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.

miRNAs play essential regulatory roles in many aspects of plant development and responses to abiotic and biotic environments. Here, we characterized Pu-miR172d, which acts as a negative regulator of stomatal density by directly repressing the expression of PuGTL1 in Populus ussuriensis. quantitative real-time PCR (RT-qPCR) and GUS reporter analyses showed that Pu-miR172d was strongly expressed in the guard cells of young leaves. Pu-miR172d overexpression significantly decreased stomatal density, resulting in the increase of WUE and drought tolerance by reducing net photosynthetic rate, stomatal conductance, and transpiration. Molecular analysis showed that PuGTL1 was a major target of Pu-miR172d cleavage. Moreover, PuGTL1-SRDX plants, in which PuGTL1 is suppressed, phenocopied Pu-miR172d overexpression lines with reduced stomatal density and enhanced WUE. In addition, the expression level of PuSDD1, a negative regulator of stomatal development, was significantly increased in young leaves of both Pu-miR172d overexpression and PuGTL1-SRDX plants. RNA-seq analysis of mature leaves revealed that Pu-miR172d overexpression decreased the expression of many genes related to photosynthesis. Our findings show that the Pu-miR172d/PuGTL1/PuSDD1 module plays an important role in stomatal differentiation, indicating its ability to improve the engineering of drought tolerance in poplar.

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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.