Transcriptome differences between a transgenic poplar tolerant to drought and the control in different growth conditions

Transgenic hybrid poplars engineered to express ectopically the heterologous pine cytosolic GS1a display a number of significant pleiotropic phenotypes including enhanced growth, enhanced nitrogen use efficiency (NUE), and resistance to drought stress. The present study was undertaken in order to assess mechanisms whereby ectopic expression of pine GS1a in transgenic poplars results in enhanced agronomic phenotypes. Microarray analysis using the Agilent Populus whole genome array has allowed identification of genes differentially expressed between wild type and GS transgenics in four tissues (sink leaves, source leaves, stems, and roots) under three growth conditions (well-watered, drought, and recovery). Genes whose expression is significantly altered showing a minimum of 2‐fold differential expression between transgenic and wild type samples were selected for further analysis. Significantly over-represented functional categories among differentially-expressed genes have been identified. Two weighted co-expression networks were constructed analyzing separately GS transgenic and wild type transcript profile data. Differential network behavior and differential gene connectivities between GS and the wild type (WT) expression profiles have been analyzed. Transcript levels for some differentially expressed candidates were validated by q-PCR. The results show that ectopic expression of the pine GS1a in poplars results in transcriptomic rewiring affecting expression of genes belonging to relevant functional categories, including hormone metabolism, regulation of transcription, and stress related genes. Two different genotypes were used, hybrid poplar (Populus tremula X P. alba, INRA 717-1B4) expressing ectopically the pine glutamine synthetase gene (GS1a)(transgenic line 4-29), and the wild type (non-transformed control). RNA was extracted from two biological replicates consisting of pooled samples from 5 individual plants from two replicate experiments. Four tissues were collected, young sink leaves (leaves in positions 2, 3 and 4, numbered from the top of the plant, considering leaf 1 the first leaf with at least 1 cm width), mature source leaves (second and third fully expanded leaves, corresponding to leaves in positions 8 and 9), stem (from nodes 4 to 8), and root. Three growth conditions were performed, well-watered, drought and recovery from drought. Genotypes (2), tissues (4), conditions (3) and replicates (2) made a total of 48 samples.