Understanding Poplar response to abiotic stress: Exploring mechanisms of salt tolerances in Populus alba L. “Villafranca” in wild type and transgenic lines.

Poplar is studied as a model tree for genetics and biotechnology research since it has a relatively small genome size approximately 400-500Mbp, and it was the first tree species to have the entire genome sequenced. Poplar is known to be the fastest-growing tree in the group of woody perennial plants and possesses many important qualities. Namely, their flexible adaptability, large production of biomass, smooth propagation, and their tendency to hybridize. They are also known as providers of forest commodities such as wood, fuel wood and fiber. In addition, they are involved in the reintegration and renewal of degraded landscapes. Most importantly, poplars’ huge carbon storage capacity is crucial in mitigating the earth’s climate issues .The Populus alba L. “villafranca” clone known as white poplar, main focus of this research found in central and southern Europe, North Africa, Asia and in Middle east has gained scientific attention because of its crucial traits such as rapid growth, resistance to various biotic and abiotic stresses as well as enhanced water use efficiency under stress conditions like zinc. It is also known for its important role in phytoremediation. Over three years. research on this clone emplyed multiple approaches, including studying its response to salinity, stress tolerance mechanisms, CRISPR-cas9 gene editing and whole genome sequencing analysis to gain critical insight into P. alba L. “villafranca” adaptability and resilience mechanisms. Salinity is one of the main abiotic stresses affecting plant survival. To investigate salinity response, P. alba L. clone ‘Villafranca’ transgenic plants over-expressing aqua1, an aquaporin of Populus x euramericana clone ‘I-214’ (GeneBank: GQ918138) were compared to wild-type plants (WT). Two lines with a different level of over-expression were selected. Plants were grown under control conditions (0 mM NaCl) and salinity (100 mM NaCl) for 27 days. At the end of the experiment, transgenic and WT plants were phenotypically indistinguishable. Shoot elongation and total leaf area were reduced equally in all treated plants, even if the relative reduction was lower in transgenic Line 16. The other morphological and physiological parameters measured did not evidence a response correlated to aqua1 over-expression. Interestingly, we observed a decrease in Na concentration in the treated roots of Line 16 compared to WT. Higher polyphenols content in roots and leaves of transgenic Line 16 under control conditions was observed suggesting that aqua1 over-expression has contributed to improving ion regulation and oxidative stress mitigation. These findings revealed that the aqua 1 over-expression helps maintain osmotic balance and efficient water transport contributing to the stability of polyphenol profiles under salinity stress, thereby enhancing tolerance of transgenic lines. Also, our results suggest aqua1 involvement in cell and plant growth and a possible buffering effect of aqua1 over-expression by transcriptional and post-translational modification that must be further investigated. Based on these findings, CRISPR/Cas9 gene editing technique was utilized in P.alba L. “Villafranca” to explore and manipulate genes associated with stress tolerance. The SOS2 gene from Salt Overly Sensitive (SOS) pathway was targeted alongside the Phytoene Desaturase (PDS) gene, a gene involved in the biosynthetic pathway of carotenoids is used as a reporter gene since its knock-out mutant develop an albino phenotype. Bioinformatics characterization of SOS and PDS genes was made using public databases to select the specific sequence. In vitro, plantlets were prepared to be transformed at the stage of four to six true leaves. Two different copies of the SOS gene were selected as the candidate target for knock-out, and the PDS gene was selected in a single copy form. Specific primers were designed to target conserved regions of interest. E. coli (DH10B) carrying the pJET 1.2 vector was used to transform the genomic DNA as the first step of the cloning procedure. Positive colonies were selected, and total DNA was sequenced using the Sanger approach, later different mutations of PDS and SOS were analyzed. The following step was designing dual guide RNAs (gRNA) for each gene, designed on the genomic sequences. A. tumefaciens and the PBV[2CRISPR]-Neo/Kana-zCas9-AtU6-26 plasmid construct, a binary vector containing gRNA sequences for targeted genome editing, selectable markers, and a modified Cas9 protein, were used to transform the plantlets. CRISPR-Cas9 SOS knocked-out genes are obtained. Lines were analyzed to check for mutations by extraction, purification, and sequencing (Sanger sequencing) of the genomic DNA. Stable CRISPR knock-out mutants were achieved after several generations. This approach demonstrated the feasibility of CRISPR/Cas9 in understanding molecular pathways underlying salinity tolerance, complementing insight from transgenic studies. To provide the comprehensive genomic foundation for these experiments, whole genome sequencing of P.alba L. “Villafranca” clone was conducted using PacBio sequencing technology. Previous contig level draft assembly of P. alba (GCF-005239225.1) sequenced using PacBio RSII and Illumina technology with SMART De-novo method was released in 2019 by the Chinese Academy of Forestry. It has provided significant insight into genomic structure and annotation such as genome size is estimated to be 416.9 Mb with 32,959 protein-coding genes (“Populus alba genome assembly ASM523922v1,” 2019), however understanding of salt stress tolerance mechanisms remained limited. This study identified repeat elements in assembly and performed gene prediction using tools like Augustus with omicsbox alongside structural variant analysis. The assembled genome provided an important resource for studying stress response, guiding CRISPR target design , and interpreting transcriptomic and phenotypic data Together this study utilizes a diverse array of cutting-edge techniques to comprehensively investigate the mechanisms underlying their resilience and demonstrate the power of integrating phenotypic, molecular, and genomic technologies to unravel complex plant stress mechanisms and lay the foundation for future applications in forestry, environmental restoration, and climate mitigation.