PTI-ETI synergistic signal mechanisms in plant immunity.

Plants face a relentless onslaught from a diverse array of pathogens in their natural environment, to which they have evolved a myriad of strategies that unfold across various temporal scales. Cell surface pattern recognition receptors (PRRs) detect conserved elicitors from pathogens or endogenous molecules released during pathogen invasion, initiating the first line of defence in plants, known as pattern-triggered immunity (PTI), which imparts a baseline level of disease resistance. Inside host cells, pathogen effectors are sensed by the nucleotide-binding/leucine-rich repeat (NLR) receptors, which then activate the second line of defence: effector-triggered immunity (ETI), offering a more potent and enduring defence mechanism. Moreover, PTI and ETI collaborate synergistically to bolster disease resistance and collectively trigger a cascade of downstream defence responses. This article provides a comprehensive review of plant defence responses, offering an overview of the stepwise activation of plant immunity and the interactions between PTI-ETI synergistic signal transduction.

Comparative genomic analysis of the Growth-Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves.

BACKGROUND: The growth-regulating factor-interacting factor (GIF) gene family plays a vital role in regulating plant growth and development, particularly in controlling leaf, seed, and root meristem homeostasis. However, the regulatory mechanism of heteromorphic leaves by GIF genes in Populus euphratica as an important adaptative trait of heteromorphic leaves in response to desert environment remains unknown. RESULTS: This study aimed to identify and characterize the GIF genes in P. euphratica and other five Salicaceae species to investigate their role in regulating heteromorphic leaf development. A total of 27 GIF genes were identified and characterized across six Salicaceae species (P. euphratica, Populus pruinose, Populus deltoides, Populus trichocarpa, Salix sinopurpurea, and Salix suchowensis) at the genome-wide level. Comparative genomic analysis among these species suggested that the expansion of GIFs may be derived from the specific Salicaceae whole-genome duplication event after their divergence from Arabidopsis thaliana. Furthermore, the expression data of PeGIFs in heteromorphic leaves, combined with functional information on GIF genes in Arabidopsis, indicated the role of PeGIFs in regulating the leaf development of P. euphratica, especially PeGIFs containing several cis-acting elements associated with plant growth and development. By heterologous expression of the PeGIF3 gene in wild-type plants (Col-0) and atgif1 mutant of A. thaliana, a significant difference in leaf expansion along the medial-lateral axis, and an increased number of leaf cells, were observed between the overexpressed plants and the wild type. CONCLUSION: PeGIF3 enhances leaf cell proliferation, thereby resulting in the expansion of the central-lateral region of the leaf. The findings not only provide global insights into the evolutionary features of Salicaceae GIFs but also reveal the regulatory mechanism of PeGIF3 in heteromorphic leaves of P. euphratica.

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.

Leaf Senescence Database v5.0: A Comprehensive Repository for Facilitating Plant Senescence Research.

Through an extensive literature survey, we have upgraded the Leaf Senescence Database (LSD v5.0; https://ngdc.cncb.ac.cn/lsd/), a curated repository of comprehensive senescence-associated genes (SAGs) and their corresponding mutants. Since its inception in 2010, LSD undergoes frequent updates to encompass the latest advances in leaf senescence research and its current version comprises a high-quality collection of 31,740 SAGs and 1,209 mutants from 148 species, which were manually searched based on robust experimental evidence and further categorized according to their functions in leaf senescence. Furthermore, LSD was greatly enriched with comprehensive annotations for the SAGs through meticulous curation using both manual and computational methods. In addition, it was equipped with user-friendly web interfaces that facilitate text queries, BLAST searches, and convenient download of SAG sequences for localized analysis. Users can effortlessly navigate the database to access a plethora of information, including literature references, mutants, phenotypes, multi-omics data, miRNA interactions, homologs in other plants, and cross-links to various databases. Taken together, the upgraded version of LSD stands as the most comprehensive and informative plant senescence-related database to date, incorporating the largest collection of SAGs and thus bearing great utility for a wide range of studies related to plant senescence.

Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Spliceosomal protein U2B″ delays leaf senescence by enhancing splicing variant JAZ9ß expression to attenuate jasmonate signaling in Arabidopsis.

The regulatory framework of leaf senescence is gradually becoming clearer; however, the fine regulation of this process remains largely unknown. Here, genetic analysis revealed that U2 small nuclear ribonucleoprotein B (U2B″), a component of the spliceosome, is a negative regulator of leaf senescence. Mutation of U2B″ led to precocious leaf senescence, whereas overexpression of U2B″ extended leaf longevity. Transcriptome analysis revealed that the jasmonic acid (JA) signaling pathway was activated in the u2b″ mutant. U2B″ enhances the generation of splicing variant JASMONATE ZIM-DOMAIN 9ß (JAZ9ß) with an intron retention in the Jas motif, which compromises its interaction with CORONATINE INSENSITIVE1 and thus enhances the stability of JAZ9ß protein. Moreover, JAZ9ß could interact with MYC2 and obstruct its activity, thereby attenuating JA signaling. Correspondingly, overexpression of JAZ9ß rescued the early senescence phenotype of the u2b″ mutant. Furthermore, JA treatment promoted expression of U2B″ that was found to be a direct target of MYC2. Overexpression of MYC2 in the u2b″ mutant resulted in a more pronounced premature senescence than that in wild-type plants. Collectively, our findings reveal that the spliceosomal protein U2B″ fine-tunes leaf senescence by enhancing the expression of JAZ9ß and thereby attenuating JA signaling.

Manipulating microRNA miR408 enhances both biomass yield and saccharification efficiency in poplar.

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop.

Histone variant HTB4 delays leaf senescence by epigenetic control of Ib bHLH transcription factor-mediated iron homeostasis.

Leaf senescence is an orderly process regulated by multiple internal factors and diverse environmental stresses including nutrient deficiency. Histone variants are involved in regulating plant growth and development. However, their functions and underlying regulatory mechanisms in leaf senescence remain largely unclear. Here, we found that H2B histone variant HTB4 functions as a negative regulator of leaf senescence. Loss of function of HTB4 led to early leaf senescence phenotypes that were rescued by functional complementation. RNA-seq analysis revealed that several Ib subgroup basic helix-loop-helix (bHLH) transcription factors (TFs) involved in iron (Fe) homeostasis, including bHLH038, bHLH039, bHLH100, and bHLH101, were suppressed in the htb4 mutant, thereby compromising the expressions of FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER (IRT1), two important components of the Fe uptake machinery. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that HTB4 could bind to the promoter regions of Ib bHLH TFs and enhance their expression by promoting the enrichment of the active mark H3K4me3 near their transcriptional start sites. Moreover, overexpression of Ib bHLH TFs or IRT1 suppressed the premature senescence phenotype of the htb4 mutant. Our work established a signaling pathway, HTB4-bHLH TFs-FRO2/IRT1-Fe homeostasis, which regulates the onset and progression of leaf senescence.

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.

Crucial Abiotic Stress Regulatory Network of NF-Y Transcription Factor in Plants.

Nuclear Factor-Y (NF-Y), composed of three subunits NF-YA, NF-YB and NF-YC, exists in most of the eukaryotes and is relatively conservative in evolution. As compared to animals and fungi, the number of NF-Y subunits has significantly expanded in higher plants. The NF-Y complex regulates the expression of target genes by directly binding the promoter CCAAT box or by physical interaction and mediating the binding of a transcriptional activator or inhibitor. NF-Y plays an important role at various stages of plant growth and development, especially in response to stress, which attracted many researchers to explore. Herein, we have reviewed the structural characteristics and mechanism of function of NF-Y subunits, summarized the latest research on NF-Y involved in the response to abiotic stresses, including drought, salt, nutrient and temperature, and elaborated the critical role of NF-Y in these different abiotic stresses. Based on the summary above, we have prospected the potential research on NF-Y in response to plant abiotic stresses and discussed the difficulties that may be faced in order to provide a reference for the in-depth analysis of the function of NF-Y transcription factors and an in-depth study of plant responses to abiotic stress.

Orthologs of Human-Disease-Associated Genes in Plants Are Involved in Regulating Leaf Senescence.

As eukaryotes, plants and animals have many commonalities on the genetic level, although they differ greatly in appearance and physiological habits. The primary goal of current plant research is to improve the crop yield and quality. However, plant research has a wider aim, exploiting the evolutionary conservatism similarities between plants and animals, and applying discoveries in the field of botany to promote zoological research that will ultimately serve human health, although very few studies have addressed this aspect. Here, we analyzed 35 human-disease-related gene orthologs in plants and characterized the genes in depth. Thirty-four homologous genes were found to be present in the herbaceous annual plant Arabidopsis thaliana and the woody perennial plant Populus trichocarpa, with most of the genes having more than two exons, including the ATM gene with 78 exons. More surprisingly, 27 (79.4%) of the 34 homologous genes in Arabidopsis were found to be senescence-associated genes (SAGs), further suggesting a close relationship between human diseases and cellular senescence. Protein-protein interaction network analysis revealed that the 34 genes formed two main subnetworks, and genes in the first subnetwork interacted with 15 SAGs. In conclusion, our results show that most of the 34 homologs of human-disease-associated genes in plants are involved in the leaf senescence process, suggesting that leaf senescence may offer a means to study the pathogenesis of human diseases and to screen drugs for the treat of diseases.

Genome-wide analysis of autophagy-related gene family and PagATG18a enhances salt tolerance by regulating ROS homeostasis in poplar.

Autophagy is the process by which intracellular components are delivered to lysosomes or vacuoles for degradation and recycling, which can promote the tolerance of organisms to biotic/abiotic stresses. However, autophagy-related genes (ATG) are not well studied in woody plants. Here, 48 ATG genes were identified in the poplar genome and divided into 14 subfamilies according to the phylogenetic tree. Collinearity analysis showed that 26 pairs of genes were derived by segmental duplication in poplars. The isogenous gene pairs of the ATG family between P. trichocarpa and other six species were analyzed by synteny analysis. Moreover, the ATG promoters contain a large number of phytohormone response elements and stress-response elements. Both phytohormone and salt treatments can induce the expression of PagATG18 subfamily genes. Overexpression of PagATG18a significantly improved the salt tolerance of poplar and reducing the oxidative damage of the membrane. Further research verified that PagATG18a interacted with the light-harvesting complex LHCB1 and APX2, indicating PagATG18a might be involved in regulating photosynthesis and antioxidant activity under stress. This study provides valuable information for further research on the functional characteristics of ATG genes in poplar and the theoretical basis for poplar stress resistance breeding.

PePYL4 enhances drought tolerance by modulating water-use efficiency and ROS scavenging in Populus.

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.

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.

Metabolic control of arginine and ornithine levels paces the progression of leaf senescence.

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.

CLE42 delays leaf senescence by antagonizing ethylene pathway in Arabidopsis.

Leaf senescence is the final stage of leaf development and is influenced by numerous internal and environmental factors. CLE family peptides are plant-specific peptide hormones that regulate various developmental processes. However, the role of CLE in regulating Arabidopsis leaf senescence remains unclear. Here, we found that CLE42 is a negative regulator of leaf senescence by using a CRISPR/Cas9-produced CLE mutant collection. The cle42 mutant displayed earlier senescence phenotypes, while overexpression of CLE42 delayed age-dependent and dark-induced leaf senescence. Moreover, application of the synthesized 12-amino-acid peptide (CLE42p) also delayed leaf senescence under natural and dark conditions. CLE42 and CLE41/44 displayed functional redundancy in leaf senescence, and the cle41 cle42 cle44 triple mutant displayed more pronounced earlier senescence phenotypes than any single mutant. Analysis of differentially expressed genes obtained by RNA-Seq methodology revealed that the ethylene pathway was suppressed by overexpressing CLE42. Moreover, CLE42 suppressed ethylene biosynthesis and thus promoted the protein accumulation of EBF, which in turn decreased the function of EIN3. Accordingly, mutation of EIN3/EIL1 or overexpression of EBF1 suppressed the earlier senescence phenotypes of the cle42 mutant. Together, our results reveal that the CLE peptide hormone regulates leaf senescence by communicating with the ethylene pathway.

Regulation of cytokinin biosynthesis using PtRD26pro -IPT module improves drought tolerance through PtARR10-PtYUC4/5-mediated reactive oxygen species removal in Populus.

Drought is a critical environmental factor which constrains plant survival and growth. Genetic engineering provides a credible strategy to improve drought tolerance of plants. Here, we generated transgenic poplar lines expressing the isopentenyl transferase gene (IPT) under the driver of PtRD26 promoter (PtRD26pro -IPT). PtRD26 is a senescence and drought-inducible NAC transcription factor. PtRD26pro -IPT plants displayed multiple phenotypes, including improved growth and drought tolerance. Transcriptome analysis revealed that auxin biosynthesis pathway was activated in the PtRD26pro -IPT plants, leading to an increase in auxin contents. Biochemical analysis revealed that ARABIDOPSIS RESPONSE REGULATOR10 (PtARR10), one of the type-B ARR transcription factors in the cytokinin pathway, was induced in PtRD26pro -IPT plants and directly regulated the transcripts of YUCCA4 (PtYUC4) and YUCCA5 (PtYUC5), two enzymes in the auxin biosynthesis pathway. Overexpression of PtYUC4 enhanced drought tolerance, while simultaneous silencing of PtYUC4/5 evidently attenuated the drought tolerance of PtRD26pro -IPT plants. Intriguingly, PtYUC4/5 displayed a conserved thioredoxin reductase activity that is required for drought tolerance by deterring reactive oxygen species accumulation. Our work reveals the molecular basis of cytokinin and auxin interactions in response to environmental stresses, and shed light on the improvement of drought tolerance without a growth penalty in trees by molecular breeding.

Preparation and Transfection of Populus tomentosa Mesophyll Protoplasts.

Mesophyll protoplasts freshly isolated from leaves are a useful research system in plants. However, cell walls in woody plants contain more pectin, making mesophyll protoplasts isolation difficult in Populus. This has limited their application in biochemical, molecular, cellular, genetic, genomic, transcriptomic, and proteomic assays. In this protocol, a simple and efficient method to prepare and transfect mesophyll protoplasts of Populus tomentosa is presented in detail. Leaves of P. tomentosa plants grown in tissue culture media were pre-treated in D-mannitol solution and then digested with an enzyme solution. After washing with W5 and MMg buffers, the protoplasts were incubated in PEG/Ca2+ solution with plasmid for transfection. The mesophyll protoplasts isolated were used to express the histone variant H2B fused with green fluorescent protein (GFP) for confocal microscopy imaging. This "P. tomentosa mesophyll protoplasts preparation and transfection" system provides a useful tool for studying woody plants using a variety of applications, including gene expression, subcellular localization, protein-protein interaction, chromatin immunoprecipitation, western blot, single-cell sequencing, and genome editing.

Genome-Wide Comprehensive Analysis of the GASA Gene Family in Populus.

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.