Since the roots are the very organ where plants first sense and respond drought stress, it is of great importance to better understand root responses to drought. Yet the underlying molecular mechanisms governing root responses to drought stress have been poorly understood.Here, we identified and functionally characterized a CCCH type transcription factor, PuC3H35, and its targets, anthocyanin reductase (PuANR) and early Arabidopsis aluminum induced1 (PuEARLI1), which are involved in mediating proanthocyanidin (PA) and lignin biosynthesis in response to drought stress in Populus ussuriensis root.PuC3H35 was root-specifically induced upon drought stress. Overexpressing PuC3H35 promoted PA and lignin biosynthesis and vascular tissue development, resulting in enhanced tolerance to drought stress by the means of anti-oxidation and mechanical supporting. We further demonstrated that PuC3H35 directly bound to the promoters of PuANR and PuEARLI1 and overexpressing PuANR or PuEARLI1 increased root PA or lignin levels, respectively, under drought stress.Taken together, these results revealed a novel regulatory pathway for drought tolerance, in which PuC3H35 mediated PA and lignin biosynthesis by collaboratively regulating 'PuC3H35-PuANR-PA' and 'PuC3H35-PuEARLI1-PuCCRs-lignin' modules in poplar roots.

The AT-hook motif nuclear-localized (AHL) family is a plant transcription factor family, which plays an important role in growth and development and stress responses. We identified and analyzed 37 AHL genes in poplar (Populus trichocarpa). Phylogenetic analysis classified the PtrAHL members into three subfamilies based on their conserved domain. All PtrAHL paralogous pairs evolved under purifying selection. The promoter analysis revealed the presence of stress-related and phytohormone-related cis-elements of the PtrAHL genes. Our analysis of the tissue-specific expression pattern of PtrAHL genes indicated their significance in tissue and organ development. Network-based prediction suggested that PtrAHL genes may interact with histone deacetylases (HDAC) and participate in the development of organs, such as roots. Drought negatively impacts plant growth and development. ABA is produced under osmotic stress condition, and it takes an important part in the stress response and tolerance of plants. Real-time quantitative PCR (qRT-PCR) showed that PtrAHL genes were induced by drought stress and ABA treatment. These insights into the expression of PtrAHL genes under stress provide a basis for PtrAHL gene functional analysis. Our study will help develop new breeding strategies to improve drought tolerance in poplar.

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

In eukaryotes, alternative splicing (AS) is a crucial regulatory mechanism that modulates mRNA diversity and stability. The contribution of AS to stress are known in many species related to stress. But the post-transcriptional mechanism in poplar under cold stress is still unclear. Recent studies have utilized the advantages of Single Molecular Real Time (SMRT) sequencing technology from Pacific Bioscience (PacBio) to identify full-length transcripts. We, therefore, used a combination of single-molecule long-read sequencing and Illumina RNA sequencing (RNA-Seq) for a global analysis of AS in two poplar species (Populus trichocarpa and P. ussuriensis) under cold stress. We further identified 1261 AS events in P. trichocarpa and 2101 in P. ussuriensis, among which intron retention, with a frequency of more than 30%, was the most prominent type under cold stress. RNA-Seq data analysis and annotation revealed the importance of calcium, abscisic acid, and reactive oxygen species signaling in cold stress response. Besides, the low temperature rapidly induced multiple splicing factors, transcription factors, and differentially expressed genes through AS. In P. ussuriensis, there was a rapid occurrence of AS events. This study provides new insight into the complexity and regulation of AS during cold stress response in two poplar species.