ABI5 Expression Research Articles

Developmental and stress regulation on expression of a novel miRNA, Fan-miR73, and its target ABI5 in strawberry.

Abscisic acid (ABA) is a critical plant hormone for fruit ripening and adaptive stress responses in strawberry. Previous high-throughput sequencing results indicated that ABA-insensitive (ABI)5, an important transcription factor in the ABA signaling pathway, was a target for a novel microRNA (miRNA), Fan-miR73. In the present study, exogenous ABA treatment was found to accelerate fruit ripening through differentially regulating the transcripts of ABA metabolism and signal transduction related genes, including NCED1, PYR1, ABI1, and SnRK2.2. Expression of Fan-miR73 was down-regulated in response to exogenous ABA treatment in a dosage-dependent manner, which resulted in an accumulation of ABI5 transcripts in the ripening-accelerated fruits. In addition, both UV-B radiation and salinity stress reduced the transcript levels of Fan-miR73, whereas promoted ABI5 expression. Furthermore, high negative correlations between the transcriptional abundance of Fan-miR73 and ABI5 were observed during ripening and in response to stress stimuli. These results enriched the possible regulatory role of miRNA involved in the post-transcriptional modification of ABI5 during strawberry ripening, as well as responses to environmental stresses.

Transcriptional regulation of the potential tumor suppressor ABI3 gene in thyroid carcinomas: interplay between methylation and NKX2-1 availability.

We previously reported that ABI3 expression was decreased in thyroid cancer tissues and that ectopic expression of ABI3 in a follicular thyroid carcinoma cell line delayed cell cycle progression and inhibited cell proliferation, invasion, migration and tumor formation in athymic mice. These data indicated that ABI3 is a tumor suppressor gene; however the mechanism through which ABI3 is silenced in thyroid carcinomas is unknown. We here show that treatment of four follicular thyroid carcinoma cell lines with 5-aza-dC induced demethylation of a specific region of the ABI3 promoter and restored ABI3 expression. In contrast, 5-aza-dC treatment did not restore ABI3 expression in a non-thyroid cell line, suggesting a tissue-specific regulation. We additionally show that 8 CpG sites located within the ABI3 promoter are hypermethylated in most thyroid carcinoma samples and the degree of methylation correlated with ABI3 expression. Narrowing the region to specific CpG sites, the CpG4-6 sites showed the largest difference between benign and malignant lesions. In silico analysis revealed that these CpG sites flank a canonical binding site for NKX2-1, a thyroid specific transcriptional factor. Analysis of thyroid samples shows a correlation between NKX2-1 and ABI3 expression. In vitro assays demonstrate that NKX2-1 was required for ABI3 expression. Luciferase assay further confirmed the promoter activity of this region, which was increased when the cells were co-transfected with NKX2-1. Our study shows that the transcriptional silencing of ABI3 in cancer cells occurs via methylation and uncovered a previously unrecognized role for NKX2-1 in the regulation of ABI3.

Modulation of the Fragrance of Kam Sweet Rice by Jasmonic Acid and Abscisic Acid Might Involve the MYC2 and ABI5 Transcription Factors

Volatile compounds were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O). Results showed that nonanal had the highest odor activity value in the filling grains. Nonanal was enhanced by application of JA and inhibited by ABA in the seedlings of Kam sweet rice. C9-aldehydes are significant odorants in many rice types and known products of the 9-lipoxygenase and 9-hydroperoxide lyases encoded by the OsLOX3 and OsHPL1 genes, respectively. OsLOX3 mRNA expression was higher in aromatic rice compared to a nonaromatic cultivar. mRNA expression of ABI5 and MYC2 negatively correlated with OsLOX3 expression (r = −0.841, P < 0.01; and r = −0.512, P < 0.05). In conclusion, the transcription factors ABI5 and MYC2 might be involved in ABA and JA modulation of nonanal biosynthesis, which could be used to improve rice quality. Practical Applications Nonanal and 2-acetyl-1-pyrroline are important and potent flavor compounds in aromatic Kam sweet rice. The OsLOX3 gene was potentially identified as a critical gene in aromatic Kam sweet rice and the enhancing effect of JA on nonanal biosynthesis was confirmed. The transcription factors ABI5 and MYC2 seem to regulate the expression of the fragrance gene OsLOX3 while MYC2 act as a positive regulator involved in JA-mediated nonanal biosynthesis. ABI5 participate in the ABA signaling pathway to down-regulate the expression of OsLOX3. These results will help understand the pathways involved in the biosynthesis of rice fragrance, which could be used to improve the quality of rice.

Arabidopsis AtDjA3 Null Mutant Shows Increased Sensitivity to Abscisic Acid, Salt, and Osmotic Stress in Germination and Post-germination Stages.

DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid (ABA). The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher ABA sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signaling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 μM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance.

Arabidopsis WRKY6 Transcription Factor Acts as a Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development.

The phytohormone abscisic acid (ABA) plays important roles during seed germination and early seedling development. Here, we characterized the function of the Arabidopsis WRKY6 transcription factor in ABA signaling. The transcript of WRKY6 was repressed during seed germination and early seedling development, and induced by exogenous ABA. The wrky6-1 and wrky6-2 mutants were ABA insensitive, whereas WRKY6-overexpressing lines showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The expression of RAV1 was suppressed in the WRKY6-overexpressing lines and elevated in the wrky6 mutants, and the expression of ABI3, ABI4, and ABI5, which was directly down-regulated by RAV1, was enhanced in the WRKY6-overexpressing lines and repressed in the wrky6 mutants. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that WRKY6 could bind to the RAV1 promoter in vitro and in vivo. Overexpression of RAV1 in WRKY6-overexpressing lines abolished their ABA-hypersensitive phenotypes, and the rav1 wrky6-2 double mutant showed an ABA-hypersensitive phenotype, similar to rav1 mutant. Together, the results demonstrated that the Arabidopsis WRKY6 transcription factor played important roles in ABA signaling by directly down-regulating RAV1 expression.

The Arabidopsis DELAY OF GERMINATION 1 gene affects ABSCISIC ACID INSENSITIVE 5 (ABI5) expression and genetically interacts with ABI3 during Arabidopsis seed development.

The seed expressed gene DELAY OF GERMINATION (DOG) 1 is absolutely required for the induction of dormancy. Next to a non-dormant phenotype, the dog1-1 mutant is also characterized by a reduced seed longevity suggesting that DOG1 may affect additional seed processes as well. This aspect however, has been hardly studied and is poorly understood. To uncover additional roles of DOG1 in seeds we performed a detailed analysis of the dog1 mutant using both transcriptomics and metabolomics to investigate the molecular consequences of a dysfunctional DOG1 gene. Further, we used a genetic approach taking advantage of the weak aba insensitive (abi) 3-1 allele as a sensitized genetic background in a cross with dog1-1. DOG1 affects the expression of hundreds of genes including LATE EMBRYOGENESIS ABUNDANT and HEAT SHOCK PROTEIN genes which are affected by DOG1 partly via control of ABI5 expression. Furthermore, the content of a subset of primary metabolites, which normally accumulate during seed maturation, was found to be affected in the dog1-1 mutant. Surprisingly, the abi3-1 dog1-1 double mutant produced green seeds which are highly ABA insensitive, phenocopying severe abi3 mutants, indicating that dog1-1 acts as an enhancer of the weak abi3-1 allele and thus revealing a genetic interaction between both genes. Analysis of the dog1 and dog1 abi3 mutants revealed additional seed phenotypes and therefore we hypothesize that DOG1 function is not limited to dormancy but that it is required for multiple aspects of seed maturation, in part by interfering with ABA signalling components.

Cloning and characterization of ChiMYB in Chrysanthemum indicum with an emphasis on salinity stress tolerance.

v-myb avianmyeloblastosis viral oncogene homolog (MYB) transcription factors are key regulators of stress responsive gene expression in plants. In this study, the MYB gene, ChiMYB (GenBank accession No. KT948997), was isolated from Chrysanthemum indicum, and was functionally characterized with an emphasis on salinity stress tolerance. The full ChiMYB cDNA sequence (948 bp) encoded a typical R2R3 MYB transcription factor that contained 315 amino acid residues and two MYB domains. The temporal expression pattern of ChiMYB was noted in C. indicum, and the highest level was detected in the roots, followed by leaves and stems. ChiMYB expression was induced by NaCl treatments, and transient expression of the fusion of ChiMYB and green fluorescent protein (GFP) indicated that the protein was targeted to the nuclei of onion epidermal cells. Arabidopsis plants overexpressing ChiMYB displayed improved tolerance to drought and salt stress. When under salt stress conditions, transgenic Arabidopsis plants had higher survival rates than non-transgenic wild-type plants. Chlorophyll content, intercellular CO2 concentration, photosynthetic rate, and stomatal conductance were higher in the transgenic Arabidopsis plants than in non-transgenic control plants. Further investigation revealed that ChiMYB was able to regulate the expression of RD29A, RAB18, COR15, ABI1, and ABA genes, which are involved in salt stress signaling pathways. Our findings demonstrated that ChiMYB is essential for plant responses to salt stress, and it may have great potential for the improvement of salt tolerance in crops.

Downregulation of Mir-181a Restores Chemosensitivity to Imatinib Mesylate in Chronic Myeloid Leukemia Cells Resistant to Tyrosine Kinase Inhibitors

Abelson Interactor protein 1 (Abi-1) is a negative regulator of Abelson kinase and interacts with its oncogenic form Bcr-Abl1 (Brehme et al., PNAS 2009:106;7414). We have recently shown that Abi-1 also modulates interactions between chronic myeloid leukemia (CML) stem/progenitor cells and their microenvironment, and has a role in acquired drug resistance. Our data indicated that abnormally low levels of Abi-1 in CML cells correlate with increased expression of integrin α4, deregulation of Fak/Akt/Erk signaling, enhanced adhesive properties, quiescence, and resistance to imatinib mesylate (IM, Chorzalska et al., Leukemia 2014:28;2165). We have found miR-181a to be significantly upregulated in IM-resistant CML cells, and miRNA expression profiling identified miR-181a as a potential regulator of Abi-1 expression. Furthermore, a bioinformatics analysis matching the miR-181a seed region to conserved ABI1 sequences using TargetScan 6.2 (Lewis, Burge, & Bartel, Cell 2005;120.15) determined that a 3'-untranslated region of ABI1 (position 131-137) is a target for miR-181a. This suggested that miR-181a may regulate Abi-1 levels in IM-resistant CML cells. We hypothesized that decreasing the levels of miR-181a in those cells would restore the intracellular levels of Abi-1, and consequently reduce the expression of integrin α4, decrease the adhesion and quiescence, and restore their sensitivity to IM.To test this hypothesis, we designed and constructed a miRNA sponge (Figure 1) using the methodology established by Kluvier et al. (Methods, 2012:58;113). The sponge can endogenously sequester and functionally reduce levels of miR-181a in CML IM-resistant cells (K562-STI-R cell line), thus allowing to measure the effect of miR-181a loss on Abi-1, integrin α4 levels and chemoresistance. K562-STI-R CML cell line was electroporated with pcDNA3-SanDI vector encoding the miR-181a sponge or a control mismatched miRNA sponge.Using qPCR, we confirmed a 35% percent decrease in miR-181a transcript levels in IM-resistant K562-STI-R CML cell line expressing the design miR-181a sponge, compared with controls (p=0.0015). This was paralleled by a 38% increase in ABI1 mRNA levels (p=0.046) and about 40% increase in Abi-1 protein levels (p=0.03) as evaluated by immunoblotting followed by densitometry, indicating that functionally decreased intracellular levels of miR-181a resulted in increased expression of ABI1. Because we previously demonstrated that down-regulation of ABI1 increases expression of integrin α4 and adhesion of IM-resistant CML cells while decreasing their proliferative potential, we then evaluated ITGA4 transcript levels and expression of integrin α4 in CML cells transfected with the miR-181a sponge. We observed a 20% decrease in ITGA4 transcript and integrin α4 protein levels compared with controls (p=0.051). Phenotypic evaluation of the chemoresistant K562-STI-R cells expressing the miR-181a sponge showed a 36% (p=0.04) increase in the cell number. Decreased number of Annexin V-positive cells suggested that miR-181a sequestration had a modest inhibitory effect on apoptosis. We then evaluated chemosensitivity of the miR-181a sponge-expressing K562-STI-R cells by culturing them in increased concentrations of IM (10, 100 and 200 µM), and measuring apoptosis by Annexin V staining using flow cytometry. We observed 30% (p=0.01), 46% (p=0.023) and 68% (p=0.018) increase in the number of apoptotic cells in miR-181a sponge-expressing cells compared with control cultured at 10, 100 and 200 µM of IM, respectively.In summary, this study provides the first piece of evidence that miR-181a is an epigenetic regulator of ABI1, and that downregulation of miR-181a by a miRNA sponge can effectively increase the intracellular levels of Abi-1 in IM-resistant CML cells, consequently increasing their sensitivity to Bcr-Abl inhibition. Utilization of targeted miRNA inhibitors may represent a new approach to restore chemosensitivity of IM-resistant CML cells. [Display omitted] DisclosuresOlszewski:Bristol-Myers Squibb, Inc.: Consultancy; Genentech, Inc.: Research Funding.

Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana.

MiRNAs play crucial roles in many aspects of plant development and the response to the environment. The miR172 family has been shown to participate in the control of flowering time and the response to abiotic stress. This family regulates the expression of APETALA2 (AP2)-like transcription factors in Arabidopsis. In the present study, soybean (Glycine max L. Merr.) miR172c, a member of the miR172 family, and its target gene were investigated for abiotic stress responses in transgenic Arabidopsis. gma-miR172c was induced by abscisic acid (ABA) treatments and abiotic stresses, including salt and water deficit. 5'-RACE (5'-rapid amplification of cDNA ends) assays indicated that miR172c directed Glyma01g39520 mRNA cleavage in soybeans. Overexpression of gma-miR172c in Arabidopsis resulted in reduced leaf water loss and increased survival rate under stress conditions. Meanwhile, the root length, germination rate, and cotyledon greening of transgenic plants were improved during both high salt and water deficit conditions. In addition, transgenic plants exhibited hypersensitivity to ABA during both the seed germination and post-germination seedling growth stages. Stress-related physiological indicators and the expression of stress/ABA-responsive genes were affected by abiotic treatments. The overexpression of gma-miR172c in Arabidopsis promoted earlier flowering compared with the wild type through modulation of the expression of flowering genes, such as FT and LFY during long days, especially under drought conditions. Glyma01g39520 weakened ABA sensitivity and reduced the tolerance to drought stress in the snz mutant of Arabidopsis by reducing the expression of ABI3 and ABI5. Overall, the present results demonstrate that gma-miR172c confers water deficit and salt tolerance but increased ABA sensitivity by regulating Glyma01g39520, which also accelerates flowering under abiotic stresses.

Transcription factor WRKY46 modulates the development of Arabidopsis lateral roots in osmotic/salt stress conditions via regulation of ABA signaling and auxin homeostasis.

The development of lateral roots (LR) is known to be severely inhibited by salt or osmotic stress. However, the molecular mechanisms underlying LR development in osmotic/salt stress conditions are poorly understood. Here we show that the gene encoding the WRKY transcription factor WRKY46 (WRKY46) is expressed throughout lateral root primordia (LRP) during early LR development and that expression is subsequently restricted to the stele of the mature LR. In osmotic/salt stress conditions, lack of WRKY46 (in loss-of-function wrky46 mutants) significantly reduces, while overexpression of WRKY46 enhances, LR development. We also show that exogenous auxin largely restores LR development in wrky46 mutants, and that the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits LR development in both wild-type (WT; Col-0) and in a line overexpressing WRKY46 (OV46). Subsequent analysis of abscisic acid (ABA)-related mutants indicated that WRKY46 expression is down-regulated by ABA signaling, and up-regulated by an ABA-independent signal induced by osmotic/salt stress. Next, we show that expression of the DR5:GUS auxin response reporter is reduced in roots of wrky46 mutants, and that both wrky46 mutants and OV46 display altered root levels of free indole-3-acetic acid (IAA) and IAA conjugates. Subsequent RT-qPCR and ChIP-qPCR experiments indicated that WRKY46 directly regulates the expression of ABI4 and of genes regulating auxin conjugation. Finally, analysis of wrky46 abi4 double mutant plants confirms that ABI4 acts downstream of WRKY46. In summary, our results demonstrate that WRKY46 contributes to the feedforward inhibition of osmotic/salt stress-dependent LR inhibition via regulation of ABA signaling and auxin homeostasis.

An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4.

The molecular mechanisms of abscisic acid (ABA) signalling have been studied for many years; however, how mitochondria-localized proteins play roles in ABA signalling remains unclear. Here an Arabidopsis mitochondria-localized protein RRL (RETARDED ROOT GROWTH-LIKE) was shown to function in ABA signalling. A previous study had revealed that the Arabidopsis mitochondria-localized protein RRG (RETARDED ROOT GROWTH) is required for cell division in the root meristem. RRL shares 54% and 57% identity at the nucleotide and amino acid sequences, respectively, with RRG; nevertheless, RRL shows a different function in Arabidopsis. In this study, disruption of RRL decreased ABA sensitivity whereas overexpression of RRL increased ABA sensitivity during seed germination and seedling growth. High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by β-glucuronidase (GUS) staining of ProRRL-GUS transgenic lines. The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production. Previous studies have revealed that the expression of alternative oxidase (AOX) in the alternative respiratory pathway is increased by mitochondrial retrograde regulation to regain ROS levels when the mitochondrial electron transport chain is impaired. The APETALA2 (AP2)-type transcription factor ABI4 is a regulator of ALTERNATIVE OXIDASE1a (AOX1a) in mitochondrial retrograde signalling. This study showed that ABA-induced AOX1a and ABI4 expression was inhibited in the rrl mutant, suggesting that RRL is probably involved in ABI4-mediated mitochondrial retrograde signalling. Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth.

Overexpression of a maize SNF-related protein kinase gene, ZmSnRK2.11, reduces salt and drought tolerance in Arabidopsis

Sucrose non-fermenting-1 related protein kinase 2 (SnRK2) is a unique family of protein kinases associated with abiotic stress signal transduction in plants. In this study, a maize SnRK2 gene ZmSnRK2.11 was cloned and characterized. The results showed that ZmSnRK2.11 is up-regulated by high-salinity and dehydration treatment, and it is expressed mainly in maize mature leaf. A transient expression assay using onion epidermal cells revealed that ZmSnRK2.11-GFP fusion proteins are localized to both the nucleus and cytoplasm. Overexpressing-ZmSnRK2.11 in Arabidopsis resulted in salt and drought sensitivity phenotypes that exhibited an increased rate of water loss, reduced relative water content, delayed stoma closure, accumulated less free proline content and increased malondialdehyde (MDA) content relative to the phenotypes observed in wild-type (WT) control. Furthermore, overexpression of ZmSnRK2.11 up-regulated the expression of the genes ABI1 and ABI2 and decreased the expression of DREB2A and P5CS1. Taken together, our results suggest that ZmSnRK2.11 is a possible negative regulator involved in the salt and drought stress signal transduction pathways in plants.

Upregulation of Abelson interactor protein 1 predicts tumor progression and poor outcome in epithelial ovarian cancer.

Abelson interactor protein 1 (Abi1) is a key regulator of actin reorganization and lamellipodia formation. Because of its role in cell migration, Abi1 has been implicated in tumor progression. In the present study, we investigated the role of Abi1 in epithelial ovarian cancer (EOC) by analyzing its expression and correlation with clinicopathological and survival data. We evaluated the expression of Abi1 in 223 paraffin-embedded EOC specimens by immunohistochemistry and 46 frozen EOC samples by Western blot and real-time reverse transcription polymerase chain reaction analysis. Results showed that Abi1 protein and mRNA expression was significantly higher in EOC tissue compared with noncancerous tumors and normal ovaries (P < .05). Moreover, high level of Abi1 expression was significantly correlated with advanced stage, high grade, elevated Ca-125 level, and suboptimal surgical debulking (P < .05). By Western blot analysis, Abi1 was expressed in highly invasive cells compared with weakly invasive cells (P < .05). Immunofluorescence was performed to demonstrate Abi1 expression in SKOV3 cells. Additionally, upregulation of Abi1 significantly correlated with shorter survival (P < .05). Most importantly, multivariate analysis showed that Abi1 overexpression is an independent prognostic factor, complementary to clinical stage and residual tumor size. In conclusion, our findings suggest that Abi1 acts as a tumor-promoting gene in EOC progression, which may lead to unfavorable prognosis. Abi1 may serve as a potential effective prognostic marker for EOC.

Arabidopsis putative MAP kinase kinase kinases Raf10 and Raf11 are positive regulators of seed dormancy and ABA response.

We isolated an Arabidopsis ABA-insensitive mutant, ais143, by activation tagging screen. T-DNA was found to be located in the coding region of a putative mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) gene, Raf10, thereby abolishing its expression in the mutant. ais143 exhibited reduced seed dormancy as well as reduced ABA sensitivity. The phenotypes were complemented by the wild-type Raf10 gene, and the overexpression (OX) of Raf10 resulted in delayed seed germination and enhanced ABA sensitivity. Raf10 has high sequence identity to another MAP3K, Raf11. Parallel analysis of Raf11 knockout (KO) and OX lines showed that their phenotypes were similar to those of Raf10 KO and OX lines. An ais143 raf11 double mutant exhibited stronger phenotypes than single mutants, indicating the functional redundancy between Raf10 and Raf11. Transcript analysis revealed that the expression of many ABA-associated genes, including the key regulatory genes ABI3 and ABI5, was altered in the Raf10 and Raf11 OX lines. Recombinant Raf10 and Raf11 proteins exhibited kinase activity, which was inhibited by the MAP3K inhibitor BAY 43-9006 but not by the MAP2K inhibitor U0126. Collectively, our data indicate that Raf10 and Raf11 kinases are important regulators of seed dormancy and ABA response and that they affect the expression of ABI3, ABI5 and other ABA-regulated genes.

New cross talk between ROS, ABA and auxin controlling seed maturation and germination unraveled in APX6 deficient Arabidopsis seeds

Successful execution of germination program greatly depends on the seeds’ oxidative homeostasis. We recently identified new roles for the H2O2-reducing enzyme ascorbate peroxidase 6 (APX6) in germination control and seeds’ stress tolerance. APX6 replaces APX1 as the dominant APX in dry seeds, and its loss-of-function results in reduced germination due to over accumulation of ROS and oxidative damage. Metabolic analyses in dry apx6 seeds, revealed altered homeostasis of primary metabolites including accumulation of TCA cycle metabolites, ABA and auxin, supporting a novel role for APX6 in regulating cellular metabolism. Increased sensitivity of apx6 mutants to ABA or IAA in germination assays indicated impaired perception of these signals. Relative suppression of ABI3 and ABI5 expression, and induction of ABI4, suggested the activation of a signaling route inhibiting germination in apx6 seeds that is independent of ABI3. Here we provide additional evidence linking ABI4 with ABA- and auxin-controlled inhibition of germination and suggest a hypothetical model for the role of APX6 in the regulation of the crosstalk between these hormones and ROS.

Arabidopsis RAV1 transcription factor, phosphorylated by SnRK2 kinases, regulates the expression of ABI3, ABI4, and ABI5 during seed germination and early seedling development.

The phytohormone abscisic acid (ABA) modulates a number of processes during plant growth and development. In this study, the molecular mechanism of Arabidopsis RAV (Related to ABI3/VP1) transcription factor RAV1 involving ABA signaling was investigated. RAV1-underexpressing lines were more sensitive to ABA than wild-type plants during seed germination and early seedling development, whereas RAV1-overexpressing lines showed strong ABA-insensitive phenotypes. Overexpression of RAV1 repressed ABI3, ABI4, and ABI5 expression, and RAV1 bound to the ABI3, ABI4, and ABI5 promoters in vitro and in vivo, indicating that RAV1 directly down-regulates the expression of ABI3, ABI4, and ABI5. The interruption of ABI5 function in RAV1-U abi5 plants abolished the ABA-hypersensitive phenotype of RAV1-U plants, demonstrating that ABI5 is epistatic to RAV1. RAV1 interacted with SNF1-RELATED PROTEIN KINASE SnRK2.2, SnRK2.3 and SnRK2.6 in the nucleus. In vitro kinase assays showed that SnRK2.2, SnRK2.3 and SnRK2.6 phosphorylated RAV1. Transient expression assays revealed that SnRK2.2, SnRK2.3 and SnRK2.6 reduced the RAV1-dependent repression of ABI5, and the ABA-insensitive phenotype of the RAV1-overexpressing line was impaired by overexpression of SnRK2.3 in the RAV1 OE3 plants. Together, these results demonstrated that the Arabidopsis RAV1 transcription factor plays an important role in ABA signaling by modulating the expression of ABI3, ABI4, and ABI5, and that its activity is negatively affected by SnRK2s.

Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis.

Plants initiate senescence to shed photosynthetically inefficient leaves. Light deprivation induces leaf senescence, which involves massive transcriptional reprogramming to dismantle cellular components and remobilize nutrients. In darkness, intermittent pulses of red light can inhibit senescence, likely via phytochromes. However, the precise molecular mechanisms transducing the signals from light perception to the inhibition of senescence remain elusive. Here, we show that in Arabidopsis, dark-induced senescence requires phytochrome-interacting transcription factors PIF4 and PIF5 (PIF4/PIF5). ELF3 and phytochrome B inhibit senescence by repressing PIF4/PIF5 at the transcriptional and post-translational levels, respectively. PIF4/PIF5 act in the signalling pathways of two senescence-promoting hormones, ethylene and abscisic acid, by directly activating expression of EIN3, ABI5 and EEL. In turn, PIF4, PIF5, EIN3, ABI5 and EEL directly activate the expression of the major senescence-promoting NAC transcription factor ORESARA1, thus forming multiple, coherent feed-forward loops. Our results reveal how classical light signalling connects to senescence in Arabidopsis.

ASCORBATE PEROXIDASE6 protects Arabidopsis desiccating and germinating seeds from stress and mediates cross talk between reactive oxygen species, abscisic acid, and auxin.

A seed's ability to properly germinate largely depends on its oxidative poise. The level of reactive oxygen species (ROS) in Arabidopsis (Arabidopsis thaliana) is controlled by a large gene network, which includes the gene coding for the hydrogen peroxide-scavenging enzyme, cytosolic ASCORBATE PEROXIDASE6 (APX6), yet its specific function has remained unknown. In this study, we show that seeds lacking APX6 accumulate higher levels of ROS, exhibit increased oxidative damage, and display reduced germination on soil under control conditions and that these effects are further exacerbated under osmotic, salt, or heat stress. In addition, ripening APX6-deficient seeds exposed to heat stress displayed reduced germination vigor. This, together with the increased abundance of APX6 during late stages of maturation, indicates that APX6 activity is critical for the maturation-drying phase. Metabolic profiling revealed an altered activity of the tricarboxylic acid cycle, changes in amino acid levels, and elevated metabolism of abscisic acid (ABA) and auxin in drying apx6 mutant seeds. Further germination assays showed an impaired response of the apx6 mutants to ABA and to indole-3-acetic acid. Relative suppression of abscisic acid insensitive3 (ABI3) and ABI5 expression, two of the major ABA signaling downstream components controlling dormancy, suggested that an alternative signaling route inhibiting germination was activated. Thus, our study uncovered a new role for APX6, in protecting mature desiccating and germinating seeds from excessive oxidative damage, and suggested that APX6 modulate the ROS signal cross talk with hormone signals to properly execute the germination program in Arabidopsis.

The ABI4-Induced Arabidopsis ANAC060 Transcription Factor Attenuates ABA Signaling and Renders Seedlings Sugar Insensitive when Present in the Nucleus

Seedling establishment is inhibited on media containing high levels (∼6%) of glucose or fructose. Genetic loci that overcome the inhibition of seedling growth on high sugar have been identified using natural variation analysis and mutant selection, providing insight into sugar signaling pathways. In this study, a quantitative trait locus (QTL) analysis was performed for seedling sensitivity to high sugar in a Col/C24 F2 population of Arabidopsis thaliana. A glucose and fructose-sensing QTL, GSQ11, was mapped through selective genotyping and confirmed in near-isogenic lines in both Col and C24 backgrounds. Allelism tests and transgenic complementation showed that GSQ11 lies within the ANAC060 gene. The Col ANAC060 allele confers sugar insensitivity and was dominant over the sugar-sensitive C24 allele. Genomic and mRNA analyses showed that a single-nucleotide polymorphism (SNP) in Col ANAC060 affects the splicing patterns of ANAC060 such that 20 additional nucleotides are present in the mRNA. The insertion created a stop codon, resulting in a truncated ANAC60 protein lacking the transmembrane domain (TMD) that is present in the C24 ANAC060 protein. The absence of the TMD results in the nuclear localization of ANAC060. The short version of the ANAC060 protein is found in ∼12% of natural Arabidopsis accessions. Glucose induces GSQ11/ANAC060 expression in a process that requires abscisic acid (ABA) signaling. Chromatin immunoprecipitation-qPCR and transient expression analysis showed that ABI4 directly binds to the GSQ11/ANAC060 promoter to activate transcription. Interestingly, Col ANAC060 reduced ABA sensitivity and Glc-induced ABA accumulation, and ABI4 expression was also reduced in Col ANAC060 lines. Thus, the sugar-ABA signaling cascade induces ANAC060 expression, but the truncated Col ANAC060 protein attenuates ABA induction and ABA signaling. This negative feedback from nuclear ANAC060 on ABA signaling results in sugar insensitivity.

Convergence of Light and ABA Signaling on the ABI5 Promoter

Light is one of the most important environmental cues regulating multiple aspects of plant growth and development, and abscisic acid (ABA) is a plant hormone that plays important roles during many phases of the plant life cycle and in plants' responses to various environmental stresses. How plants integrate the external light signal with endogenous ABA pathway for better adaptation and survival remains poorly understood. Here, we show that BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX) protein previously shown to positively regulate seedling photomorphogenesis, is also involved in ABA signaling. Our genetic data show that BBX21 may act upstream of several ABA INSENSITIVE (ABI) genes and ELONGATED HYPOCOTYL 5 (HY5) in ABA control of seed germination. Previous studies showed that HY5 acts as a direct activator of ABI5 expression, and that BBX21 interacts with HY5. We further demonstrate that BBX21 negatively regulates ABI5 expression by interfering with HY5 binding to the ABI5 promoter. In addition, ABI5 was shown to directly activate its own expression, whereas BBX21 negatively regulates this activity by directly interacting with ABI5. Together, our study indicates that BBX21 coordinates with HY5 and ABI5 on the ABI5 promoter and that these transcriptional regulators work in concert to integrate light and ABA signaling in Arabidopsis thaliana.