ABA Treatment Research Articles

Reference genes selection of Paeonia ostii 'Fengdan' under osmotic stresses and hormone treatments by RT-qPCR.

Tree peony possess significant ornamental, medicinal and oil values. Osmotic stresses including dehydratiuon and salinity limit the expansion of cultivation area of tree peony. Information on reference genes selection under osmotic stress and hormone stimulation of tree peony still limited. This study aimed to determine the stable reference genes suitable for tree peony under osmotic stresses and hormone treatments, and provide a theoretical basis for the molecular biology research. Twelve candidate reference genes were evaluated in Paeonia ostii 'Fengdan' under osmotic stress and hormone treatments by RT-qPCR. Delta Ct method, geNorm, and NormFinder were used for the comprehensive expression stability ranking comparison. The results revealed that tubulin-α was the preferred internal reference genes for drought and ABA treatment, tubulin-β was identified as the most suitable reference gene under drought and OPDA induction, 18s-rRNA was regarded as the most stable gene for salinity and JA treatment, eIF-5A was listed as the most stable gene for JA and MeJA treatments. The experiments also displayed that EF1-α were comparatively unstable under ABA and BR hormone treatments. These preferred reference genes could be useful in qPCR studies involving osmotic or hormonal stresses in Paeonia ostii 'Fengdan'. It is anticipated that the results will benefit tree peony functional genomics studies and molecular breeding research in the future.

Ectopic expression of NAC transcription factor HaNAC3 from Haloxylon ammodendron increased abiotic stress resistance in tobacco.

HaNAC3 is a transcriptional activator located in the nucleus that may be involved in the response to high temperature, high salt and drought stresses as well as phytohormone IAA and ABA treatments. Our study demonstrated that HaNAC3 increased the tolerance of transgenic tobacco to abiotic stress and was involved in the regulation of a range of downstream genes and metabolic pathways. This also indicates the potential application of HaNAC3 as a plant tolerance gene. NAC transcription factors play a key role in plant growth and development and plant responses to biotic and abiotic stresses. However, the biological functions of NAC transcription factors in the desert plant Haloxylon ammodendron are still poorly understood. In this study, the NAC transcription factor HaNAC3 was isolated and cloned from a typical desert plant H. ammodendron, and its possible biological functions were investigated. Bioinformatics analysis showed that HaNAC3 has the unique N-terminal NAC structural domain of NAC transcription factor. Quantitative real-time fluorescence analysis showed that HaNAC3 was able to participate in the response to simulated drought, high temperature, high salt, and phytohormone IAA and ABA treatments, and was very sensitive to simulated high temperature and phytohormone ABA treatments. Subcellular localization analysis showed that the GFP-HaNAC3 fusion protein was localized in the nucleus of tobacco epidermal cells. The transcriptional self-activation assay showed that HaNAC3 had transcriptional self-activation activity, and the truncation assay confirmed that the transcriptional activation activity was located at the C-terminus. HaNAC3 gene was expressed exogenously in wild-type Nicotiana benthamiana, and the physiological function of HaNAC3 was verified by simulating drought and other abiotic stresses. The results indicated that transgenic tobacco had better resistance to abiotic stresses than wild-type B. fuminata. Further transcriptome analysis showed that HaNAC3 was involved in the regulation of a range of downstream resistance genes, wax biosynthesis and other metabolic pathways. These results suggest that HaNAC3 may have a stress resistance role in H. ammodendron and has potential applications in plant molecular breeding.

Genome-Wide Identification of Strawberry C2H2-ZFP C1-2i Subclass and the Potential Function of FaZAT10 in Abiotic Stress.

C2H2-type zinc finger proteins (C2H2-ZFPs) play a key role in various plant biological processes and responses to environmental stresses. In Arabidopsisthaliana, C2H2-ZFP members with two zinc finger domains have been well-characterized in response to abiotic stresses. To date, the functions of these genes in strawberries are still uncharacterized. Here, 126 C2H2-ZFPs in cultivated strawberry were firstly identified using the recently sequenced Fragaria × ananassa genome. Among these C2H2-ZFPs, 46 members containing two zinc finger domains in cultivated strawberry were further identified as the C1-2i subclass. These genes were unevenly distributed on 21 chromosomes and classified into five groups according to the phylogenetic relationship, with similar physicochemical properties and motif compositions in the same group. Analyses of conserved domains and gene structures indicated the evolutionary conservation of the C1-2i subclass. A Ka/Ks analysis indicated that the C1-2i members were subjected to purifying selection during evolution. Furthermore, FaZAT10, a typical C2H2-ZFP, was isolated. FaZAT10 was expressed the highest in roots, and it was induced by drought, salt, low-temperature, ABA, and MeJA treatments. It was localized in the nucleus and showed no transactivation activity in yeast cells. Overall, these results provide useful information for enriching the analysis of the ZFPs gene family in strawberry, and they provide support for revealing the mechanism of FaZAT10 in the regulatory network of abiotic stress.

Genome-Wide Identification, Evolution and Expressional Analysis of OSCA Gene Family in Barley (Hordeum vulgare L.).

The hyperosmolality-gated calcium-permeable channel gene family (OSCA) is one kind of conserved osmosensors, playing a crucial role in maintaining ion and water homeostasis and protecting cellular stability from the damage of hypertonic stress. Although it has been systematically characterized in diverse plants, it is necessary to explore the role of the OSCA family in barley, especially its importance in regulating abiotic stress response. In this study, a total of 13 OSCA genes (HvOSCAs) were identified in barley through an in silico genome search method, which were clustered into 4 clades based on phylogenetic relationships with members in the same clade showing similar protein structures and conserved motif compositions. These HvOSCAs had many cis-regulatory elements related to various abiotic stress, such as MBS and ARE, indicating their potential roles in abiotic stress regulation. Furthermore, their expression patterns were systematically detected under diverse stresses using RNA-seq data and qRT-PCR methods. All of these 13 HvOSCAs were significantly induced by drought, cold, salt and ABA treatment, demonstrating their functions in osmotic regulation. Finally, the genetic variations of the HvOSCAs were investigated using the re-sequencing data, and their nucleotide diversity in wild barley and landrace populations were 0.4966 × 10-3 and 0.391 × 10-3, respectively, indicating that a genetic bottleneck has occurred in the OSCA family during the barley evolution process. This study evaluated the genomic organization, evolutionary relationship and genetic expression of the OSCA family in barley, which not only provides potential candidates for further functional genomic study, but also contributes to genetically improving stress tolerance in barley and other crops.

Cloning and in silico characterization of an abiotic stress-inducible U-box domain-containing protein gene GsPUB8 from Glycine soja

The ubiquitination pathway is involved in the posttranslational modification of cellular proteins. However, the role of E3 ubiquitin ligase family proteins under abiotic stress conditions remains unclear, particularly in soybean. The core objective of the current study was to isolate and functionally characterize the GsPUB8 protein gene from wild soybean (Glycine soja) by using a homologous cloning method to investigate its abiotic stress responses. The GsPUB8 is a 40,562 Da molecular weight protein with 373 amino acid residues. The sequence alignment revealed the presence of U-box domain while the phylogenetic analysis showed an abundance of PUB8 proteins in both monocot and dicot plants. Analysis of gene structure predicted the absence of introns along with the presence of one exon. Furthermore, the activity of the GsPUB8 protein was anticipated in the plasma membrane and its expression was persuaded with NaCl, ABA, PEG6000, and NaHCO3 treatments with considerably higher manifestation in roots than leaves although, expressed in both vegetative and reproductive parts of G. soja. GsPUB8 protein showed 54% and 32% sequence identity to U-box domain containing 8 and 12 proteins from Arabidopsis thaliana and Oryza sativa subsp. japonica, respectively. GsPUB8 exhibited relatively higher expression under saline and drought stress particularly in roots. Whereas, the 3D model of GsPUB8 protein was generated using the SWISS-MODEL. This study can be used to manipulate the GsPUB8 protein or GsPUB8 gene for transformation purposes and its functional characterization under abiotic stress conditions.

Genome-Wide Identification of C2H2 ZFPs and Functional Analysis of BRZAT12 under Low-Temperature Stress in Winter Rapeseed (Brassica rapa).

Zinc-finger protein (ZFP) transcription factors are among the largest families of transcription factors in plants. They participate in various biological processes such as apoptosis, autophagy, and stemness maintenance and play important roles in regulating plant growth and development and the response to stress. To elucidate the functions of ZFP genes in the low-temperature response of winter (Brassica rapa L.) B. rapa, this study identified 141 members of the C2H2 ZFP gene family from B. rapa, which are heterogeneously distributed on 10 chromosomes and have multiple cis-acting elements related to hormone regulation and abiotic stress of adversity. Most of the genes in this family contain only one CDS, and genes distributed in the same evolutionary branch share mostly the same motifs and are highly conserved in the evolution of cruciferous species. The genes were significantly upregulated in the roots and growth cones of ‘Longyou-7’, indicating that they play a role in the stress-response process of winter B. rapa. The expression level of the Bra002528 gene was higher in the strongly cold-resistant varieties than in the weakly cold-resistant varieties after low-temperature stress. The survival rate and BrZAT12 gene expression of trans-BrZAT12 Arabidopsis thaliana (Arabidopsis) were significantly higher than those of the wild-type plants at low temperature, and the enzyme activities in vivo were higher than those of the wild-type plants, indicating that the BrZAT12 gene could improve the cold resistance of winter B. rapa. BrZAT12 expression and superoxide dismutase and ascorbate peroxidase enzyme activities were upregulated in winter B. rapa after exogenous ABA treatment. BrZAT12 expression and enzyme activities decreased after the PD98059 treatment, and BrZAT12 expression and enzyme activities were higher than in the PD98059 treatment but lower than in the control after both treatments together. It is speculated that BrZAT12 plays a role in the ABA signaling process in which MAPKK is involved. This study provides a theoretical basis for the resolution of cold-resistance mechanisms in strong winter B. rapa.

Trihelix Transcriptional Factor GhGT26 of Cotton Enhances Salinity Tolerance in Arabidopsis.

Cotton (Gossypium hirsutum L.), the most important textile crop worldwide, often encounters abiotic stress during its growing season and its productivity is significantly limited by adverse factors. Trihelix transcription factors (also known as GT factors) are important proteins involved in the morphological development and responses to abiotic stress in plants. However, their functions and molecular mechanisms in the cotton toward abiotic stress response remain unclear. In this study, a member (GhGT26) of the cotton Trihelix family was functionally characterized in the model plant Arabidopsis. This protein containing a SANT domain belongs to the GT-1 subgroup of trihelix proteins. GhGT26 was widely expressed in tissues (with the highest level in flower) and responded to high salt and ABA treatments at the transcriptional level. Using the Arabidopsis protoplast assay system, we found that the GhGT26 protein was located in the cell nuclei. The EMSA assay revealed that the GhGT26 protein could bind to the Site1-type GT cis elements (GT-3a) and MYB elements MRE3 and MRE4. The overexpression of GhGT26 improved plant tolerance to salt stress in transgenic Arabidopsis plants. Although ABA inhibits root elongation, the statistical analysis revealed that the root lengths of GhGT26-overexpressing Arabidopsis were the same as the wild plants after ABA treatment. Our results demonstrate that GhGT26 positively regulates salt stress via ABA-independent pathways. This evidence suggests that the GhGT26 may participate in the regulation of stress tolerance in cotton.

The IbPYL8-IbbHLH66-IbbHLH118 complex mediates the abscisic acid-dependent drought response in sweet potato.

Drought limits crop development and yields. bHLH (basic helix-loop-helix) transcription factors play critical roles in regulating the drought response in many plants, but their roles in this process in sweet potato are unknown. Here, we report that two bHLH proteins, IbbHLH118 and IbbHLH66, play opposite roles in the ABA-mediated drought response in sweet potato. ABA treatment repressed IbbHLH118 expression but induced IbbHLH66 expression in the drought-tolerant sweet potato line Xushu55-2. Overexpressing IbbHLH118 reduced drought tolerance, whereas overexpressing IbbHLH66 enhanced drought tolerance, in sweet potato. IbbHLH118 directly binds to the E-boxes in the promoters of ABA-insensitive 5 (IbABI5), ABA-responsive element binding factor 2 (IbABF2) and tonoplast intrinsic protein 1 (IbTIP1) to suppress their transcription. IbbHLH118 forms homodimers with itself or heterodimers with IbbHLH66. Both of the IbbHLHs interact with the ABA receptor IbPYL8. ABA accumulates under drought stress, promoting the formation of the IbPYL8-IbbHLH66-IbbHLH118 complex. This complex interferes with IbbHLH118's repression of ABA-responsive genes, thereby activating ABA responses and enhancing drought tolerance. These findings shed light on the role of the IbPYL8-IbbHLH66-IbbHLH118 complex in the ABA-dependent drought response of sweet potato and identify candidate genes for developing elite crop varieties with enhanced drought tolerance.

Transcriptome and functional analyses reveal ERF053 from Medicago falcata as key regulator in drought resistances.

Medicago falcata L. is an important legume forage grass with strong drought resistant, which could be utilized as an important gene pool in molecular breed of forage grass. In this study, M. falcata seedlings were treated with 400 mM mannitol to simulate drought stress, and the morphological and physiological changes were investigated, as well as the transcriptome changes of M. falcata seedlings at different treatment time points (0 h, 2 h, 6 h, 12 h, 24 h, 36 h and 48 h). Transcriptome analyses revealed four modules were closely related with drought response in M. falcata by WGCNA analysis, and four ERF transcription factor genes related with drought stress were identified (MfERF053, MfERF9, MfERF034 and MfRAP2.1). Among them, MfERF053 was highly expressed in roots, and MfERF053 protein showed transcriptional activation activity by transient expression in tobacco leaves. Overexpression of MfERF053 in Arabidopsis improved root growth, number of lateral roots and fresh weight under drought, salt stress and exogenous ABA treatments. Transgenic Arabidopsis over-expressing MfERF053 gene grew significantly better than the wild type under both drought stress and salt stress when grown in soil. Taken together, our strategy with transcriptome combined WGCNA analyses identified key transcription factor genes from M. falcata, and the selected MfERF053 gene was verified to be able to enhance drought and salt resistance when over-expressed in Arabidopsis.

Cloning, Subcellular Location and Expression Analysis of Grape Myb Gene

MYB gene plays an important role in plant growth, development and response to abiotic stress. In the present study, ‘Yatomi Rosa’ grape (Vitis vinifera L.) was used as the test material. Two MYB, VvMYBB1 and VvMYBA3 gene were obtained by homologous cloning, and their subcellular location, different organs, and expression patterns under stress treatment and hormone induction were obtained. VvMYBB1 and VvMYBA3 proteins were located in the nucleus and belonged to nuclear proteins. They were highly expressed in roots and flowers. These genes might have a negative effect on the formation of peel color. VvMYB1 gene and VvMYBA3 gene might play an important role in drought resistance and salt stress resistance. Under the induction of exogenous hormones, relative expression of VvMYBB1 gene was higher than that of VvMYBA3 gene under IAA, ETH, SA and MeJA treatment. Relative expression of VvMYBB1 gene was lower than that of VvMYBA3 gene under 6-BA, GA3 and ABA treatment. It showed that MYB gene played an important role in the development of different organs of grapes, fruit color, and response to abiotic stress and exogenous hormones induction in grape. Bangladesh J. Bot. 51(3): 499-506, 2022 (September)

Comprehensive genome-wide analysis of polyamine and ethylene pathway genes in Cleistogenes songorica and CsSAMDC2 function in response to abiotic stress

Polyamine and ethylene pathway genes (PEGs) are widely involved in regulating plant abiotic stresses. However, the PEGs in Cleistogenes songorica have not yet been thoroughly studied. In the present study, 17 polyamine-, 12 ethylene- and 6 S-adenosylmethionine (SAM)- related genes were identified at the genome-wide level in C. songorica . Phylogenetic analysis showed that genes in the ethylene and polyamine pathways and SAM family genes were not clearly separated. Promoter regions of these genes were rich in stress-related cis -regulatory elements. Several PEG and SAM genes coexpressed together and were responsive to abiotic stress treatment. In particular, qRT-PCR analysis showed that the expression of CsSAMDC2 increased under salt, dehydration and ABA treatment conditions. Arabidopsis plants transformed with CsSAMDC2 driven by a stress-inducible RD29A or constitutive promoter 35S exhibited enhanced dehydration tolerance and maintained higher chlorophyll content and photosynthetic capacity. The expression of CsSAMDC2 was generally higher in lines harboring RD29A::CsSAMDC2 compared to 35 S::CsSAMDC2 lines under drought stress treatment. Seed germination of RD29A::CsSAMDC2 or 35 S::CsSAMDC2 transgenic plants under salt stress treatment was superior to their wild-type counterparts, and the leaves of the transgenic plants exhibited a more compact structure. In addition, under salt stress, application of ABA improved lateral root development of RD29A::CsSAMDC2 Arabidopsis plants. The qRT-PCR analysis indicated that the expression of well-known abiotic stress-responsive genes AtPEGs, AtERF1 and AtRD29A, increased in both RD29A::CsSAMDC2 and 35 S::CsSAMDC2 transgenic Arabidopsis after drought, salt stress and salt stress plus ABA treatments. Our results suggest that CsSAMDC2 enhances drought and salt tolerance in Arabidopsis and therefore may contribute to abiotic stress tolerance in forage and food crops. • Genome wide analysis of ethylene and polyamine pathway and SAM family genes in C. songorica. • Evolutionary and functional similarities between ethylene and polyamine pathway genes. • CsSAMDC2 driven by RD29A promoter improved drought and salt tolerance of transgenic Arabidopsis. • Application of ABA on transgenic lines under salt stress further improved the expression of polyamine and ethylene pathway genes.

Identification and expression analysis of the grape pentatricopeptide repeat (PPR) gene family in abiotic stress.

The pentatricopeptide repeat (PPR) is one of the largest gene family in plants, and play important role in regulating plant growth, development and abiotic stress response. However, PPR genes have been poorly studied in grapes. In this study, based on the grape genome database, bioinformatics methods and quantitative real-time PCR (qRT-PCR) were used to identify the VvPPR family and the response to abiotic stress. A total of 181 PPR genes were identified in grape and divided into two subfamilies. Subcellular localization predicted that this gene family mainly functions in chloroplasts, nucleus, and mitochondria. Protein-protein interaction prediction indicated that there may be interaction between VvPPR44,53 and VvPPR44. The promoter region of VvPPR gene family contained various cis-acting elements, which were related to light and hormone. Expression pattern analysis showed that the VvPPR gene family was highly expressed in grape leaves, buds and carpel tissues. qRT-PCR results showed that the expression of VvPPR genes in roots was higher than stems and leaves under NAA, SA, ABA, MeJA and GA3 treatments. VvPPR8 was significantly up-regulated after GA3 and MeJA treatment for 24h, VvPPR53 was significantly up-regulated after SA, NAA, ABA and MeJA treatment. In addition, In grape leaves, VvPPR53 was up-regulated under PEG, Nacl and 4℃ treatments. These data indicate that VvPPR gene family members are responsive to hormones and abiotic stresses, and that there are some differences in the degree of response and expression in different grape tissues. This study provides a certain theoretical basis for grape resistance breeding.

Systematic analysis of the pectin methylesterase gene family in Nicotiana tabacum and reveal their multiple roles in plant development and abiotic stresses.

The pectin methylesterases (PMEs) play multiple roles in regulating plant development and responses to various stresses. In our study, a total of 121 PME genes were identified in the tobacco genome, which were clustered into two groups based on phylogenetic analysis together with Arabidopsis members. The investigations of gene structure and conserved motif indicated that exon/intron and motif organizations were relatively conserved in each group. Additionally, several stress-related elements were identified in the promoter region of these genes. The survey of duplication events revealed that segmental duplications were critical to the expansion of the PME gene family in tobacco. The expression profiles analysis revealed that these genes were expressed in various tissues and could be induced by diverse abiotic stresses. Notably, NtPME029 and NtPME043, were identified as homologues with AtPME3 and AtPME31, respectively. Furthermore, NtPME029 was highly expressed in roots and the over-expression of the NtPME029 gene could promote the development of roots. While NtPME043 could be induced by salt and ABA treatments, and the over-expression of the NtPME043 gene could significantly enhance the salt-stress tolerance in tobacco. Overall, these findings may shed light on the biological and functional characterization of NtPME genes in tobacco.

Effects of cytokinin and abscisic acid on heat resistance of Vetiveria zizanioides

Vetiveria zizanioides is ideal in maintaining soil and water, and is widely used for remediation of soil contaminated by heavy metals. However, it is affected by high-temperature stress. In this study, Vetiveria zizanioides plants were sprayed with 6-BA and ABA in a growth chamber 1 d before heat stress treatment, then the plants were subjected to high-temperature conditions. Relative water content, relative electrical conductivity, contents of ascorbic acid (AsA) and reduced glutathione (GSH) as the antioxidative substances, and content of malondiadehyde (MDA) were determined. Also, the antioxidative enzyme activities and the osmoprotectants levels were detected. Diaminobenzidine (DAB) staining of leaves and roots in Vetiveria zizanioides was observed for determination of hydrogen peroxide accumulation. The results showed that relative water content was decreased, relative electrical conductivity and MDA content were increased by the heat stress treatment. Under high-temperature conditions, relative water content was increased and relative electrical conductivity was decreased by 6-BA and ABA treatments. At the middle and the late stages of the heat stress treatment, activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and the contents of ASA and GSH in leaves of the 6-BA-treated and ABA-treated plants were significantly higher, whereas the MDA content was significantly lower than those in the high-temperature controls. Hydrogen peroxide accumulation levels in the 6-BA-treated and ABA-treated leaves and roots were lower than in the high-temperature controls. Contents of soluble sugar, sucrose, and glucose in the 6-BA-treated and ABA-treated leaves were significantly higher than those in the high-temperature controls. Proline content in the 6-BA-treated and ABA-treated leaves was stable. The results suggested that the enhancement of the heat resistance by 6-BA and ABA treatments was correlated with the activation of the antioxidant system, as well as the sugar-based osmoprotectant.

The OsWRKY63-OsWRKY76-OsDREB1B module regulates chilling tolerance in rice.

Rice (Oryza sativa) is sensitive to low temperatures, which affects the yield and quality of rice. Therefore, uncovering the molecular mechanisms behind chilling tolerance is a critical task for improving cold tolerance in rice cultivars. Here, we report that OsWRKY63, a WRKY transcription factor with an unknown function, negatively regulates chilling tolerance in rice. OsWRKY63-overexpressing rice lines are more sensitive to cold stress. Conversely, OsWRKY63-knockout mutants generated using a CRISPR/Cas9 genome editing approach exhibited increased chilling tolerance. OsWRKY63 was expressed in all rice tissues, and OsWRKY63 expression was induced under cold stress, dehydration stress, high salinity stress, and ABA treatment. OsWRKY63 localized in the nucleus plays a role as a transcription repressor and downregulates many cold stress-related genes and reactive oxygen species scavenging-related genes. Molecular, biochemical, and genetic assays showed that OsWRKY76 is a direct target gene of OsWRKY63 and that its expression is suppressed by OsWRKY63. OsWRKY76-knockout lines had dramatically decreased cold tolerance, and the cold-induced expression of five OsDREB1 genes was repressed. OsWRKY76 interacted with OsbHLH148, transactivating the expression of OsDREB1B to enhance chilling tolerance in rice. Thus, our study suggests that OsWRKY63 negatively regulates chilling tolerance through the OsWRKY63-OsWRKY76-OsDREB1B transcriptional regulatory cascade in rice.

Genome-wide identification of calcineurin B-like protein-interacting protein kinase gene family reveals members participating in abiotic stress in the ornamental woody plant Lagerstroemia indica.

Calcineurin B-like protein-interacting protein kinases (CIPKs) play important roles in plant responses to stress. However, their function in the ornamental woody plant Lagerstroemia indica is remains unclear. In this study, the LiCIPK gene family was analyzed at the whole genome level. A total of 37 LiCIPKs, distributed across 17 chromosomes, were identified. Conserved motif analysis indicated that all LiCIPKs possess a protein kinase motif (S_TKc) and C-terminal regulatory motif (NAF), while seven LiCIPKs lack a protein phosphatase interaction (PPI) motif. 3D structure analysis further revealed that the N-terminal and C-terminal 3D-structure of 27 members are situated near to each other, while 4 members have a looser structure, and 6 members lack intact structures. The intra- and interspecies collinearity analysis, synonymous substitution rate (Ks) peaks of duplicated LiCIPKs, revealed that ∼80% of LiCIPKs were retained by the two whole genome duplication (WGD) events that occurred approximately 56.12–61.16 million year ago (MYA) and 16.24–26.34 MYA ago. The promoter of each LiCIPK contains a number of auxin, abscisic acid, gibberellic acid, salicylic acid, and drought, anaerobic, defense, stress, and wound responsive cis-elements. Of the 21 members that were successfully amplified by qPCR, 18 LiCIPKs exhibited different expression patterns under NaCl, mannitol, PEG8000, and ABA treatments. Given that LiCIPK30, the AtSOS2 ortholog, responded to all four types of stress it was selected for functional verification. LiCIPK30 complements the atsos2 phenotype in vivo. 35S:LiCIPK-overexpressing lines exhibit increased leaf area increment, chlorophyll a and b content, reactive oxygen species scavenging enzyme activity, and expression of ABF3 and RD22, while the degree of membrane lipid oxidation decreases under NaCl treatment compared to WT. The evolutionary history, and potential mechanism by which LiCIPK30 may regulate plant tolerance to salt stress were also discussed. In summary, we identified LiCIPK members involved in abiotic stress and found that LiCIPK30 transgenic Arabidopsis exhibits more salt and osmotic stress tolerance than WT. This research provides a theoretical foundation for further investigation into the function of LiCIPKs, and for mining gene resources to facilitate the cultivation and breeding of new L. indica varieties in coastal saline-alkali soil.

LcNAC90 transcription factor regulates biosynthesis of anthocyanin in harvested litchi in response to ABA and GA3

The antagonism between ABA and GA in regulating accumulation of anthocyanin has been well documented. Lately we showed both ABA and GA3 alleviated litchi browning, but the underlying molecular mechanism remains unclear. Here we showed 6-d storage caused obvious browning in litchi, but ABA and GA3 increased anthocyanin and reduced color fading relative to the control. LC-MS analysis identified 21 anthocyanins from litchi, of which seven were upregulated and 14 downregulated for ABA treatment after storage relative to before, and four upregulated and 17 downregulated for GA3 treatment. Among the upregulated anthocyanins were three shared by ABA and GA3 treatments. RNA-seq revealed that ABA and GA3 upregulated genes for 7 anthocyanin biosynthetic enzymes, including LcPAL and LcCHS, whose expression patterns were consistent with that of LcNAC90. Moreover, ABA and GA3 activated transcription of LcNAC90 transiently expressed in tobacco, verifying the responsiveness of LcNAC90 to ABA and GA3. The regulatory network analysis suggested that LcPAL and LcCHS transcripts were highly correlated with LcNAC90. Sequence analysis revealed NAC binding sites in promoters of LcPAL and LcCHS. Biochemical evidences showed LcNAC90 specifically bound to the promoters and activated transcription of LcPAL and LcCHS. LcNAC90 overexpression in Arabidopsis increased leaf pigmentation and upregulated anthocyanin biosynthetic genes in Arabidopsis, e.g. AtPAL and AtCHS. Collectively, this work suggests that ABA and GA3 activate LcNAC90 to enhance anthocyanin biosynthesis in litchi during storage. The findings provide novel insight into mechanism underlying anthocyanin accumulation regulated by ABA and GA.

Transcriptome-wide identification of walnut PP2C family genes in response to external stimulus

Walnut is an important economic tree species while confronting with global environmental stress, resulting in decline in quality and yield. Therefore, it is urgent to elucidate the molecular mechanism for the regulation of walnut response to adversity. The protein phosphatase 2C (PP2C) gene family participates in cellular processes in eukaryotes through reversible phosphorylation of proteins and signal transduction regulation. However, the stress response function of PP2C genes was far to be clarified. Therefore, to understand the stress response mechanism of walnut tree, in this study, a total of 41 PP2C genes with complete ORFs were identified from Juglans regia, whose basic bio-information and expression patterns in response to multiple stresses and ABA were confirmed. The results showed that the ORFs of JrPP2Cs were 495 ~ 3231 bp in length, the predicted JrPP2C proteins contained 164 to 1076 amino acids and the molecular weights were 18,581.96 ~ 118,853.34 Da, the pI was 4.55 ~ 9.58. These JrPP2C genes were unevenly distributed on 14 chromosomes, among which Chr11 and Chr13 contained the most genes. Phylogenetic analysis found that these JrPP2C proteins were classed into 9 subfamilies, among which group F covered most JrPP2Cs. The JrPP2Cs in the same subfamily exhibited similarities in the composition of conserved domains, amino acid sequences of motifs and exon/intron organization in DNA sequences. Each JrPP2C includes 4 ~ 10 motifs and each motif contained 15 ~ 37 amino acids. Among the motifs, motif1, motif2, motif3 and motif8 were most abundant. Most of the JrPP2C genes diversely response to osmotic, cadmium, and Colletotrichum gloeosporioide stress as well as ABA treatments, among which JrPP2C28, JrPP2C17, JrPP2C09, JrPP2C36 were more obvious and deserves further attention. All these results indicated that JrPP2C genes play potential vital roles in plant response to multiple stimulus, and are possibly involved in ABA-dependent signaling pathway.

Characterization of walnut JrWOX11 and its overexpression provide insights into adventitious root formation and development and abiotic stress tolerance.

The well-developed root system enables plant survival under various environmental stresses. WUSCHEL-RELATED HOMEOBOX GENE 11 (WOX11) plays a critical role in adventitious root formation and development in rice, Arabidopsis, and easy-to-root tree poplar. However, in difficult-to-root trees, the knowledge of WOX11 during adventitious root formation and development remains scarce. In this study, the JrWOX11 gene was isolated from a difficult-to-root tree walnut and heterologously expressed in the “84K” poplar. The results showed that JrWOX11 contained a similar structure and sequence to the homologous genes in rice, Arabidopsis, and poplar, but had different numbers and types of motifs and cis-elements. JrWOX11 lacked the motif GGAIQY compared to that in easy-to-root trees. In addition, JrWOX11 expression was induced by ABA, PEG, and NaCl treatments. Overexpression of JrWOX11 in poplar promoted root initiation and significantly increased adventitious root (ARs) number, lateral roots (LRs) number, and root hair (RH) length. Furthermore, the aboveground biomass was notably increased under NaCl and PEG treatments in transgenic plants. When NaCl and PEG were removed, the survival rate, aerial shoot development, and de novo root organogenesis were also markedly enhanced in transgenic shoot cuttings. The study provides valuable information on the differences between JrWOX11 and the homologous genes in rice, Arabidopsis, and poplar, and supports the critical role of JrWOX11 in the formation of AR and tolerance to salt and osmotic stresses.

Comprehensive Analysis of GRAS Gene Family and Their Expression under GA3, Drought Stress and ABA Treatment in Larix kaempferi

The GRAS family transcription factors play important roles in regulating plant growth and responses to abiotic stress, which can be utilized to breed novel plants with improved abiotic stress resistance. However, the GRAS gene family has been largely unexplored for tree species, particularly for Larix kaempferi, which has high economic and ecological values, challenging practices for breeding abiotic stress-resistant L. kaempferi. In order to improve the stress resistance by regulating the transcription factors in L. kaempferi, we identified 11 GRAS genes in L. kaempferi and preliminarily characterized them through comprehensive analyses of phylogenetic relationships, conserved motifs, promoter cis-elements, and expression patterns, as well as protein interaction network prediction. The phylogenetic analysis showed that the LkGRAS family proteins were classified into four subfamilies, including DELLA, HAM, SCL, and PAT1, among which the SCL subfamily was the largest one. Conserved motif analysis revealed many putative motifs such as LHRI-VHIID-LHRII-PFYRE-SAW at C-terminals of the LkGRAS proteins; we discovered a unique motif of the LkGRAS genes. Promoter cis-acting element analysis exhibited several putative elements associated with abiotic stresses and phytohormones; the abscisic acid-responsive elements (ABRE) and G-box are the most enriched elements in the promoters. Through expression profiles of LkGRAS genes in different tissues and under drought-stress and phytohormones (GA3 and ABA) treatments, it was demonstrated that LkGRAS genes are most active in the needles, and they rapidly respond to environmental cues such as drought-stress and phytohormone treatments within 24 h. Protein interaction network prediction analysis revealed that LkGRAS proteins interact with various proteins, among which examples are the typical GA, ABA, and drought-stress signaling factors. Taken together, our work identifies the novel LkGRAS gene family in L. kaempferi and provides preliminary information for further in-depth functional characterization studies and practices of breeding stress-resistant L. kaempferi.