Comprehensive analysis of MAPK gene family in Populus trichocarpa and physiological characterization of PtMAPK3-1 in response to MeJA induction.

Mitogen-activated protein kinases (MAPKs) play important roles in plant growth and development, as well as hormone and stress responses by signaling to eukaryotic cells, through MAPK cascade, the presence of various cues; thereby, regulating various responses. The MAPK cascade consists mainly of three gene families, MAPK, MAPKK, and MAPKKK, which activate downstream signaling pathways through sequential phosphorylation. Although the MAPK cascade gene family has been reported in several species, there is a lack of comprehensive analysis in poplar. We identified 21 MAPK genes, 11 MAPKK genes, and 104 MAPKKK genes in Populus trichocarpa. The phylogenetic classification was supported by conservative motif, gene structure and motif analysis. Whole genome duplication has an important role in the expansion of MAPK cascade genes. Analysis of promoter cis-elements and expression profiles indicates that MAPK cascade genes have important roles in plant growth and development, abiotic and biotic stresses, and phytohormone response. Expression profiling revealed a significant upregulation of PtMAPK3-1 expression in response to drought, salt and disease stresses. Poplar transiently overexpressing PtMAPK3-1 and treated with methyl jasmonic acid (MeJA) had higher catalase and peroxidase levels than non-overexpressing poplar. This work represents the first complete inventory of the MAPK cascade in P. trichocarpa, which reveals that PtMAPK3-1 is induced by the MeJA hormone and participates in the MeJA-induced enhancement of the antioxidant enzyme system.

Transcriptome analysis reveals key genes regulating signaling and metabolic pathways during the growth of moso bamboo (Phyllostachys edulis) shoots.

Moso bamboo (Phyllostachys edulis), a high-value bamboo used to produce food (young shoots), building, and industrial goods. To explore key candidate genes regulating signal transduction and metabolic processes during the initiation of stem elongation in moso bamboo, a transcriptome analysis of the shoots during three successive early elongation stages was performed. From cluster and differential expression analyses, 2984 differentially expressed genes (DEGs) were selected for an enrichment analysis. The DEGs were significantly enriched in the plant hormone signal transduction, sugar and starch metabolism, and energy metabolism pathways. Consequently, the DEG expression patterns of these pathways were analyzed, and the plant endogenous hormone and carbon metabolite (including sucrose, total soluble sugar, and starch) contents for each growth stage, of the shoot, were determined. The cytokinin-signaling pathway was continuously active in the three successive elongation stages, in which several cytokinin-signaling genes played indispensable roles. Additionally, many key DEGs regulating sugar, starch metabolism, and energy conversion, which are actively involved in energy production and substrate synthesis during the continuous growth of the shoots, were found. In summary, our study lays a foundation for understanding the mechanisms of moso bamboo growth and provides useful gene resources for breeding through genetic engineering.

Geranyl diphosphate synthase large subunits OfLSU1/2 interact with small subunit OfSSUII and are involved in aromatic monoterpenes production in Osmanthus fragrans.

Osmanthus fragrans is a famous ornamental tree species for its pleasing floral fragrance. Monoterpenoids are the core floral volatiles of O. fragrans flowers, which have tremendous commercial value. Geranyl diphosphate synthase (GPPS) is a key enzyme that catalyzes the formation of GPP, the precursor of monoterpenoids. However, there are no reports of GPPSs in O. fragrans. Here, we performed RNA sequencing on the O. fragrans flowers and identified three GPPSs. Phylogenetic tree analysis showed that OfLSU1/2 belonged to the GPPS.LSU branch, while the OfSSUII belonged to the GPPS.SSU branch. OfLSU1, OfLSU2 and OfSSUII were all localized in chloroplasts. Y2H and pull-down assays showed that OfLSU1 or OfLSU2 interacted with OfSSUII to form heteromeric GPPSs. Site mutation experiments revealed that the conserved CXXXC motifs of OfLSU1/2 and OfSSUII were essential for the interaction between OfLSU1/2 and OfSSUII. Transient expression experiments showed that OfLSU1, OfLSU2 and OfSSUII co-expressed with monoterpene synthase genes OfTPS1 or OfTPS2 improved the biosynthesis of monoterpenoids (E)-ß-ocimene and linalool. The heteromeric GPPSs formed by OfLSU1/2 interacting with OfSSUII further improves the biosynthesis of monoterpenoids. Overall, these preliminary results suggested that the GPPSs play a key role in regulating the production of aromatic monoterpenes in O. fragrans.

Comprehensive investigation of BZR gene family in four dicots and the function of PtBZR9 and PtBZR12 under drought stress.

Brassinazole-resistant (BZR) transcription factor plays an important role in plant growth and stress resistance through brassinosteroid (BR) signal transduction. However, systematic analysis of the BZR family in dicots remains limited. In this study, we conducted a genome-wide study of four typical dicots: Arabidopsis thaliana, Carica papaya, Vitis vinifera and Populus trichocarpa. Thirty-four BZR gene family members were identified and classified them into three subfamilies. Analysis of promoter and expression patterns revealed crucial role of a pair of homologous BZR genes, PtBZR9 and PtBZR12, in poplar may play a critical role under abiotic stress. PtBZR9 and PtBZR12 were localised in the nucleus and exhibited mutual interactions. Moreover, transient overexpression (OE) of PtBZR9 and PtBZR12 in poplar enhanced tolerance to drought stress. The phenotypic and physiological characteristics of PtBZR9 and PtBZR12 OE in Arabidopsis mirrored those of transient OE in the poplar. Additionally, PtBZR9 and PtBZR12 can bind to the E-box element. Under exogenous BR treatment, transgenic lines displayed a greater decrease in root length than the wild type. Thus, these findings provide a solid foundation for future research on the complex regulatory mechanisms of BZR genes.

CiAP2/ERF65 and CiAP2/ERF106, a pair of homologous genes in pecan (Carya illinoensis), regulate plant responses during submergence in transgenic Arabidopsis thaliana.

When plants are entirely submerged, photosynthesis and respiration are severely restricted, affecting plant growth and potentially even causing plant death. The AP2/ERF superfamily has been widely reported to play a vital role in plant growth, development and resistance to biotic and abiotic stresses. However, no relevant studies exist on flooding stress in pecan. In this investigation, we observed that CiAP2/ERF65 positively modulated the hypoxia response during submergence, whereas CiAP2/ERF106 was sensitive to submergence. The levels of physiological and biochemical indicators, such as POD, CAT and among others, in CiAP2/ERF65-OE lines were significantly higher than those in wild-type Arabidopsis thaliana, indicating that the antioxidant capacity of CiAP2/ERF65-OE lines was enhanced under submergence. The RNA-seq results revealed that the maintenance of the expression levels of the antenna protein gene, different signaling pathways for regulation, as well as the storage and consumption of ATP, might account for the opposite phenotypes of CiAP2/ERF65 and CiAP2/ERF106. Furthermore, the expression of some stress-related genes was altered during submergence and reoxygenation. Overall, these findings enhance our understanding of submergence stress in pecan, providing important candidate genes for the molecular design and breeding of hypoxia resistant in plants.

The R2R3-MYB transcription factor OfMYB21 positively regulates linalool biosynthesis in Osmanthus fragrans flowers.

Osmanthus fragrans is a well-known landscape ornamental tree species for its pleasing floral fragrance and abundance of flowers. Linalool, the core floral volatiles of O. fragrans, has tremendous economic value in the pharmaceuticals, cleaning products and cosmetics industries. However, the transcriptional regulatory network for the biosynthesis of linalool in O. fragrans remains unclear. Here, OfMYB21, a potential transcription factor regulating the linalool synthetase OfTPS2, was identified using RNA-seq data and qRT-PCR analysis. Yeast one-hybrid, dual-luciferase and EMSA showed that OfMYB21 directly binds to the promoter of OfTPS2 and activates its expression. Overexpression of OfMYB21 in the petals of O. fragrans led to up-regulation of OfTPS2 and increased accumulation of linalool, while silencing of OfMYB21 led to down-regulation of OfTPS2 and decreased biosynthesis of linalool. Subsequently, yeast two-hybrid, pull-down and BiFC experiments showed that OfMYB21 interacts with JA signaling factors OfJAZ2/3 and OfMYC2. Interestingly, the interaction between OfMYC2 and OfMYB21 further enhanced the transcription of OfTPS2, whereas OfJAZ3 attenuated this effect. Overall, our studies provided novel finding on the regulatory mechanisms responsible for the biosynthesis of the volatile monoterpenoid linalool in O. fragrans.

Whole-genome identification and multiple abiotic stresses expression pattern profiling analysis of PLATZ transcription factor family members in Pecan (Carya illinoensis).

Plant AT-rich sequence and zinc-binding (PLATZ), as a plant-specific transcription factor, have been identified and studied in a variety of plants. However, there are no reports about PLATZ proteins in Carya illinoensis (pecan). Here, 24 C. illinoensis CiPLATZs have been identified and divided into 4 groups. Gene structure, motif composition, conserved domain and cis-acting elements analysis indicated that the PLATZ gene family was highly conserved. Transcriptome data combined with qRT-PCR analysis revealed that CiPLATZ6, CiPLATZ12, CiPLATZ13, CiPLATZ14 and CiPLATZ23 were highly expressed in multiple tissues of C. illinoensis and strongly responded to drought, salt and heat stress. Among them, CiPLATZ6, CiPLATZ12 and CiPLATZ23 were all located in the nucleus and had no transcriptional autoactivation ability in yeast cells, and acted as transcriptional suppressors in plants. In addition, the CiPLATZ23-overexpressing transgenic Arabidopsis thaliana showed enhanced tolerance to drought. Measurements of physiological indicators and analysis of stress-related genes expression levels in transgenic A. thaliana were used to support this conclusion. The results of this study are helpful to understand the structural feature and function of CiPLATZs, and provide candidate genes for molecular breeding of drought tolerance of C. illinoensis.

Systematic analysis of the Serine/Arginine-Rich Protein Splicing Factors (SRs) and focus on salt tolerance of PtSC27 in Populus trichocarpa.

Serine/Arginine-Rich Protein Splicing Factors (SRs) are indispensable splicing factors, which play significant roles in spliceosome assembly, splicing regulation and regulation of plant stress. However, a comprehensive analysis and function research of SRs in the woody plant is still lacking. In this report, we conducted the identification and comprehensive analysis of the 71 SRs in poplar and three other dicots, including basic characterization, phylogenetic, conserved motifs, gene duplication, promoter and splice isoform of these genes. Based on the publicly available transcriptome data, expression pattern of SRs in poplar under low temperature, high temperature, drought and salt stress were further analyzed. Subsequently, a key candidate gene PtSC27 that responded to salt stress was screened. More importantly, overexpression of PtSC27 increased plant survival rate under salt stress, and enhanced salt tolerance by regulating malondialdehyde (MDA) content, peroxidase (POD) and catalase (CAT) enzyme activities in transgenic plants. Meanwhile, overexpression of PtSC27 made transgenic plants insensitive to exogenous ABA and improved the expression of some ABA signal-related genes under salt stress. Overall, our studies lay a foundation for understanding the structure and function of SRs in the poplar and provide useful gene resources for breeding through genetic engineering.

GEPSdb: The Gene Expression Database of Poplar under Stress.

As a model tree species, poplar (Populus L.) has important economic and ecological value. Here, we constructed the GEPSdb (Gene Expression Database of Poplar under Stress; http://gepsdb.ahau-edu.cn/), which is an integrated database of poplar gene expression profiles derived from RNA-seq and microarray library data. This database provides a comprehensive collection of gene expression data from poplar exposed to 14 types of environmental stress from 11 high-quality RNA-seq experiments and 51 microarray libraries. The GEPSdb includes 56 genes from previous literature that have been examined in poplar and functionally verified. By incorporating data from numerous expression analyses, GEPSdb provides a user-friendly web interface for querying, browsing, and visualizing the expression profiles of related genes. Consequently, GEPSdb can be used to link transcription data with phenotypes and can enhance our understanding of important biological processes and mechanisms underlying complex agronomic traits in poplar.

Comparative analysis of the stellacyanins (SCs) family and focus on drought resistance of PtSC18 in Populus trichocarpa.

Stellacyanin (SC) is a type I (blue) copper protein, which plays a crucial role in plant growth and stress response. However, the comprehensive analysis and functional research of SCs in the woody plant is still lacking. Here, a total of 74 SCs were collected and identified from Arabidopsis, papaya, grape, rice and poplar. Bioinformatics was used to analyze the gene structure, protein structure and evolutionary relationship of 74 genes, especially 19 SCs in Populus trichocarpa. Based on the RNA-seq data, expression pattern of SCs in poplar under cold, high temperature, drought and salt stress were further analyzed. Subsequently, a key candidate gene PtSC18 that strongly responded to drought stress was screened. Subcellular localization experiment exhibited that PtSC18 was localized in the nucleus and plasma membrane. Overexpression of PtSC18 enhanced drought tolerance of transgenic Arabidopsis by improving water retention and reducing oxidative damage. Measurements of physiological indicators, including chlorophyll, H2O2, malondialdehyde content, peroxidase and catalase enzyme activities and electrical conductivity, all supported this conclusion. More importantly, PtSC18 enhanced the expression of some stress-related genes in transgenic Arabidopsis. Overall, our results lay a foundation for understanding the structure and function of PtSCs and provide useful gene resources for breeding through genetic engineering.

PhePLATZ1, a PLATZ transcription factor in moso bamboo (Phyllostachys edulis), improves drought resistance of transgenic Arabidopsis thaliana.

Drought is one of the most serious environmental stresses. Plant AT-rich sequence and zinc-binding (PLATZ) proteins perform indispensable functions to regulate plant growth and development and to respond to environmental stress. In this present study, we identified PhePLATZ1 in moso bamboo and found that its expression was up-regulated in response to 20% PEG-6000 and abscisic acid (ABA) treatments. Next, transgenic PhePLATZ1-overexpressing Arabidopsis lines were generated. Overexpression of PhePLATZ1 improved drought stress resistance of transgenic plants by mediating osmotic regulation, enhancing water retention capacity and reducing membrane and oxidative damage. These findings were corroborated by analysing physiological indicators including chlorophyll, relative water content, leaf water loss rate, electrolyte leakage, H2O2, proline, malondialdehyde content and the enzyme activities of peroxidase and catalase. Subsequent seed germination and seedling root length experiments that included exposure to exogenous ABA treatments showed that ABA sensitivity decreased in transgenic plants relative to wild-type plants. Moreover, transgenic PhePLATZ1-overexpressing plants promoted stomatal closure in response to ABA treatment, suggesting that PhePLATZ1 might play a positive regulatory role in the drought resistance of plants via the ABA signaling pathway. In addition, the transgenic PhePLATZ1-OE plants showed altered expression of some stress-related genes when grown under drought conditions. Taken together, these findings improve our understanding of the drought response of moso bamboo and provide a key candidate gene for the molecular breeding of this species for drought tolerance.