The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in Populus.

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees.

Aspartyl tRNA-synthetase (AspRS) gene family enhances drought tolerance in poplar through BABA-PtrIBIs-PtrVOZ signaling module.

BACKGROUND: Drought stress is a prevalent abiotic stress that significantly hinders the growth and development of plants. According to studies, ß-aminobutyric acid (BABA) can influence the ABA pathway through the AtIBI1 receptor gene to enhance cold resistance in Arabidopsis. However, the Aspartate tRNA-synthetase (AspRS) gene family, which acts as the receptor for BABA, has not yet been investigated in poplar. Particularly, it is uncertain how the AspRS gene family (PtrIBIs)r can resist drought stress after administering various concentrations of BABA to poplar. RESULTS: In this study, we have identified 12 AspRS family genes and noted that poplar acquired four PtrIBI pairs through whole genome duplication (WGD). We conducted cis-action element analysis and found a significant number of stress-related action elements on different PtrIBI genes promoters. The expression of most PtrIBI genes was up-regulated under beetle and mechanical damage stresses, indicating their potential role in responding to leaf damage stress. Our results suggest that a 50 mM BABA treatment can alleviate the damage caused by drought stress in plants. Additionally, via transcriptome sequencing, we observed that the partial up-regulation of BABA receptor genes, PtrIBI2/4/6/8/11, in poplars after drought treatment. We hypothesize that poplar responds to drought stress through the BABA-PtrIBIs-PtrVOZ coordinated ABA signaling pathway. Our research provides molecular evidence for understanding how plants respond to drought stress through external application of BABA. CONCLUSIONS: In summary, our study conducted genome-wide analysis of the AspRS family of P. trichocarpa and identified 12 PtrIBI genes. We utilized genomics and bioinformatics to determine various characteristics of PtrIBIs such as chromosomal localization, evolutionary tree, gene structure, gene doubling, promoter cis-elements, and expression profiles. Our study found that certain PtrIBI genes are regulated by drought, beetle, and mechanical damage implying their crucial role in enhancing poplar stress tolerance. Additionally, we observed that external application of low concentrations of BABA increased plant drought resistance under drought stress. Through the BABA-PtrIBIs-PtrVOZ signaling module, poplar plants were able to transduce ABA signaling and regulate their response to drought stress. These results suggest that the PtrIBI genes in poplar have the potential to improve drought tolerance in plants through the topical application of low concentrations of BABA.

Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress.

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

PeTGA1 enhances disease resistance against Colletotrichum gloeosporioides through directly regulating PeSARD1 in poplar.

Basic leucine zipper (bZIP) proteins play important roles in responding to biotic and abiotic stresses in plants. However, the molecular mechanisms of plant resistance to pathogens remain largely unclear in poplar. The present study isolated a TGACG-binding (TGA) transcription factor, PeTGA1, from Populus euphratica. PeTGA1 belongs to subgroup D of the bZIP family and was localized to the nucleus. To study the role PeTGA1 plays in response to Colletotrichum gloeosporioides, transgenic triploid white poplars overexpressing PeTGA1 were generated. Results showed that poplars with overexpressed PeTGA1 showed a higher effective defense response to C. gloeosporioides than the wild-type plants. A yeast one-hybrid assay and an electrophoretic mobility shift assay revealed that PeTGA1 could directly bind to the PeSARD1 (P. euphratica SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1) promoter, an important regulator for salicylic acid biosynthesis. The transactivation assays indicated that PeTGA1 activated the expression of PeSARD1, and PR1 (PATHOGENESIS-RELATED 1), a SA marker gene involved in SA signaling. Subsequently, we observed that the PeTGA1 overexpression lines showed elevated SA levels, thereby resulting in the increased resistance to C. gloeosporioides. Taken together, our results indicated that PeTGA1 may exert a key role in plant immunity not only by targeting PeSARD1 thus participating in the SA biosynthesis pathway but also by involving in SA signaling via activating the expression of PR1.

Genome-Wide Investigation of the PtrCHLP Family Reveals That PtrCHLP3 Actively Mediates Poplar Growth and Development by Regulating Photosynthesis.

Chlorophyll (Chl) plays a crucial role in plant photosynthesis. The geranylgeraniol reductase gene (CHLP) participates in the terminal hydrogenation of chlorophyll biosynthesis. Although there are many studies related to the genome-wide analysis of Populus trichocarpa, little research has been conducted on CHLP family genes, especially those concerning growth and photosynthesis. In this study, three CHLP genes were identified in Populus. The evolutionary tree indicated that the CHLP family genes were divided into six groups. Moreover, one pair of genes was derived from segmental duplications in Populus. Many elements related to growth were detected by cis-acting element analysis of the promoters of diverse PtrCHLPs. Furthermore, PtrCHLPs exhibit different tissue expression patterns. In addition, PtrCHLP3 is preferentially expressed in the leaves and plays an important role in regulating chlorophyll biosynthesis. Silencing of PtrCHLP3 in poplar resulted in a decrease in chlorophyll synthesis in plants, thus blocking electron transport during photosynthesis. Furthermore, inhibition of PtrCHLP3 expression in poplar can inhibit plant growth through the downregulation of photosynthesis. Ultimately, PtrCHLP3 formed a co-expression network with photosynthesis and chlorophyll biosynthesis-related genes, which synergistically affected the growth and photosynthesis of poplars. Thus, this study provides genetic resources for the improved breeding of fast-growing tree traits.

Genome-wide analysis and expression profiling of Cation/H+ exchanger (CAX) family genes reveal likely functions in cadmium stress responses in poplar.

Cadmium, a toxic heavy metal, seriously affects human health and ecological security. The cation/H+ exchanger (CAX) family is a unique metal transporter that plays a crucial role in Cd acquisition, transfer, and remission in plants. Although there are many studies related to the genome-wide analysis of Populus trichocarpa, little research has been done on the CAX family genes, especially concerning Cd stress. In this study, genome-wide analysis of the Populus CAX family identified seven stress-related CAX genes. The evolutionary tree indicated that the CaCA family genes were grouped into four clusters. Moreover, seven pairs of genes were derived by segmental duplication in poplars. Cis-acting element analysis identified numerous stress-related elements in the promoters of diverse PtrCAXs. Furthermore, some PtrCAXs were up-regulated by drought, beetle, and mechanical damage, indicating their possible function in regulating stress response. Under cadmium stress, all CAX genes in the roots were up-regulated. Our findings suggest that plants may regulate their response to Cd stress through the TF-CAXs module. Comprehensively investigating the CAX family provides a scientific basis for the phytoremediation of heavy metal pollution by Populus.

ABF3 enhances drought tolerance via promoting ABA-induced stomatal closure by directly regulating ADF5 in Populus euphratica.

Water availability is a main limiting factor for plant growth, development, and distribution throughout the world. Stomatal movement mediated by abscisic acid (ABA) is particularly important for drought adaptation, but the molecular mechanisms in trees are largely unclear. Here, we isolated an ABA-responsive element binding factor, PeABF3, in Populus euphratica. PeABF3 was preferentially expressed in the xylem and young leaves, and was induced by dehydration and ABA treatments. PeABF3 showed transactivation activity and was located in the nucleus. To study its functional mechanism in poplar responsive to drought stress, transgenic triploid white poplars (Populus tomentosa 'YiXianCiZhu B385') overexpressing PeABF3 were generated. PeABF3 overexpression significantly enhanced stomatal sensitivity to exogenous ABA. When subjected to drought stress, PeABF3 overexpression maintained higher photosynthetic activity and promoted cell membrane integrity, resulting in increased water-use efficiency and enhanced drought tolerance compared with wild-type controls. Moreover, a yeast one-hybrid assay and an electrophoretic mobility shift assay revealed that PeABF3 activated the expression of Actin-Depolymerizing Factor-5 (PeADF5) by directly binding to its promoter, promoting actin cytoskeleton remodeling and stomatal closure in poplar under drought stress. Taken together, our results indicate that PeABF3 enhances drought tolerance via promoting ABA-induced stomatal closure by directly regulating PeADF5 expression.

PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in Populus.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) plays an important role in stress responses, but the transcriptional regulation of ZAT12 in response to abiotic stress remains unclear. In this study, we confirmed that a SALT TOLERANCE ZINC FINGER1 transcription factor from Populus euphratica (PeSTZ1) could regulate the expression of PeZAT12 by dual-luciferase reporter (DLR) assay and electrophoretic mobility shift assay. The expression of PeSTZ1 was rapidly induced by NaCl and hydrogen peroxide (H2O2) treatments. Overexpressing PeSTZ1 in poplar 84K (Populus alba × Populus glandulosa) plant was endowed with a strong tolerance to salt stress. Under salt stress, transgenic poplar exhibited higher expression levels of PeZAT12 and accumulated a larger amount of antioxidant than the wild-type plants. Meanwhile, ASCORBATE PEROXIDASE2 (PeAPX2) can be activated by PeZAT12 and PeSTZ1, promoting the accumulation of cytosolic ascorbate peroxidase (APX) to scavenge reactive oxygen species (ROS) under salt stress. This new regulatory model (PeSTZ1-PeZAT12-PeAPX2) was found in poplar, providing a new idea and insight for the interpretation of poplar resistance. Transgenic poplar reduced the accumulation of ROS, restrained the degradation of chlorophyll and guaranteed the photosynthesis and electron transport system. On the other hand, transgenic poplar slickly adjusted K+/Na+ homeostasis to alleviate salt toxicity in photosynthetic organs of plants under salt stress and then increased biomass accumulation. In summary, PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in poplar.

PeSTZ1, a C2H2-type zinc finger transcription factor from Populus euphratica, enhances freezing tolerance through modulation of ROS scavenging by directly regulating PeAPX2.

In the present study, PeSTZ1, a cysteine-2/histidine-2-type zinc finger transcription factor, was isolated from the desert poplar, Populus euphratica, which serves as a model stress adaptation system for trees. PeSTZ1 was preferentially expressed in the young stems and was significantly up-regulated during chilling and freezing treatments. PeSTZ1 was localized to the nucleus and bound specifically to the PeAPX2 promoter. To examine the potential functions of PeSTZ1, we overexpressed it in poplar 84K hybrids (Populus alba × Populus glandulosa), which are known to be stress-sensitive. Upon exposure to freezing stress, transgenic poplars maintained higher photosynthetic activity and dissipated more excess light energy (in the form of heat) than wild-type poplars. Thus, PeSTZ1 functions as a transcription activator to enhance freezing tolerance without sacrificing growth. Under freezing stress, PeSTZ1 acts upstream of ASCORBATE PEROXIDASE2 (PeAPX2) and directly regulates its expression by binding to its promoter. Activated PeAPX2 promotes cytosolic APX that scavenges reactive oxygen species (ROS) under cold stress. PeSTZ1 may operate in parallel with C-REPEAT-BINDING FACTORS to regulate COLD-REGULATED gene expression. Moreover, PeSTZ1 up-regulation reduces malondialdehyde and ROS accumulation by activating the antioxidant system. Taken together, these results suggested that overexpressing PeSTZ1 in 84K poplar enhances freezing tolerance through the modulation of ROS scavenging via the direct regulation of PeAPX2 expression.

Poplar miR472a targeting NBS-LRRs is involved in effective defence against the necrotrophic fungus Cytospora chrysosperma.

The hemibiotroph Colletotrichum gloeosporioides and the necrotroph Cytospora chrysosperma cause poplar foliage and stem disease, respectively, resulting in substantial economic losses. In this study, Populus trichocarpa ptc-miR472a was down-regulated in leaves treated with salicylic acid, jasmonic acid (JA) or bacterial flagellin (flg22). Here, ptc-miR472a and a short tandem target mimic (STTM) of miR472a were overexpressed in P. alba × P. glandulosa, and overexpression lines of miR472a and silenced lines of STTM472a were generated. Compared with the STTM472a and wild type lines, lower reactive oxygen species accumulation was detected in miR472a overexpressing plants treated with flg22, C. gloeosporioides or C. chrysosperma. In addition, the miR472a overexpressing lines exhibited the highest susceptibility to the hemibiotroph, C. gloeosporioides, but the highest effective defence response to the necrotroph, C. chrysosperma. The JA/ethylene marker gene ERF1 was rapidly up-regulated in miR472a overexpressing plants. Furthermore, five phased, secondary, small interfering RNAs (phasiRNAs) were confirmed in the miR472a overexpressing and STTM472a lines, triggering phasiRNAs predicted to enhance NBS-LRR silencing. Taken together, our results revealed that ptc-miR472a exerts a key role in plant immunity to C. gloeosporioides and C. chrysosperma by targeting NBS-LRR transcripts. This study provides a new strategy and method in plant breeding to improve plant disease resistance.

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus.

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H2 O2 production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild-type) controls. Moreover, up-regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA-induced stomatal closure caused by hydrogen peroxide (H2 O2 ) production in transgenic poplar plants.