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

Abstract\ud \ud Background\ud 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.\ud \ud \ud Results\ud 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), β-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.\ud \ud \ud Conclusion\ud 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.CAPESFAPES

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