Jeong Dong Bahk

Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus

Protein N-glycosylation in the endoplasmic reticulum (ER) and in the Golgi apparatus is an essential process in eukaryotic cells. Although the N-glycosylation pathway in the ER has been shown to regulate protein quality control, salt tolerance, and cellulose biosynthesis in plants, no biological roles have been linked functionally to N-glycan modifications that occur in the Golgi apparatus. Herein, we provide evidence that mutants defective in N-glycan maturation, such as complex glycan 1 (cgl1), are more salt-sensitive than wild type. Salt stress caused growth inhibition, aberrant root-tip morphology, and callose accumulation in cgl1, which were also observed in an ER oligosaccharyltransferase mutant, staurosporin and temperature sensitive 3a (stt3a). Unlike stt3a, cgl1 did not cause constitutive activation of the unfolded protein response. Instead, aberrant modification of the plasma membrane glycoprotein KORRIGAN 1/RADIALLY SWOLLEN 2 (KOR1/RSW2) that is necessary for cellulose biosynthesis occurred in cgl1 and stt3a. Genetic analyses identified specific interactions among rsw2, stt3a, and cgl1 mutations, indicating that the function of KOR1/RSW2 protein depends on complex N-glycans. Furthermore, cellulose deficient rsw1-1 and rsw2-1 plants were also salt-sensitive. These results establish that plant protein N-glycosylation functions beyond protein folding in the ER and is necessary for sufficient cell-wall formation under salt stress.

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress‐elicited changes in plants at the proteome level. Hydroponically grown 2‐wk‐old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H2O2 contents in roots. A total of 23 As‐regulated proteins including predicted and novel ones were identified using 2‐DE coupled with MS analyses. The expression levels of S‐adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST‐tau, and tyrosine‐specific protein phosphatase proteins (TSPP) were markedly up‐regulated in response to arsenate, whereas treatment by H2O2 also regulated the levels of CS suggesting that its expression was certainly regulated by As or As‐induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose‐dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

Loss of Halophytism by Interference with SOS1 Expression

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na+) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na+ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na+ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na+ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na+ as Arabidopsis.

Chilling stress-induced proteomic changes in rice roots.

Roots are highly sensitive organs in plants. To gain a better knowledge of the chilling stress responses of plants, it is imperative to analyze the tissue-specific proteome patterns under chilling stress. In the present study, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry, has been adopted to investigate the protein expression patterns of rice roots in response to chilling stress. Rice seedlings were subjected to 10 degrees C and samples were collected 24 and 72h after treatment. To identify the low-abundant proteins in root tissues, samples were fractionated by 15% polyethylene glycol (PEG), separated by 2-DE, and visualized by silver or CBB staining. A total of 27 up-regulated proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry or electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analysis. Together with the previously identified cold-stress-responsive proteins, a group of novel proteins were identified including acetyl transferase, phosphogluconate dehydrogenase, NADP-specific isocitrate dehydrogenase, fructokinase, PrMC3, putative alpha-soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, and glyoxalase 1. These proteins are involved in several cellular processes, including energy production and metabolism, vesicular trafficking, and detoxification. Gene expression at the mRNA level of some selected proteins revealed that transcription levels are not always concomitant to the translational level. Thus, investigation of root proteome expression and identification of some novel proteins could be useful in better understanding the molecular basis of chilling stress responses in plants.

Identification of a Novel Divergent Calmodulin Isoform from Soybean Which Has Differential Ability to Activate Calmodulin-dependent Enzymes

Calmodulin plays pivotal roles in the transduction of various Ca-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to −5, SCaM-4 and −5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca-dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions

The H2O2‐catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site‐directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D‐hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)‐ and T90A‐hPrxI, the T90D‐hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT‐hPrxI.

Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach

Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. In the present work, the protein expression profile of rice leaves exposed to glyphosate was analyzed in order to investigate the alternative effects of glyphosate on plants. Two-week-old rice leaves were subjected to glyphosate or a reactive oxygen species (ROS) inducing herbicide paraquat, and total soluble proteins were extracted and analyzed by two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. A total of 25 differentially expressed proteins were identified from the glyphosate treated sample, wherein 18 proteins were up-regulated and 7 proteins were down-regulated. These proteins had shown a parallel expression pattern in response to paraquat. Results from the 2-DE analysis, combined with immunoblotting, clearly revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit was significantly decreased by the treatment of both herbicides. An increased accumulation of antioxidant enzymes including ascorbate peroxidase, glutathione S-transferase, thioredoxin h-type, nucleoside diphosphate kinase 1, peroxiredoxin and a superoxide dismutase [Cu-Zn] chloroplast precursor in the glyphosate-treated sample suggests that a glyphosate treatment possibly generates oxidative stress in plants. Moreover, a gene expression analysis of five antioxidant enzymes by Northern blot confirmed their mRNA levels in the rice leaves. A histo-cytochemical investigation with DAB (3,3-diaminobenzidine) to localize H(2)O(2) and increases of the thiobarbituric acid reactive substances (TBARS) concentration revealed that the glyphosate application generates ROS, which resulted in the peroxidation and destruction of lipids in the rice leaves.

Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes

An Arabidopsisprotoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsismesophyll protoplasts expressing murine BaxcDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivotargeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N-acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsisnucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of ArabidopsisBax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.

Chromium-induced physiological and proteomic alterations in roots of Miscanthus sinensis.

Despite the widespread occurrence of chromium toxicity, its molecular mechanism is poorly documented in plants compared to other heavy metals. To investigate the molecular mechanisms that regulate the response of Miscanthus sinensis roots to elevated level of chromium, seedlings were grown for 4 weeks and exposed to potassium dichromate for 3 days. Physiological, biochemical and proteomic changes in roots were investigated. Lipid peroxidation and H₂O₂ content in roots were significantly increased. Protein profiles analyzed by two-dimensional gel electrophoresis revealed that 36 protein spots were differentially expressed in chromium-treated root samples. Of these, 13 protein spots were up-regulated, 21 protein spots were down-regulated and 2 spots were newly induced. These differentially displayed proteins were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified proteins included known heavy metal-inducible proteins such as carbohydrate and nitrogen metabolism, molecular chaperone proteins and novel proteins such as inositol monophosphatase, nitrate reductase, adenine phosphoribosyl transferase, formate dehydrogenase and a putative dihydrolipoamide dehydrogenase that were not known previously as chromium-responsive. Taken together, these results suggest that Cr toxicity is linked to heavy metal tolerance and senescence pathways, and associated with altered vacuole sequestration, nitrogen metabolism and lipid peroxidation in Miscanthus roots.

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.