Green Phenotype Research Articles

ISSR Based Study of Fine Fescue (Festuca ovina L.) Highlighted the Genetic Diversity of Iranian Accessions

Festuca species have been used for forage, turf, soil conservation and ornamental purpose, which highlighted the importance of these species. This study was conducted to determine genetic variability among the 48 accessions of fine-leaved fescue from different regions of Iran using ISSR markers. To understand genetic relationships among these accessions, unweight pair-group method (UPGMA) were applied to the ISSR marker data set. The ten ISSR primers amplified in total 189 loci for 48 Festuca ovina accessions. All of the amplified loci were polymorphic. A high level of genetic diversity was detected in F. ovina accessions with an average genetic diversity index of 1.522. The expected heterozygosity (He) for the primers varied from 0.153 to 0.347. According to the ISSR data, accessions grouped in two main and seven sub clusters. Results indicated that each of the main clusters include plants that demonstrate Green and Silver phenotypes. Little congruence between the groupings patterns of accessions of F. ovina based on ISSR markers data and categorization based on phenotypes as a Green and Silver, was observed. In conclusion results reveal the high genetic diversity among F. ovina accessions, which can be used for alternative conservation and breeding programs.

Mutation of chloroplast CHLM contributes to down-regulation of multiple stress response genes in Arabidopsis

Mg-protoporphyrin IX methyltransferase (CHLM) plays a crucial role in chlorophyll biosynthesis and converts Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester. Previous research has indicated that down-expression of CHLM causes a pale green phenotype and Mg-protoporphyrin IX accumulation in Arabidopsis thaliana plants. Here, we report that a novel missense mutation (G to A transition) in the CHLM gene, results in the Gly59 → Glu amino acid substitution in the corresponding protein of Arabidopsis chlm-4 mutant plants. In silico modelling studies suggested that this substitution may disrupt membrane association of CHLM. Functional studies showed that the mutant form of CHLM leads to the excessive accumulation of superoxide anion radicals (O2−), although elimination of O2− could alleviate the yellow phenotype observed in chlm-4. In addition, qRT-PCR and western blotting analysis demonstrated that the expression of O2− scavengers, FSD1 and CSD2, were repressed in chlm-4 plants. The accumulation of photosystem II proteins was also decreased due to the CHLM mutation. Furthermore, we found that chlm-4 plants exhibited hypersensitive salt-stress during seed germination and down-expression of the salt stress-responsive CBFs-family genes. These results suggest CHLM positively regulates the expression of multiple photosynthetic-related and stress-responsive nuclear genes.

QTL-Seq and Sequence Assembly Rapidly Mapped the Gene BrMYBL2.1 for the Purple Trait in Brassica rapa

Anthocyanins have strong antioxidant activity and are believed to be healthy for human beings. The Brassica rapa L. ssp. chinensis var. purpurea “Zicaitai” is rich in anthocyanins. We constructed an F2 population of Zicaitai and “Caixin” (Brassica rapa ssp. parachinensis) and it shows clear segregation of the purple phenotype (i.e., variation in anthocyanin enrichment). Here, quantitative trait locus (QTL)-Seq was performed with two sample groups from the F2 population: one exhibiting an intense purple phenotype and the other showed a completely green phenotype. The results showed that the QTL-Seq and linkage analysis located different major loci. This indicates that there are two major genetic factors that plays different roles in regulating anthocyanin enrichment in Zicaitai. This was further supported by the data simulation of an in silico F2 population that QTL-Seq and linkage analysis can locate different major loci. Furthermore, the draft genomes of the two parents (Zicaitai and Caixin) were assembled and utilized to search for mutations in candidate genes. A ~100-bp insertion was found in the third exon of gene BrMYBL2.1 in Zicaitai. BrMYBL2.1 is a negative regulator of anthocyanin biosynthesis, while BrEGL3.2—previously located by linkage mapping—is a positive regulator. For these populations with multiple genes contributing large effects to a trait, a strategy of low depth re-sequencing of F2 individuals followed by QTL-Seq analysis with the free combination of sample groups is proposed. Furthermore, draft-sequence assembly of parental genomes together with QTL mapping is suggested as an efficient means for fine-mapping genes rapidly in segregating populations.

GmPGL1, a Thiamine Thiazole Synthase, Is Required for the Biosynthesis of Thiamine in Soybean.

Thiamine is an essential cofactor in several enzymatic reactions for all living organisms. Animals cannot synthesize thiamine and depend on their diet. Enhancing the content of thiamine is one of the most important goals of plant breeding to solve the thiamine deficiency associated with the low-thiamin staple crops. In this study, a Glycine max pale green leaf 1 (Gmpgl1) mutant was isolated from the EMS mutagenized population of soybean cultivar, Williams 82. Map-based cloning of the GmPGL1 locus revealed a single nucleotide deletion at the 292th nucleotide residue of the first exon of Glyma.10g251500 gene in Gmpgl1 mutant plant, encoding a thiamine thiazole synthase. Total thiamine contents decreased in both seedlings and seeds of the Gmpgl1 mutant. Exogenous application of thiazole restored the pale green leaf phenotype of the mutant. The deficiency of thiamine in Gmpgl1 mutant led to reduced activities of the pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC), and decreased contents of six amino acids as compared to that in the wild type plants. These results revealed that GmPGL1 played an essential role in thiamine thiazole biosynthesis.

Functional Dissection of the Chickpea (Cicer arietinum L.) Stay-Green Phenotype Associated with Molecular Variation at an Ortholog of Mendel's I Gene for Cotyledon Color: Implications for Crop Production and Carotenoid Biofortification.

“Stay-green” crop phenotypes have been shown to impact drought tolerance and nutritional content of several crops. We aimed to genetically describe and functionally dissect the particular stay-green phenomenon found in chickpeas with a green cotyledon color of mature dry seed and investigate its potential use for improvement of chickpea environmental adaptations and nutritional value. We examined 40 stay-green accessions and a set of 29 BC2F4-5 stay-green introgression lines using a stay-green donor parent ICC 16340 and two Indian elite cultivars (KAK2, JGK1) as recurrent parents. Genetic studies of segregating populations indicated that the green cotyledon trait is controlled by a single recessive gene that is invariantly associated with the delayed degreening (extended chlorophyll retention). We found that the chickpea ortholog of Mendel’s I locus of garden pea, encoding a SGR protein as very likely to underlie the persistently green cotyledon color phenotype of chickpea. Further sequence characterization of this chickpea ortholog CaStGR1 (CaStGR1, for carietinum stay-green gene 1) revealed the presence of five different molecular variants (alleles), each of which is likely a loss-of-function of the chickpea protein (CaStGR1) involved in chlorophyll catabolism. We tested the wild type and green cotyledon lines for components of adaptations to dry environments and traits linked to agronomic performance in different experimental systems and different levels of water availability. We found that the plant processes linked to disrupted CaStGR1 gene did not functionality affect transpiration efficiency or water usage. Photosynthetic pigments in grains, including provitaminogenic carotenoids important for human nutrition, were 2–3-fold higher in the stay-green type. Agronomic performance did not appear to be correlated with the presence/absence of the stay-green allele. We conclude that allelic variation in chickpea CaStGR1 does not compromise traits linked to environmental adaptation and agronomic performance, and is a promising genetic technology for biofortification of provitaminogenic carotenoids in chickpea.

Genetic mapping of green curd gene Gr in cauliflower.

Gr5.1 is the major locus for cauliflower green curd color and mapped to an interval of 236 Kbp with four most likely candidate genes. Cauliflower with colored curd enhances not only the visual appeal but also the nutritional value of the crop. Green cauliflower results from ectopic development of chloroplasts in the normal white curd. However, the underlying genetic basis is unknown. In this study, we employed QTL-seq analysis to identify the loci that were associated with green curd phenotype in cauliflower. A F2 population was generated following a cross between a white curd (Stovepipe) and a green curd (ACX800) cauliflower plants. By whole-genome resequencing and SNP analysis of green and white F2 bulks, two QTLs were detected on chromosomes 5 (Gr5.1) and 7 (Gr7.1). Validation by traditional genetic mapping with CAPS markers suggested that Gr5.1 represented a major QTL, whereas Gr7.1 had a minor effect. Subsequent high-resolution mapping of Gr5.1 in the second large F2 population with additional CAPS markers narrowed down the target region to a genetic and physical distance of 0.3cM and 236Kbp, respectively. This region contained 35 genes with four of them representing the best candidates for the green curd phenotype in cauliflower. They are LOC106295953, LOC106343833, LOC106345143, and LOC106295954, which encode UMP kinase, DEAD-box RNA helicase 51-like, glutathione S-transferase T3-like, and protein MKS1, respectively. These findings lay a solid foundation for the isolation of the Gr gene and provide a potential for marker-assisted selection of the green curd trait in cauliflower breeding. The eventual isolation of Gr will also facilitate better understanding of chloroplast biogenesis and development in plants.

Genetic analysis and fine mapping of a qualitative trait locus wpb1 for albino panicle branches in rice

Chloroplast plays an important role in the plant life cycle. However, the details of its development remain elusive in rice. In this study, we report the fine-mapping of a novel rice gene wpb1 (white panicle branch 1), which affects chloroplast biogenesis, from a tropical japonica variety that results in an albino panicle branches at and after the heading stage. The wpb1 variety was crossed with Nipponbare to generate the F2 and BC1F2 populations. Green and white panicle branch phenotypes with a 3:1 segregation ratio was observed in the F2 population. Bulked segregant analysis (BSA) based on whole genome resequencing was conducted to determine the wpb1 locus. A candidate interval spanning from 11.35 to 23.79M (physical position) on chromosome 1 was identified. The results of BSA analysis were verified by a 40K rice SNP-array using the BC1F2 population. A large-scale F2 population was used to pinpoint wpb1, and the locus was further narrowed down to a 95-kb interval. Furthermore, our results showed that the expression levels of the majority of the genes involved in Chl biosynthesis, photosynthesis and chloroplast development were remarkably affected in wpb1 variety and in F2 plants with a white panicle branch phenotype. In line with the results mentioned above, anatomical structural examination and chlorophyll (Chl) content measurement suggested that wpb1 might play an important role in the regulation of chloroplast development. Further cloning and functional characterization of the wpb1 gene will shed light on the molecular mechanism underlying chloroplast development in rice.

An artificial miRNA as a new tool to silence and explore gene functions in apple.

Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar 'Gala'. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.

A Suppressor Mutation Partially Reverts the xantha Trait via Lowered Methylation in the Promoter of Genomes Uncoupled 4 in Rice.

The xantha trait of a yellow leaf rice mutant (HYB), controlled epigenetically by elevated CHG methylation of the genomes uncoupled 4 (OsGUN4) promoter, has reduced chlorophyll content, altered tetrapyrrole biosynthesis, and deregulated transcription of photosynthesis-associated nuclear genes (PhANGs) compared to its wild-type progenitor Longtefu B (LTB). In the present study, we identified a suppressor mutant (CYB) of HYB and characterized its genetic, molecular, and physiological basis of the mutant phenotype. We found that the light-green phenotype of CYB was caused by a suppressor mutation in an unknown gene other than OsGUN4. Compared to HYB, the CHG methylation in the OsGUN4 promoter was reduced, while OsGUN4 transcript and protein abundance levels were increased in CYB. The contents of total chlorophyll and its intermediate metabolites (except protoporphyrin IX) in CYB plants were intermediate between HYB and LTB. The expression levels of 30 genes involved in tetrapyrrole biosynthesis in CYB were all partially reverted to those of LTB, so were the PhANGs. In summary, a suppressor mutation caused the reversion of the xantha trait via reducing CHG methylation in OsGUN4 promoter.

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

SummaryMolecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M2) family derived from a heterozygous (M1) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M2) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M3) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M2) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.

Physical and physiological changes in phenotypes of nance [Byrsonima crassifolia (L.) H.B.K.] with different harvest maturity

Nance [Byrsonima crassifolia (L.) H.B.K.] belongs to a subgenus of Byrsonima Rich. ex Kunth that comprises approximately 200 species distributed in tropical and subtropical climates of Mexico, Central America and South America. Some of these species are endangered. Nance fruit contain functional nutrients but have not been commercially used. Local residents usually collect natural abscission fruit from ground and the shelf life is very short, only one or two days. Five nance phenotypes produced in Tabasco, Mexico, were studied at different stages of maturity to determine physical and physiological changes to establish harvest criteria. The results showed that the phenotypes yellow, yellow chintul and red chintul grew in direction of the equatorial diameter, while the green phenotype and green chintul grew in direction of the polar diameter. All the phenotypes studied showed significant changes in peel color, a decrease in peel firmness and increase in the content of total soluble solids of the pulp. The evidence found in these physical changes, as well as in the change in kinetics and production of carbon dioxide and ethylene, lead us to conclude that the phenotypes studied have a ripening climacteric pattern dependent on ethylene. Consequently, it is possible to harvest nance fruit directly from the tree to ensure a fruit visual, physical and eating quality to establish an industry.

Genome-Wide Analysis of Coding and Long Non-Coding RNAs Involved in Cuticular Wax Biosynthesis in Cabbage (Brassica oleracea L. var. capitata).

Cuticular wax is a mixture of very long chain fatty acids (VLCFAs) and their derivatives, which determines vital roles for plant growth. In cabbage, the cuticular wax content of leaf blades is an important trait influencing morphological features of the head. Understanding the molecular basis of cuticular wax biosynthesis can help breeders develop high quality cabbage varieties. Here, we characterize a cabbage non-wax glossy (nwgl) plant, which exhibits glossy green phenotype. Cryo-scanning electron microscope analysis showed abnormal wax crystals on the leaf surfaces of nwgl plants. Cuticular wax composition analyzed by GC-MS displayed severely decreased in total wax loads, and individual wax components in nwgl leaves. We delimited the NWGL locus into a 99-kb interval between the at004 marker and the end of chromosome C08 through fine mapping. By high-throughput RNA sequencing, we identified 1247 differentially expressed genes (DEGs) and 148 differentially expressed lncRNAs in nwgl leaves relative to the wild-type. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the DEGs and cis-regulated target genes for differentially expressed lncRNAs were significantly enriched in wax and lipid biosynthetic or metabolic processes. Our results provide the novel foundation to explore the complex molecular basis of cuticular wax biosynthesis.

Transcriptome analysis based on a combination of sequencing platforms provides insights into leaf pigmentation in Acer rubrum

BackgroundRed maple (Acer rubrum L.) is one of the most common and widespread trees with colorful leaves. We found a mutant with red, yellow, and green leaf phenotypes in different branches, which provided ideal materials with the same genetic relationship, and little interference from the environment, for the study of complex metabolic networks that underly variations in the coloration of leaves. We applied a combination of NGS and SMRT sequencing to various red maple tissues.ResultsA total of 125,448 unigenes were obtained, of which 46 and 69 were thought to be related to the synthesis of anthocyanins and carotenoids, respectively. In addition, 88 unigenes were presumed to be involved in the chlorophyll metabolic pathway. Based on a comprehensive analysis of the pigment gene expression network, the mechanisms of leaf color were investigated. The massive accumulation of Cy led to its higher content and proportion than other pigments, which caused the redness of leaves. Yellow coloration was the result of the complete decomposition of chlorophyll pigments, the unmasking of carotenoid pigments, and a slight accumulation of Cy.ConclusionsThis study provides a systematic analysis of color variations in the red maple. Moreover, mass sequence data obtained by deep sequencing will provide references for the controlled breeding of red maple.

Improved camouflage through ontogenetic colour change confers reduced detection risk in shore crabs.

Animals from many taxa, from snakes and crabs to caterpillars and lobsters, change appearance with age, but the reasons why this occurs are rarely tested.We show the importance that ontogenetic changes in coloration have on the camouflage of the green shore crabs (Carcinus maenas), known for their remarkable phenotypic variation and plasticity in colour and pattern.In controlled conditions, we reared juvenile crabs of two shades, pale or dark, on two background types simulating different habitats for 10 weeks.In contrast to expectations for reversible colour change, crabs did not tune their background match to specific microhabitats, but instead, and regardless of treatment, all developed a uniform dark green phenotype. This parallels changes in shore crab appearance with age observed in the field.Next, we undertook a citizen science experiment at the Natural History Museum London, where human subjects (“predators”) searched for crabs representing natural colour variation from different habitats, simulating predator vision.In concert, crabs were not hardest to find against their original habitat, but instead, the dark green phenotype was hardest to detect against all backgrounds.The evolution of camouflage can be better understood by acknowledging that the optimal phenotype to hide from predators may change over the life history of many animals, including the utilization of a generalist camouflage strategy. A plain language summary is available for this article.

Chlorella vulgaris integrates photoperiod and chloroplast redox signals in response to growth at high light.

Photoacclimation to variable light and photoperiod regimes in C. vulgaris represents a complex interplay between "biogenic" phytochrome-mediated sensing and "operational" redox sensing signaling pathways. Chlorella vulgaris Beijerinck UTEX 265 exhibits a yellow-green phenotype when grown under high light (HL) in contrast to a dark green phenotype when grown at low light (LL). The redox state of the photosynthetic electron transport chain (PETC) as estimated by excitation pressure has been proposed to govern this phenotypic response. We hypothesized that if the redox state of the PETC was the sole regulator of the HL phenotype, C. vulgaris should photoacclimate in response to the steady-state excitation pressure during the light period regardless of the length of the photoperiod. As expected, LL-grown cells exhibited a dark green phenotype, low excitation pressure (1 - qP = 0.22 ± 0.02), high chlorophyll (Chl) content (375 ± 77fg Chl/cell), low Chl a/b ratio (2.97 ± 0.18) as well as high photosynthetic efficiency and photosynthetic capacity regardless of the photoperiod. In contrast, C. vulgaris grown under continuous HL developed a yellow-green phenotype characterized by high excitation pressure (1 - qP = 0.68 ± 0.01), a relatively low Chl content (180 ± 53fg Chl/cell), high Chl a/b ratio (6.36 ± 0.54) with concomitantly reduced light-harvesting polypeptide abundance, as well as low photosynthetic capacity and efficiency measured on a per cell basis. Although cells grown under HL and an 18h photoperiod developed a typical yellow-green phenotype, cells grown at HL but a 12h photoperiod exhibited a dark green phenotype comparable to LL-grown cells despite exhibiting growth under high excitation pressure (1 - qP = 0.80 ± 0.04). The apparent uncoupling of excitation pressure and phenotype in HL-grown cells and a 12h photoperiod indicates that chloroplast redox status cannot be the sole regulator of photoacclimation in C. vulgaris. We conclude that photoacclimation in C. vulgaris to HL is dependent upon growth history and reflects a complex interaction of endogenous systems that sense changes in photoperiod as well as photosynthetic redox balance.

Leaf metabolites profiling between red and green phenotypes of Suaeda salsa by widely targeted metabolomics.

The Chenopodiaceae Suaeda salsa (L.) Pall. is a traditional Chinese medicine and food with green and red phenotypes in the Yellow River Delta. We identified 521 metabolites using widely targeted metabolomics, of which 165 were selected as significantly differential metabolites which could be related to the leaf traits of different phenotypes of S. salsa. Two anthocyanins (i.e. cyanidin O-acetylhexoside and delphinidin-3-O-(6'-O-α-rhamnopyranosy l-β-glucopyranoside)) were responsible for red colour in red leaves of S. salsa. Gallic acid, which existed only in red one, was the main reason for leaf succulence. D-arabitol and ribitol were two significantly upregulated carbohydrates in red phenotype. Four alkaloids (i.e. harmaline, aminophylline, pipecolate and trigonelline) were upregulated in red leaves. Hormonal changed involved a decrease in indoleacetic acid-valine (IAA-Val), N6-isopentenyladenosine-5'-monophosphate (iPRMP), isopentenyladenineriboside (iPR), trans-abscisic acid (S-ABA), salicylic acid O-hexoside, methyl jasmonate, N6-isopentenyladenine (iP), trans-zeatin riboside-O-glucoside iso2, trans-zeatin riboside-O-glucoside, and a tendency for dihydrozeatin 9-O-glucoside (DZ9G) down accumulation. In addition, the regulation of amino acids and lipids also contributed to the adaptation of red phenotype to harsh environment. Generally, our findings provide a comprehensive comparison of the metabolites between two phenotypes of S. salsa and an interpretation of phenotypic differences from the point of metabolomics.

Cottonseed Protein, Oil, and Mineral Nutrition in Near-Isogenic <i>Gossypium hirsutum</i> Cotton Lines Expressing Leaf Color Phenotypes under Field Conditions

Information about the effects of phenotype traits on cottonseed protein, oil, and nutrients is scarce. The objective of this research was to investigate the effects of leaf color trait on seed nutrition in near-isogenic Gossypium hirsutum cotton expressing green (G) and yellow (Y) leaf color phenotypes. Our hypothesis was that leaf color can influence the accumulation of nutrients in seeds. Sets of isogenic lines were: DES 119 (G) and DES 119 (Y); DP 5690 (G) and DP 5690 (Y); MD 51ne (G) and MD 51ne (Y); SG 747 (G) and SG 747 (Y). Each NIL set is 98.44 % identical. Parent line SA 30 (P) was used as the control. The experiment was repeated for two years (2014 and 2015). The results showed that, in 2014, seed oil in DES 119 (G) and SG 747 (G) were significantly higher than their equivalent yellow lines. Green lines showed higher content of phosphorus compared with yellow lines. Higher levels of Cu, Fe, Mn, Ni, and Zn were recorded in DES 119 (G) and MD 51ne (G). In 2015, seed protein, oil, C, N, P, B, Cu, and Fe were higher in green lines than in yellow lines. There was a significant correlation between protein and nutrients, and between oil and nutrients in 2015, but not in 2014 as the temperature was warmer in 2015 than in 2014. This research demonstrated that leaf color can alter seed composition and mineral nutrition under certain environmental growing conditions such as temperature.

Genome-wide analysis of superoxide dismutase genes in Larix kaempferi

Superoxide dismutase is a key enzyme that scavenges superoxide anion and plays vital roles in plant antioxidant system. This study identified six SOD genes from the deciduous conifer Larix kaempferi, which is widely distributed across the cooler regions of the northern hemisphere. These six SOD genes were classified into three types: Cu/Zn-SOD (LkSOD1, 2, 3 and 4), Fe-SOD (LkSOD5) and Mn-SODs (LkSOD6). Three Cu/Zn-SOD proteins (LkSOD1, 3 and 4) were cytosolic-localized, while the other three proteins (LkSOD2, 5 and 6) were chloroplast-localized. Larix SOD proteins displayed catalytic activities toward superoxide anion, and retained >55% of its maximum enzymatic activity between 10 °C and 40 °C. Over expressions of three Larix SOD genes (LkSOD2, 4 and 6) in Arabidopsis thaliana, respectively, showed increased germination rates and root lengths during salt stress. LkSOD5 gene could rescue pale green and dwarf phenotype of Arabidopsis atfsd2-2 mutant. Taken together, this study provided comprehensive insight into the gymnosperm SOD gene family.

Identification of a gene encoding glutathione S-transferase that is related to anthocyanin accumulation in buckwheat (Fagopyrum esculentum)

Anthocyanins are a group of flavonoids found in buckwheat (Fagopyrum esculentum) and many other plant species; however; little is known about their mechanisms of synthesis and regulation in buckwheat. We previously reported a spontaneous mutant buckwheat line that shows the green stem phenotype; this line does not accumulate anthocyanins but synthesizes flavonol and proanthocyanidin in the stem. Here, we used this line and lines developed by this line to search for genes related to anthocyanin accumulation in buckwheat. The lines with green stem showed flavonoid-3-O-glucosyltransferase activity against UDP-glucose, indicating that the flavonoid-3-O-glucosyltransferase gene was not controlling the green stem trait. We therefore searched the buckwheat genome database for a gene encoding glutathione S-transferase (GST), a flavonoid-binding protein that transports flavonoids to the vacuole, and identified a candidate gene, FeGST1. Expression analysis showed that FeGST1 was expressed in wild type buckwheat but not in the green stem lines. Linkage analysis with an F2 segregating population produced by crossing between the green stem line and a self-compatible line showed that FeGST1 segregated with stem color without any recombination. This indicates that the green stem trait could be caused by homozygous non-functional alleles of the FeGST1 locus.

Redox-control of chlorophyll biosynthesis mainly depends on thioredoxins.

In order to maintain enzyme stability and activity, chloroplasts use two systems of thiol-disulfide reductases for the control of redox-dependent properties of proteins. Previous studies have revealed that plastid-localized thioredoxins (TRX) and the NADPH-dependent thioredoxin reductase C (NTRC) are important for the reduction of cysteine residues of enzymes involved in chlorophyll synthesis. Very recently, it was shown that the palegreen phenotype of the ntrc mutant is suppressed when the contents of 2-cysteine peroxiredoxins (2CP) A and B are decreased. Here, we show that suppression of the ntrc phenotype results from a recovery of wild-type-like redox control of chlorophyll biosynthesis enzymes in ntrc/2cp mutants. The presented results support the conclusion that TRXs rather than NTRC are the predominant reductases mediating the redox-regulation of these enzymes.