Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis.

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K(+) uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K(+) channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using (86)radioactive rubidium ion ((86)Rb(+)) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.

TET3-OGT interaction increases the stability and the presence of OGT in chromatin.

Gene expression is controlled by alterations in the epigenome, including DNA methylation and histone modification. Recently, it was reported that 5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by proteins in the ten-eleven translocation (TET) family. This conversion is believed to be part of the mechanism by which methylated DNA is demethylated. Moreover, histones undergo modifications such as phosphorylation and acetylation. In addition, modification with O-linked-N-acetylglucosamine (O-GlcNAc) by O-GlcNAc transferase (OGT) was recently identified as a novel histone modification. Herein, we focused on TET3, the regulation of which is still unclear. We attempted to elucidate the mechanism of its regulation by biochemical approaches. First, we conducted mass spectrometric analysis in combination with affinity purification of FLAG-TET3, which identified OGT as an important partner of TET3. Co-immunoprecipitation assays using a series of deletion mutants showed that the C-terminal H domain of TET3 was required for its interaction with OGT. Furthermore, we showed that TET3 is GlcNAcylated by OGT, although the GlcNAcylation did not affect the global hydroxylation of methylcytosine by TET3. Moreover, we showed that TET3 enhanced its localization to chromatin through the stabilization of OGT protein. Taken together, we showed a novel function of TET3 that likely supports the function of OGT.

Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis.

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.

Multiple post-translational modifications in hepatocyte nuclear factor 4α.

To investigate the role of post-translational modifications (PTMs) in the hepatocyte nuclear factor 4α (HNF4α)-mediated transcription, we took a comprehensive survey of PTMs in HNF4α protein by mass-spectrometry and identified totally 8 PTM sites including newly identified ubiquitilation and acetylation sites. To assess the impact of identified PTMs in HNF4α-function, we introduced point mutations at the identified PTM sites and, tested transcriptional activity of the HNF4α. Among the point-mutations, an acetylation site at lysine 458 was found significant in the HNF4α-mediated transcriptional control. An acetylation negative mutant at lysine 458 showed an increased transcriptional activity by about 2-fold, while an acetylation mimic mutant had a lowered transcriptional activation. Furthermore, this acetylation appeared to be fluctuated in response to extracellular nutrient conditions. Thus, by applying an comprehensive analysis of PTMs, multiple PTMs were newly identified in HNF4α and unexpected role of an HNF4α acetylation could be uncovered.

Biochemical Analysis of Histone Succinylation.

Posttranslational modification (PTM) of proteins is used to regulate protein activity and stability. Histone PTMs are regarded as some of the most important, as they can directly regulate gene expression through chromatin reorganization. Recently, histone proteins were found to undergo succinylation, adding to other well-known PTMs such as acetylation, methylation, and phosphorylation. However, there is little information regarding the enzyme which catalyzes histone lysine succinylation. In fact, it is unclear whether this reaction is enzymatic. In this study, we tested histone succinylation activity in vitro using cell nuclear extracts of HepG2 cells. Although whole nuclear extracts did not show histone succinylation activity, we found that an SP 1.0 M KCl fraction of nuclear extracts indeed had such activity. These data offer the first direct evidence that histone succinylation is an enzymatic PTM as are other histone codes in the nucleus.

Identification of Posttranslational Modifications in Peroxisome Proliferator-Activated Receptor γ Using Mass Spectrometry.

Posttranslational modification (PTM) of proteins is critical for various cellular processes. However, there are few studies examining PTMs in specific proteins using unbiased approaches. Here we report the attempt to identify the PTMs in peroxisome proliferator-activated receptor γ (PPARγ) proteins using our previously established PTM analysis system. In this study, we identified several PTMs in exogenously expressed PPARγ2 proteins from 293T cells as well as endogenous PPARγ1 proteins from a Caco-2 colon cancer cell line. The identified PTMs include phosphorylation of serine 112 and serine 81 in PPARγ2 and PPARγ1, respectively, both of which are well-known mitogen-activated protein kinase- (MAP kinase-) mediated PTMs in PPARγ proteins, thus confirming our experimental approach. Furthermore, previously unknown PTMs were also identified, demonstrating that this method can be applied to find previously unidentified PTMs in PPARγ proteins and other proteins including nuclear receptors.