term 'response to organic substance' and KEGG pathway 'nitrogen metabolism' were enriched for drought-stressed leaves, while GO term 'cell wall organization or biogenesis' and KEGG pathway 'zeatin biosynthesis' were enriched for drought-stressed roots. The enrichment of the GO term 'enzyme linked receptor protein signalling pathway' in both leaf and root drought responses suggests that these tissues may also have similar mechanisms. However, roots under drought stress for four hs responded by activating programed cell death. The KEGG pathway 'plant hormone signal transduction' was detected for 4-and 12-h droughtstressed leaves and 12-h drought-stressed roots, suggesting that plant hormone signal transduction plays an important role in both roots and leaves. GO enrichment of upregulated and downregulated genes for leaves and roots reflect differentially regulatory mechanisms of response to drought stress via different biological processes such as the regulation of photosynthesis and auxin signalling pathway in leaves, and the regulation of defence response and water homeostasis in roots. Fifteen candidate genes, including transcription factors, protein kinase, transporter, late embryogenesis abundant protein and mannitol dehydrogenase, were further selected to determine their response to drought using qRT-PCR. The results show that the expression patterns of 13 of the 15 genes correspond to the RNA-seq results. This study provides new insights into the drought response mechanism of P. euphratica and suggests new candidate gene resources.

Despite the high economic and ecological importance of forests, our knowledge of the adaptive evolution of leaf traits remains very limited. Euphrates poplar (Populus euphratica), which has high tolerance to arid environment, has evolved four heteromorphic leaf forms, including narrow (linear and lanceolate) and broad (ovate and broad-ovate) leaves on different crowns. Here, we revealed the significant functional divergence of four P. euphratica heteromorphic leaves at physiological and cytological levels. Through global analysis of transcriptome and DNA methylation across tree and leaf developmental stages, we revealed that gene expression and DNA epigenetics differentially regulated key processes involving development and functional adaptation of heteromorphic leaves, such as hormone signaling pathways, cell division, and photosynthesis. Combined analysis of gene expression, methylation, ATAC-seq and Hi-C-seq revealed longer interaction of 3D genome, hypomethylation, and open chromatin state upregulates IAA-related genes (such as PIN-FORMED1 and ANGUSTIFOLIA3) and promotes the occurrence of broad leaves while narrow leaves were associated with highly concentrated heterochromatin, hypermethylation and upregulated ABA pathway genes (such as Pyrabactin Resistance1-like10). Therefore, development of P. euphratica heteromorphic leaves along with functional divergence was regulated by differentially expressed genes, DNA methylation, chromatin accessibility, and 3D genome remodeling to adapt to the arid desert. This study advances our understanding of differential regulation on development and functional divergence of heteromorphic leaves in P. euphratica at the multi-omics level and provides a valuable resource for investigating the adaptive evolution of heteromorphic leaves in Populus.

Reference-quality genomes of both sexes are essential for studying sex determination and sex-chromosome evolution, as their gene contents and expression profiles differ. Here, we present independent chromosome-level genome assemblies for the female (XX) and male (XY) genomes of desert poplar (Populus euphratica), resolving a 22.7-Mb X and 24.8-Mb Y chromosome. We also identified a relatively complete 761-kb sex-linked region (SLR) in the peritelomeric region on chromosome 14 (Y). Within the SLR, recombination around the partial repeats for the feminizing factor ARR17 (ARABIDOPSIS RESPONSE REGULATOR 17) was potentially suppressed by flanking palindromic arms and the dense accumulation of retrotransposons. The inverted small segments S1 and S2 of ARR17 exhibited relaxed selective pressure and triggered sex determination by generating 24-nt small interfering RNAs that induce male-specific hyper-methylation at the promoter of the autosomal targeted ARR17. We also detected two male-specific fusion genes encoding proteins with NB-ARC domains at the breakpoint region of an inversion in the SLR that may be responsible for the observed sexual dimorphism in immune responses. Our results show that the SLR appears to follow proposed evolutionary dynamics for sex chromosomes and advance our understanding of sex determination and the evolution of sex chromosomes in Populus.