Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Overexpression of a Poplar RING-H2 Zinc Finger, Ptxerico, Confers Enhanced Drought Tolerance via Reduced Water Loss and Ion Leakage in Populus.

Drought stress is one of the major environmental problems in the growth of crops and woody perennials, but it is getting worse due to the global climate crisis. XERICO, a RING (Really Interesting New Gene) zinc-finger E3 ubiquitin ligase, has been shown to be a positive regulator of drought tolerance in plants through the control of abscisic acid (ABA) homeostasis. We characterized a poplar (Populus trichocarpa) RING protein family and identified the closest homolog of XERICO called PtXERICO. Expression of PtXERICO is induced by both salt and drought stress, and by ABA treatment in poplars. Overexpression of PtXERICO in Arabidopsis confers salt and ABA hypersensitivity in young seedlings, and enhances drought tolerance by decreasing transpirational water loss. Consistently, transgenic hybrid poplars overexpressing PtXERICO demonstrate enhanced drought tolerance with reduced transpirational water loss and ion leakage. Subsequent upregulation of genes involved in the ABA homeostasis and drought response was confirmed in both transgenic Arabidopsis and poplars. Taken together, our results suggest that PtXERICO will serve as a focal point to improve drought tolerance of woody perennials.

Wood forming tissue-specific bicistronic expression of PdGA20ox1 and PtrMYB221 improves both the quality and quantity of woody biomass production in a hybrid poplar.

With the exponential growth of the human population and industrial developments, research on renewable energy resources is required to alleviate environmental and economic impacts caused by the consumption of fossil fuels. In this study, we present a synthetic biological application of a wood forming tissue-specific bicistronic gene expression system to improve both the quantity and quality of woody biomass to minimize undesirable growth penalties. Our transgenic poplars, designed to express both PdGA20ox1 (a GA20-oxidase from Pinus densiflora producing bioactive gibberellin, GA) and PtrMYB221 (a MYB transcription factor negatively regulating lignin biosynthesis) under the developing xylem (DX) tissue-specific promoter (i.e., DX15::PdGA20ox1-2A-PtrMYB221 poplar), resulted in a 2-fold increase in biomass quantity compared to wild-type (WT), without undesirable growth defects. A similar phenotype was observed in transgenic Arabidopsis plants harboring the same gene constructs. These phenotypic consequences were further verified in the field experiments. Importantly, our transgenic poplars exhibited an improved quality of biomass with reduced lignin content (~16.0 wt%) but increased holocellulose content (~6.6 wt%). Furthermore, the saccharification efficiency of our transgenic poplar increased significantly by up to 8%. Our results demonstrate that the controlled production of both GA and a secondary wall modifying regulator in the same spatio-temporal manner can be utilized as an efficient biotechnological tool for producing the desired multi-purpose woody biomass.

Loliolide Presents Antiapoptosis and Antiscratching Effects in Human Keratinocytes.

Loliolide is a monoterpenoid hydroxylactone present in freshwater algae that has anti-inflammatory and antiaging activity; however, its effects on ultraviolet-damaged skin have yet to be elucidated. This study investigated the antiapoptosis and wound-healing effects of loliolide using HaCaT cells (a human keratinocyte cell line). Loliolide inhibited the expression of reactive oxygen species (ROS) induced by ultraviolet radiation as well as wrinkle formation-related matrix metalloproteinase genes and increased the expression of the damage repair-related gene SIRT1. The apoptosis signaling pathway was confirmed by Western blot analysis, which showed that loliolide was able to reduce the expression of caspases 3, 8, and 9, which are related to ROS-induced apoptosis. In addition, Western blotting, reverse-transcription polymerase chain reaction (PCR), and real-time PCR analyses showed that loliolide enhanced the expression of the epidermal growth factor receptor signaling pathway (PI3K, AKT) and migration factors, such as K6, K16, and K17; keratinocyte growth factor; and inflammatory cytokines, such as interleukin (IL)-1, IL-17, and IL-22 expressed during the cellular scratching process, suggesting a putative wound-healing ability. Because of the antiapoptosis and antiscratching effects on skin of both loliolide and loliolide-rich Prasiola japonica ethanol extract, we consider the former to be an important compound used in the cosmeceutical industry.

Down-regulation of GIGANTEA-like genes increases plant growth and salt stress tolerance in poplar.

The flowering time regulator GIGANTEA (GI) connects networks involved in developmental stage transitions and environmental stress responses in Arabidopsis. However, little is known about the role of GI in growth, development and responses to environmental challenges in the perennial plant poplar. Here, we identified and functionally characterized three GI-like genes (PagGIa, PagGIb and PagGIc) from poplar (Populus alba × Populus glandulosa). PagGIs are predominantly nuclear localized and their transcripts are rhythmically expressed, with a peak around zeitgeber time 12 under long-day conditions. Overexpressing PagGIs in wild-type (WT) Arabidopsis induced early flowering and salt sensitivity, while overexpressing PagGIs in the gi-2 mutant completely or partially rescued its delayed flowering and enhanced salt tolerance phenotypes. Furthermore, the PagGIs-PagSOS2 complexes inhibited PagSOS2-regulated phosphorylation of PagSOS1 in the absence of stress, whereas these inhibitions were eliminated due to the degradation of PagGIs under salt stress. Down-regulation of PagGIs by RNA interference led to vigorous growth, higher biomass and enhanced salt stress tolerance in transgenic poplar plants. Taken together, these results indicate that several functions of Arabidopsis GI are conserved in its poplar orthologues, and they lay the foundation for developing new approaches to producing salt-tolerant trees for sustainable development on marginal lands worldwide.

Developing xylem-preferential expression of PdGA20ox1, a gibberellin 20-oxidase 1 from Pinus densiflora, improves woody biomass production in a hybrid poplar.

Woody biomass has gained popularity as an environmentally friendly, renewable and sustainable resource for liquid fuel production. Here, we demonstrate biotechnological improvement of the quantity and quality of woody biomass by employing developing xylem (DX)-preferential production of gibberellin (GA), a phytohormone that positively regulates stem growth. First, for the proof of concept experiment, we produced transgenic Arabidopsis plants expressing GA20-oxidase, a key enzyme in the production of bioactive GAs, from Pinus densiflora (PdGA20ox1) under the control of either a constitutive 35S promoter, designated 35S::PdGA20ox1, or a DX-specific promoter (originated from poplar), designated DX15::PdGA20ox1. As we hypothesized, both transgenic Arabidopsis plants (35S::PdGA20ox1 and DX15::PdGA20ox1) exhibited an accelerated stem growth that resulted in a large increase of biomass, up to 300% compared to wild-type control plants, together with increased secondary wall thickening and elongation of fibre cells. Next, we applied our concept to the production of transgenic poplar trees. Both transgenic poplar trees (35S::PdGA20ox1 and DX15::PdGA20ox1) showed dramatic increases in biomass, up to 300%, with accelerated stem growth and xylem differentiation. Cell wall monosaccharide composition analysis revealed that in both Arabidopsis and poplar, glucose and xylose contents were significantly increased. However, undesirable phenotypes of 35S::PdGA20ox1 poplar, including poor root growth and leaf development, were found. Interestingly, DX15::PdGA20ox1 poplar resulted in a reduction of undesirable phenotypes. Our results indicate that the controlled production of GAs through a tissue-specific promoter can be utilized as an efficient biotechnological tool for producing enhanced plant biomass, minimizing unwanted effects.

The poplar basic helix-loop-helix transcription factor BEE3 - Like gene affects biomass production by enhancing proliferation of xylem cells in poplar.

Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

Differential regulation of cytokinin oxidase genes and cytokinin-induced auxin biosynthesis by cellular cytokinin level in transgenic poplars.

KEY MESSAGE: The present work with transgenic poplar lines producing varying levels of trans -zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin. Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4 appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis.

Knockdown of PagSAP11 Confers Drought Resistance and Promotes Lateral Shoot Growth in Hybrid Poplar (Populus alba × Populus tremula var. glandulosa).

Plants have evolved defense mechanisms to overcome unfavorable climatic conditions. The growth and development of plants are regulated in response to environmental stress. In this study, we investigated the molecular and physiological characteristics of a novel gene PagSAP11 in hybrid poplar (Populus alba × Populus tremula var. glandulosa) under drought stress. PagSAP11, a stress-associated protein (SAP) family gene, encodes a putative protein containing an A20 and AN1 zinc-finger domain at its N- and C-termini, respectively. Knockdown of PagSAP11 transgenic poplars (SAP11-Ri) enhanced their tolerance to drought stress compared with wild type plants. Moreover, the RNAi lines showed increased branching of lateral shoots that led to a gain in fresh weight, even when grown in the living modified organism (LMO) field. In SAP11-Ri transgenic plants, the expression levels of genes involved in axillary bud outgrowth and cell proliferation such as DML10, CYP707A and RAX were increased while the DRM gene which involved in bud dormancy was down-regulated. Taken together, these results indicate that PagSAP11 represents a promising candidate gene for engineering trees with improved stress tolerance and growth during unfavorable conditions.

Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance.

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (Populus alba × Poplus glandulosa) expressing the AtNDPK2 gene under the control of a SWPA2 promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant's tolerance to MV-mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV-treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin-response genes IAA2 and IAA5. These results suggest that enhanced AtNDPK2 expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar.

Effect of genetically modified poplars on soil microbial communities during the phytoremediation of waste mine tailings.

The application of transgenic plants to clean up environmental pollution caused by the wastes of heavy metal mining is a promising method for removing metal pollutants from soils. However, the effect of using genetically modified organisms for phytoremediation is a poorly researched topic in terms of microbial community structures, despite the important role of microorganisms in the health of soil. In this study, a comparative analysis of the bacterial and archaeal communities found in the rhizosphere of genetically modified (GM) versus wild-type (WT) poplar was conducted on trees at different growth stages (i.e., the rhizospheres of 1.5-, 2.5-, and 3-year-old poplars) that were cultivated on contaminated soils together with nonplanted control soil. Based on the results of DNA pyrosequencing, poplar type and growth stages were associated with directional changes in the structure of the microbial community. The rate of change was faster in GM poplars than in WT poplars, but the microbial communities were identical in the 3-year-old poplars. This phenomenon may arise because of a higher rate and greater extent of metal accumulation in GM poplars than in naturally occurring plants, which resulted in greater changes in soil environments and hence the microbial habitat.

Field evaluation of transgenic hybrid poplars with desirable wood properties and enhanced growth for biofuel production by bicistronic expression of PdGA20ox1 and PtrMYB3 in wood-forming tissue.

BACKGROUND: To create an ideotype woody bioenergy crop with desirable growth and biomass properties, we utilized the viral 2A-meidated bicistronic expression strategy to express both PtrMYB3 (MYB46 ortholog of Populus trichocarpa, a master regulator of secondary wall biosynthesis) and PdGA20ox1 (a GA20-oxidase from Pinus densiflora that produces gibberellins) in wood-forming tissue (i.e., developing xylem). RESULTS: Transgenic Arabidopsis plants expressing the gene construct DX15::PdGA20ox1-2A-PtrMYB3 showed a significant increase in both stem fresh weight (threefold) and secondary wall thickening (1.27-fold) relative to wild-type (WT) plants. Transgenic poplars harboring the same gene construct grown in a greenhouse for 60 days had a stem fresh weight up to 2.6-fold greater than that of WT plants. In a living modified organism (LMO) field test conducted for 3 months of active growing season, the stem height and diameter growth of the transgenic poplars were 1.7- and 1.6-fold higher than those of WT plants, respectively, with minimal adverse growth defects. Although no significant changes in secondary wall thickening of the stem tissue of the transgenic poplars were observed, cellulose content was increased up to 14.4 wt% compared to WT, resulting in improved saccharification efficiency of the transgenic poplars. Moreover, enhanced woody biomass production by the transgenic poplars was further validated by re-planting in the same LMO field for additional two growing seasons. CONCLUSIONS: Taken together, these results show considerably enhanced wood formation of our transgenic poplars, with improved wood quality for biofuel production.

Drought Stress-Mediated Transcriptome Profile Reveals NCED as a Key Player Modulating Drought Tolerance in Populus davidiana.

Populus trichocarpa has been studied as a model poplar species through biomolecular approaches and was the first tree species to be genome sequenced. In this study, we employed a high throughput RNA-sequencing (RNA-seq) mediated leaf transcriptome analysis to investigate the response of four different Populus davidiana cultivars to drought stress. Following the RNA-seq, we compared the transcriptome profiles and identified two differentially expressed genes (DEGs) with contrasting expression patterns in the drought-sensitive and tolerant groups, i.e., upregulated in the drought-tolerant P. davidiana groups but downregulated in the sensitive group. Both these genes encode a 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme required for abscisic acid (ABA) biosynthesis. The high-performance liquid chromatography (HPLC) measurements showed a significantly higher ABA accumulation in the cultivars of the drought-tolerant group following dehydration. The Arabidopsis nced3 loss-of-function mutants showed a significantly higher sensitivity to drought stress, ~90% of these plants died after 9 days of drought stress treatment. The real-time PCR analysis of several key genes indicated a strict regulation of drought stress at the transcriptional level in the P. davidiana drought-tolerant cultivars. The transgenic P. davidiana NCED3 overexpressing (OE) plants were significantly more tolerant to drought stress as compared with the NCED knock-down RNA interference (RNAi) lines. Further, the NCED OE plants accumulated a significantly higher quantity of ABA and exhibited strict regulation of drought stress at the transcriptional level. Furthermore, we identified several key differences in the amino acid sequence, predicted structure, and co-factor/ligand binding activity of NCED3 between drought-tolerant and susceptible P. davidiana cultivars. Here, we presented the first evidence of the significant role of NCED genes in regulating ABA-dependent drought stress responses in the forest tree P. davidiana and uncovered the molecular basis of NCED3 evolution associated with increased drought tolerance.

PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathway in a hybrid poplar.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.

Ginseng-derived patatin-related phospholipase PgpPLAIIIß alters plant growth and lignification of xylem in hybrid poplars.

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIß derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIß plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

Wood Transcriptome Profiling Identifies Critical Pathway Genes of Secondary Wall Biosynthesis and Novel Regulators for Vascular Cambium Development in Populus.

Wood, the most abundant biomass on Earth, is composed of secondary xylem differentiated from vascular cambium. However, the underlying molecular mechanisms of wood formation remain largely unclear. To gain insight into wood formation, we performed a series of wood-forming tissue-specific transcriptome analyses from a hybrid poplar (Populus alba × P. glandulosa, clone BH) using RNA-seq. Together with shoot apex and leaf tissue, cambium and xylem tissues were isolated from vertical stem segments representing a gradient of secondary growth developmental stages (i.e., immature, intermediate, and mature stem). In a comparative transcriptome analysis of the 'developing xylem' and 'leaf' tissue, we could identify critical players catalyzing each biosynthetic step of secondary wall components (e.g., cellulose, xylan, and lignin). Several candidate genes involved in the initiation of vascular cambium formation were found via a co-expression network analysis using abundantly expressed genes in the 'intermediate stem-derived cambium' tissue. We found that transgenic Arabidopsis plants overexpressing the PtrHAM4-1, a GRAS family transcription factor, resulted in a significant increase of vascular cambium development. This phenotype was successfully reproduced in the transgenic poplars overexpressing the PtrHAM4-1. Taken together, our results may serve as a springboard for further research to unravel the molecular mechanism of wood formation, one of the most important biological processes on this planet.

Anti-Apoptotic and Anti-Inflammatory Activities of Edible Fresh Water Algae Prasiola japonica in UVB-Irradiated Skin Keratinocytes.

Skin is the outer tissue layer and is a barrier protecting the body from various external stresses. The fresh water green edible algae Prasiola japonica has antiviral, antimicrobial, and anti-inflammatory properties; however, few studies of its effects on skin-protection have been reported. In this study, Prasiola japonica ethanol extract (Pj-EE) was prepared, and its skin-protective properties were investigated in skin keratinocytes. Pj-EE inhibited ROS production in UVB-irradiated HaCaT cells without cytotoxicity. Pj-EE also suppressed the apoptotic death of UVB-irradiated HaCaT cells by decreasing the generation of apoptotic bodies and the proteolytic activation of apoptosis caspase-3, -8, and -9. Moreover, Pj-EE downregulated the mRNA expression of the inflammatory gene cyclooxygenase-2 (COX-2), the pro-inflammatory cytokine genes interleukin (IL)-1ß, IL-8, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, and the tissue remodeling genes matrix metalloproteinase (MMP)-1, -2, -3, and -9. The Pj-EE-induced anti-inflammatory effect was mediated by suppressing the activation of nuclear factor-kappa B (NF-κB) signaling pathway in the UVB-irradiated HaCaT cells. Taken together, these results suggest that Pj-EE exerts skin-protective effects through anti-oxidant, anti-apoptotic, and anti-inflammatory activities in skin keratinocytes.

Poplar MYB transcription factor PtrMYB012 and its Arabidopsis AtGAMYB orthologs are differentially repressed by the Arabidopsis miR159 family.

A phenotype-based screening of the T1 transgenic Arabidopsis population transformed by overexpression constructs of the entire poplar MYB transcription factor family found that overexpression of a poplar MYB transcription factor, PtrMYB012, in Arabidopsis resulted in upwardly curled rosette leaves, dwarfism and male sterility. Sequence analysis identified that PtrMYB012 is homologous to the Arabidopsis GAMYB genes (e.g., AtMYB65 and AtMYB33). Gene expression analysis revealed that PtrMYB012 is specifically expressed in floral tissues, especially in male catkins, similar to AtMYB65. It was well known that Arabidopsis GAMYBs are negatively regulated by microRNA159 (miR159) during vegetative growth; thus, the typical phenotypes of upwardly curled leaves, dwarfism and male sterility were only shown in overexpression of GAMYBs with mutations in the miR159 target sequence. To confirm our phenotypic consequences, we independently re-produced transgenic Arabidopsis plants overexpressing PtrMYB012 without mutations in the miR159 target sequence. The resulting 35 S::PtrMYB012 Arabidopsis plants phenocopied the previous transgenic Arabidopsis plants, suggesting that PtrMYB012 is probably not a target of Arabidopsis miR159 despite containing the conserved miR159 target sequence. To gain further insight, we produced transgenic poplars overexpressing the intact PtrMYB012. As a result, no conspicuous phenotype was found in 35 S::PtrMYB012 poplar plants. These results suggest that PtrMYB012 transcripts are down-regulated by miR159 in poplar but not in Arabidopsis. Indeed, subsequent 5'-RACE analysis confirmed that PtrMYB012 transcripts are completely degraded in poplar, probably by miR159, but not in Arabidopsis. These results suggest that species-specific family members of miR159 are important for the regulation of normal growth and development in plants.

Evaluation of a novel promoter from Populus trichocarpa for mature xylem tissue specific gene delivery.

Wood (i.e., secondary xylem) is an important raw material for many industrial applications. Mature xylem (MX) tissue-specific genetic modification offers an effective means to improve the chemical and physical properties of the wood. Here, we describe a promoter that drives strong gene expression in a MX tissue-specific manner. Using whole-transcriptome genechip analyses of different tissue types of poplar, we identified five candidate genes that had strong expression in the MX tissue. The putative promoter sequences of the five MX-specific genes were evaluated for their promoter activity in both transgenic Arabidopsis and poplar. Among them, we found the promoter of Potri.013G007900.1 (called the PtrMX3 promoter) had the strongest activity in MX and thus was further characterized. In the stem and root tissues of transgenic Arabidopsis plants, the PtrMX3 promoter activity was found exclusively in MX tissue. MX-specific activity of the promoter was reproduced in the stem tissue of transgenic poplar plants. The PtrMX3 promoter activity was not influenced by abiotic stresses or exogenously applied growth regulators, indicating the PtrMX3 promoter is bona fide MX tissue-specific. Our study provides a strong MX-specific promoter for MX-specific modifications of woody biomass.