Novel Insights into Anthocyanin Metabolism and Molecular Characterization of Associated Genes in Sugarcane Rinds Using the Metabolome and Transcriptome

Saccharum officinarum (sugarcane) is the fifth major cultivated crop around the world. Sugarcane rind is a promising source for anthocyanin pigments; however, limited information is available on the anthocyanin and its biosynthesis in sugarcane rinds. In this study, we have quantified 49 compounds including 6 flavonoids and 43 anthocyanins in the rind of 6 sugarcane cultivars by using LCMS/MS approach. Thirty of them were quantified for the first time in sugarcane. The 43 anthocyanins included 10 cyanidin (Cya), 11 pelargonidin (Pel), 9 peonidin (Peo), 5 malvidin (Mal), 4 delphinidin (Del), and 4 petunidin (Pet) metabolites. High contents of Cya derivatives were observed in the rind of YT71/210 (dark purple rind), such as cya-3-O-(6-O-malonyl)-glu 1283.3 µg/g and cya-3-O-glu 482.67 µg/g followed by ROC22 (red rind) 821.3 µg/g and 409 µg/g, respectively, whereas the YT93/159 (green rind) showed a minimum level of these compounds. Among six cultivars, ROC22 rind has high levels of Peo derivatives such as peo-3-O-glu (197 µg/g), peo-3-O-(6-O-malonyl)-glu (69 µg/g) and peo-3-O-(6-O-p-coumaryl)-glu (55.17 µg/g). The gene expression analysis revealed that some genes, including a MYB(t) gene, were highly associated with the color phenotype. Thus, we cloned and overexpressed the gene in Arabidopsis and found the pinkish brown color in the hypocotyl of all transgenic lines compared with the wild type. Hence, we have quantified a wide range of anthocyanins in major sugarcane cultivars, reported many new anthocyanins for the first time, and concluded that Cya and Peo derivatives are the major contributing factor of dissimilar colors in sugarcane. The finding and the verification of a novel MYB gene involved in anthocyanin biosynthesis have demonstrated that our study was very valuable for gene discovery and genetic improvement of sugarcane cultivars to harvest high anthocyanin contents.

Download Full-text

Genetic characterization of Rhizobium sp. strain TAL1145 that nodulates tree legumes

Canadian Journal of Microbiology ◽

10.1139/m94-034 ◽

1994 ◽

Vol 40 (3) ◽

pp. 208-215 ◽

Cited By ~ 16

Author(s):

M. L. C. George ◽

J. P. W. Young ◽

D. Borthakur

Keyword(s):

Rhizobium Leguminosarum ◽

Genetic Characterization ◽

Rhizobium Meliloti ◽

Wild Type ◽

Tree Legumes ◽

Wide Range ◽

The Common ◽

Rrna Sequences ◽

Rhizobium Sp

Rhizobium sp. strain TALI 145 nodulates Leucaena ieucocephaia and Phaseolus vulgaris, in addition to a wide range of tropical tree legumes. Six overlapping clones that complemented nodulation defects in leucaena and bean rhizobia were isolated and a 40-kb map of the symbiosis region was constructed. The common nod and nifA genes were situated approximately 17 kb apart, with the nodlJ genes in between. These clones enabled a derivative of TAL1145 carrying a partially deleted pSym to form ineffective nodules on both leucaena and bean, and a similar derivative of Rhizobium etli TAL182 to form ineffective nodules on bean. When two representative clones, pUHR9 and pUHR114, were each transferred to wild-type rhizobial strains, they allowed ineffective nodulation by Rhizobium meliloti on both leucaena and bean and by Rhizobium leguminosarum bv. viciae on bean. Transconjugants of R. leguminosarum bv. trifolii formed effective nodules on leucaena and ineffective nodules on bean. Tn5 mutagenesis of the symbiosis region resulted in a variety of nodulation and fixation phenotypes on leucaena and bean. On the basis of 16S rRNA sequences, TAL1145 was found to be distinct from both R. tropici and NGR234, the two groups of leucaena symbionts that were previously described.Key words: Rhizobium, Leucaena leucocephala, nodulation, nitrogen fixation.

Download Full-text

Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases

Biochemical Journal ◽

10.1042/bj20031575 ◽

2004 ◽

Vol 381 (1) ◽

pp. 295-306 ◽

Cited By ~ 80

Author(s):

Senarath B. P. ATHAUDA ◽

Koji MATSUMOTO ◽

Sanath RAJAPAKSHE ◽

Masayuki KURIBAYASHI ◽

Masaki KOJIMA ◽

...

Keyword(s):

Amino Acid ◽

Computer Modelling ◽

Structural Characteristics ◽

Amino Acid Sequences ◽

Cysteine Residues ◽

Aspartic Proteinases ◽

Protein Databases ◽

Wide Range ◽

First Time

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 °C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named ‘the nepenthesin-type AP-specific insertion’, that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

Download Full-text

Ethylene-inducible Expression of ipt Gene Produces a Dramatic Increase in Fower Bud Count in Transgenic Plants

HortScience ◽

10.21273/hortsci.39.4.821b ◽

2004 ◽

Vol 39 (4) ◽

pp. 821B-821

Author(s):

Richard J. McAvoy* ◽

Mariya V. Khodakovskaya ◽

Hong Liu ◽

Yi Li

Keyword(s):

Transgenic Plants ◽

Vegetative State ◽

Crop Improvement ◽

Marked Change ◽

Acc Oxidase ◽

Wild Type ◽

Flower Bud ◽

Vegetative Development ◽

Wide Range ◽

Transgenic Lines

Cytokinins play an important role in regulating plant growth and development. The cytokinin gene, isopentenyl transferase (ipt), was placed under the control of the ACC oxidase promoter from the LEACO1 gene from Lycopersicon esculentum and introduced into Nicotiana tabacum (cv. Havana) and chrysanthemum (Dendranthema × grandiflorum `Iridon'). Transformants were confirmed by PCR reaction and Southern blot and analyzed for phenotypical changes under both greenhouse and growth chamber conditions. With both species, LEACO1-ipt transgenic plants displayed a wide range of vegetative and generative phenotypes. With plants growing in the vegetative state, some LEACO1-ipt transgenic lines appeared similar to the non-transgenic wild-type cultivars while other lines showed excessive lateral branch development and short internodes. With plants grown under generative conditions, several LEACO1-ipt transgenic lines showed a 2 to 10-fold increase in the number of flower buds relative to the wild-type cultivars. With chrysanthemum, dramatic increases in bud count were observed on transgenic lines that otherwise displayed a morphology similar to the non-transgenic lines. Analysis of ipt expression indicated a marked change in gene expression between the most extreme phenotypes observed in this study. LEACO1-ipt lines that express normal vegetative development but increased flower bud counts appear to have great potential for ornamental crop improvement.

Download Full-text

First Apocarotenoids Profiling of Four Microalgae Strains

Antioxidants ◽

10.3390/antiox8070209 ◽

2019 ◽

Vol 8 (7) ◽

pp. 209 ◽

Cited By ~ 5

Author(s):

Mariosimone Zoccali ◽

Daniele Giuffrida ◽

Fabio Salafia ◽

Carmen Socaciu ◽

Kari Skjånes ◽

...

Keyword(s):

Supercritical Fluid ◽

Fatty Acid Esters ◽

Nannochloropsis Gaditana ◽

Tetraselmis Chuii ◽

Chromatography Tandem Mass Spectrometry ◽

Wide Range ◽

First Time ◽

Chlamydomonas Sp ◽

Acid Esters

Both enzymatic or oxidative carotenoids cleavages can often occur in nature and produce a wide range of bioactive apocarotenoids. Considering that no detailed information is available in the literature regarding the occurrence of apocarotenoids in microalgae species, the aim of this study was to study the extraction and characterization of apocarotenoids in four different microalgae strains: Chlamydomonas sp. CCMP 2294, Tetraselmis chuii SAG 8-6, Nannochloropsis gaditana CCMP 526, and Chlorella sorokiniana NIVA-CHL 176. This was done for the first time using an online method coupling supercritical fluid extraction and supercritical fluid chromatography tandem mass spectrometry. A total of 29 different apocarotenoids, including various apocarotenoid fatty acid esters, were detected: apo-12’-zeaxanthinal, β-apo-12’-carotenal, apo-12-luteinal, and apo-12’-violaxanthal. These were detected in all the investigated strains together with the two apocarotenoid esters, apo-10’-zeaxanthinal-C4:0 and apo-8’-zeaxanthinal-C8:0. The overall extraction and detection time for the apocarotenoids was less than 10 min, including apocarotenoids esters, with an overall analysis time of less than 20 min. Moreover, preliminary quantitative data showed that the β-apo-8’-carotenal content was around 0.8% and 2.4% of the parent carotenoid, in the C. sorokiniana and T. chuii strains, respectively. This methodology could be applied as a selective and efficient method for the apocarotenoids detection.

Download Full-text

GISH characterization of Erianthus arundinaceus chromosomes in three generations of sugarcane intergeneric hybrids

Genome ◽

10.1139/g10-010 ◽

2010 ◽

Vol 53 (5) ◽

pp. 331-336 ◽

Cited By ~ 36

Author(s):

Nathalie Piperidis ◽

Jian-wen Chen ◽

Hai-hua Deng ◽

Li-Ping Wang ◽

Phillip Jackson ◽

...

Keyword(s):

Saccharum Officinarum ◽

Intergeneric Hybrids ◽

Chromosome Loss ◽

Biotic And Abiotic Stresses ◽

Breeding Programs ◽

Sugarcane Cultivar ◽

Chromosome Transmission ◽

Long Time ◽

First Time

Within Erianthus , a genus close to Saccharum , the species E. arundinaceus has the potential to contribute valuable traits to sugarcane, including adaptation to biotic and abiotic stresses and ratooning ability. Sugarcane breeders have tried for a long time to use Erianthus species in their breeding programs but until recently were constrained by a lack of fertile Saccharum × Erianthus hybrids. We report here for the first time the chromosome composition of fertile Saccharum officinarum × E. arundinaceus F1, BC1 (F1 × sugarcane cultivar), and BC2 (BC1 × sugarcane cultivar) hybrids. The F1 and BC2 resulted from n + n chromosome transmission, while the BC1 resulted from 2n + n transmission. In the BC1 clones, the number of E. arundinaceus chromosomes ranged from 21 to 30, and in the BC2 clones, the number ranged from 14 to 15, revealing cases of chromosome loss. No recombination events between Saccharum and Erianthus chromosomes were observed in either the BC1 or BC2 clones. The implications of these results for introgression of genes from E. arundinaceus in sugarcane breeding programs are discussed. We propose a strategy to identify the agronomic value of chromosomes from E. arundinaceus and to conduct targeted breeding based on this information.

Download Full-text

Transgenic lines of Begonia maculata generated by ectopic expression of PttKN1

Biologia ◽

10.2478/s11756-011-0008-3 ◽

2011 ◽

Vol 66 (2) ◽

Cited By ~ 1

Author(s):

Quan-le Xu ◽

Jiang-ling Dong ◽

Nan Gao ◽

Mei-yu Ruan ◽

Hai-yan Jia ◽

...

Keyword(s):

Transgenic Plants ◽

Ectopic Expression ◽

Class I ◽

Wild Type Plant ◽

Wild Type ◽

Knox Gene ◽

Knox Genes ◽

Pttkn1 Gene ◽

Transgenic Lines ◽

First Time

AbstractKNOX (KNOTTED1-like homeobox) genes encode homeodomain-containing transcription factors which play crucial roles in meristem maintenance and proper patterning of organ initiation. PttKN1 gene, isolated from the vascular cambium of hybrid aspen (Populus tremula × P. tremuloides), is a member of class I KNOX gene family. In order to understand the roles of PttKN1 gene in meristem activity and morphogenesis as well as to explore the possibility to generate novel ornamental lines via its ectopic expression, it was introduced into the genome of Begonia maculata Raddi by Agrobacterium tumefasciens-mediated gene transformation here. Four types of transgenic plants were observed, namely coral-like (CL) type, ectopic foliole (EF) type, phyllotaxy-irregular (IP) type and cup-shaped (CS) type, which were remarkably different from corresponding wild type and were not also observed in the regenerated plantlets of wild type plant. Among these four types of transgenic plants, the phenotype of coral-like was observed for the first time in the transformants ectopically expressed KNOX genes. The observation of scanning electron microscope (SEM) showed ectopic meristems on the adaxial leaf surface of the transformants. Interestingly, the plantlets with ectopic foliole could generate new ectopic folioles from the original ectopic folioles again, and the plants regenerated from the EF-type transformants could also maintain the original morphology. The same specific RT-PCR band of the four types of transgenic plantlets showed that PttKN1 was ectopically expressed. All these data demonstrated that the ectopic expression of PttKN1 caused a series of alterations in morphology which provided possibilities producing novel ornamental lines and that PttKN1 played important roles in meristem initiation, maintenance and organogenesis events as other class I KNOX genes.

Download Full-text

Cytogenetic and Array-CGH Characterization of a Simple Case of Reciprocal t(3;10) Translocation Reveals a Hidden Deletion at 5q12

Genes ◽

10.3390/genes12060877 ◽

2021 ◽

Vol 12 (6) ◽

pp. 877

Author(s):

Angelo Cellamare ◽

Nicoletta Coccaro ◽

Maria Cristina Nuzzi ◽

Paola Casieri ◽

Marilina Tampoia ◽

...

Keyword(s):

Growth Retardation ◽

Array Cgh ◽

Clinical Manifestations ◽

Postnatal Growth ◽

Facial Dysmorphism ◽

Conventional Cytogenetic Analysis ◽

Wide Range ◽

Conventional Cytogenetics ◽

First Time

Chromosome deletions, including band 5q12, have rarely been reported and have been associated with a wide range of clinical manifestations, such as postnatal growth retardation, intellectual disability, hyperactivity, nonspecific ocular defects, facial dysmorphism, and epilepsy. In this study, we describe for the first time a child with growth retardation in which we identified a balanced t(3;10) translocation by conventional cytogenetic analysis in addition to an 8.6 Mb 5q12 deletion through array-CGH. Our results show that the phenotypic abnormalities of a case that had been interpreted as “balanced” by conventional cytogenetics are mainly due to a cryptic deletion, highlighting the need for molecular investigation in subjects with an abnormal phenotype before assuming the cause is an apparently simple cytogenetic rearrangement. Finally, we identify PDE4D and PIK3R1 genes as the two major candidates responsible for the clinical features expressed in our patient.

Download Full-text

A multifunctional GH39 glycoside hydrolase from the anaerobic gut fungus Orpinomyces sp. strain C1A

10.7287/peerj.preprints.2076 ◽

2016 ◽

Author(s):

Jessica M Morrison ◽

Mostafa S Elshahed ◽

Noha Youssef

Keyword(s):

Active Site ◽

Glycoside Hydrolase ◽

Biochemical Characterization ◽

Xylanase Activity ◽

Comparative Modeling ◽

Glycoside Hydrolase Family ◽

Wide Range ◽

Promising Source ◽

Substrate Competition

Background. The anaerobic gut fungi (phylum Neocallimastigomycota) represent a promising source of novel lignocellulolytic enzymes. Here, we report on the cloning, expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme (Bgxg1) that is highly transcribed by the anaerobic fungus Orpinomyces sp. strain C1A under different growth conditions. This represents the first study of a GH39-family enzyme from the anaerobic fungi. Methods. Using enzyme activity assays, we performed a biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH and temperature values to identify the optimal enzyme conditions and the specificity of the enzyme. In addition, substrate competition studies and comparative modeling efforts were completed. Results. Contrary to the narrow range of activities (β-xylosidase or α-L-iduronidase) observed in previously characterized GH39 enzymes, Bgxg1 is unique in that it is multifunctional, exhibiting strong β-xylosidase, β-glucosidase, β-galactosidase activities (11.5 ± 1.2, 73.4 ± 7.15, and 54.6 ± 2.26 U/mg, respectively) and a weak xylanase activity (10.8 ± 1.25 U/mg), strength determined as compared to previously characterized enzymes. Physiological characterization revealed that Bgxg1 is active over a wide range of pH (3-8, optimum 6) and temperatures (25-60°C, optimum 39°C), and possesses excellent temperature and thermal stability. Substrate competition assays suggest that all observed activities occur at a single active site. Using comparative modeling and bioinformatics approaches, we putatively identified ten amino acid differences between Bgxg1 and previously biochemically characterized GH39 β-xylosidases that we speculate could impact active site architecture, size, charge, and/or polarity. The putative contributions of these changes to the observed relaxed specificities in Bgxg1 are discussed. Discussion. Collectively, the unique capabilities and multi-functionality of Bgxg1 render it an excellent candidate for inclusion in enzyme cocktails mediating cellulose and hemicellulose saccharification from lignocellulosic biomass.

Download Full-text

Genetic and Global Epigenetic Modification, Which Determines the Phenotype of Transgenic Rice?

International Journal of Molecular Sciences ◽

10.3390/ijms21051819 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1819

Author(s):

Xiaoru Fan ◽

Jingguang Chen ◽

Yufeng Wu ◽

CheeHow Teo ◽

Guohua Xu ◽

...

Keyword(s):

Tissue Culture ◽

Transgenic Plants ◽

Plant Height ◽

Epigenetic Modification ◽

Epigenetic Changes ◽

Wild Type ◽

Global Methylation ◽

Phenotypic Changes ◽

Wide Range ◽

Transgenic Lines

Transgenic technologies have been applied to a wide range of biological research. However, information on the potential epigenetic effects of transgenic technology is still lacking. Here, we show that the transgenic process can simultaneously induce both genetic and epigenetic changes in rice. We analyzed genetic, epigenetic, and phenotypic changes in plants subjected to tissue culture regeneration, using transgenic lines expressing the same coding sequence from two different promoters in transgenic lines of two rice cultivars: Wuyunjing7 (WYJ7) and Nipponbare (NP). We determined the expression of OsNAR2.1 in two overexpression lines generated from the two cultivars, and in the RNA interference (RNAi) OsNAR2.1 line in NP. DNA methylation analyses were performed on wild-type cultivars (WYJ7 and NP), regenerated lines (CK, T0 plants), segregation-derived wild-type from pOsNAR2.1-OsNAR2.1 (SDWT), pOsNAR2.1-OsNAR2.1, pUbi-OsNAR2.1, and RNAi lines. Interestingly, we observed global methylation decreased in the T0 regenerated line of WYJ7 (CK-WJY7) and pOsNAR2.1-OsNAR2.1 lines but increased in pUbi-OsNAR2.1 and RNAi lines of NP. Furthermore, the methylation pattern in SDWT returned to the WYJ7 level after four generations. Phenotypic changes were detected in all the generated lines except for SDWT. Global methylation was found to decrease by 13% in pOsNAR2.1-OsNAR2.1 with an increase in plant height of 4.69% compared with WYJ7, and increased by 18% in pUbi-OsNAR2.1 with an increase of 17.36% in plant height compared with NP. This suggests an absence of a necessary link between global methylation and the phenotype of transgenic plants with OsNAR2.1 gene over-expression. However, epigenetic changes can influence phenotype during tissue culture, as seen in the massive methylation in CK-WYJ7, T0 regenerated lines, resulting in decreased plant height compared with the wild-type, in the absence of a transformed gene. We conclude that in the transgenic lines the phenotype is mainly determined by the nature and function of the transgene after four generations of transformation, while the global epigenetic modification is dependent on the genetic background. Our research suggests an innovative insight in explaining the reason behind the occurrence of transgenic plants with random and undesirable phenotypes.

Download Full-text

A multifunctional GH39 glycoside hydrolase from the anaerobic gut fungusOrpinomycessp. strain C1A

PeerJ ◽

10.7717/peerj.2289 ◽

2016 ◽

Vol 4 ◽

pp. e2289 ◽

Cited By ~ 12

Author(s):

Jessica M. Morrison ◽

Mostafa S. Elshahed ◽

Noha Youssef

Keyword(s):

Active Site ◽

Glycoside Hydrolase ◽

Biochemical Characterization ◽

Xylanase Activity ◽

Comparative Modeling ◽

Glycoside Hydrolase Family ◽

Wide Range ◽

Promising Source ◽

Substrate Competition

Background.The anaerobic gut fungi (phylum Neocallimastigomycota) represent a promising source of novel lignocellulolytic enzymes. Here, we report on the cloning, expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme (Bgxg1) that is highly transcribed by the anaerobic fungusOrpinomycessp. strain C1A under different growth conditions. This represents the first study of a GH39-family enzyme from the anaerobic fungi.Methods.Using enzyme activity assays, we performed a biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH and temperature values to identify the optimal enzyme conditions and the specificity of the enzyme. In addition, substrate competition studies and comparative modeling efforts were completed.Results.Contrary to the narrow range of activities (β-xylosidase or α-L-iduronidase) observed in previously characterized GH39 enzymes, Bgxg1 is unique in that it is multifunctional, exhibiting strong β-xylosidase, β-glucosidase, β-galactosidase activities (11.5 ± 1.2, 73.4 ± 7.15, and 54.6 ± 2.26 U/mg, respectively) and a weak xylanase activity (10.8 ± 1.25 U/mg), as compared to previously characterized enzymes. Further, Bgxg1 possesses extremely high affinity (as evident by the lowestKmvalues), compared to all previously characterized β-glucosidases, β-galactosidases, and xylanases. Physiological characterization revealed that Bgxg1 is active over a wide range of pH (3–8, optimum 6) and temperatures (25–60 °C, optimum 39 °C), and possesses excellent temperature and thermal stability. Substrate competition assays suggest that all observed activities occur at a single active site. Using comparative modeling and bioinformatics approaches, we putatively identified ten amino acid differences between Bgxg1 and previously biochemically characterized GH39 β-xylosidases that we speculate could impact active site architecture, size, charge, and/or polarity.Discussion.Collectively, the unique capabilities and multi-functionality of Bgxg1 render it an excellent candidate for inclusion in enzyme cocktails mediating cellulose and hemicellulose saccharification from lignocellulosic biomass.

Download Full-text