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Drought is one of the major limiting factors in the growth of terrestrial plants. Abscisic acid (ABA) and pyrabactin resistance 1/prabactin resistance-1 like/regulatory components of ABA receptors (PYR/PYL/RCARs) play a key role in response to drought stress. However, the underlying mechanisms of this control remain largely elusive in trees. In this study, PePYL4, a potential ortholog of the PYR/PYL/RCARs gene, was cloned from Populus euphratica. It was localized in the cytoplasm and nucleus, induced by ABA, osmotic and dehydration treatments. To study the potential biological functions of PePYL4, transgenic triploid white poplars (Populus tomentosa ‘YiXianCiZhu B38’) overexpressing PePYL4 were generated. PePYL4 overexpression significantly increased ABA sensitivity and reduced stomatal aperture. Compared with wild-type plants, transgenic plants had higher water-use efficiency (WUE) and lower transpiration. When exposed to drought stress, PePYL4 overexpression plants maintained higher photosynthetic activity and accumulated more biomass. Moreover, overexpression of PePYL4 improved antioxidant enzyme activity and ascorbate content to accelerate reactive oxygen species scavenging. Meanwhile, upregulation expression of the stress-related genes also contributed to improving the drought tolerance of transgenic plants. In conclusion, our data suggest that PePYL4 is a promising gene target for regulating WUE and drought tolerance in Populus.

Regulation of abscisic acid (ABA) biosynthesis helps plants adapt to drought stress, but the underlying molecular mechanisms are largely unclear. Here, a drought-induced transcription factor XsAGL22 was isolated from yellowhorn (Xanthoceras sorbifolium Bunge). Yeast one-hybrid and electrophoretic mobility shift assays indicated that XsAGL22 can physically bind to the promoters of the ABA biosynthesis-related genes XsNCED6 and XsBG1, and a dual-luciferase assay showed that XsAGL22 activates the promoters of the later two genes. Transient overexpression of XsAGL22 in yellowhorn leaves also increased the expression of XsNCED6 and XsBG1 and increased cellular ABA levels. Finally, heterologous overexpression of XsAGL22 in poplar increased ABA content, reduced stomatal aperture and increased drought resistance. Our results suggest that XsAGL22 is a powerful regulator of ABA biosynthesis and plays a critical role in drought resistance in plants.

Drought is one of the primary abiotic stresses, seriously implicating plant growth and productivity. Stomata play a crucial role in regulating drought tolerance. However, the molecular mechanism on stomatal movement-mediated drought tolerance remains unclear. Using genetic, molecular and biochemical techniques, we identified that the PdGNC directly activating the promoter of PdHXK1 by binding the GATC element, a hexokinase (HXK) synthesis key gene. Here, PdGNC, a member of the GATA transcription factor family, was greatly induced by abscisic acid and dehydration. Overexpressing PdGNC in poplar (Populus clone 717) resulted in reduced stomatal aperture with greater water-use efficiency and increased water deficit tolerance. By contrast, CRISPR/Cas9-mediated poplar mutant gnc exhibited increased stomatal aperture and water loss with reducing drought resistance. PdGNC activates PdHXK1 (a hexokinase synthesis key gene), resulting in a remarkable increase in hexokinase activity in poplars subjected to water deficit. Furthermore, hexokinase promoted nitric oxide (NO) and hydrogen peroxide (H2 O2 ) production in guard cells, which ultimately reduced stomatal aperture and increased drought resistance. Together, PdGNC confers drought stress tolerance by reducing stomatal aperture caused by NO and H2 O2 production via the direct regulation of PdHXK1 expression in poplars.

Root growth is essential for plant survival under drought stress. Root growth includes diameter growth and longitudinal growth, which play different roles in the regulation of drought tolerance. However, the specific role of root growth in woody and herbaceous plants under drought stress remains largely uncharacterized. In a previous study, we confirmed that a root-specific transcription factor, NUCLEAR FACTOR Y subunit B21 (NF-YB21), positively regulated drought resistance by promoting root diameter growth and longitudinal growth in poplar. In the present study, we further analyzed induction of NF-YB21 in Arabidopsis in response to drought and exogenous abscisic acid (ABA) treatment. In PdNF-YB21 promoter-driven GUS transgenic Arabidopsis plants, PdNF-YB21 was predominantly expressed in the root and was induced by osmotic stress. Overexpression of PdNF-YB21 in Arabidopsis enhanced drought resistance by promoting growth in root length but not in root diameter. Physiological analysis suggested that PdNF-YB21 promoted increase in root length, root hydraulic conductivity, root water-use efficiency (WUE), chlorophyll content, photosynthetic rate, and biomass accumulation under drought stress. The present findings suggest that PdNF-YB21 plays an important role in regulation of root length and drought resistance in a herbaceous plant species. The results provide an explanation for the different mechanisms in herbaceous and woody plants involved in regulation of drought resistance by promoting growth in root length or diameter, and will be useful for breeding cultivars with enhanced drought tolerance. This study reported the important role of root-specific transcription factor PdNF-YB21 in regulating root length and improving drought tolerance in Arabidopsis thaliana.

Somatic cells of higher plants possess the remarkable ability to regenerate new individuals via reestablishing apical meristems. Reconstitution of shoot meristem is the vital process and is required for application of plant biotechnology. Under

Plants have evolved complex mechanisms to cope with the fluctuating environmental availability of nitrogen. However, potential genes modulating plant responses to nitrate are yet to be characterized. Here, a poplar GATA transcription factor gene PdGNC (GATA nitrate-inducible carbon-metabolism-involved) was found to be strongly induced by low nitrate. Overexpressing PdGNC in poplar clone 717-1B4 (P. tremula × alba) significantly improved nitrate uptake, remobilization, and assimilation with higher nitrogen use efficiency (NUE) and faster growth, particularly under low nitrate conditions. Conversely, CRISPR/Cas9-mediated poplar mutant gnc exhibited decreased nitrate uptake, relocation, and assimilation, combined with lower NUE and slower growth. Assays with yeast one-hybrid, electrophoretic mobility shift, and a dual-luciferase reporter showed that PdGNC directly activated the promoters of nitrogen pathway genes PdNRT2.4b, PdNR, PdNiR, and PdGS2, leading to a significant increase in nitrate utilization in poplar. As expected, the enhanced NUE promoted growth under low nitrate availability. Taken together, our data show that PdGNC plays an important role in the regulation of NUE and growth in poplar by improving nitrate acquisition, remobilization, and assimilation, and provide a promising strategy for molecular breeding to improve productivity under nitrogen limitation in trees.

Background Wood provides an important biomass resource for biofuel production around the world. The radial growth of tree stems is central to biomass production for forestry and biofuels, but it is challenging to dissect genetically because it is a complex trait influenced by many genes. In this study, we adopted methods of physiology, transcriptomics and genetics to investigate the regulatory mechanisms of tree radial growth and wood development. Results Physiological comparison showed that two Populus genotypes presented different rates of radial growth of stems and accumulation of woody biomass. A comparative transcriptional network approach was used to define and characterize functional differences between two Populus genotypes. Analyses of transcript profiles from wood-forming tissue of the two genotypes showed that 1542, 2295 and 2110 genes were differentially expressed in the pre-growth, fast-growth and post-growth stages, respectively. The co-expression analyses identified modules of co-expressed genes that displayed distinct expression profiles. Modules were further characterized by correlating transcript levels with genotypes and physiological traits. The results showed enrichment of genes that participated in cell cycle and division, whose expression change was consistent with the variation of radial growth rates. Genes related to secondary vascular development were up-regulated in the faster-growing genotype in the pre-growth stage. We characterized a BEL1-like (BELL) transcription factor, PeuBELL15, which was up-regulated in the faster-growing genotype. Analyses of transgenic Populus overexpressing as well as CRISPR/Cas9-induced mutants for BELL15 showed that PeuBELL15 improved accumulation of glucan and lignin, and it promoted secondary vascular growth by regulating the expression of genes relevant for cellulose synthases and lignin biosynthesis. Conclusions This study illustrated that active division and expansion of vascular cambium cells and secondary cell wall deposition of xylem cells contribute to stem radial increment and biomass accumulation, and it identified relevant genes for these complex growth traits, including a BELL transcription factor gene PeuBELL15. This provides genetic resources for improving and breeding elite genotypes with fast growth and high wood biomass.

Gibberellic acid-stimulated Arabidopsis (GASA) proteins, as cysteine-rich peptides (CRPs), play roles in development and reproduction and biotic and abiotic stresses. Although the GASA gene family has been identified in plants, the knowledge about GASAs in Populus euphratica, the woody model plant for studying abiotic stress, remains limited. Here, we referenced the well-sequenced Populus trichocarpa genome, and identified the GASAs in the whole genome of P. euphratica and P. trichocarpa. 21 candidate genes in P. trichocarpa and 19 candidate genes in P. euphratica were identified and categorized into three subfamilies by phylogenetic analysis. Most GASAs with signal peptides were located extracellularly. The GASA genes in Populus have experienced multiple gene duplication events, especially in the subfamily A. The evolution of the subfamily A, with the largest number of members, can be attributed to whole-genome duplication (WGD) and tandem duplication (TD). Collinearity analysis showed that WGD genes played a leading role in the evolution of GASA genes subfamily B. The expression patterns of P. trichocarpa and P. euphratica were investigated using the PlantGenIE database and the real-time quantitative PCR (qRT-PCR), respectively. GASA genes in P. trichocarpa and P. euphratica were mainly expressed in young tissues and organs, and almost rarely expressed in mature leaves. GASA genes in P. euphratica leaves were also widely involved in hormone responses and drought stress responses. GUS activity assay showed that PeuGASA15 was widely present in various organs of the plant, especially in vascular bundles, and was induced by auxin and inhibited by mannitol dramatically. In summary, this present study provides a theoretical foundation for further research on the function of GASA genes in P. euphratica.

Drought is one of the primary abiotic stresses, seriously implicating plant growth and productivity. Stomata play a crucial role in regulating drought tolerance. However, the molecular mechanism on stomatal movement-mediated drought tolerance remains unclear. Using genetic, molecular and biochemical techniques, we identified that the PdGNC directly activating the promoter of PdHXK1 by binding the GATC element, a hexokinase (HXK) synthesis key gene. Here, PdGNC, a member of the GATA transcription factor family, was greatly induced by abscisic acid and dehydration. Overexpressing PdGNC in poplar (Populus clone 717) resulted in reduced stomatal aperture with greater water-use efficiency and increased water deficit tolerance. By contrast, CRISPR/Cas9-mediated poplar mutant gnc exhibited increased stomatal aperture and water loss with reducing drought resistance. PdGNC activates PdHXK1 (a hexokinase synthesis key gene), resulting in a remarkable increase in hexokinase activity in poplars subjected to water deficit. Furthermore, hexokinase promoted nitric oxide (NO) and hydrogen peroxide (H

Additional file 6: Figure S3. Plots for Gene Ontology enrichment analysis of differential expressed genes between two neighbouring growth stages for Neva and I-214.

Poplar is a key player in biomass resource and a dominant species for lignocellulosic biofuel production around the world. Today, the use of poplars is mainly concentrated in the aboveground parts. However, as an important part of trees, the poplar root system developed, roots account for about 20 % of the volume of the tree, and the root material volume is very considerable and its utilization potential is huge. The root lignin is central to biomass production in poplar. However, there are very few studies on root lignin structure. Recently, we found a root-specific transcription factor, NUCLEAR FACTOR Y subunit B21 (NF-YB21), is involved in regulating root growth and lignification. In the present study, physiology, transgenosis, transcriptomics, metabolome, Confocal Raman Microscopy (CRM) and Nuclear Magnetic Resonance (NMR) techniques were performed to further investigate the regulatory mechanisms of PdNF-YB21 on lignin structure in poplar roots. The results of transcriptome and metabolome showed that there were more genes and metabolites related to S-lignin synthesis in transgenic plants. Root firmness measurements, CRM and NMR results showed that overexpression of PdNF-YB21 dramatically improved root hardness, increased root lignin content and S/G ratio, and increased the concentrations of glucan, xylan and lignin in roots, which promoted root growth. In short, this present study not only illustrated the regulatory mechanisms of PdNF-YB21 on lignin structure in poplar roots, but also proposed a directive breeding of new poplar species for current biorefinery process.

Root growth control plays an important role in plant adaptation to drought stress, but the underlying molecular mechanisms of this control remain largely elusive. Here, a root-specific nuclear factor Y (NF-Y) transcription factor PdNF-YB21 was isolated from Populus. The functional mechanism of PdNF-YB21 was characterised by various morphological, physiological, molecular, biochemical and spectroscopy techniques. Overexpression of PdNF-YB21 in poplar promoted root growth with highly lignified and enlarged xylem vessels, resulting in increased drought resistance. By contrast, CRISPR/Cas9-mediated poplar mutant nf-yb21 exhibited reduced root growth and drought resistance. PdNF-YB21 interacted with PdFUSCA3 (PdFUS3), a B3 domain transcription factor. PdFUS3 directly activated the promoter of the abscisic acid (ABA) synthesis key gene PdNCED3, resulting in a significant increase in root ABA content in poplars subjected to water deficit. Coexpression of poplar NF-YB21 and FUS3 significantly enhanced the expression of PdNCED3. Furthermore, ABA promoted indoylacetic acid transport in root tips, which ultimately increased root growth and drought resistance. Taken together, our data indicate that NF-YB21-FUS3-NCED3 functions as an important avenue in auxin-regulated poplar root growth in response to drought.

Thirty-nine GATA transcription factor genes were identified in the poplar genome. The GATA transcription factor PdGNC positively regulates photosynthesis and plant growth by promoting chloroplast development in poplar., GATA transcription factors are involved in the regulation of diverse growth processes and environmental responses in Arabidopsis and rice. In this study, we conducted a comprehensive bioinformatic survey of the GATA family in the woody perennial Populus trichocarpa. Thirty-nine Populus GATA genes were classified into four subfamilies based on gene structure and phylogenetic relationships. Predicted cis-elements suggested potential roles of poplar GATA genes in light, phytohormone, development, and stress responses. A poplar GATA gene, PdGATA19/PdGNC (GATA nitrate-inducible carbon-metabolism-involved), was identified from a fast growing poplar clone. PdGNC expression was significantly up-regulated in leaves under both high (50 mM) and low (0.2 mM) nitrate concentrations. The CRISPR/Cas9-mediated mutant crispr-GNC showed severely retarded growth and enhanced secondary xylem differentiation. PdGNC-overexpressing transformants exhibited 25–30% faster growth, 20–28% higher biomass accumulation, and ~25% increase in chlorophyll content, photosynthetic rate, and plant height, compared with the wild type. Transcriptomic analysis showed that PdGNC was involved in photosynthetic electron transfer and carbon assimilation in the leaf, cell division and carbohydrate utilization in the stem, and nitrogen uptake in the root. These data indicated that PdGNC plays a crucial role in plant growth and is potentially useful in tree molecular breeding.

Drought is a severe environmental stress that limits plant growth and productivity. Plant hormones play a key role in response to biological and abiotic stresses. And phytohormones can regulate stomatal opening and transpiration for improving water-use efficiency under water stress conditions. In this study, PtrWRKY75, a member of the WRKY transcription factor family, which is involved in many abiotic stress responses, was cloned from Populus trichocarpa (clone ‘Nisqually-1′). PtrWRKY75 was mainly expressed in senescent leaves and could be induced by both dehydration and treatment with salicylic acid (SA). PtrWRKY75 was localized to the nucleus and bound specifically to the PHENYLALANINE AMMONIA LYASE 1 (PAL1) promoter. To investigate the biological functions of PtrWRKY75, it was overexpressed in triploid white poplar (Populus tomentosa). Compared with wild-type plants, PtrWRKY75-overexpressing poplar lines (OE) maintained a higher photosynthetic rate and growth under drought stress. Under drought stress, PtrWRKY75 acted upstream of PAL1 and directly regulated PAL1 expression by binding to its promoter. Under drought stress, activated PAL1 increased reactive oxygen species (ROS) accumulation by promoting SA biosynthesis, ultimately leading to smaller stomatal aperture. Under drought conditions, the OE lines showed improved water-use efficiency by reducing their stomatal conductance and transpiration rate, and thus drought tolerance was enhanced. In conclusion, PtrWRKY75 is a promising gene target for improving the drought tolerance of Populus by SA.