginine (R) methylation that is critical for transcriptional and post-transcriptional gene regulation. In Arabidopsis, PRMT5 that catalyzes symmetric R dimethylation is best characterized. PRMT5 mutants are late-flowering and show altered responses to environmental stress. Among PRMT5 targets are Arabidopsis thaliana GLYCINE RICH RNA BINDING PROTEIN 7 (AtGRP7) and AtGRP8 that promote the transition to flowering. AtGRP7 R141 has been shown to be modified by PRMT5. Here, we tested whether this symmetric dimethylation of R141 is important for AtGRP7's physiological role in flowering time control. We constructed AtGRP7 mutant variants with non-methylable R141 (R141A, R141K). Genomic clones containing these variants complemented the late-flowering phenotype of the grp7-1 mutant to the same extent as wild-type AtGRP7. Furthermore, overexpression of AtGRP7 R141A or R141K promoted flowering similar to overexpression of the wild-type protein. Thus, flowering time does not depend on R141 and its modification. However, germination experiments showed that R141 contributes to the activity of AtGRP7 in response to abiotic stress reactions mediated by abscisic acid during early development. Immunoprecipitation of AtGRP7-GFP in the prmt5 background revealed that antibodies against dimethylated arginine still recognized AtGRP7, suggesting that additional methyltransferases may be responsible for modification of AtGRP7.

Foxe1 mutant zebrafish show indications of a hypothyroid phenotype and increased sensitivity to ethanol for craniofacial malformations.

FOXE1 mutations in humans are associated with cleft palate and hypothyroidism. We previously developed a foxe1 mutant zebrafish demonstrating mineralization defects in larvae. In the present study, we investigate the thyroid status and skeletal phenotype of adult foxe1 mutants. Mutant fish have increased expression of tshβ in the pituitary, and of hepatic dio1 and dio2. In plasma, we found higher Mg levels. Together these findings are indicative of hypothyroidism. We further observed mineralization defects in scales due to enhanced osteoclast activity as measured by increased expression levels of tracp, ctsk, and rankl. Gene-environment interactions in the etiology of FOXE1-related craniofacial abnormalities remain elusive, which prompts the need for models to investigate genotype-phenotype associations. We here investigated whether ethanol exposure increases the risk of developing craniofacial malformations in foxe1 mutant larvae that we compared to wild types. We found in ethanol-exposed mutants an increased incidence of developmental malformations and marked changes in gene expression patterns of cartilage markers (sox9a), apoptotic markers (casp3b), retinoic acid metabolism (cyp26c1), and tissue hypoxia markers (hifaa, hifab). Taken together, this study shows that the foxe1 mutant zebrafish recapitulates phenotypes associated with FOXE1 mutations in human patients and a clear foxe1-ethanol interaction.

Nonfunctional coq10 mutants maintain the ERMES complex and reveal true phenotypes associated with the loss of the coenzyme Q chaperone protein Coq10

Coenzyme Q (CoQ) is a redox-active lipid molecule that acts as an electron carrier in the mitochondrial electron transport chain. In Saccharomyces cerevisiae, CoQ is synthesized in the mitochondrial matrix by a multisubunit protein-lipid complex termed the CoQ synthome, the spatial positioning of which is coordinated by the endoplasmic reticulum-mitochondria encounter structure (ERMES). The MDM12 gene encoding the cytosolic subunit of ERMES is coexpressed with COQ10, which encodes the putative CoQ chaperone Coq10, via a shared bidirectional promoter. Deletion of COQ10 results in respiratory deficiency, impaired CoQ biosynthesis, and reduced spatial coordination between ERMES and the CoQ synthome. While Coq10 protein content is maintained upon deletion of MDM12, we show that deletion of COQ10 by replacement with a HIS3 marker results in diminished Mdm12 protein content. Since deletion of individual ERMES subunits prevents ERMES formation, we asked whether some or all of the phenotypes associated with COQ10 deletion result from ERMES dysfunction. To identify the phenotypes resulting solely due to the loss of Coq10, we constructed strains expressing a functionally impaired (coq10-L96S) or truncated (coq10-R147∗) Coq10 isoform using CRISPR-Cas9. We show that both coq10 mutants preserve Mdm12 protein content and exhibit impaired respiratory capacity like the coq10Δ mutant, indicating that Coq10's function is vital for respiration regardless of ERMES integrity. Moreover, the maintenance of CoQ synthome stability and efficient CoQ biosynthesis observed for the coq10-R147∗ mutant suggests these deleterious phenotypes in the coq10Δ mutant result from ERMES disruption. Overall, this study clarifies the role of Coq10 in modulating CoQ biosynthesis.