Unraveling genetic variation among white spruce families generated through different breeding strategies: Heritability, growth, physiology, hormones and gene expression.

Tree improvement programs select genotypes for faster growth, at both early and late stages, to increase yields over unimproved material, and the improvement is frequently attributed to genetic control in growth parameters among genotypes. Underutilized genetic variability among genotypes also has the potential to ensure future gains are possible. However, the genetic variation in growth, physiology and hormone control among genotypes generated from different breeding strategies has not been well characterized in conifers. We assessed growth, biomass, gas exchange, gene expression and hormone levels in white spruce seedlings obtained from three different breeding strategies (controlled crosses, polymix pollination, open pollination) using parents grafted into a clonal seed orchard in Alberta, Canada. A pedigree-based best linear unbiased prediction (ABLUP) mixed model was implemented to quantify variability and narrow-sense heritability for target traits. The levels of several hormones and expression of gibberellin-related genes in apical internodes were also determined. Over the first two years of development, the estimated heritabilities for height, volume, total dry biomass, above ground dry biomass, root:shoot ratio and root length, varied between 0.10 and 0.21, with height having the highest value. The ABLUP values showed large genetic variability in growth and physiology traits both between families from different breeding strategies, and within families. The principal component analysis showed that developmental and hormonal traits explained 44.2% and 29.4% of the total phenotypic variation between the three different breeding strategies and two growth groups. In general, controlled crosses from the fast growth group showed the best apical growth, with more accumulation of indole-3-acetic acid, abscisic acid, phaseic acid, and a 4-fold greater gene expression of PgGA3ox1 in genotypes from controlled crosses versus those from open pollination. However, in some cases, open pollination from the fast and slow growth groups showed the best root development, higher water use efficiency (iWUE and δ13C) and more accumulation of zeatin and isopentenyladenosine. In conclusion, tree domestication can lead to trade-offs between growth, carbon allocation, photosynthesis, hormone levels and gene expression, and we encourage the use of this phenotypic variation identified in improved and unimproved trees to advance white spruce tree improvement programs.

Overexpression of TgERF1, a Transcription Factor from Tectona grandis, Increases Tolerance to Drought and Salt Stress in Tobacco.

Teak (Tectona grandis) is one of the most important wood sources, and it is cultivated in tropical regions with a significant market around the world. Abiotic stresses are an increasingly common and worrying environmental phenomenon because it causes production losses in both agriculture and forestry. Plants adapt to these stress conditions by activation or repression of specific genes, and they synthesize numerous stress proteins to maintain their cellular function. For example, APETALA2/ethylene response factor (AP2/ERF) was found to be involved in stress signal transduction. A search in the teak transcriptome database identified an AP2/ERF gene named TgERF1 with a key AP2/ERF domain. We then verified that the TgERF1 expression is rapidly induced by Polyethylene Glycol (PEG), NaCl, and exogenous phytohormone treatments, suggesting a potential role in drought and salt stress tolerance in teak. The full-length coding sequence of TgERF1 gene was isolated from teak young stems, characterized, cloned, and constitutively overexpressed in tobacco plants. In transgenic tobacco plants, the overexpressed TgERF1 protein was localized exclusively in the cell nucleus, as expected for a transcription factor. Furthermore, functional characterization of TgERF1 provided evidence that TgERF1 is a promising candidate gene to be used as selective marker on plant breeding intending to improve plant stress tolerance.

Overexpression of the Tectona grandis TgNAC01 regulates growth, leaf senescence and confer salt stress tolerance in transgenic tobacco plants.

NAC transcription factors play critical roles in xylem secondary development and in regulation of stress response in plants. NAC proteins related to secondary cell wall development were recently identified and characterized in Tectona grandis (teak), one of the hardwood trees of highest economic importance in the world. In this work, we characterized the novel TgNAC01 gene, which is involved in signaling pathways that mediate teak response to stress. Abscisic acid (ABA) increases TgNAC01 expression in teak plants. Therefore, this gene may have a role in signaling events that mediate ABA-dependent osmotic stress responsive in this plant species. Stable expression in tobacco plants showed that the TgNAC01 protein is localized in the cell nucleus. Overexpression of TgNAC01 in two out three independent transgenic tobacco lines resulted in increased growth, leaf senescence and salt tolerance compared to wild type (WT) plants. Moreover, the stress tolerance of transgenic plants was affected by levels of TgNAC01 gene expression. Water potential, gas exchange and chlorophyll fluorescence were used to determine salt stress tolerance. The 35S:TgNAC01-6 line under 300 mM NaCl stress responded with a significant increase in photosynthesis rate, stomatal conductance, transpiration and carboxylation efficiency, but lower water potential compared to WT plants. The data indicate that the TgNAC01 transcription factor acts as a transcriptional activator of the ABA-mediated regulation and induces leaf senescence.

SNP-based analysis reveals unexpected features of genetic diversity, parental contributions and pollen contamination in a white spruce breeding program.

Accurate monitoring of genetic diversity levels of seedlots and mating patterns of parents from seed orchards are crucial to ensure that tree breeding programs are long-lasting and will deliver anticipated genetic gains. We used SNP genotyping to characterize founder trees, five bulk seed orchard seedlots, and trees from progeny trials to assess pollen contamination and the impact of severe roguing on genetic diversity and parental contributions in a first-generation open-pollinated white spruce clonal seed orchard. After severe roguing (eliminating 65% of the seed orchard trees), we found a slight reduction in the Shannon Index and a slightly negative inbreeding coefficient, but a sharp decrease in effective population size (eightfold) concomitant with sharp increase in coancestry (eightfold). Pedigree reconstruction showed unequal parental contributions across years with pollen contamination levels between 12 and 51% (average 27%) among seedlots, and 7-68% (average 30%) among individual genotypes within a seedlot. These contamination levels were not correlated with estimates obtained using pollen flight traps. Levels of pollen contamination also showed a Pearson's correlation of 0.92 with wind direction, likely from a pollen source 1 km away from the orchard under study. The achievement of 5% genetic gain in height at rotation through eliminating two-thirds of the orchard thus generated a loss in genetic diversity as determined by the reduction in effective population size. The use of genomic profiles revealed the considerable impact of roguing on genetic diversity, and pedigree reconstruction of full-sib families showed the unanticipated impact of pollen contamination from a previously unconsidered source.

Effect of elevated gibberellic acid application on growth and gene expression patterns in white spruce families from a tree improvement program in Alberta, Canada.

Nine open-pollinated families of Picea glauca (Moench) Voss from the Region D1 Controlled Parentage Program (Alberta, Canada) were systematically chosen from fast, medium and slow-growth rankings based on breeding values for height from field progeny tests at age 30 years. Seeds from these families were sown and grown to age 3 years to analyze the performance and correlations of growth, physiological traits and expression of gibberellin-related genes, with and without elevated gibberellic acid 3 (GA3) application, under greenhouse conditions. We observed a significant interaction effect between families and growth groups subjected to 50 µg µl-1 of GA3 treatment, causing a decrease in apical internode length, diameter, volume and absolute transcript level for fast-growing families but an increase for families in the slow-growth group for the same traits. We also observed that in the apical internode, the gene PgGA20ox1 had significantly more relative expression under the elevated GA3 treatment than the control trees. In the stem, PgGA3ox1 showed a significantly higher relative expression under elevated GA3 treatment compared with control trees. Also, the slow-growth group showed more relative expression of PgGA20ox1 (in the apical internode) and PgGA3ox1 (in the stem) than the fast-growth group. The apical internode length and diameter significantly increased by 24% and 16%, respectively, with the hormone treatment in the slow growing group. In general, the PgGID1 and PgDELLA1 genes were upregulated and downregulated respectively, in spruce shoots under the GA3 treatment, meaning a positive feedback regulation by those genes were influencing PgGA20ox1 and PgGA3ox1 expression in that tissue type. Moreover, there was a significant correlation between absolute transcript levels of PgGA20ox1 in the apical internode and apical internode length, and absolute transcript levels of PgGA3ox1 in the stem and the diameter, in the fast-growth group families. This study shows that expression of GA genes is a limiting factor for growth in certain white spruce families with a complex feedback mechanism. Finally, absolute transcript levels of endogenous GA relative to growth parameters in juvenile seedlings could potentially be used to accelerate the early selection of families with inherently rapid apical and radial growth expansion.

Physiological and molecular responses to drought stress in teak (Tectona grandis L.f.).

Drought stress is an increasingly common and worrying phenomenon because it causes a loss of production in both agriculture and forestry. Teak is a tropical tree which needs alternating rainy and dry seasons to produce high-quality wood. However, a robust understanding about the physiological characteristics and genes related to drought stress in this species is lacking. Consequently, after applying moderate and severe drought stress to teak seedlings, an infrared gas analyzer (IRGA) was used to measure different parameters in the leaves. Additionally, using the root transcriptome allowed finding and analyzing the expression of several drought-related genes. As a result, in both water deficit treatments a reduction in photosynthesis, transpiration, stomatal conductance and leaf relative water content was found. As well, an increase in free proline levels and intrinsic water use efficiency was found when compared to the control treatment. Furthermore, 977 transcripts from the root contigs showed functional annotation related to drought stress, and of these, TgTPS1, TgDREB1, TgAREB1 and TgPIP1 were selected. The expression analysis of those genes along with TgHSP1, TgHSP2, TgHSP3 and TgBI (other stress-related genes) showed that with moderate treatment, TgTPS1, TgDREB1, TgAREB1, TgPIP1, TgHSP3 and TgBI genes had higher expression than the control treatment, but with severe treatment only TgTPS1 and TgDREB1 showed higher expression than the control treatment. At the end, a schematic model for the physiological and molecular strategies under drought stress in teak from this study is provided. In conclusion, these physiological and biochemical adjustments in leaves and genetic changes in roots under severe and prolonged water shortage situations can be a limiting factor for teak plantlets' growth. Further studies of those genes under different biotic and abiotic stress treatments are needed.

ANÁLISIS in silico Y EXPRESIÓN GÉNICA DEL FACTOR DE TRANSCRIPCIÓN TgNACO1 IMPLICADO EN XILOGÉNESIS Y ESTRÉS ABIÓTICO EN Tectona grandis / In silico Analysis and Gene Expression of TgNACO1 Transcription Factor Involved in Xylogenesis and Abiotic Stress in Tectona grandis

RESUMEN El xilema secundario es el componente más abundante de la biomasa vegetal. Por tanto, conocer los genes que regulan su formación ayudaría a diseñar estrategias para el mejoramiento genético de la madera. Así, el objetivo de este trabajo fue realizar el análisis computacional de la estructura primaria y secundaria del factor de transcripción (FT) TgNACO1 de Tectona grandis, además de evaluar su historia evolutiva, dominios conservados y expresión génica en tejidos lignificados de árboles de 12 y 60 años. Para ello, se realizó una evaluación del potencial de interacción ion-electrón (PIIE), mediante el método del espectro de la información (MEI) utilizando la librería SFAPS de R-Project, seguido del modelamiento estructural utilizando el software MODELLER y visualizado mediante PyMol. Además, el análisis de alineamiento de secuencia múltiple y filogenia fue mediante el software Bioedit y MrBayes respectivamente. También se evaluó los niveles de síntesis del FT TgNACO1 mediante qRT-PCR. Como resultados, se evidenció que el FT mantiene una estructura (3-hoja antiparalela retorcida, que se compacta contra una a-hélice en la región N-terminal, teniendo así tres dominios a hélice y siete dominios (3 plegada. Asimismo, mediante el MEI se demostró que tiene alrededor de cinco funciones biológicas y mutaciones sobre los aminoácidos con mayor PIIE, lo que conlleva a evoluciones sobre las redes de regulación genética. Finalmente, el FT TgNACO1 podría presentar un papel fundamental en la organización y desarrollo de las partes que componen la albura, como las células radiales de la zona cambial, los vasos, fibras y los anillos de crecimiento.

ABSTRACT Secondary xylem is the most abundant component of plant biomass. Therefore, knowing the genes that regulate its formation would help to design strategies for wood genetic improvement. Thus, the objective of this work was to perform computational analysis of the primary and secondary structure of the TgNACO1 transcription factor (FT) of Tectona grandis, and to evaluate its evolutionary history, conserved domains and gene expression in lignified tissues of trees with 12 and 60 years old. For this, an ion-electron interaction potential (IEP) was evaluated using the information-spectrum method (IEM) using the R-Project and SFAPS library, followed by structural modeling using the MODELLER software and visualized by PyMol program. In addition, the analysis of multiple sequence alignment and phylogeny was performed using Bioedit and MrBayes software, respectively. We also evaluated the qRT-PCR levels of TgNACO1. As results, it was found that TgNACO1 maintains a twisted antiparallel 3-sheet structure, which is compacted against an a-helix in the N-terminal region, having three a-helix domains and seven folded ((-domains. Also, through the IEM, it was demonstrated that it has about five biological functions, and mutations on amino acids with higher IEP, which leads to evolutions on genetic regulation networks. Finally, the FT TgNACO1 could play an esential role in the organization and development of the parts that make up the sapwood, such as the radial cells of the cambial zone, the vessels, fibers and the growth rings.

Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide.

Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

Large-scale transcriptional profiling of lignified tissues in Tectona grandis.

BACKGROUND: Currently, Tectona grandis is one of the most valuable trees in the world and no transcript dataset related to secondary xylem is available. Considering how important the secondary xylem and sapwood transition from young to mature trees is, little is known about the expression differences between those successional processes and which transcription factors could regulate lignin biosynthesis in this tropical tree. Although MYB transcription factors are one of the largest superfamilies in plants related to secondary metabolism, it has not yet been characterized in teak. These results will open new perspectives for studies of diversity, ecology, breeding and genomic programs aiming to understand deeply the biology of this species. RESULTS: We present a widely expressed gene catalog for T. grandis using Illumina technology and the de novo assembly. A total of 462,260 transcripts were obtained, with 1,502 and 931 genes differentially expressed for stem and branch secondary xylem, respectively, during age transition. Analysis of stem and branch secondary xylem indicates substantial similarity in gene ontologies including carbohydrate enzymes, response to stress, protein binding, and allowed us to find transcription factors and heat-shock proteins differentially expressed. TgMYB1 displays a MYB domain and a predicted coiled-coil (CC) domain, while TgMYB2, TgMYB3 and TgMYB4 showed R2R3-MYB domain and grouped with MYBs from several gymnosperms and flowering plants. TgMYB1, TgMYB4 and TgCES presented higher expression in mature secondary xylem, in contrast with TgMYB2, TgHsp1, TgHsp2, TgHsp3, and TgBi whose expression is higher in young lignified tissues. TgMYB3 is expressed at lower level in secondary xylem. CONCLUSIONS: Expression patterns of MYB transcription factors and heat-shock proteins in lignified tissues are dissimilar when tree development was evaluated, obtaining more expression of TgMYB1 and TgMYB4 in lignified tissues of 60-year-old trees, and more expression in TgHsp1, TgHsp2, TgHsp3 and TgBi in stem secondary xylem of 12-year-old trees. We are opening a door for further functional characterization by reverse genetics and marker-assisted selection with those genes. Investigation of some of the key regulators of lignin biosynthesis in teak, however, could be a valuable step towards understanding how rigidity of teak wood and extractives content are different from most other woods. The obtained transcriptome data represents new sequences of T. grandis deposited in public databases, representing an unprecedented opportunity to discover several related-genes associated with secondary xylem such as transcription factors and stress-related genes in a tropical tree.

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.).

BACKGROUND: Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. RESULTS: Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), ß-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. CONCLUSION: This study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1α have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided.