Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant.

We recently reported that a genetic transformation of the RNA-Binding-Protein (McRBP), an RNA chaperone gene derived from common ice plant (Mesembryanthemum crystallinum), alleviated injury and loss of biomass production by salt stress in Eucalyptus camaldulensis in a semi-confined screen house trial. In this study, we assessed the potential environmental impact of the transgenic Eucalyptus in a manner complying with Japanese biosafety regulatory framework required for getting permission for experimental confined field trials. Two kinds of bioassays for the effects of allelopathic activity on the growth of other plants, i.e., the sandwich assay and the succeeding crop assay, were performed for three transgenic lines and three non-transgenic lines. No significant differences were observed between transgenic and non-transgenic plants. No significant difference in the numbers of cultivable microorganisms analyzed by the spread plate method were observed among the six transgenic and non-transgenic lines. These results suggested that there is no significant difference in the potential impact on biodiversity between the transgenic McRBP-E. camaldulensis lines and their non-transgenic comparators.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.).

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA-Binding-Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened-house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants' acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high-stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non-transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate-stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non-transgenic status survived the 70 mM NaCl treatment, but after 6-month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non-transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non-transgenic plants after 15-week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.

RNA-seq analysis of lignocellulose-related genes in hybrid Eucalyptus with contrasting wood basic density.

BACKGROUND: Wood basic density (WBD), the biomass of plant cell walls per unit volume, is an important trait for elite tree selection in kraft pulp production. Here, we investigated the correlation between WBD and wood volumes or wood properties using 98 open-pollinated, 2.4 to 2.8 year-old hybrid Eucalyptus (Eucalyptus urophylla x E. grandis). Transcript levels of lignocellulose biosynthesis-related genes were studied. RESULTS: The progeny plants had average WBD of 516 kg/m3 with normal distribution and did not show any correlations between WBD and wood volume or components of α-cellulose, hemicellulose and Klason lignin content. Transcriptomic analysis of two groups of five plants each with high (570-609 kg/m3) or low (378-409 kg/m3) WBD was carried out by RNA-Seq analysis with total RNAs extracted from developing xylem tissues at a breast height. Lignocellulose biosynthesis-related genes, such as cellulose synthase, invertase, cinnamate-4-hydroxylase and cinnamoyl-CoA reductase showed higher transcript levels in the high WBD group. Among plant cell wall modifying genes, increased transcript levels of several expansin and xyloglucan endo-transglycosylase/hydrolase genes were also found in high WBD plants. Interestingly, strong transcript levels of several cytoskeleton genes encoding tubulin, actin and myosin were observed in high WBD plants. Furthermore, we also found elevated transcript levels of genes encoding NAC, MYB, basic helix-loop-helix, homeodomain, WRKY and LIM transcription factors in the high WBD plants. All these results indicate that the high WBD in plants has been associated with the increased transcription of many genes related to lignocellulose formation. CONCLUSIONS: Most lignocellulose biosynthesis related genes exhibited a tendency to transcribe at relatively higher level in high WBD plants. These results suggest that lignocellulose biosynthesis-related genes may be associated with WBD.

Responses to environmental stresses in woody plants: key to survive and longevity.

Environmental stresses have adverse effects on plant growth and productivity, and are predicted to become more severe and widespread in decades to come. Especially, prolonged and repeated severe stresses affecting growth and development would bring down long-lasting effects in woody plants as a result of its long-term growth period. To counteract these effects, trees have evolved specific mechanisms for acclimation and tolerance to environmental stresses. Plant growth and development are regulated by the integration of many environmental and endogenous signals including plant hormones. Acclimation of land plants to environmental stresses is controlled by molecular cascades, also involving cross-talk with other stresses and plant hormone signaling mechanisms. This review focuses on recent studies on molecular mechanisms of abiotic stress responses in woody plants, functions of plant hormones in wood formation, and the interconnection of cell wall biosynthesis and the mechanisms shown above. Understanding of these mechanisms in depth should shed light on the factors for improvement of woody plants to overcome severe environmental stress conditions.

Transcriptional enhancement of a bacterial choline oxidase A gene by an HSP terminator improves the glycine betaine production and salinity stress tolerance of Eucalyptus camaldulensis trees.

Novel transgenic Eucalyptus camaldulensis trees expressing the bacterial choline oxidase A (codA) gene by the Cauliflower mosaic virus (CaMV) 35S promoter and the Arabidopsis thaliana heat shock protein (HSP) terminator was developed. To evaluate the codA transcription level and the metabolic products and abiotic stress tolerance of the transgenic trees, a six-month semi-confined screen house cultivation trial was conducted under a moderate-stringency salt-stress condition. The transcription level of the CaMV 35S promoter driven-codA was more than fourfold higher, and the content of glycine betaine, the metabolic product of codA, was twofold higher, with the HSP terminator than with the nopaline synthase (NOS) terminator. Moreover, the screen house cultivation revealed that the growth of transgenic trees under the salt stress condition was alleviated in correlation with the glycine betaine concentration. These results suggest that the enhancement of codA transcription by the HSP terminator increased the abiotic stress tolerance of Eucalyptus plantation trees.

Environmental risk assessment of impacts of transgenic Eucalyptus camaldulensis events highly expressing bacterial Choline Oxidase A gene.

Under the Japanese biosafety regulatory framework for transgenic plants, data for assessing a transgenic plant's impact on biodiversity must be submitted in order to obtain approval for a confined field trial. We recently reported the development of four novel transgenic Eucalyptus camaldulensis clones expressing the bacterial choline oxidase A (codA) gene, i.e., codAH-1, codAH-2, codAN-1, and codAN-2, and evaluated their abiotic tolerance by semiconfined screen house trial cultivation. Here we evaluated the impacts of the transgenic E. camaldulensis clones on productivities of harmful substances from those clones to affect soil microorganisms and/or other plants in the environment. A comparison of the assessment data between the transgenic trees and non-transgenic comparators showed no significant difference in potential impacts on biodiversity. The results contribute to sound-science evidence ensuring substantial equivalence between transgenic and non-transgenic E. camaldulensis.

Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition.

Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen.

We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.

Ntlim1, a PAL-box binding factor, controls promoter activity of the horseradish wound-inducible peroxidase gene.

To understand molecular mechanisms underlying wound-induced expression of plant peroxidase genes, the promoter of a horseradish C2 peroxidase (prxC2) gene was analyzed. We had previously isolated a tobacco nuclear protein, Ntlim1, as a trans factor binding to a PAL-box motif of the prxC2 promoter; however, the function of the Ntlim1 trans factor and the PAL-box motif in wound-responsive expression of the prxC2 gene remains unclear. Here, we found that the prxC2 promoter without the intact PAL-box motif failed to direct a normal level of both the basal and the wound-induced expression of beta-glucuronidase (GUS) reporter gene in transgenic tobacco plants, indicating that the PAL-box motif functions as an essential cis element of the prxC2 promoter. We also found that antisense expression of Ntlim1 in transgenic plants carrying the prxC2 promoter::GUS chimeric construct decreased not only the level of the basal and the wound-induced expression of the GUS reporter gene but also the extent of wound inducibility of the prxC2 promoter itself. This result indicates that Ntlim1 is required for the basal level of prxC2 promoter activity as well as its up-regulation under wound stress. Moreover, consistent with the results obtained in planta, result from super-shift assay indicates that the Ntlim1 binds to the PAL-box motif independently of wound stress.