PagWOX11/12a from hybrid poplar enhances drought tolerance by modulating reactive oxygen species.

WOX11/12 is a homeobox gene of WOX11 and WOX12 in Arabidopsis that plays important roles in crown root development and growth. It has been reported that WOX11/12 participates in adventitious root (AR) formation and different abiotic stress responses, but the downstream regulatory network of WOX11/12 in poplar remains to be further investigated. In this study, we found that PagWOX11/12a is strongly induced by PEG-simulated drought stress. PagWOX11/12a-overexpressing poplar plantlets showed lower oxidative damage levels, greater antioxidant enzyme activities and reactive oxygen species (ROS) scavenging capacity than non-transgenic poplar plants, whereas PagWOX11/12a dominant repression weakened root biomass accumulation and drought tolerance in poplar. RNA-seq analysis revealed that several differentially expressed genes (DEGs) regulated by PagWOX11/12a are involved in redox metabolism and drought stress response. We used RT-qPCR and yeast one-hybrid (Y1H) assays to validate the downstream target genes of PagWOX11/12a. These results provide new insights into the biological function and molecular regulatory mechanism of WOX11/12 in the abiotic resistance processes of poplar.

PagWOX11/12a positively regulates the PagSAUR36 gene that enhances adventitious root development in poplar.

Adventitious root (AR) development is an extremely complex biological process that is affected by many intrinsic factors and extrinsic stimuli. Some WUSCHEL-related homeobox (WOX) transcription factors have been reported to play important roles in AR development, but their functional relationships with auxin signaling are poorly understood, especially the developmental plasticity of roots in response to adversity stress. Here, we identified that the WOX11/12a-SMALL AUXIN UP RNA36 (SAUR36) module mediates AR development through the auxin pathway in poplar, as well as under salt stress. PagWOX11/12a displayed inducible expression during AR development, and overexpression of PagWOX11/12a significantly promoted AR development and increased salt tolerance in poplar, whereas dominant repression of PagWOX11/12a produced the opposite phenotype. PagWOX11/12a proteins directly bind to the SAUR36 promoter to regulate SAUR36 transcription, and this binding was enhanced during salt stress. Genetic modification of PagWOX11/12a-PagSAUR36 expression revealed that the PagWOX11/12a-PagSAUR36 module is crucial for controlling AR development via the auxin pathway. Overall, our results indicate that a novel WOX11-SAUR-auxin signaling regulatory module is required for AR development in poplar. These findings provide key insights and a better understanding of the involvement of WOX11 in root developmental plasticity in saline environments.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. '84K'). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and ß-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

ThHSFA1 Confers Salt Stress Tolerance through Modulation of Reactive Oxygen Species Scavenging by Directly Regulating ThWRKY4.

Heat shock transcription factors (HSFs) play critical roles in several types of environmental stresses. However, the detailed regulatory mechanisms in response to salt stress are still largely unknown. In this study, we examined the salt-induced transcriptional responses of ThHSFA1-ThWRKY4 in Tamarix hispida and their functions and regulatory mechanisms in salt tolerance. ThHSFA1 protein acts as an upstream regulator that can directly activate ThWRKY4 expression by binding to the heat shock element (HSE) of the ThWRKY4 promoter using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. ThHSFA1 and ThWRKY4 expression was significantly induced by salt stress and abscisic acid (ABA) treatment in the roots and leaves of T. hispida. ThHSFA1 is a nuclear-localized protein with transactivation activity at the C-terminus. Compared to nontransgenic plants, transgenic plants overexpressing ThHSFA1 displayed enhanced salt tolerance and exhibited reduced reactive oxygen species (ROS) levels and increased antioxidant enzyme activity levels under salt stress. Therefore, we further concluded that ThHSFA1 mediated the regulation of ThWRKY4 in response to salt stress in T. hispida.

Poplar PsnICE1 enhances cold tolerance by binding to different cis-acting elements to improve reactive oxygen species-scavenging capability.

Low temperature is a major stress that severely affects plant growth and development. Inducer of CBF expression 1 (ICE1) plays a key role in plant cold tolerance by regulating the expression of cold stress-responsive genes. In the present study, we characterized the function and underlying regulatory mechanism of PsnICE1 from Xiaohei poplar (Populus simonii × Populus nigra). PsnICE1 was significantly induced in response to cold stress in the roots, stems and leaves. PsnICE1 proteins were found to localize to the nucleus and exert transactivation activity via their N-terminal transactivation domain. Compared with non-transgenic poplar, transgenic poplar overexpressing PsnICE1 showed substantially enhanced tolerance to cold stress, with higher survival rates and antioxidant enzyme activity levels and reduced reactive oxygen species (ROS) accumulation. In contrast, plants with RNA inhibition-mediated silencing of PsnICE1 showed the opposite phenotype. PsnICE1 can bind to H-box element and abscisic acid-responsive element (ABRE), and more importantly, it mainly binds to IBS1 (a newly discovered cis-acting element) and E-box elements to regulate stress-related genes involved in ROS scavenging. Overall, these results indicated that PsnICE1 functions as a positive regulator of cold tolerance and serves as a potential candidate gene for plant cold tolerance improvement via molecular breeding.

PagWOX11/12a activates PagCYP736A12 gene that facilitates salt tolerance in poplar.

The WUSCHEL-related homeobox (WOX) transcription factors WOX11 and WOX12 regulate adventitious rooting and responses to stress. The underlying physiological and molecular regulatory mechanisms in salt stress tolerance remain largely unexplored. Here, we characterized the roles of PagWOX11/12a from 84K poplar (Populus alba × P. glandulosa) and the underlying regulatory mechanism in salt stress. PagWOX11/12a was strongly induced by salt stress in roots. Overexpression of PagWOX11/12a in poplar enhanced salt tolerance, as evident by the promotion of growth-related biomass. In contrast, salt-treated PagWOX11/12a dominant repression plants displayed reduced biomass growth. Under salt stress conditions, PagWOX11/12a-overexpressed lines showed higher reactive oxygen species (ROS) scavenging capacity and lower accumulation of hydrogen peroxide (H2 O2 ) than non-transgenic 84K plants, whereas the suppressors displayed the opposite phenotype. In addition, PagWOX11/12a directly bound to the promoter region of PagCYP736A12 and regulated PagCYP736A12 expression. The activated PagCYP736A12 could enhance ROS scavenging, thus reducing H2 O2 levels in roots under salt stress in PagWOX11/12a-overexpressed poplars. The collective results support the important role of PagWOX11/12a in salt acclimation of poplar trees, indicating that PagWOX11/12a enhances salt tolerance through modulation of ROS scavenging by directly regulating PagCYP736A12 expression in poplar.

ThNAC12 from Tamarix hispida directly regulates ThPIP2;5 to enhance salt tolerance by modulating reactive oxygen species.

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant development and abiotic stress responses, and aquaporins have diverse functions in environmental stress responses. In this study, we described the salt-induced transcriptional responses of ThNAC12 and ThPIP2;5 in Tamarix hispida, and their regulatory mechanisms in response to salt stress. Using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays, we identified that ThNAC12 directly binds to the NAC recognition sequence (NACRS) of the ThPIP2;5 promoter and then activates the ThPIP2;5 expression. Subcellular localization and transcriptional activation assays demonstrated that ThNAC12 was a nuclear protein with a C-terminal transactivation domain. Compared with the corresponding control plants, transgenic plants overexpressing ThNAC12 exhibited enhanced salt tolerance and displayed increased reactive oxygen species (ROS) scavenging capability and antioxidant enzyme activity levels under salt stress. All results suggested that overexpression of ThNAC12 in plants enhanced salt tolerance through modulation of ROS scavenging via direct regulation of ThPIP2;5 expression in T. hispida.

WUSCHEL-related homeobox gene PagWOX11/12a is involved in drought tolerance through modulating reactive oxygen species scavenging in poplar.

WUSCHEL-related homeobox (WOX) transcription factors play essential roles in key developmental processes and in response to different abiotic stresses. In a recent study, we have refined a molecular regulation mechanism that drought-induced PagERF35 directly activates the expression of PagWOX11/12a thus to promote root elongation and biomass, especially under drought conditions, and resulting in enhanced drought tolerance in poplar. In this study, we further found that PagWOX11/12a overexpression significantly enhanced drought tolerance and improved survival rate. Interestingly, transgenic poplars overexpressing PagWOX11/12a exhibited higher ability in scavenging reactive oxygen species (ROS) under drought stress. Combined with these and previous findings, we proposed the mechanism that PagWOX11/12a could not only promote root elongation and biomass growth to increase drought tolerance but also improve plant drought tolerance by regulating ROS level through possibly modulating the expression of ROS scavenging related genes.

WUSCHEL-related homeobox gene PagWOX11/12a responds to drought stress by enhancing root elongation and biomass growth in poplar.

In plants, a large root system improves the uptake of water and nutrients, and is important for responding to drought stress. The poplar WUSCHEL-related homeobox (WOX) transcription factor promotes adventitious rooting, but its regulation of root growth in response to drought stress remains elusive. In this study, we found that PagWOX11/12a from hybrid poplar 84K (Populus alba×Populus glandulosa) is expressed predominantly in the roots and is strongly induced by drought stress. Compared with non-transgenic 84K plants, transgenic poplar plants overexpressing PagWOX11/12a displayed increased root biomass and enhanced drought tolerance, while opposite phenotypes were observed for PagWOX11/12a dominant repression plants. PagWOX11/12a functions as a nuclear transcriptional activator with a transactivation domain at the C-terminus. In addition, PagERF35 was found to specifically bind to a dehydration-responsive element (DRE) within the PagWOX11/12a promoter and activate PagWOX11/12a gene expression. These results indicate that PagERF35 may activate PagWOX11/12a expression in response to drought stress by promoting root elongation and biomass, thereby increasing drought tolerance of poplar.