Genome-wide identification of the TGA transcription factor family in Populus alba × Populus glandulosa and functional validation of PagTGA7b in salt tolerance

The Ethylene Responsive Factor (ERF) transcription factor family is important for regulating plant growth and stress responses. Although the expression patterns of ERF family members have been reported in many plant species, their role in Populus alba × Populus glandulosa, an important model plant for forest research, remains unclear. In this study, we identified 209 PagERF transcription factors by analyzing the P. alba × P. glandulosa genome. We analyzed their amino acid sequences, molecular weight, theoretical pI (Isoelectric point), instability index, aliphatic index, grand average of hydropathicity, and subcellular localization. Most PagERFs were predicted to localize in the nucleus, with only a few PagERFs localized in the cytoplasm and nucleus. Phylogenetic analysis divided the PagERF proteins into ten groups, Class I to X, with those belonging to the same group containing similar motifs. Cis-acting elements associated with plant hormones, abiotic stress responses, and MYB binding sites were analyzed in the promoters of PagERF genes. We used transcriptome data to analyze the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa, including axillary buds, young leaves, functional leaves, cambium, xylem, and roots, and the results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots. Quantitative verification results were consistent with transcriptome data. When P. alba × P. glandulosa seedlings were treated with 6% polyethylene glycol 6000 (PEG6000), the results of RT-qRCR showed that nine PagERF genes responded to drought stress in various tissues. This study provides a new perspective on the roles of PagERF family members in regulating plant growth and development, and responses to stress in P. alba × P. glandulosa. Our study provides a theoretical basis for ERF family research in the future.

Genome-wide identification of poplar GSTU gene family and its PtrGSTU23 and PtrGSTU40 to improve salt tolerance in poplar

Soil salinity is an important determinant of crop productivity and triggers salt stress response pathways in plants. The salt stress response is controlled by transcriptional regulatory networks that maintain regulatory homeostasis through combinations of transcription factor (TF)-DNA and TF-TF interactions. We investigated the transcriptome of poplar 84 K (Populus alba × Populus glandulosa) under salt stress using samples collected at 4- or 6-h intervals within 2 days of salt stress treatment. We detected 24,973 differentially expressed genes, including 2,231 TFs that might be responsive to salt stress. To explore these interactions and targets of TFs in perennial woody plants, we combined gene regulatory networks, DNA affinity purification sequencing, yeast two-hybrid-sequencing, and multi-gene association approaches. Growth-regulating factor 15 (PagGRF15) and its target, high-affinity K+ transporter 6 (PagHAK6), were identified as an important regulatory module in the salt stress response. Overexpression of PagGRF15 and PagHAK6 in transgenic lines improved salt tolerance by enhancing Na+ transport and modulating H2O2 accumulation in poplar. Yeast two-hybrid assays identified more than 420 PagGRF15-interacting proteins, including ETHYLENE RESPONSE FACTOR TFs and a zinc finger protein (C2H2) that are produced in response to a variety of phytohormones and environmental signals and are likely involved in abiotic stress. Therefore, our findings demonstrate that PagGRF15 is a multifunctional TF involved in growth, development, and salt stress tolerance, highlighting the capability of a multifaceted approach in identifying regulatory nodes in plants.

Soil salinization is increasingly recognized as a critical environmental challenge that significantly threatens plant survival and agricultural productivity. To elucidate the mechanism of salt resistance in poplar, physiological and transcriptomic analyses were conducted on 84K poplar (Populus alba × Populus glandulosa) under varying salt concentrations (0, 100, 200 and 300mM NaCl). As salt levels increased, observable damage to poplar progressively intensified. Differentially expressed genes under salt stress were primarily enriched in photosynthesis, redox activity and glutathione metabolism pathways. Salt stress reduced chlorophyll content and net photosynthetic rate, accompanied by the downregulation of photosynthesis-related genes. NaCl (300 mM) significantly inhibited the photochemical activity of photosystems. The higher photochemical activity under 100 and 200mM NaCl was attributed to the activated PGR5-cyclic electron flow photoprotective mechanism. However, the NAD(P)H dehydrogenase-like (NDH)-cyclic electron flow was inhibited under all salt levels. Salt stress led to reactive oxygen species accumulation, activating the ASA-GSH cycle and antioxidant enzymes to mitigate oxidative damage. Weighted gene co-expression network analysis showed that five photosynthesis-related hub genes (e.g., FNR and TPI) were down-regulated and nine antioxidant-related hub genes (e.g., GRX, GPX and GST) were up-regulated under salt stress conditions. PagGRXC9 encodes glutaredoxin and was found to be differentially expressed during the salt stress condition. Functional studies showed that overexpressing PagGRXC9 enhanced salt tolerance in yeast, and in poplar, it improved growth, FV/FM, non-photochemical quenching values and resistance to H2O2-induced oxidative stress under salt stress. This study constructed the photosynthetic and antioxidant response network for salt stress in poplar, revealing that PagGRXC9 enhances salt tolerance by reducing photoinhibition and increasing antioxidant capacity. These findings provide valuable insights for breeding salt-tolerant forest trees.

The rhizosphere microbiomes of halophytes are crucial for plant adaptation to high-salinity soil conditions, but how to harness rhizosphere microbes to confer salt stress resistance to plants remains obscure. This study aimed to establish a framework (isolate-select-construct) for tailoring simplified salt-tolerant synthetic microbial communities (SynComs) and explore how they confer salt stress resistance to the plant. First, a total of 512 strains were isolated from the high-salt rhizosphere soil of Populus euphratica through high-throughput cultivation. Among these, nine strains were further selected for their salt-tolerant and growth-promoting abilities, with three isolates identified as key microbes, including hub microbes, keystone taxa and biomarkers. Guided by a function-driven strategy, we constructed five distinct SynComs, with SynCom5, SynCom7 and SynCom9 showing the most significant improvement in the growth of hybrid Poplar 84K (Populus alba × Populus glandulosa). Mechanistic investigations revealed that these SynComs can increase resistance to salt stress by directly reducing oxidative stress, adjusting osmolytes and balancing ions. Additionally, these SynComs were observed to recruit specific root-associated bacterial consortia that enhance the adaptability of poplar to salt stress. Overall, this study lays the groundwork for designing SynComs that promote plant growth and offers insights into harnessing specific microbial communities to boost plants' salt resistance.

PagWRKY12 promotes salt and drought tolerance in 84K poplar.

Ethylene response factor (ERF) gene family plays an important role in abiotic stress responses. In this study, we isolated a salt-inducible ERF gene, ERF38 (Potri.006G138900.1), from the 84K poplar (Populus alba × Populus glandulosa) and investigated its functions in salt and osmotic tolerance. We identified that ERF38 protein was targeted to nucleus and had no self-activation. Results from yeast-one-hybrid indicated that the ERF38 protein can specifically bind to the dehydration responsive element (DRE). We then successfully transferred the ERF38 gene into the 84K poplar. Under respective salt and polyethylene glycol (PEG)-6000 stresses, four of the physiological traits, including peroxidase (POD) and superoxide dismutase (SOD) activities, soluble protein content, and proline content, increased significantly in the transgenic plants, compared to the wild type. Regarding the other two parameters, hydrogen peroxide (H2O2) and malondialdehyde (MDA) content, their increments in the transgenic lines under the stresses, which were compared to the water control, were significantly low than that of the wild type. In addition, reactive oxygen species (ROS) are scavenged in the transgenic lines under the stresses, but not in the wild type (WT). Interestingly, when challenged with the stresses, expression levels of a few genes associated with POD and SOD metabolism were significantly increased in the transgenic poplars. In all, evidence from morphological, physiological, and biochemical analyses indicated that over-expression of ERF38 gene can improve salt and osmotic tolerance in the transgenic poplar.

Salt stress is a major abiotic stress restrict plant growth and distribution. In our previous study, we found the ABI5-BINDING PROTEIN 2a (PagAFP2a) gene was rapidly and significantly induced by salt stress in hybrid poplar (Populus alba × Populus glandulosa), however, its function in salt stress responses was unclear. In this study, we further demonstrated that the PagAFP2a gene expression is significantly induced by salt and ABA treatments. Additionally, the ABA-responsive element (ABRE) binding proteins (PagAREB1s) directly bind to PagAFP2a promoter and activate its expression. Physiological analysis showed that PagAFP2a overexpression (PagAFP2aOE) or PagAREB1-3 knockout (PagAREB1-3KO) significantly reduced salt tolerance whereas PagAFP2a knockout (PagAFP2aKO) or PagAREB1-3 overexpression (PagAREB1-3OE) significantly enhanced salt tolerance in poplar. Correspondingly, salt stress responsive genes were significantly upregulated in PagAFP2aKO and PagAREB1-3OE plants while downregulated in PagAFP2aOE and PagAREB1-3KO plants. Furthermore, we demonstrated that PagAFP2a directly interacts with PagAREB1s and represses its transcriptional activity at the target genes. In summary, our results unveil the PagAFP2a-PagAREB1s module form a negative feedback loop in ABA signaling to fine-tune salt stress responses in Populus.

Salt is an important environmental stress factor, which seriously affects the growth, development and distribution of plants. Chlorophyllase plays an important role in stress response. Nevertheless, little is known about the physiological and molecular mechanism of chlorophyll (Chlase, CLH) genes in plants. We cloned PeCLH2 from Populus euphratica and found that PeCLH2 was differentially expressed in different tissues, especially in the leaves of P. euphratica. To further study the role of PeCLH2 in salt tolerance, PeCLH2 overexpression and RNA interference transgenic lines were established in Populus alba × Populus glandulosa and used for salt stress treatment and physiologic indexes studies. Overexpressing lines significantly improved tolerance to salt treatment and reduced reactive oxygen species production. RNA interference lines showed the opposite. Transcriptome analysis was performed on leaves of control and transgenic lines under normal growth conditions and salt stress to predict genes regulated during salt stress. This provides a basis for elucidating the molecular regulation mechanism of PeCLH2 in response to salt stress and improving the tolerance of poplar under salt stress.

The basic helix-loop-helix (bHLH) family of transcription factors is one of the largest and oldest transcription factor families in plants. Members of the bHLH family regulate various growth and metabolic processes in plants. We used quantitative trait locus (QTL) mapping and transcriptome sequencing (RNA-seq) to identify PRE1 as a candidate bHLH transcription factor associated with root dry weight (RDW) in poplar. PRE1 was highly expressed in the roots and xylem, and was responsive to gibberellin, salicylic acid, drought, and salt stress. We cloned the PRE1 homolog from Populus simonii 'Tongliao1', referred to as PsPRE1, and transformed it into 84K poplar (Populus alba × Populus glandulosa). The overexpression of PsPRE1 in 84K poplar increased adventitious root development, fresh weight, total root number, and maximum root length. Poplar lines overexpressing PsPRE1 also exhibited enhanced salt tolerance while retaining a normal growth phenotype in the presence of salt stress. Catalase (CAT) activity in the PsPRE1 overexpression lines was higher than that of the wild-type, which may play a role in detoxifying stress-induced hydrogen peroxide production. An RNA-seq analysis of the PsPRE1 overexpression line revealed several differentially expressed genes (DEGs) involved in or related to auxin-, gibberellin-, and salicylic acid pathways, which indicates that the regulation of root development in poplar by PsPRE1 may involve multiple hormones.

The Jumonji C (JMJ-C) domain-containing gene family regulates epigenetic and developmental processes in plants. We identified 55 JMJ-C genes in Populus alba × Populus glandulosa using HMM and BLASTp analyses. Chromosomal mapping revealed an asymmetric distribution with conserved synteny. Phylogenetic reconstruction revealed that PagJMJ genes segregate into five evolutionarily conserved subfamilies, exhibiting classification patterns identical to those of Arabidopsis thaliana and Populus trichocarpa. Synteny analysis indicated a closer relationship with P. trichocarpa than with A. thaliana. Motif and promoter analyses highlighted subfamily-specific features and diverse cis-elements, particularly light-responsive motifs. Expression profiling revealed tissue-specific patterns, with key genes enriched in roots, vascular tissues, and leaves. Developmental analysis in cambium and xylem identified four expression clusters related to wood formation. Co-expression analysis identified six key PagJMJ genes (PagJMJ6, 29, 34, 39, 53, and 55) strongly associated with wood formation-related transcription factors. ChIP-qPCR analysis revealed that key genes co-expressed with PagJMJ genes were marked by H3K4me3 and H3K9me2 modifications. These findings provide insights into the evolutionary and functional roles of PagJMJ genes in poplar vascular development and wood formation.

PagEXPA1 combines with PagCDKB2;1 to regulate plant growth and the elongation of fibers in Populus alba × Populus glandulosa

Physiological mechanism of the enhanced drought tolerance in transgenic poplar (Populus alba × Populus glandulosa) with codA gene

Drought stress affects plant productivity by altering plant responses at the morphological, physiological, and molecular levels. In this study, we identified physiological and genetic responses in Populus alba × Populus glandulosa hybrid clones 72-30 and 72-31 after 12 days of exposure to drought treatment. After 12 days of drought treatment, glucose, fructose, and sucrose levels were significantly increased in clone 72-30 under drought stress. The Fv/Fo and Fv/Fm values in both clones also decreased under drought stress. The changes in proline, malondialdehyde, and H2O2 levels were significant and more pronounced in clone 72-30 than in clone 72-31. The activities of antioxidant-related enzymes, such as catalase and ascorbate peroxidase, were significantly higher in the 72-31 clone. To identify drought-related genes, we conducted a transcriptomic analysis in P. alba × P. glandulosa leaves exposed to drought stress. We found 883 up-regulated and 305 down-regulated genes in the 72-30 clone and 279 and 303 in the 72-31 clone, respectively. These differentially expressed genes were mainly in synthetic pathways related to proline, abscisic acid, and antioxidants. Overall, clone 72-31 showed better drought tolerance than clone 72-30 under drought stress, and genetic changes also showed different patterns.

The microRNA169 (miR169) family and NF-YA transcription factors (TFs) are crucial for drought stress responses. However, the mechanisms by which these factors regulate reactive oxygen species (ROS) homeostasis under drought conditions remain inadequately characterized in Populus. Here, we identified an NF-YA TF, PagNF-YA5, from hybrid poplar 84 K (Populus alba × Populus glandulosa). Knockout of PagNF-YA5 reduced drought tolerance in transgenic poplars, while its overexpression enhanced tolerance. Tobacco transient co-expression, 5' RACE, and dual-luciferase reporter assays confirmed that miR169z specifically cleaved PagNF-YA5 transcripts. Overexpressing miR169z decreased drought tolerance in transgenic poplars, whereas repressing its expression using short tandem target mimics improved tolerance. Transcriptomic and biochemical analyses revealed that NF-YA5 directly activates glycerol-3-phosphate dehydrogenase 1 (PagGPDHc1) expression. PagGPDHc1 upregulation elevates NAD+ levels, thereby inhibiting ROS production and enhancing drought tolerance. Conversely, gpdhc1-knockout poplars displayed opposing phenotypic effects. Collectively, this study elucidates a molecular mechanism by which the miR169z-NF-YA5-GPDHc1 module enhances drought tolerance through NAD+-mediated inhibit ROS production in Populus. These findings advance our understanding of drought adaptation mechanisms in woody plants and establish a molecular framework for the genetic improvement of forest trees under water deficit conditions.