Structural characterization of the lignin-carbohydrate complex in biomass pretreated with Fenton oxidation and hydrothermal treatment and consequences on enzymatic hydrolysis efficiency.

In this study, lignin-carbohydrate complexes (LCCs) were isolated from biomass (raw and pretreated) to investigate the structural changes in biomass pretreated by Fenton oxidation and hydrothermal treatment, and their effect on enzymatic hydrolysis. The composition and structure of the LCCs fractions were investigated via carbohydrate analysis, XRD, FT-IR, and 2D HSQC NMR. The biomass degradation rate of yellow poplar and larch during Fenton oxidation and hydrothermal treatment was approximately 30%. Most of the hemicellulose was degraded during pretreatment, while xylan remained in the yellow poplar, and galactan, mannan, and xylan remained in the larch. The fractional yield of glucan-rich LCC (LCC1) in the yellow poplar (raw and pretreated biomass) was high, while that of glucomannan-rich LCC (LCC3) in larch was higher than the yield yellow poplar. Phenyl glycoside, γ-ester, and benzyl ether linkages were observed in the LCCs of yellow poplar, while phenyl glycoside and γ-ester were detected in those of larch. Following pretreatment, the frequencies of ß-ß', ß-5, and γ-ester in the LCCs of larch were found to be higher than in those of yellow poplar. The efficiencies of enzymatic hydrolysis for the pretreated yellow poplar and larch were 93.53% and 26.23%, respectively. These finding indicated that the ß-ß', ß-5, and γ-ester linkages included in the pretreated biomass affected the efficiency of enzymatic hydrolysis.

Functional Characteristics of Caffeoyl Shikimate Esterase in Larix Kaempferi and Monolignol Biosynthesis in Gymnosperms.

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 µM and 14.44 nM min-1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

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.

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase.

The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined "pseudolaratriene." Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene.

Biochemical functions of the glutathione transferase supergene family of Larix kaempferi.

Glutathione transferases (GSTs), which are ubiquitous in plants, play a major role in the detoxification of xenobiotics and oxidative stress metabolism. Due to their role in herbicide detoxification, previous studies of plant GSTs have mainly focused on agricultural plants. In contrast, functional information regarding gymnosperm GSTs is scarce. In this study, we cloned 27 full-length GST genes from the deciduous conifer Larix kaempferi, which is widely distributed across the cooler regions of the northern hemisphere. As with the angiosperm GST gene family, Larix GSTs are divided into eight classes, and tau class GSTs are the most numerous. Compared to the other seven classes of GSTs, Larix tau GST genes show substantially more variation in their expression patterns. The purified Larix GST proteins showed different substrate specificities, substrate activities, and kinetic characteristics. The pH and temperature profiles of purified Larix GST proteins showed broad optimum pH and temperature ranges for enzymatic activity, suggesting that Larix GSTs have evolutionary adaptations to various adverse environments. Taken together, this study provides comprehensive insight into the gymnosperm GST gene family.

Overexpression of UDP-glucose pyrophosphorylase from Larix gmelinii enhances vegetative growth in transgenic Arabidopsis thaliana.

KEY MESSAGE: A UDP-glucose pyrophosphorylase gene ( LgUGPase ) was identified from Larix gmelinii, and its function in enhancing vegetative growth and cellulose biosynthesis was confirmed by analyzing transgenic Arabidopsis thaliana overexpressed LgUGPase . UDP-glucose pyrophosphorylase (UGPase), an important regulatory enzyme in carbohydrate metabolism, catalyzes the reversible production of glucose 1-phosphate and the conversion of uridine triphosphate to uridine diphosphate glucose and pyrophosphate. In this study, a larch UGPase (LgUGPase) gene was isolated from Larix gmelinii. The 1,443-bp open reading frame encodes a protein of 480 amino acids with a predicted molecular weight of 53.7 kDa and shows striking sequence similarity to UGPase proteins from Pinus taeda and Picea sitchensis. Semiquantitative reverse transcription-polymerase chain reaction showed that the LgUGPase gene was expressed primarily in the larch stem in addition to its root and leaf. Southern blot analysis indicated that LgUGPase is encoded by two genes in the L. gmelinii genome. Overexpression of LgUGPase enhanced vegetative growth in transgenic Arabidopsis and increased the contents of soluble sugars and cellulose, and thickened parenchyma cell walls. These results revealed that L. gmelinii UGPase participates in sucrose/polysaccharide metabolism and cell wall biosynthesis, suggesting that LgUGPase may be a good candidate gene for improvement of fiber cell development in plants.

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir.

Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

[Genetic diversity and differentiation of Gmelin larch Larix gmelinii populations from Evenkia (Central Siberia)]. / Geneticheskoe raznoobrazie i differentsiatsiia populiatsii listvennitsy gmelina v évenkii ( Sredniaia Sibir').

Within- and among-population diversity of Gmelin larch Larix gmelinii (Rupr.) Rupr. from Evenkia was inferred from data on 17 genes determining allozyme diversity of ten enzymes. More than 50% of the genes proved to be polymorphic. On average, each tree was heterozygous at 9.2% genes. Heterozygosity expected from the Hardy-Weinberg proportions was higher, 12.5%. A deficit of heterozygous genotypes was observed in all populations under study and attributed to inbreeding. With Wright's F statistics, average individual inbreeding was estimated at 26.6% relative to the population (F(IS)) and at 27.8% relative to the species (F(IT)). The greatest deficit of heterozygosity was observed for the youngest population. Within-population variation accounted for more than 98% of the total variation, while the contribution of among-population variation was 1.66%. Genetic distance between populations varied from 0.0025 to 0.0042, averaging 0.0035.