Linkage Mapping of Stem Saccharification Digestibility in Rice

Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties

Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host

Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana

LPAAT activity (sn-1-18:1-LPA and¹⁴C-labeled 18:1-CoA as substrates) of microsomal preparation from yeast overexpressed with empty plasmid (control), NbLPCAT2 and NbLPCAT1.

<p>LPAAT activity (sn-1-18:1-LPA and¹⁴C-labeled 18:1-CoA as substrates) of microsomal preparation from yeast overexpressed with empty plasmid (control), NbLPCAT2 and NbLPCAT1.</p

Expression patterns of <i>NbLPCAT</i> genes in roots (A,B), stems (C,D), flowers and seeds (E,F) during different developing periods of tissue-cultivated <i>N</i>. <i>benthamiana</i>.

<p>The expression levels of <i>NbLPCAT</i>s were analyzed by the real-time quantitative RT-PCR method. X-axis indicates different developing periods and y-axis indicates relative expression levels. Gene encoding elongation factor 1α signal was used as the reference gene. Error bars represent standard deviations of mean value from three technical replicates. W, week.</p

Sequence alignment of NbLPCATs with the related LPCATs from higher plants.

<p>The amino acid sequences of NbLPCATs were aligned, using the software Clustal X v1.83 with those of characterized LPCATs from <i>B</i>. <i>napus</i> and <i>A</i>. <i>thaliana</i>. The Jalview v2.8.2 program was used to highlight the homology between LPCAT protein sequences. Conserved motifs and the putative ER signal are boxed. Invariant residues are marked with black triangle stars.</p

Tissue-specific expression patterns of <i>NbLPCAT</i> genes in different tissues of 13-week-old (A, B), 16-week-old (C, D), and 19-week-old (E, F), tissue-cultivated <i>N</i>. <i>benthamiana</i>.

<p>The expression levels of <i>NbLPCAT</i>s were analyzed by the real-time quantitative RT-PCR method. X-axis indicates different tissues and y-axis indicates relative expression levels. Gene encoding elongation factor 1α signal was used as the reference gene. Error bars represent standard deviations of mean value from three technical replicates. W, week.</p

LysoPAF sensitivity test for yeast mutant Y02431 (<i>lca1</i>△) expressing NbLPCAT or harbouring the pESC-Ura empty vector.

<p>Yeast cells grown overnight and induced for the expression of NbLPCAT for 24 h were suspended in sterile distilled water and adjusted to an OD₆₀₀ of 2, 1, 0.5, and 0.1. The resulting 2 μL yeast solution was spotted on a SC-Ura agar plate containing 5, 10, 25, and 30 μg/mL lysoPAF. The growth of yeast cells was evaluated after 72 h at 28°C.</p

LPAAT activity of microsomal preparation from yeast overexpressed with empty plasmid (control) and with NbLPCAT2 towards different acyl-CoAs.

<p>A, expressed as pmol of <i>de novo</i> synthesised [¹⁴C] PA/mg microsomal protein/min. B, expressed as % of activity towards 18:1-CoA.</p