Reinterpretation of anthocyanins biosynthesis in developing black rice seeds through gene expression analysis

The biosynthesis of anthocyanins is still questionable in regulating the quantities of anthocyanins biosynthesized in rice seeds and the expression levels of transcription factors and the structural genes involved in the biosynthetic pathway of anthocyanins. We herein investigated the relationship between the accumulated anthocyanin contents and the expression levels of genes related to the biosynthesis of anthocyanins in rice seeds. Liquid chromatography/mass spectrometry-mass spectrometry analysis of cyanidin 3-glucoside (C3G) in rice seeds showed no accumulation of C3G in white and red rice cultivars, and the differential accumulation of C3G among black rice cultivars. RNA-seq analysis in rice seeds, including white, red, and black rice cultivars, at twenty days after heading (DAH) further exhibited that the genes involved in the biosynthesis of anthocyanins were differentially upregulated in developing seeds of black rice. We further verified these RNA-seq results through gene expression analysis by a quantitative real-time polymerase chain reaction in developing seeds of white, red, and black rice cultivars at 20 DAH. Of these genes related to the biosynthesis of anthocyanins, bHLHs, MYBs, and WD40, which are regulators, and the structural genes, including chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3´-hydroxylase (F3´H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were differentially upregulated in black rice seeds. The correlation analysis revealed that the quantities of C3G biosynthesized in black rice seeds were positively correlated to the expression levels of bHLHs, MYBs and WD40, CHS, F3H, F3´H, DFR, and ANS. In addition, we present bHLH2 (LOC_Os04g47040) and MYBs (LOC_Os01g49160, LOC_Os01g74410, and LOC_Os03g29614) as new putative transcription factor genes for the biosynthesis of anthocyanins in black rice seeds. It is expected that this study will help to improve the understanding of the molecular levels involved in the biosynthesis of anthocyanins in black rice seeds

Transcriptional changes in the developing rice seeds under salt stress suggest targets for manipulating seed quality

Global sea-level rise, the effect of climate change, poses a serious threat to rice production owing to saltwater intrusion and the accompanying increase in salt concentration. The reclaimed lands, comprising 22.1% of rice production in Korea, now face the crisis of global sea-level rise and a continuous increase in salt concentration. Here, we investigated the relationship between the decrease in seed quality and the transcriptional changes that occur in the developing rice seeds under salt stress. Compared to cultivation on normal land, the japonica rice cultivar, Samgwang, grown on reclaimed land showed a greatly increased accumulation of minerals, including sodium, magnesium, potassium, and sulfur, in seeds and a reduced yield, delayed heading, decreased thousand grain weight, and decreased palatability and amylose content. Samgwang showed phenotypical sensitivity to salt stress in the developing seeds. Using RNA-seq technology, we therefore carried out a comparative transcriptome analysis of the developing seeds grown on reclaimed and normal lands. In the biological process category, gene ontology enrichment analysis revealed that the upregulated genes were closely associated with the metabolism of biomolecules, including amino acids, carboxylic acid, lignin, trehalose, polysaccharide, and chitin, and to stress responses. MapMan analysis revealed the involvement of upregulated genes in the biosynthetic pathways of abscisic acid and melatonin and the relationship of trehalose, raffinose, and maltose with osmotic stress. Interestingly, many seed storage protein genes encoding glutelins and prolamins were upregulated in the developing seeds under salt stress, indicating the negative effect of the increase of storage proteins on palatability. Transcription factors upregulated in the developing seeds under salt stress included, in particular, bHLH, MYB, zinc finger, and heat shock factor, which could act as potential targets for the manipulation of seed quality under salt stress. Our study aims to develop a useful reference for elucidating the relationship between seed response mechanisms and decreased seed quality under salt stress, providing potential strategies for the improvement of seed quality under salt stress

MONOLIGNOL AND LIGNAN BIOSYNTHETIC STUDIES FROM REACTION MECHANISMS TO NEXT GENERATION SEQUENCING

Three objectives were studied: determination of the biochemical mechanisms of an Arabidopsis thaliana allylic double bond reductase (AtDBR1) producing stress/defense metabolites, dihydrocinnamyl aldehydes, and the Arabidopsis cinnamyl alcohol dehydrogenase (AtCAD5), as well as exploring the potential of utilizing next-generation sequencing to help identify unknown biochemical steps in formation of the lignan, podophyllotoxin, in Podophyllum species

Monolignol and lignan biosynthetic studies: From reaction mechanisms to next generation sequencing

Three objectives were studied: determination of the biochemical mechanisms of an Arabidopsis thaliana allylic double bond reductase (AtDBR1) producing stress/defense metabolites, dihydrocinnamyl aldehydes, and the Arabidopsis cinnamyl alcohol dehydrogenase (AtCAD5), as well as exploring the potential of utilizing next-generation sequencing to help identify unknown biochemical steps in formation of the lignan, podophyllotoxin, in Podophyllum species. Apo and binary structures of AtCAD5 were solved at 2.0 and 2.6 Å resolution, respectively, and ternary complexes were modeled with p-coumaryl aldehyde. A putative proton shuttle mechanism for AtCAD5 involving Thr49, His52 and Asp57 was evaluated, based on a proposed comparable mechanism for horse liver alcohol dehydrogenase. Site-directed mutants of each were prepared with corresponding mutant proteins characterized by kinetic and isothermal titration calorimetry (ITC) analyses. It was established that Thr49 was important in overall catalysis, whereas His52 and Asp57 were not. No evidence was obtained for a putative extended proton relay mechanism in AtCAD5. Apo, binary and ternary complexes of AtDBR1 were obtained at 2.5 (apo) and 2.8 (binary and ternary) Å resolution, respectively. Analysis of the ternary structure indicated a concerted catalytic mechanism involving hydride transfer to C-7 of p-coumaryl aldehyde (C-3 in 4-HNE), with the Tyr260 hydrogen-bonded to the aldehydic group of p-coumaryl aldehyde and the 2´-OH of nicotine amide ribose. Site-directed mutation of the Tyr260 residue further confirmed an essential role in catalysis through kinetic and ITC analyses. Illumina-based short reads/next-generation sequencing and bioinformatics analyses, together with a targeted metabolomics approach, of Podophyllum hexandrum and P. peltatum, were used to explore the potential of these technologies to deduce unknown biosynthetic steps to podophyllotoxin. Genes encoding steps in shikimate/chorismate, aromatic amino acid pathways, phenylpropanoid and lignan pathways were assembled, and putative enzymes catalyzing methylene-dioxy bridge formation were identified. Of these, recombinant proteins encoding by two genes CYP719A23 (P. hexandrum ) and CYP719A24 (P. peltatum) were capable of converting (–)-matairesinol into (–)-pluviatolide

Identification and Characterization of LARGE EMBRYO, a New Gene Controlling Embryo Size in Rice (Oryza sativa L.)

Abstract Background Although embryo accounts for only 2–3% of the total weight of a rice grain, it is a good source of various nutrients for human health. Because enlarged embryo size causes increase of the amount of nutrients and bioactive compounds stored within rice grain, giant embryo mutants of rice (Oryza sativa L.) are excellent genetic resources for improving the nutritional value of rice grains. Results Three giant embryo mutants, including large embryo (le), giant embryo (ge) and super-giant embryo (ge s ), with variable embryo size were used in this study. We investigated whether genes controlling embryo size in these mutants (le, ge and ge s ) were allelic to each other. Although ge and ge s was allelic to GIANT EMBRY (GE), le was not allelic to ge and ge s in allelism test. The GE gene carried a unique nucleotide substitution in each of the two mutants (ge and ge s ), resulting in non-synonymous mutations in exon 2 of GE in both mutants. However, the GE gene of the le mutant did not carry any mutation, suggesting that the enlarged embryo phenotype of le was governed by another gene. Using map-based cloning, we mapped the LE gene to the short arm of chromosome 3. The le mutant showed mild enlargement in embryo size, which resulted from an increase in the size of scutellar parenchyma cells. The LE encodes a C3HC4-type RING finger protein and was expressed to relatively high levels in seeds at a late developmental stage. Knockdown of LE expression using RNA interference increased the embryo size of rice grains, confirming the role of LE in determining the embryo size. Conclusion Overall, we identified a new gene controlling embryo size in rice. Phenotypic and molecular characterization results suggest that the le mutant will serve as a valuable resource for developing new rice cultivars with large embryos and nutrient-dense grains

Novel allelic variant of Lpa1 gene associated with a significant reduction in seed phytic acid content in rice (Oryza sativa L.).

In plants, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also known as phytic acid (PA), is a major component of organic phosphorus (P), and accounts for up to 85% of the total P in seeds. In rice (Oryza sativa L.), PA mainly accumulates in rice bran, and chelates mineral cations, resulting in mineral deficiencies among brown rice consumers. Therefore, considerable efforts have been focused on the development of low PA (LPA) rice cultivars. In this study, we performed genetic and molecular analyses of OsLpa1, a major PA biosynthesis gene, in Sanggol, a low PA mutant variety developed via chemical mutagenesis of Ilpum rice cultivar. Genetic segregation and sequencing analyses revealed that a recessive allele, lpa1-3, at the OsLpa1 locus (Os02g0819400) was responsible for a significant reduction in seed PA content in Sanggol. The lpa1-3 gene harboured a point mutation (C623T) in the fourth exon of the predicted coding region, resulting in threonine (Thr) to isoleucine (Ile) amino acidsubstitution at position 208 (Thr208Ile). Three-dimensional analysis of Lpa1 protein structure indicated that myo-inositol 3-monophosphate [Ins(3)P1] could bind to the active site of Lpa1, with ATP as a cofactor for catalysis. Furthermore, the presence of Thr208 in the loop adjacent to the entry site of the binding pocket suggests that Thr208Ile substitution is involved in regulating enzyme activity via phosphorylation. Therefore, we propose that Thr208Ile substitution in lpa1-3 reduces Lpa1 enzyme activity in Sanggol, resulting in reduced PA biosynthesis

Next Generation Sequencing in Predicting Gene Function in Podophyllotoxin Biosynthesis

Background: Biosynthetic pathways to structurally complex plant medicinals are incomplete or unknown. Results: Next generation sequencing/bioinformatics and metabolomics analysis of Podophyllum tissues gave putative unknown genes in podophyllotoxin biosynthesis. Conclusion: Regio-specific methylenedioxy bridge-forming CyP450s were identified catalyzing pluviatolide formation. Significance: Database of several medicinal plant transcriptome assemblies and metabolic profiling are made available for scientific community. Podophyllum species are sources of (−)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (−)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum . Both enzymes were capable of converting (−)-matairesinol into (−)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (−)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways

Mechanistic and structural studies of apoform, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase At5g16970

In this study, we determined the crystal structures of the apoform, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase encoded by At5g16970. This protein, one of 11 homologues in Arabidopsis thaliana, is most closely related to the Pinus taeda phenylpropenal double bond reductase, involved in, for example, heartwood formation. Both enzymes also have essential roles in plant defense, and can function by catalyzing the reduction of the 7-8-double bond of phenylpropanal substrates, such as p-coumaryl and coniferyl aldehydes in vitro. At5g16970 is also capable of reducing toxic substrates with the same alkenal functionality, such as 4-hydroxy-(2E)-nonenal. The overall fold of At5g16970 is similar to that of the zinc-independent medium chain dehydrogenase/reductase superfamily, the members of which have two domains and are dimeric in nature, i.e. in contrast to their original classification as being zinc-containing oxidoreductases. As provisionally anticipated from the kinetic data, the shape of the binding pocket can readily accommodate p-coumaryl aldehyde, coniferyl aldehyde, 4-hydroxy-(2E)-nonenal, and 2-alkenals. However, the enzyme kinetic data among these potential substrates differ, favoring p-coumaryl aldehyde. Tyr-260 is provisionally proposed to function as a general acid/base for hydride transfer. A catalytic mechanism for this reduction, and its applicability to related important detoxification mammalian proteins, is also proposed

Arachis hypogaea resveratrol synthase 3 alters the expression pattern of UDP-glycosyltransferase genes in developing rice seeds.

The resveratrol-producing rice (Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut (Arachis hypogaea) resveratrol synthase 3 (AhRS3) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes (UGTs) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGTs, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGTs showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes (PAL, C4H and 4CL) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL, C4H and 4CL in developing rice seeds

Dirigent Protein-Mediated Lignan and Cyanogenic Glucoside Formation in Flax Seed: Integrated Omics and MALDI Mass Spectrometry Imaging

An integrated omics approach using genomics, transcriptomics, metabolomics (MALDI mass spectrometry imaging, MSI), and bioinformatics was employed to study spatiotemporal formation and deposition of health-protecting polymeric lignans and plant defense cyanogenic glucosides. Intact flax (Linum usitatissimum) capsules and seed tissues at different development stages were analyzed. Transcriptome analyses indicated distinct expression patterns of dirigent protein (DP) gene family members encoding (−)- and (+)-pinoresinol-forming DPs and their associated downstream metabolic processes, respectively, with the former expressed at early seed coat development stages. Genes encoding (+)-pinoresinol-forming DPs were, in contrast, expressed at later development stages. Recombinant DP expression and DP assays also unequivocally established their distinct stereoselective biochemical functions. Using MALDI MSI and ion mobility separation analyses, the pinoresinol downstream derivatives, secoisolariciresinol diglucoside (SDG) and SDG hydroxymethylglutaryl ester, were localized and detectable only in early seed coat development stages. SDG derivatives were then converted into higher molecular weight phenolics during seed coat maturation. By contrast, the plant defense cyanogenic glucosides, the monoglucosides linamarin/lotaustralin, were detected throughout the flax capsule, whereas diglucosides linustatin/neolinustatin only accumulated in endosperm and embryo tissues. A putative biosynthetic pathway to the cyanogens is proposed on the basis of transcriptome coexpression data. Localization of all metabolites was at ca. 20 μm resolution, with the web based tool OpenMSI enabling not only resolution enhancement but also an interactive system for real-time searching for any ion in the tissue under analysis