SNF1-related Protein Kinase Research Articles

The Sucrose Non-Fermenting 1-Related Protein Kinase 2 (SnRK2) Genes Are Multifaceted Players in Plant Growth, Development and Response to Environmental Stimuli.

Reversible protein phosphorylation orchestrated by protein kinases and phosphatases is a major regulatory event in plants and animals. The SnRK2 subfamily consists of plant-specific protein kinases in the Ser/Thr protein kinase superfamily. Early observations indicated that SnRK2s are mainly involved in response to abiotic stress. Recent evidence shows that SnRK2s are multifarious players in a variety of biological processes. Here, we summarize the considerable knowledge of SnRK2s, including evolution, classification, biological functions and regulatory mechanisms at the epigenetic, post-transcriptional and post-translation levels.

Rice SnRK protein kinase OsSAPK8 acts as a positive regulator in abiotic stress responses

SnRK2 (sucrose non-fermenting 1-related protein kinases 2) protein kinase family involves in several abiotic stress response in plants. Although the regulatory mechanism of SnRK2 have been well demonstrated in Arabidopsis thaliana, their functions in rice are still largely unknown. Here, we report a SnRK2 family gene, OsSAPK8, can be strongly induced by abiotic stresses, including low-temperature, drought and high salt stress. The ossapk8 mutants showed lower tolerance to low-temperature, high salinity and drought stresses at the vegetative stages. Moreover, the expressions of marker genes for those abiotic stresses, e.g. OsDREB1, OsDREB2, OsNCED and OsRAB21, were downregulated in the ossapk8 mutants. We further confirmed that the yield was reduced in ossapk8 mutant lines compared with the wild type. Our results provide evidence for OsSAPK8 acting as a positive regulator in cold, drought, and salt stress responses.

Expression, Subcellular Localization, and Interactions of CPK Family Genes in Maize.

Calcium-dependent protein kinase (CPKs) is a key player in the calcium signaling pathway to decode calcium signals into various physiological responses. cDNA sequences of 9 ZmCPK genes were successfully cloned from all four phylogenetic groups in maize. qRT-PCR analysis showed the expression variation of these selected genes under abscisic acid (ABA) and calcium chloride (CaCl2) treatment. Due to the presence of N-myristoylation/palmitoylation sites, the selected ZmCPK members were localized in a plasma membrane. To clarify whether ZmCPK, a key player in calcium signaling, interacts with key players of ABA, protein phosphatase 2Cs (PP2Cs) and the SNF1-related protein kinase 2s (SnRK2s) and mitogen-activated protein kinase (MAPK) signaling pathways in maize, we examined the interaction between 9 CPKs, 8 PP2Cs, 5 SnRKs, and 20 members of the MPK family in maize by using yeast two-hybrid assay. Our results showed that three ZmCPKs interact with three different members of ZmSnRKs while four ZmCPK members had a positive interaction with 13 members of ZmMPKs in different combinations. These four ZmCPK proteins are from three different groups in maize. These findings of physical interactions between ZmCPKs, ZmSnRKs, and ZmMPKs suggested that these signaling pathways do not only have indirect influence but also have direct crosstalk that may involve the defense mechanism in maize. The present study may improve the understanding of signal transduction in plants.

A ligand-independent origin of abscisic acid perception

Land plants are considered monophyletic, descending from a single successful colonization of land by an aquatic algal ancestor. The ability to survive dehydration to the point of desiccation is a key adaptive trait enabling terrestrialization. In extant land plants, desiccation tolerance depends on the action of the hormone abscisic acid (ABA) that acts through a receptor-signal transduction pathway comprising a PYRABACTIN RESISTANCE 1-like (PYL)-PROTEIN PHOSPHATASE 2C (PP2C)-SNF1-RELATED PROTEIN KINASE 2 (SnRK2) module. Early-diverging aeroterrestrial algae mount a dehydration response that is similar to that of land plants, but that does not depend on ABA: Although ABA synthesis is widespread among algal species, ABA-dependent responses are not detected, and algae lack an ABA-binding PYL homolog. This raises the key question of how ABA signaling arose in the earliest land plants. Here, we systematically characterized ABA receptor-like proteins from major land plant lineages, including a protein found in the algal sister lineage of land plants. We found that the algal PYL-homolog encoded by Zygnema circumcarinatum has basal, ligand-independent activity of PP2C repression, suggesting this to be an ancestral function. Similarly, a liverwort receptor possesses basal activity, but it is further activated by ABA. We propose that co-option of ABA to control a preexisting PP2C-SnRK2-dependent desiccation-tolerance pathway enabled transition from an all-or-nothing survival strategy to a hormone-modulated, competitive strategy by enabling continued growth of anatomically diversifying vascular plants in dehydrative conditions, enabling them to exploit their new environment more efficiently.

Stress-Activated Protein Kinase OsSAPK9 Regulates Tolerance to Salt Stress and Resistance to Bacterial Blight in Rice

BackgroundSalt stress and bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) are key limiting factors of rice (Oryza sativa L.) yields. Members of sucrose non-fermenting 1 (SNF1)-related protein kinase 2 (SnRK2), which is a family of plant-specific Ser/Thr kinases, are important components of signaling pathways involved in plant developmental processes and responses to stresses. There are 10 members of the SnRK2 family in rice; however, their functions are poorly understood, as are the underlying molecular mechanisms.ResultsIn this study, we found that OsSAPK9, which belongs to the SnRK2 family, positively regulated salt-stress tolerance and strain-specific resistance to bacterial blight in rice. RNA sequencing revealed that there were 404 and 1324 genes differentially expressed in OsSAPK9-RNAi in comparison with wild-type plants under salt-stress conditions and after Xoo inoculation, respectively, which participate in basic metabolic processes. In total, 65 common differentially expressed genes involved mainly in defense responses were detected both under salt-stress conditions and after Xoo inoculation. Moreover, in vivo and in vitro experiments demonstrated that OsSAPK9 forms a protein complex with the molecular chaperones OsSGT1 and OsHsp90, and transgenic plants overexpressing OsSGT1 exhibited decreased tolerances to salt stress and significantly increased resistance levels to bacterial blight. Thus, OsSAPK9 may function as a center node regulator of salt-stress responses and disease-resistance pathways through its interaction with OsSGT1 in rice.ConclusionThis study confirms that OsSAPK9 functions as a positive regulator of salt-stress responses and disease resistance through its interaction with OsSGT1 in rice.

Grafting Cucumber Onto Pumpkin Induced Early Stomatal Closure by Increasing ABA Sensitivity Under Salinity Conditions.

During early periods of salt stress, reduced stomatal opening can prevent water loss and wilting. Abscisic acid (ABA) signal plays an important role in this process. Here, we show that cucumber grafted onto pumpkin exhibits rapid stomatal closure, which helps plants to adapt to osmotic stress caused by salinity. Increased ABA contents in the roots, xylem sap, and leaves were evaluated in two grafting combinations (self-grafted cucumber and cucumber grafted onto pumpkin rootstock). The expression levels of ABA biosynthetic or signaling related genes NCED2 (9-cis-epoxycarotenoid dioxygenase gene 2), ABCG22 (ATP-binding cassette transporter genes 22), PP2C (type-2C protein phosphatases), and SnRK2.1 (sucrose non-fermenting 1-related protein kinases 2) were investigated. Results showed that a root-sourced ABA signal led to decreased stomatal opening and transpiration in the plants grafted onto pumpkin. Furthermore, plants grafted onto pumpkin had increased sensitivity to ABA, compared with self-grafted cucumbers. The inhibition of ABA biosynthesis with fluridon in roots increased the transpiration rate (Tr) and stomatal conductance (Gs) in the leaves. Our study demonstrated that the roots of pumpkin increases the sensitivity of the scion to ABA delivered from the roots to the shoots, and enhances osmotic tolerance under NaCl stress. Such a mechanism can be greatly exploited to benefit vegetable production particularly in semiarid saline regions.

Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms.

SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis (Arabidopsis thaliana) AtSCS gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca2+ Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing 35S::AtSCS-A-c-myc or 35S::AtSCS-B-c-myc in the scs-1 knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins.

Sugar sensing responses to low and high light in leaves of the C4 model grass Setaria viridis.

Although sugar regulates photosynthesis, the signalling pathways underlying this process remain elusive, especially for C4 crops. To address this knowledge gap and identify potential candidate genes, we treated Setaria viridis (C4 model) plants acclimated to medium light intensity (ML, 500 µmol m-2 s-1) with low (LL, 50 µmol m-2 s-1) or high (HL, 1000 µmol m-2 s-1) light for 4 d and observed the consequences on carbon metabolism and the transcriptome of source leaves. LL impaired photosynthesis and reduced leaf content of signalling sugars (glucose, sucrose, and trehalose-6-phosphate). In contrast, HL strongly induced sugar accumulation without repressing photosynthesis. LL more profoundly impacted the leaf transcriptome, including photosynthetic genes. LL and HL contrastingly altered the expression of hexokinase (HXK) and sucrose-non-fermenting 1 (Snf1)-related protein kinase 1 (SnRK1) sugar sensors and trehalose pathway genes. The expression of key target genes of HXK and SnRK1 were affected by LL and sugar depletion, while surprisingly HL and strong sugar accumulation only slightly repressed the SnRK1 signalling pathway. In conclusion, we demonstrate that LL profoundly impacted photosynthesis and the transcriptome of S. viridis source leaves, while HL altered sugar levels more than LL. We also present the first evidence that sugar signalling pathways in C4 source leaves may respond to light intensity and sugar accumulation differently from C3 source leaves.

AKINβ1, a subunit of SnRK1, regulates organic acid metabolism and acts as a global modulator of genes involved in carbon, lipid, and nitrogen metabolism.

The sucrose non-fermenting-1-related protein kinase 1 (SnRK1) is a highly conserved heterotrimeric protein kinase in plants. It possesses a catalytic subunit (α) and two regulatory subunits (β and γ). The effects of altered expression of AKINβ1 on carbohydrate metabolism and gene expression in leaves were investigated in an Arabidopsis T-DNA insertion mutant. The contents of key intermediates in the tricarboxylic acid (TCA) cycle of the mutant leaves were markedly reduced throughout the diurnal cycle, coupled with a decrease in measurable respiration rate. Compared with the wild type, 2485 genes and 188 genes were differentially expressed in leaves of the akinβ1 mutant in response to light and darkness, respectively. Among these, several genes exhibited very substantial decreases in expression. Notably, expression of particular isoforms of multigene families involved in malate and lipid metabolism and nitrate uptake showed decreases of 40- to 240-fold during the light period, but not during darkness. The subcellular localization of AKINβ1 and the regulatory function of N-myristoylation for this localization were investigated, showing that AKINβ1 localizes to the Golgi. A model is hypothesized to explain the effects of AKINβ1 on metabolism and gene expression in Arabidopsis.

Genetic Analyses of the Arabidopsis ATG1 Kinase Complex Reveal Both Kinase-Dependent and Independent Autophagic Routes during Fixed-Carbon Starvation.

Under nutrient and energy-limiting conditions, plants up-regulate sophisticated catabolic pathways such as autophagy to remobilize nutrients and restore energy homeostasis. Autophagic flux is tightly regulated under these circumstances through the AuTophaGy-related1 (ATG1) kinase complex, which relays upstream nutrient and energy signals to the downstream components that drive autophagy. Here, we investigated the role(s) of the Arabidopsis (Arabidopsis thaliana) ATG1 kinase during autophagy through an analysis of a quadruple mutant deficient in all four ATG1 isoforms. These isoforms appear to act redundantly, including the plant-specific, truncated ATG1t variant, and like other well-characterized atg mutants, homozygous atg1abct quadruple mutants display early leaf senescence and hypersensitivity to nitrogen and fixed-carbon starvations. Although ATG1 kinase is essential for up-regulating autophagy under nitrogen deprivation and short-term carbon starvation, it did not stimulate autophagy under prolonged carbon starvation. Instead, an ATG1-independent response arose requiring phosphatidylinositol-3-phosphate kinase (PI3K) and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), possibly through phosphorylation of the ATG6 subunit within the PI3K complex by the catalytic KIN10 subunit of SnRK1. Together, our data connect ATG1 kinase to autophagy and reveal that plants engage multiple pathways to activate autophagy during nutrient stress, which include the ATG1 route as well as an alternative route requiring SnRK1 and ATG6 signaling.plantcell;31/12/2973/FX1F1fx1.

AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs.

Cell surface receptors perceive signals from the environment and transfer them to the interior of the cell. The Arabidopsis thaliana PR5 receptor-like kinase (AtPR5K) subfamily consists of three members with extracellular domains that share sequence similarity with the PR5 proteins. In this study, we characterized the role of AtPR5K2 in plant drought-stress signaling. AtPR5K2 is predominantly expressed in leaves and localized to the plasma membrane. The atpr5k2-1 mutant showed tolerance to dehydration stress, while AtPR5K2-overexpressing plants was hypersensitive to drought. Bimolecular fluorescence complementation assays showed that AtPR5K2 physically interacted with the type 2C protein phosphatases ABA-insensitive 1 (ABI1) and ABI2 and the SNF1-related protein kinase 2 (SnRK2.6) proteins, all of which are involved in the initiation of abscisic acid (ABA) signaling; however, these interactions were inhibited by treatments of exogenous ABA. Moreover, AtPR5K2 was found to phosphorylate ABI1 and ABI2, but not SnRK2.6. Taken together, these results suggest that AtPR5K2 participates in ABA-dependent drought-stress signaling through the phosphorylation of ABI1 and ABI2.

Arabidopsis CTP:phosphocholine cytidylyltransferase 1 is phosphorylated and inhibited by sucrose nonfermenting 1–related protein kinase 1 (SnRK1)

De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis.

SnRK2 Protein Kinases and mRNA Decapping Machinery Control Root Development and Response to Salt

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.

Interaction between wheat translationally controlled tumor protein TCTP and SNF1-related protein kinase SnRK1

Translationally controlled tumor proteins (TCTP) and SNF1- related protein kinase (SnRK1) are conserved and widely present in eukaryotic cells. TCTP regulates cell division, plant growth and development, and mediates plant resistance against pathogen infection. SnRK1 participates in a range of physiological processes including sugar metabolism and resistance to abiotic and biotic stresses. Previous work in our laboratory demonstrated that wheat TCTP can respond to Puccinia triticina infection and induce host defense responses. In order to further investigate the mechanism of TaTCTP in wheat resistance to Puccinia triticina infection, we used TAP (tandem affinity purification) and mass spectrometry to screen the potential interactants of TaTCTP. A SNF1- related protein kinase (SnRK1) was identified as a potential interacting protein of TaTCTP. The results of yeast two-hybrid assay showed that TCTP could interact with SnRK1 in yeast, and the yeast carrying TCTP and SnRK1 could grow on SD/-Leu/-Trp/-His/-Ade (SD/-LWHA) medium. The fluorescence signal of the interaction between TCTP and SnRK1 was found to be distributed in the cytoplasm in the Bi-fluorescense complementation experiment. Co-IP experiments further showed that TCTP and SnRK1 could interact in plant cells. This study lays an important foundation for further studying the mechanism of TaTCTP in the interaction between wheat and Puccinia triticina, and it play a great influence on further improving the molecular mechanism of wheat resistant to Puccinia triticina.

The C-Terminal Domains SnRK2 Box and ABA Box Have a Role in Sugarcane SnRK2s Auto-Activation and Activity.

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s—known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function.

GmSnRK1.1, a Sucrose Non-fermenting-1(SNF1)-Related Protein Kinase, Promotes Soybean Resistance to Phytophthora sojae.

Phytophthora root and stem rot, a destructive disease of soybean [Glycine max (L.) Merr.], is caused by the oomycete Phytophthora sojae. However, how the disease resistance mechanisms of soybean respond to P. sojae infection remains unclear. Previously, we showed that GmWRKY31, which interacts with a sucrose non-fermenting-1(SNF1)-related protein kinase (SnRK), enhances resistance to P. sojae in soybean. Here, we report that the membrane-localized SnRK GmSnRK1.1 is involved in the soybean host response to P. sojae. The overexpression of GmSnRK1.1 (GmSnRK1.1-OE) increased soybean resistance to P. sojae, and the RNA interference (RNAi)-mediated silencing of GmSnRK1.1 (GmSnRK1.1-R) reduced resistance to P. sojae. Moreover, the activities and transcript levels of the antioxidant enzymes superoxide dismutase and peroxidase were markedly higher in the GmSnRK1.1-OE transgenic soybean plants than in the wild type (WT), but were reduced in the GmSnRK1.1-R plants. Several isoflavonoid phytoalexins related genes GmPAL, GmIFR, Gm4CL and GmCHS were significantly higher in “Suinong 10” and GmSnRK1.1-OE lines than these in “Dongnong 50,” and were significantly lower in GmSnRK1.1-R lines. In addition, the accumulation of salicylic acid (SA) and the expression level of the SA biosynthesis-related gene were significantly higher in the GmSnRK1.1-OE plants than in the WT and GmSnRK1.1-R plants, moreover, SA biosynthesis inhibitor treated GmSnRK1.1-R lines plants displayed clearly increased pathogen biomass compared with H2O-treated plants after 24 h post-inoculation. These results showed that GmSnRK1.1 positively regulates soybean resistance to P. sojae, potentially functioning via effects on the expression of SA-related genes and increased accumulation of SA.

Sugar partitioning and source-sink interaction are key determinants of leaf senescence in maize.

Source-sink communication is one of the key regulators of senescence; however, the mechanisms underlying such regulation are largely unknown. We analysed senescence induced by the lack of grain sink in maize, termed source-sink regulated senescence (SSRS), and compared the associated physiological and metabolic changes with those accompanying natural senescence. Phenotypic characterization of31 diverse field-grown inbreds revealed substantial variation for both SSRS and natural senescence. Partitioning of excess carbohydrates to alternative sinks, mainly internodes and husks, emerged as a critical mechanism underlying both SSRS and stay-green. Time-course analyses of SSRS sensitive (B73) and resistant (PHG35) inbreds confirmed the role of sugar partitioning in SSRS and stay-green. Elevated hemicellulose content in PHG35 internodes highlighted the role of the cell wall as a significant alternative sink. Sugar signalling emerged as an important regulator of SSRS as evident from an increased accumulation of trehalose-6-phosphate and decreased transcript levels of snf1-related protein kinase1, two signalling components associated with senescence, in B73. These findings demonstrate a crucial role of sugar partitioning, signalling, and utilization in SSRS. Available genetic variation for SSRS and a better understanding of the underlying mechanisms would help modify sugar partitioning and senescence to enhance the productivity of maize and related grasses.

A Cryophyte Transcription Factor, CbABF1, Confers Freezing, and Drought Tolerance in Tobacco.

Abscisic acid responsive element binding factors (ABFs) play crucial roles in plant responses to abiotic stress. However, little is known about the roles of ABFs in alpine subnival plants, which can survive under extreme environmental conditions. Here, we cloned and characterized an ABF1 homolog, CbABF1, from the alpine subnival plant Chorispora bungeana. Expression of CbABF1 was induced by cold, drought, and abscisic acid. Subcellular localization analysis revealed that CbABF1 was located in the nucleus. Further, CbABF1 had transactivation activity, which was dependent on the N-terminal region containing 89 residues. A Snf1-related protein kinase, CbSnRK2.6, interacted with CbABF1 in yeast two-hybrid analysis and bimolecular fluorescence complementation assays. Transient expression assay revealed that CbSnRK2.6 enhanced the transactivation of CbABF1 on ABRE cis-element. We further found that heterologous expression of CbABF1 in tobacco improved plant tolerance to freezing and drought stress, in which the survival rates of the transgenic plants increased around 40 and 60%, respectively, compared with wild-type plants. Moreover, the transgenic plants accumulated less reactive oxygen species, accompanied by high activities of antioxidant enzymes and elevated expression of stress-responsive genes. Our results thus suggest that CbABF1 is a transcription factor that plays an important role in cold and drought tolerance and is a candidate gene in molecular breeding of stress-tolerant crops.

Expression, purification and characterization of the plant Snf1-related protein kinase 1 from Escherichia coli

The SnRK1 (SNF1 related protein kinase 1), a plant homologue of SNF1 (Sucrose non-fermenting 1)/AMPK (AMP-activated protein kinase), is an important metabolic sensor involving in catabolic and anabolic processes. SnRK1 is essential for plant metabolism regulation and response to environmental stresses. The plant SnRK1 consists of one catalytic (α1/α2 subunit) and two regulatory subunits (β1/β2/β3 and γ/βγ subunits), and functions as a heterotrimeric complex. Here we took advantage of a tricistronic expression vector and successfully purified the holoenzyme containing three subunits of SnRK1 from the E.coli. Using advantages of the E.coli system, we would be able to purify SnRK1 complex with high yield and high purity. Moreover, the complex is stable with high homogeneity. Using the purified complex, we confirmed that the βγ rather than the γ subunit of the plant SnRK1 acts as the canonical regulatory subunit. Besides, some basic characters of the SnRK1 holoenzyme was studied. Together, our results provide a convenient way for purify the plant SnRK1 complexes, and this would be helpful for follow-up study on SnRK1's structure and mechanism.

Transcript analyses reveal a comprehensive role of abscisic acid in modulating fruit ripening in Chinese jujube

BackgroundChinese jujube (Ziziphus jujuba Mill.) is a non-climacteric fruit; however, the underlying mechanism of ripening and the role of abscisic acid involved in this process are not yet understood for this species.ResultsIn the present study, a positive correlation between dynamic changes in endogenous ABA and the onset of jujube ripening was determined. Transcript analyses suggested that the expression balance among genes encoding nine-cis-epoxycarotenoid dioxygenase (ZjNCED3), ABA-8′-hydroxylase (ZjCYP707A2), and beta-glucosidase (ZjBG4, ZjBG5, ZjBG8, and ZjBG9) has an important role in maintaining ABA accumulation, while the expression of a receptor (ZjPYL8), protein phosphatase 2C (ZjPP2C4–8), and sucrose nonfermenting 1-related protein kinase 2 (ZjSnRK2–2 and ZjSnRK2–5) is important in regulating fruit sensitivity to ABA applications. In addition, white mature ‘Dongzao’ fruit were harvested and treated with 50 mg L− 1 ABA or 50 mg L− 1 nordihydroguaiaretic acid (NDGA) to explore the role of ABA in jujube fruit ripening. By comparative transcriptome analyses, 1103 and 505 genes were differentially expressed in response to ABA and NDGA applications on the 1st day after treatment, respectively. These DEGs were associated with photosynthesis, secondary, lipid, cell wall, and starch and sugar metabolic processes, suggesting the involvement of ABA in modulating jujube fruit ripening. Moreover, ABA also exhibited crosstalk with other phytohormones and transcription factors, indicating a regulatory network for jujube fruit ripening.ConclusionsOur study further elucidated ABA-associated metabolic and regulatory processes. These findings are helpful for improving strategies for jujube fruit storage and for gaining insights into understand complex non-climacteric fruit ripening processes.