Molecular And Physio-anatomical Responses Of Populus Under Drought Stress

Populus spp.consists of fast-growing forest tree species,and are extensively cultivated for paper,pulp and timber,and also have tremendous potential for biomass energy production.They contribute significantly in preventing soil-water loss or soil erosion,and in regularizing climate along with maintaining ecological stability.It is a diversified and wide spread genus,and has comprehensively been propagated.Most of the species growing in arid and semi-arid climatic zone are exposed to extended water deficit duration.We hypothesized that there would be a large set of parallel changes in the molecular,physiological and anatomical responses when plants exposed to drought stress;these changes may enhance the capability of plants to survive and grow during drought periods.Therefore,in this study,we used hybrid poplar Populus tremula×Populus alba INRA no.717–1B4（hereafter referred to as poplar 717）as a model species to investigate the molecular,physiological and anatomical basis of woody plants involved in abiotic stress tolerance especially the drought stress,in order to better understand the mechanisms that enable plants to adapt to water stress and maintain growth,development and productivity during stress periods and to aid in for drought resistance.Moreover,we also investigated different Populus species to find out reliable set of reference genes（RGs）under different developmental tissues.The main results are as following:1.The quantitative real-time polymerase chain reaction（q RT-PCR）analysis revealed that the expression of flavonoid biosynthesis genes（Pt PAL,Pt4-CL,Pt CHS,Pt FLS-1,Pt F3H,Pt DFR,and Pt ANS）gradually increased in the leaves of hybrid poplar（P.tremula×P.alba）,corresponding to the drought stress duration.In addition,the activity and capacity of antioxidants have also increased,which is positively correlated with the increment of phenolic,flavonoid,anthocyanin,and carotenoid compounds under drought stress.As the drought stress prolonged,the level of reactive oxygen species such as hydrogen peroxide（H₂O₂）and singlet oxygen（O₂^-）too increased.The concentration of phytohormone salicylic acid（SA）also increased significantly in the stressed poplar717 leaves.2.With the use of transcriptome data generated from different Populus species under developmental and abiotic stress conditions,we describe a methodology to identify candidate RGs from high-throughput sequencing data available at public platforms.We retrieved the novel and reported RGs in transcriptome datasets of different Populus species,and selected 12 novel RGs and 6 reported RGs according to the coefficient of variation（CV）and fold change（FC）value of gene expression.The expression level of candidate RGs,under different developmental and abiotic stress conditions was determined by q RT-PCR.The expression stability of these genes was evaluated using software packages and algorithms includingΔCt,ge Norm,Norm Finder and Best Keeper.Among the 18 tested candidate RGs through comprehensive ranking analysis,4 of the top5 genes are newly identified candidate RGs Potri.001G349400,Potri.005G110600,Potri.011G084400 and Potri.008G111700 are the 4 most stable RGs.Under different stress treatments,4 out of 5 top stable RGs are novel RGs including Potri.001G349400,Potri.005G110600,Potri.011G084400 and Potri.008G111700.In different tree species,4out of 5 top stable RGs are novel RGs like Potri.001G349400,Potri.011G084400,Potri.005G110600 and Potri.002G157500.3.Drought is a frequently occurring abiotic stress to forests,leading to periodic forest mortality with huge economic and environmental loses.The plant leaves wilt due to increased flexibility of petiole under drought stress.We presented an anatomical and morphological model to investigate the role of poplar 717 petiole in protecting a tree from water stress.This study showed that the xylem is more sensitive to environmental factors,specifically drought stress.The drought stress at L5 petiole position severely affected its development;probably due to young leaves have more fragile vascular tissues at L5 in comparison to petiole at L10 and L15 positions,under both moderate and severe drought stress.Further,petiole tissue is very important to understand vascular dynamics under drought,as most of the previous studies have been conducted at stem or leaf level to study anatomical implications of drought.Taken together,it can be concluded that flavonoids biosynthesis plays substantial role in plant defense against drought stress.The drought stress severely affects water conductance in Populus petiole,as suggested by disrupted vascular tissues functioning under progressive drought stress conditions.Finally,the appropriate selection of stable RGs is very crucial for normalizing q RT-PCR experiments involving abiotic stress and developmental studies.