Natural Albino Mutant of Daylily (Hemerocallis spp.) Reveals a Link between Drought Sensitivity and Photosynthetic Pigments Metabolism.

BACKGROUND: Mutant analysis remains one of the main genetic tools for characterising unclarified gene functions in plants, especially in non-model plants. Daylily (Hemerocallis spp.) is a popular perennial ornamental plant grown worldwide. Analysis of daylily mutants can enhance understanding of genes regulating the albino phenotype and improve the cultivar quality of daylily. METHODS: The natural albino mutant (Alb-⁣/-) was isolated by screening a self-pollinated progeny of daylily cultivar 'black-eyed stella'. Transmission electron microscopy was used in analysing the structure of plastids between mutant and wild-type seedlings. The content of chlorophyll, carotenoids and chlorophyll precursors in plants was measured by ultraviolet spectrophotometry. RNA sequencing and physiological measurements were performed to explore the association between drought tolerance and mutation. RESULTS: All the seedlings of the daylily albino mutants died spontaneously within fifteen days after germination when grown in soil. The carotenoid and chlorophyll content in the leaves of the mutant plants significantly decreased compared with those of the wild-type control. The mutant plants displayed stunted growth, and their leaves were white or light yellow in color. Abnormal plastids such as those showing endomembrane vesiculation and lacking stacking were discovered in the leaves of mutant plants. Furthermore, genetic analysis revealed that a single recessive nuclear gene mutation led to the albino trait, RNA sequencing and real-time quantitative PCR validation showed extensive differences in gene expression between the mutant plants and the wild-type control, and most of the genes related to chlorophyll metabolism were down-regulated, with foldchange ranging from 0.20-0.49. Additionally, the surviving homozygous plants (Alb+⁣/+), which do not contain this mutation, were also isolated by analysing the phenotype of their self-pollinated progeny. The net photosynthesis rate and light saturation point of Alb+⁣/+ were higher than those of heterozygous (Alb+⁣/-) plants. Additionally, the Alb+⁣/+ plants were more tolerant to drought conditions than the Alb+⁣/- plants, suggesting that a heterozygous Alb- mutation is sufficient to negatively affect photosynthetic efficiency and drought tolerance. CONCLUSIONS: The albino mutation negatively affects photosynthetic efficiency and drought tolerance, and homozygous mutation is required for the characteristic albino phenotype. This work highlights the link between albino mutation, photosynthetic pigment metabolism and drought sensitivity in daylily.

Impact of dark septate endophytes on salt stress alleviation of tomato plants.

Fungal endophytes can improve plant tolerance to abiotic stress conditions. Dark septate endophytes (DSEs) belong to phylogenetically non-related groups of root colonizing fungi among the Ascomycota with high melanin-producing activities. They can be isolated from roots of more than 600 plant species in diverse ecosystems. Still the knowledge about their interaction with host plants and their contribution to stress alleviation is limited. The current work aimed to test the abilities of three DSEs (Periconia macrospinosa, Cadophora sp., Leptodontidium sp.) to alleviate moderate and high salt stress in tomato plants. By including an albino mutant, the role of melanin for the interaction with plants and salt stress alleviation could also be tested. P. macrospinosa and Cadophora sp. improved shoot and root growth 6 weeks after inoculation under moderate and high salt stress conditions. No matter how much salt stress was applied, macroelement (P, N, and C) contents were unaffected by DSE inoculation. The four tested DSE strains successfully colonized the roots of tomato, but the colonization level was clearly reduced in the albino mutant of Leptodontidium sp. Any difference in the effects on plant growth between the Leptodontidium sp. wild type strain and the albino mutant could, however, not be observed. These results show that particular DSEs are able to increase salt tolerance as they promote plant growth specifically under stress condition. Increased plant biomasses combined with stable nutrient contents resulted in higher P uptake in shoots of inoculated plants at moderate and high salt conditions and higher N uptake in the absence of salt stress in all inoculated plants, in P. macrospinosa-inoculated plants at moderate salt condition and in all inoculated plants except the albino mutants at high salt condition. In summary, melanin in DSEs seems to be important for the colonization process, but does not influence growth, nutrient uptake or salt tolerance of plants.

The thylakoid membrane protein NTA1 is an assembly factor of the cytochrome b6f complex essential for chloroplast development in Arabidopsis.

The cytochrome b6f (Cyt b6f) complex is a multisubunit protein complex in chloroplast thylakoid membranes required for photosynthetic electron transport. Here we report the isolation and characterization of the new tiny albino 1 (nta1) mutant in Arabidopsis, which has severe defects in Cyt b6f accumulation and chloroplast development. Gene cloning revealed that the nta1 phenotype was caused by disruption of a single nuclear gene, NTA1, which encodes an integral thylakoid membrane protein conserved across green algae and plants. Overexpression of NTA1 completely rescued the nta1 phenotype, and knockout of NTA1 in wild-type plants recapitulated the mutant phenotype. Loss of NTA1 function severely impaired the accumulation of multiprotein complexes related to photosynthesis in thylakoid membranes, particularly the components of Cyt b6f. NTA1 was shown to directly interact with four subunits (Cyt b6/PetB, PetD, PetG, and PetN) of Cyt b6f through the DUF1279 domain and C-terminal sequence to mediate their assembly. Taken together, our results identify NTA1 as a new and key regulator of chloroplast development that plays essential roles in assembly of the Cyt b6f complex by interacting with multiple Cyt b6f subunits.

Cytological, genetic and transcriptomic characterization of a cucumber albino mutant.

Photosynthesis, a fundamental process for plant growth and development, is dependent on chloroplast formation and chlorophyll synthesis. Severe disruption of chloroplast structure results in albinism of higher plants. In the present study, we report a cucumber albino alc mutant that presented white cotyledons under normal light conditions and was unable to produce first true leaf. Meanwhile, alc mutant could grow creamy green cotyledons under dim light conditions but died after exposure to normal light irradiation. No chlorophyll and carotenoid were detected in the alc mutant grown under normal light conditions. Using transmission electron microscopy, impaired chloroplasts were observed in this mutant. The genetic analysis indicated that the albino phenotype was recessively controlled by a single locus. Comparative transcriptomic analysis between the alc mutant and wild type revealed that genes involved in chlorophyll metabolism and the methylerythritol 4-phosphate pathway were affected in the alc mutant. In addition, three genes involved in chloroplast development, including two FtsH genes and one PPR gene, were found to have negligible expression in this mutant. The quality of RNA sequencing results was further confirmed by real-time quantitative PCR analysis. We also examined 12 homologous genes from alc mutant in other plant species, but no genetic variation in the coding sequences of these genes was found between alc mutant and wild type. Taken together, we characterized a cucumber albino mutant with albinism phenotype caused by chloroplast development deficiency and this mutant can pave way for future studies on plastid development.

Analysis of genomic characteristics and their influence on metabolism in Aspergillus luchuensis albino mutants using genome sequencing.

Black Aspergillus luchuensis and its white albino mutant are essential fungi for making alcoholic beverages in Japan. A large number of industrial strains have been created using novel isolation or gene/genome mutation techniques. Such mutations influence metabolic and phenotypic characteristics in industrial strains, but few comparative studies of inter-strain mutation have been conducted. We carried out comparative genome analyses of 8 industrial strains of A. luchuensis and A. kawachii IFO 4308, the latter being the first albino strain to be isolated. Phylogenetic analysis based on 8938 concatenated genes exposed the diversity of black koji strains and uniformity among albino industrial strains, suggesting that passaged industrial albino strains have more genetic mutations compared with strain IFO 4308 and black koji strains. Comparative analysis showed that the albino strains had mutations in genes not only for conidial pigmentation but also in those that encode N-terminal acetyltransferase A and annexin XIV-like protein. The results also suggest that some mutations may have emerged through subculturing of albino strains. For example, mutations in the genes for isocitrate lyase and sugar transporters were observed only in industrial albino strains. This implies that selective pressure for increasing enzyme activity or secondary metabolites may have influenced the mutation of genes associated with environmental stress responses in A. luchuensis albino strains. Our study clarifies hitherto unknown genetic and metabolic characteristics of A. luchuensis industrial strains and provides potential applications for comparative genome analysis for breeding koji strains.

Phenotypic and proteomic characteristics of sorghum (Sorghum bicolor) albino lethal mutant sbe6-a1.

Leaf color mutants are ideal materials for chloroplast development and photosynthetic mechanism research. Here, we characterized an EMS (ethyl methane sulfonate)-mutagenized sorghum (Sorghum bicolor) mutant, sbe6-a1, in which the severe disruption in chloroplast structure and a chlorophyll deficiency promote an albino leaf phenotype and lead to premature death. The proteomic analyses of mutant and its progenitor wild-type (WT) were performed using a Q Exactive plus Orbitrap mass spectrometer and 4,233 proteins were accurately quantitated. The function analysis showed that most of up-regulated proteins in mutant sbe6-a1 had not been well characterized. GO-enrichment analysis of the differentially abundant proteins (DAPs) showed that up-regulated DAPs were significantly enriched in catabolic process and located in mitochondria, while down regulated DAPs were located in chloroplasts and participated in photosynthesis and some other processes. KEGG pathway-enrichment analyses indicated that the degradation and metabolic pathways of fatty acids, as well as some amino acids and secondary metabolites, were significantly enhanced in the mutant sbe6-a1, while photosynthesis-related pathways, some secondary metabolites' biosynthesis and ribosomal pathways were significantly inhibited. Analysis also shows that some DAPs, such as FBAs, MDHs, PEPC, ATP synthase, CABs, CHLM, PRPs, pathogenesis-related protein, sHSP, ACP2 and AOX may be closely associated with the albino phenotype. Our analysis will promote the understanding of the molecular phenomena that result in plant albino phenotypes.

GRA78 encoding a putative S-sulfocysteine synthase is involved in chloroplast development at the early seedling stage of rice.

Cysteine functions not only as an amino acid in proteins but also as a precursor for a large number of essential biomolecules. Cysteine is synthesized via the incorporation of sulfide to O-acetylserine under the catalysis of O-acetylserine(thiol)lyase (OASTL). In dicotyledonous Arabidopsis, nine OASTL genes have been reported. However, in their null mutants, only the mutant of CS26 encoding S-sulfocysteine synthase showed the visible phenotypic changes, displaying significantly small plants and pale-green leaves under long-day condition but not short-day condition. Up to now, no OASTL gene or mutant has been identified in monocotyledon. In this study, we isolated a green-revertible albino mutant gra78 in rice (Oryza sativa). Its albino phenotype at the early seedling stage was sensitive to temperature but independent of photoperiod. Map-based cloning revealed that candidate gene LOC_Os01g59920 of GRA78 encodes a putative S-sulfocysteine synthase showing significant similarity with Arabidopsis CS26. Complementation experiment confirmed that mutation in LOC_Os01g59920 accounted for the mutant phenotype of gra78. GRA78 is constitutively expressed in all tissues and its encoded protein is targeted to the chloroplast. In addition, qRT-PCR suggested that expression levels of four OASTL homolog genes and five photosynthetic genes were remarkably down-regulated.

Evaluation of a real-time impedance analysis platform on fungal infection.

End-point assays of in vitro cell proliferation and death have been employed to study the mechanisms of fungal pathogenesis and have shown the responses of host cells at individual time points. A new cell analysis technology has been developed that allows for the continuous measurement and quantification of cell activities, thus enabling the dynamic assessment of electrical impedance when various pathogens are cultured in vitro. In this study, this system was evaluated to determine the response of the cell line RAW264.7 to infection by several clinically relevant fungi in vitro, including Aspergillus fumigatus, Candida albicans, and melanized and albino mutant strains of Fonsecaea monophora. The results showed that infection resulted in rounding of the host cells with a loss of contact between individual cells and a decline in the electrical impedance of all test groups. However, changes in the electrical impedance were variable. Aspergillus fumigatus caused initial increases and later significant decreases in the electrical impedance, while for C. albicans and F. monophora, the effect was reduced. The melanized strain of F. monophora caused a faster change in the electrical impedance than the albino strain. Our data proved that this system can be used as an efficient tool for monitoring cellular responses to fungal infection.

Overview of genetic and biochemical loci in Mesocricetus auratus and its albino mutant / 中国实验动物学报

Objective To establish the genetic and biochemical loci in Mesocricetus auratus and its albino mua-tant.Methods Protein isozyme cellulose acetate electrophoresis was used to determine the genetic and biochemical loci in Mesocricetus auratus and its albino mutant, using the genetic and biochemical loci of mice and rats.Results 25 biochemi-cal markers of Mesocricetus auratus and albino mutant were established, and polymorphism of their genetic biochemical loci was analyzed.Conclusions Polymorphism of biochemical loci is present in Mesocricetus auratus.Some differences exist between the genetic biochemical loci of Mesocricetus auratus and their albino mutant.These results laid the foundation for further study on genetic mechanism of albino mutation in Mesocricetus auratus.

Cloning and sequence analysis of dystonin(Dst) variant X1 in Cricetulusbarabensis and albino mutant Cricetulusbarabensis / 中国比较医学杂志

Objective To clone and sequence the dystonin variant X1 gene of Cricetulusbarabensis and the albino mutant Cricetulusbarabensis so as to find out the difference of encoding arear of the muscular ribosome between Cricetulusbarabensis and the albino mutant Cricetulusbarabensis.Methods According to the same type of abnormal muscle tone protein of the mice and rats, we designed 6 pairs of primers, and got their cDNA genes from skins of the Cricetulusbarabensis and the albino mutant Cricetulusbarabensis by RT-PCR amplification, then cloned and sequenced. Results Sequence alignment showed 17 variances in coding areas, and 24 in amino acid, but no in key nucleic acid and protein.Conclusion The variances in coding areas will not lead to the albino, and its mechanism requires further investigation.

Changes of cytokines in the Cricetulus barabensis and their albino mutant infected with trichinella spiralis / 中国比较医学杂志

Objective To explore the differences in immune responses between Cricetulus barabensis and their albino mutant infected with Trichinella spiralis.Methods The physiological parameters of blood , expression levels of IL-2 protein and IL-6 gene in the spleen were analyzed.Results The level of immune cells and cytokines of Cricetulus barabensis was higher than that in the albino mutant .Conclusions Cricetulus barabensis is a suitable model animal for research on long-term latent infection such as infection with Trichinella spiralis.

The lens proteins in adult and embryos of the periodic albino mutant ofXenopus laevis.

The crystallins of normal and ap mutants ofX. laevis have been studied using biochemical (electrophoresis in agar and polyacrylamide gels, isoelectric focusing) and immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoabsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The immunochemical analysis was carried out with rabbit antisera prepared against electrophoretic fractions of the mutant lens.Crystallins of adultX. laevis (ap/ap; ++/++) are heterogenous as judged by electrophoretic mobility, isoelectric point, antigenic and species specificity.No qualitative nor quantitative differences were found between crystallins of normal and mutant animals at the level of the protein subunits. These conclusions, however, are valid only for those crystallins, which are solubilized at pH 9.0.Immunofluorescence studies showed that crystallins appear in the normal and mutant embryos at practically the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found.Some of the gamma and, perhaps, beta-crystallins appear first; alpha-crystallins appear later. It has been shown for the first time that some gamma-crystallins are formed at advanced developmental stages.The periodic albino mutation does not affect the function of genes coding for crystallins either in embryos or in the adultX. laevis.