Ting-Feng Yeh

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome–based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Elucidation of the structure of cellulolytic enzyme lignin

Abstract Cellulolytic enzyme lignin (CEL) and milled wood lignin (MWL) were prepared by three different ball-milling methods. The structure of CEL at various yields was elucidated and compared with MWL using wet chemical analysis, FTIR and solution-state NMR techniques. Results show that ball milling of wood degrades β-O-4 structures in lignin. However, even after extensive ball milling, less than 25% of the β-O-4 structures were degraded. The extent of degradation was less for softwood than for hardwood lignin. Extractable lignin yield, either MWL or CEL, was the best way to assess the extent and effect of ball milling. CEL is preferred over MWL, as it can be isolated in higher yield with less degradation. CEL was isolated at yields ranging from 20% to 86%. Over this range the CEL had similar structures, suggesting that lignin in the secondary wall is uniform in structure. The residual enzyme lignin (REL) was structurally different from CEL and may originate mainly from the middle lamella. In this paper we propose a new procedure for the isolation of lignin for use in structural studies, whereby wood is sufficiently milled and successively extracted to produce three lignin fractions representing the total lignin in wood.

Morphological and chemical variations between juvenile wood, mature wood, and compression wood of loblolly pine (Pinus taeda L.)

Abstract To better understand the within-tree variations between juvenile wood, mature wood, and compression wood, wood from a 35-year-old mature bent loblolly pine was separated into seven groups by different positions in the tree. Morphological and chemical structure analyses, including fiber quality, X-ray diffraction, sugar and lignin content analysis, as well as nitrobenzene oxidation, ozonation, and advanced NMR spectroscopy, were performed. Fiber properties were significantly different for tree-top juvenile normal wood and tree-bottom juvenile normal wood, juvenile normal and mature normal wood, juvenile compression and mature compression wood. However, differences in the chemical structure and composition were less significant within the specific tissues indicated above.

Rapid analysis of transgenic trees using transmittance near-infrared spectroscopy (NIR)

Abstract Genetic engineering of trees has generated a large amount of interest in the development of highly improved transgenic trees. To efficiently monitor and control the properties of the transgenic products, a rapid, mini-scale analytical method is required. Transmittance near-infrared (NIR) spectroscopy was chosen as a fast analysis tool for characterizing the chemical properties of the transgenic products. Pellets were prepared from 75 mg of wood meal and directly scanned using transmittance NIR spectroscopy. Very strong correlations were obtained between the NIR data and conventional wet-chemistry results for the lignin content, S/G ratio, cellulose and xylose content. The results indicate that transmittance NIR is a powerful tool for determining and screening the chemical properties of transgenic trees.

Comparison of morphological and chemical properties between juvenile wood and compression wood of loblolly pine

Abstract In conifers, juvenile wood (JW) is always associated with compression wood (CW). Due to their similar properties, there is a common belief that JW is the same as CW. To resolve whether JW is identical to CW, 24 rooted cuttings of one loblolly pine clone were planted in growth chambers under normal, artificial bending, and windy environments. The results show that the morphology of JW is significantly different from CW. Furthermore, chemical analyses revealed that JW and CW are significantly different in chemical composition. Our results indicate that JW is different from CW, and the wood formed under a controlled windy environment is a mild type of compression wood.

Effects of alkali pretreatment on surface properties and green color conservation of moso bamboo (Phyllostachys pubescens Mazel).

Summary Standing bamboo culms have an attractive greenish skin. Once harvested, however, they turn yellow and the color starts to decay. These adverse properties limit their applications and degrade their performance. Chromated copper arsenate (CCA) based preservatives have been reported to be good protectors for preserving the green color of bamboo culms. An alkali pretreatment was required before applying CCA. However, the mechanism of alkali pretreatment on color conservation is not fully understood. In this study, morphology, wettability, chemical and pigment changes of moso bamboo (Phyllostachys pubescens Mazel) were studied after treating with four alkali reagents. The results showed that 2% KOH effectively removed the waxes and capes of silica cells on moso bamboo surface and also increased the surface wettability much better than treatments with 4% K2CO3, 10% K2CO3, 4% Na2CO3, or 4% NaHCO3. The KOH pretreatment also results in a better penetration and reaction with the green color protector (5% Boliden K-33, type-B CCA), and subsequently preserved the green color appearance of moso bamboo.

Protection and fastness of green color of moso bamboo (Phyllostachys pubescens Mazel) treated with chromium-based reagents

The attractive green color of moso bamboo (Phyllostachys pubescens Mazel) culms fades without chemical treatment. Chromated copper arsenate (CCA) has been used to protect the green color of bamboo, but CCA is harmful to factory workers and the environment. To overcome the toxicity of arsenic in CCA, two chromium-based formulas developed by the authors, chromated copper phosphate (CCP) and chromated phosphate (CP), were evaluated for their protection of the green color of moso bamboo. The results revealed that bamboo treated with CP had a greener color than those treated with CCA or CCP. The concentration, treatment time, and CrO3/H3PO4 ratio in CP solution greatly affected the color of moso bamboo culms. The attractive green color, which resembles the color of fresh-cut moso bamboo, was obtained by treating bamboo with 2% CP solution at 60°C for 3h, using a 1∶1 CrO3/H3PO4 ratio in aqueous solution. In addition, the CP-treated moso bamboo exhibited excellent green color fastness in both accelerated ultraviolet lightfastness testing and outdoor weathering exposure.

Potential source of S-(+)-linalool from Cinnamomum osmophloeum ct. linalool leaf: essential oil profile and enantiomeric purity.

Cinnamomum osmophloeum ct. linalool is one of the chemotypes of the indigenous cinnamon in Taiwan. In this study, hydrodistillation was used for extracting the essential oils (EOs) of C. osmophloeum ct. linalool leaves collected from various plants and seasons, and GC-MS and GC-FID were used to examine variations and contents of the chemical composition in EOs. Moreover, the absolute configuration of the main constituent and its EO content were illustrated by GC-FID with a chiral column. In addition, we also investigated the effect of the extraction time (1, 2, 6, and 10 h) on the yield of EO and the contents of the main constituents. Results from this study revealed that the average EO yield of 12 plants was 3.7%, and linalool accounted for more than 90%. The linalool in the EO was proved to be pure S-(+)-linalool, and its content in the leaves ranged from 28.8 ± 0.3 to 35.1 ± 0.2 mg/g. Furthermore, there were no obvious differences in EO yield and S-(+)-linalool content from various plants and seasons. On the other hand, we also demonstrated that EO and S-(+)-linalool from C. osmophloeum ct. linalool leaves can be completely extracted out by 1 h of hydrodistillation.

Effects of chromated-phosphate treatment process on the green color protection of ma bamboo (Dendrocalamus latiflorus)

Ma bamboo (Dendrocalamus latiflorus Munro) treated with chromated phosphate (CP) exhibits an excellent green color. To understand the effects of the treatment sequence of CrO3 and H3PO4 and their interactions regarding green color protection, CrO3-H3PO4 and H3PO4-CrO3 two-step treatments plus a H3PO4-CrO3-H3PO4 three-step treatment were carried out in this study. Results revealed that the treatment sequence of CrO3 and H3PO4 definitely affects the effectiveness of bamboo color protection. Green color protection of ma bamboo culm could not be achieved by treating it with CrO3 or H3PO4 alone or with the H3PO4-CrO3 two-step treatment. Only by treating it with the CrO3-H3PO4 two-step treatment or the H3PO4-CrO3-H3PO4 three-step treatment did ma bamboo exhibit an excellent green color. The results indicated that bamboo reacts first with CrO3 and then forms an insoluble complex with H3PO4, which produces the green color on its epidermis. Chlorophyll analyses demonstrated that chlorophyll is not a key factor for green color protection. The green pigment was also formed when chlorophyll-free bamboo was treated with 2% CP at 60°C for 3h.

A Potential Low-Coumarin Cinnamon Substitute: Cinnamomum osmophloeum Leaves

The essential oils from leaves of Taiwans indigenous cinnamon (Cinnamomum osmophloeum ct. cinnamaldehyde) have similar constituents as compared to that from commercial bark cinnamons. This indigenous cinnamon has been proven to have excellent bioactivities. To understand whether this indigenous cinnamon contains a high level of the hepatotoxic compound, coumarin, as often seen in Cassia cinnamons, current research focused on determining the coumarin content in this indigenous cinnamon and screening the low-coumarin clones. The results demonstrated that the coumarin contents in all tested indigenous cinnamon clones were much lower than that found in Cassia cinnamons. In addition, this indigenous cinnamon contains about 80% (w/w) of cinnamaldehyde and 0.4-2.7% (w/w) of eugenol in its leaf essential oils. This combination could provide this indigenous cinnamon a better shelf life compared to that of regular commercial cinnamons. These results suggested that leaves of this indigenous cinnamon could be a potential resource for a safer cinnamon substitute.