The U-Box E3 Ubiquitin Ligase PUB35 Negatively Regulates ABA Signaling through AFP1-mediated Degradation of ABI5.

Abscisic acid (ABA) signaling is crucial for plant responses to various abiotic stresses. The Arabidopsis (Arabidopsis thaliana) transcription factor ABA INSENSITIVE 5 (ABI5) is a central regulator of ABA signaling. ABI5 BINDING PROTEIN 1 (AFP1) interacts with ABI5 and facilitates its 26S-proteasome-mediated degradation, although the detailed mechanism has remained unclear. Here, we report that an ABA-responsive U-box E3 ubiquitin ligase, PLANT U-BOX 35 (PUB35), physically interacts with AFP1 and ABI5. PUB35 directly ubiquitinated ABI5 in a bacterially reconstituted ubiquitination system and promoted ABI5 protein degradation in vivo. ABI5 degradation was enhanced by AFP1 in response to ABA treatment. Phosphorylation of the T201 and T206 residues in ABI5 disrupted the ABI5-AFP1 interaction and affected the ABI5-PUB35 interaction and PUB35-mediated degradation of ABI5 in vivo. Genetic analysis of seed germination and seedling growth showed that pub35 mutants were hypersensitive to ABA as well as to salinity and osmotic stresses, whereas PUB35 overexpression lines were hyposensitive. Moreover, abi5 was epistatic to pub35, whereas the pub35-2 afp1-1 double mutant showed a similar ABA response to the two single mutants. Together, our results reveal a PUB35-AFP1 module involved in fine-tuning ABA signaling through ubiquitination and 26S-proteasome-mediated degradation of ABI5 during seed germination and seedling growth.

Genome-wide identification and expression analysis of aquaporin family in Canavalia rosea and their roles in the adaptation to saline-alkaline soils and drought stress.

BACKGROUND: Canavalia rosea (Sw.) DC. (bay bean) is an extremophile halophyte that is widely distributed in coastal areas of the tropics and subtropics. Seawater and drought tolerance in this species may be facilitated by aquaporins (AQPs), channel proteins that transport water and small molecules across cell membranes and thereby maintain cellular water homeostasis in the face of abiotic stress. In C. rosea, AQP diversity, protein features, and their biological functions are still largely unknown. RESULTS: We describe the action of AQPs in C. rosea using evolutionary analyses coupled with promoter and expression analyses. A total of 37 AQPs were identified in the C. rosea genome and classified into five subgroups: 11 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, 11 Nod26-like intrinsic proteins, 4 small and basic intrinsic proteins, and 1 X-intrinsic protein. Analysis of RNA-Seq data and targeted qPCR revealed organ-specific expression of aquaporin genes and the involvement of some AQP members in adaptation of C. rosea to extreme coral reef environments. We also analyzed C. rosea sequences for phylogeny reconstruction, protein modeling, cellular localizations, and promoter analysis. Furthermore, one of PIP1 gene, CrPIP1;5, was identified as functional using a yeast expression system and transgenic overexpression in Arabidopsis. CONCLUSIONS: Our results indicate that AQPs play an important role in C. rosea responses to saline-alkaline soils and drought stress. These findings not only increase our understanding of the role AQPs play in mediating C. rosea adaptation to extreme environments, but also improve our knowledge of plant aquaporin evolution more generally.

Ectopic Expression of CrPIP2;3, a Plasma Membrane Intrinsic Protein Gene from the Halophyte Canavalia rosea, Enhances Drought and Salt-Alkali Stress Tolerance in Arabidopsis.

Aquaporins are channel proteins that facilitate the transmembrane transport of water and other small neutral molecules, thereby playing vital roles in maintaining water and nutrition homeostasis in the life activities of all organisms. Canavalia rosea, a seashore and mangrove-accompanied halophyte with strong adaptability to adversity in tropical and subtropical regions, is a good model for studying the molecular mechanisms underlying extreme saline-alkaline and drought stress tolerance in leguminous plants. In this study, a PIP2 gene (CrPIP2;3) was cloned from C. rosea, and its expression patterns and physiological roles in yeast and Arabidopsis thaliana heterologous expression systems under high salt-alkali and high osmotic stress conditions were examined. The expression of CrPIP2;3 at the transcriptional level in C. rosea was affected by high salinity and alkali, high osmotic stress, and abscisic acid treatment. In yeast, the expression of CrPIP2;3 enhanced salt/osmotic and oxidative sensitivity under high salt/osmotic and H2O2 stress. The overexpression of CrPIP2;3 in A. thaliana could enhance the survival and recovery of transgenic plants under drought stress, and the seed germination and seedling growth of the CrPIP2;3 OX (over-expression) lines showed slightly stronger tolerance to high salt/alkali than the wild-type. The transgenic plants also showed a higher response level to high-salinity and dehydration than the wild-type, mostly based on the up-regulated expression of salt/dehydration marker genes in A. thaliana plants. The reactive oxygen species (ROS) staining results indicated that the transgenic lines did not possess stronger ROS scavenging ability and stress tolerance than the wild-type under multiple stresses. The results confirmed that CrPIP2;3 is involved in the response of C. rosea to salt and drought, and primarily acts by mediating water homeostasis rather than by acting as an ROS transporter, thereby influencing physiological processes under various abiotic stresses in plants.

FABP7 is a potential biomarker to predict response to neoadjuvant chemotherapy for breast cancer.

BACKGROUND: Early prediction of response to neoadjuvant chemotherapy (NAC) is critical in choosing appropriate chemotherapeutic regimen for patients with locally advanced breast cancer. Herein, we sought to identify potential biomarkers to predict the response to neoadjuvant chemotherapy for breast cancer patients. METHODS: Three genomic profiles acquired by microarray analysis from subjects with or without residual tumors after NAC downloaded from the GEO database were used to screen the differentially expressed genes (DEGs). An array of public databases, including ONCOMINE, cBioportal, Breast Cancer Gene Expression Miner v4.0, and the Kaplan Meir-plotter, etc., were used to evaluate the potential functions, related signaling pathway, as well as prognostic values of FABP7 in breast cancer. Anti-cancer drug sensitivity assay, real-time PCR, flow cytometry and western-blotting assays were used to investigate the function of FABP7 in breast cancer cells and examine the relevant mechanism. RESULTS: Two differentially expressed genes, including FABP7 and ESR1, were identified to be potential indicators of response to anthracycline and taxanes for breast cancer. FABP7 was associated with better chemotherapeutic response, while ESR1 was associated with poorer chemotherapeutic effectiveness. Generally, the expression of FABP7 was significantly lower in breast cancer than normal tissue samples. FABP7 mainly high expressed in ER-negative breast tumor and might regulate cell cycle to enhance chemosensitivity. Moreover, elevated FABP7 expression increased the percentage of cells at both S and G2/M phase in MDA-MB-231-ADR cells, and decreased the percentage of cells at G0/G1 phase, as compared to control group. Western-blotting results showed that elevated FABP7 expression could increase Skp2 expression, while decrease Cdh1 and p27kip1 expression in MDA-MB-231-ADR cells. In addition, FABP7 was correlated to longer recurrence-free survival (RFS) in BC patients with ER-negative subtype of BC treated with chemotherapy. CONCLUSION: FABP7 is a potential favorable biomarker and predicts better response to NAC in breast cancer patients. Future study on the predictive value and detail molecular mechanisms of FABP7 in contribution to chemosensitivity in breast cancer is warranted.

Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae.

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

Ipomoea pes-caprae IpASR Improves Salinity and Drought Tolerance in Transgenic Escherichia coli and Arabidopsis.

Ipomoea pes-caprae L. is an extremophile halophyte with strong adaptability to seawater and drought. It is widely used in the ecological restoration of coastal areas or degraded islands in tropical and subtropical regions. In this study, a new abscisic acid, stressandripening (ASR) gene, IpASR, was reported, and is mainly associated with biological functions involved in salt and drought tolerance. Sequence analysis of IpASR showed that this protein contains an ABA/WDS (abscisic acid/water deficit stress) domain, which is a common feature of all plant ASR members. Overexpression of IpASR improved Escherichia coli growth performance compared with the control under abiotic stress treatment. The transgenic overexpressing IpASR Arabidopsis showed higher tolerance to salt and drought stress than the wild type and lower accumulation of hydrogen peroxide (H2O2) and superoxide (O2-) accompanied by increased antioxidant enzyme activity in vivo. IpASR exhibits transcription factor's activity. Therefore, the overexpression of IpASR in Arabidopsis is supposed to influence the expression of some genes involved in anti-oxidative and abiotic stresses. The results indicate that IpASR is involved in the plant response to salt and drought and probably acts as a reactive oxygen species scavenger or transcription factor, and therefore influences physiological processes associated with various abiotic stresses in plants.

Characterization of a phytochelatin synthase gene from Ipomoea pes-caprae involved in cadmium tolerance and accumulation in yeast and plants.

Phytochelatin synthases (PCSs) play a crucial part in heavy metal tolerance in plants via the synthesis of phytochelatins (PCs), which can chelate heavy metals (HMs) in the vacuole and decrease cell damage. Plant PCSs are commonly designated as key genes for phytoremediation. In this study, we identified a PCS gene (IpPCS1) from Ipomoea pes-caprae and investigated its role in regulating cadmium (Cd) tolerance and accumulation. The expression of a truncated IpPCS1t in yeast could complement the Cd-sensitive phenotype of the ycf1Δ mutant strain, as well as improve the Cd tolerance of the wild-type yeast strain, while promoting Cd accumulation in the yeast cells. The expression of IpPCS1 was induced in I. pes-caprae plants under Cd treatment. Compared with IpPCS1, the lack of a C-terminal in IpPCS1t did not affect its Cd tolerance, but might restrict the zinc (Zn) detoxification in yeast. The overexpression of IpPCS1t in Arabidopsis could improve the Cd tolerance slightly and had little impact on Cd accumulation in transgenic plant. Our results indicated that IpPCS1 has certain potential application value in Cd tolerance and detoxification, therefore provides a useful genetic resource for enhancing Cd tolerance and improving the Cd phytoremediation capacity of plants or organisms. In addition, our research is the first time to discover a new possible Cd activation site in the C-terminal of IpPCS1.

Isolation and characterization of an atypical LEA gene (IpLEA) from Ipomoea pes-caprae conferring salt/drought and oxidative stress tolerance.

Late embryogenesis abundant (LEA) proteins belong to a large family that exists widely in plants and is mainly involved in desiccation processes during plant development or in the response to abiotic stresses. Here, we reported on an atypical LEA gene (IpLEA) related to salt tolerance from Ipomoea pes-caprae L. (Convolvulaceae). Sequence analysis revealed that IpLEA belongs to the LEA_2 (PF03168) group. IpLEA was shown to have a cytoplasmic localization pattern. Quantitative reverse transcription PCR analysis showed that IpLEA was widely expressed in different organs of the I. pes-caprae plants, and the expression levels increased following salt, osmotic, oxidative, freezing, and abscisic acid treatments. Analysis of the 1,495 bp promoter of IpLEA identified distinct cis-acting regulatory elements involved in abiotic stress. Induction of IpLEA improved Escherichia coli growth performance compared with the control under abiotic stresses. To further assess the function of IpLEA in plants, transgenic Arabidopsis plants overexpressing IpLEA were generated. The IpLEA-overexpressing Arabidopsis seedlings and adult plants showed higher tolerance to salt and drought stress than the wild-type. The transgenic plants also showed higher oxidative stress tolerance than the wild-type Arabidopsis. Furthermore, the expression patterns of a series of stress-responsive genes were affected. The results indicate that IpLEA is involved in the plant response to salt and drought, probably by mediating water homeostasis or by acting as a reactive oxygen species scavenger, thereby influencing physiological processes under various abiotic stresses in microorganisms and plants.

Molecular Cloning and Functional Characterization of the Dehydrin (IpDHN) Gene From Ipomoea pes-caprae.

Dehydrin (DHN) genes can be rapidly induced to offset water deficit stresses in plants. Here, we reported on a dehydrin gene (IpDHN) related to salt tolerance isolated from Ipomoea pes-caprae L. (Convolvulaceae). The IpDHN protein shares a relatively high homology with Arabidopsis dehydrin ERD14 (At1g76180). IpDHN was shown to have a cytoplasmic localization pattern. Quantitative RT-PCR analyses indicated that IpDHN was differentially expressed in most organs of I. pes-caprae plants, and its expression level increased after salt, osmotic stress, oxidative stress, cold stress and ABA treatments. Analysis of the 974-bp promoter of IpDHN identified distinct cis-acting regulatory elements, including an MYB binding site (MBS), ABRE (ABA responding)-elements, Skn-1 motif, and TC-rich repeats. The induced expression of IpDHN in Escherichia coli indicated that IpDHN might be involved in salt, drought, osmotic, and oxidative stresses. We also generated transgenic Arabidopsis lines that over-expressed IpDHN. The transgenic Arabidopsis plants showed a significant enhancement in tolerance to salt/drought stresses, as well as less accumulation of hydrogen peroxide (H2O2) and the superoxide radical (O2 -), accompanied by increasing activity of the antioxidant enzyme system in vivo. Under osmotic stresses, the overexpression of IpDHN in Arabidopsis can elevate the expression of ROS-related and stress-responsive genes and can improve the ROS-scavenging ability. Our results indicated that IpDHN is involved in cellular responses to salt and drought through a series of pleiotropic effects that are likely involved in ROS scavenging and therefore influence the physiological processes of microorganisms and plants exposed to many abiotic stresses.