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Artik Elisa Angkawijaya†, Van Cam Nguyen†, Farrel Gunawan, and Yuki Nakamura* (2020) A pair of diacylglycerol kinases essential for gametogenesis and ER phospholipid metabolism in leaves and flowers of Arabidopsis. Plant Cell, http://www.plantcell.org/content/early/2020/05/29/tpc.20.00251. (†co-first author; *corresponding author)

Discovery of a new mechanism to produce a key lipid signal for pollen function
Pollen function is crucial for successful fertilization and thus propagation of the plants. A number of agricultural problems are associated with pollen infertility, so a knowledge-based solution is demanded. A lipid compound phosphatidic acid (PA) has been known an important lipid signal during pollen growth. However, how PA is generated and how it affects the pollen function remain elusive.
We hypothesized that PA for pollen function may be produced via unconventional pathway mediated by a type of enzyme called the kinases. We have successfully identified a pair of kinases, named DGK2 and DGK4, which can be found in growing pollen.
Mutation of these kinases led to plant infertility due to pollen growth defect, and the defect was rescued by supplementing PA. We concluded that PA signal produced by DGK2 and DGK4 are essential for pollen function and plant fertility.
Since this type of enzyme is widely found in agronomically important crop species, we consider that the underlying mechanism is highly conserved and the findings may be useful to improve agricultural problems in the future.

An anther of a transgenic Arabidopsis plant that expresses fluorescent DGK2 in the pollen.

Kun-Yang Lin, Wen-Der Wang, Chi-Hung Lin, Elham Rastegari, Yu-Han Su, Yu-Tzu Chang, Yung-Feng Liao, Yi-Chieh Chang, Haiwei Pi, Bo-Yi Yu, Shu-Hwa Chen, Chung-Yen Lin, Mei-Yeh Lu, Tsu-Yi Su, Fei-Yang Tzou, Chih-Chiang Chan & Hwei-Jan Hsu (2020) Piwi reduction in the aged niche eliminates germline stem cells via Toll-GSK3 signaling. Nature Communications 11, Article number: 3147

Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade β-catenin. Disruption of β-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and β-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.

Santos Mary Nia M, Cho Shu-Ting, Wu Chih-Feng, Chang Jeff, Kuo Chih-Horng and Lai Erh-Min* (2020) Redundancy and Specificity of Type VI Secretion vgrG Loci in Antibacterial Activity of Agrobacterium tumefaciens 1D1609 Strain. Front. Microbiol. 10:3004. doi: 10.3389/fmicb.2019.03004 https://www.frontiersin.org/articles/10.3389/fmicb.2019.03004/full

Type VI secretion system (T6SS) is one powerful anti-bacterial and anti-host nanoweapon employed by many Gram-negative bacteria for growth advantages or pathogenesis. A T6SS-harboring bacterium can encode one to multiple VgrG proteins for delivery of cognate effector(s) but the prevalence and biological significance of having sequence redundant vgrG genes have not been comprehensively explored. In this study, the groups of Erh-Min Lai and Chih-Horng Kuo employed phylogenomic analysis to reveal that the majority of Proteobacterial genomes harbor multiple copies of vgrGbut strikingly only a few bacterial genomes have redundant vgrG paralogs. Among them, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Further study revealed that the expansion of vgrG gene clusters in 1D1609 contributed to effector diversity and functional redundancy. The four vgrGs are functionally redundant in T6SS assembly and 1D1609 uses a nuclease as major antibacterial toxin for interbacterial competition. Importantly, the near identical VgrG proteins between two sibling strains (1D1609 and C58) can be exchanged for effector delivery in killing another competing bacterium such as T6SS-lacking Escherichia coli. This led us to propose that Agrobacterium has developed this flexible mode of T6SS effector delivery - using highly similar VgrGs, either produced endogenously or injected from its close relatives. This flexibility could allow them to be an ally to sister cells and an effective weapon against distantly related bacterial competitors. The knowledge gained from this study has advanced our understanding on the secretion mechanism and strategy evolved in agrobacteria or broadly in many bacterial pathogens to compete within microbial community.
Santos MNM, Cho S-T, Wu C-F, Chang C-J, Kuo C-H and Lai E-M (2019) Redundancy and Specificity of Type VI Secretion vgrG Loci in Antibacterial Activity of Agrobacterium tumefaciens 1D1609 Strain. Front. Microbiol. 10:3004. doi: 10.3389/fmicb.2019.03004

A shared belt-and-braces warfare strategy within Agrobacterium strains

Maria Karmella Apaya1, Tien-Fen Kuo1, Meng-Ting Yang1, Greta Yang, Chiao-Ling Hsiao, Song-Bin Chang, Yenshou Ling, Wen-Chin Yang* (2020) Phytochemicals as modulators of β-cells and immunity for the therapy of type 1 diabetes: Recent discoveries in pharmacological mechanisms and clinical potential. Pharmacological Research 156 (2020) 104754

Type 1 diabetes (T1D) is an incurable and lethal autoimmune disease. New remedies for T1D are in great demand. Phytochemicals from natural products are an extraordinary source of discovery of drug leads for diabetes. It was reported that over 90 plant compounds and extracts have beneficial effects on T1D via multiple mechanisms involving the regulation of immunity and/or β cells. Flavonoids, terpenoids, and anthranoids can inhibit starch-digesting enzymes, aldose reductase, MAP kinases, NFκB, and/or IκB kinases implicated in energy metabolism, β-cells, and immunity. Moreover, human clinical trials centering on flavonoids, isoflavonoids, terpenoids, stilbenoids, and polyynes are discussed. An overview of emerging anti-diabetic strategies using plant compounds and extracts for applications in T1D prophylaxis and therapy is also provided.

Kamal Adhikari, I-Wen Lo, Chun-Liang Chen, Yung-Lin Wang Kuan-Hung Lin, Saeid Malek Zadeh, Rajesh Rattinam, Yi-Shan Li, Chang-Jer Wu, and Tsung-Lin Li (2020). Chemoenzymatic Synthesis and Biological Evaluation for Bioactive Molecules Derived from Bacterial Benzoyl Coenzyme A Ligase and Plant Type III Polyketide Synthase Biomolecules. 2020 May; 10(5): 738. doi(10.3390/biom10050738)

Plant type III polyketide synthases produce diverse bioactive molecules with a great medicinal significance to human diseases. Here, we demonstrated versatility of a stilbene synthase (STS) from Pinus Sylvestris, which can accept various non-physiological substrates to form unnatural polyketide products. Three enzymes (4-coumarate CoA ligase, malonyl-CoA synthetase and engineered benzoate CoA ligase) along with synthetic chemistry was practiced to synthesize starter and extender substrates for STS. Of these, the crystal structures of benzoate CoA ligase (BadA) from Rhodopseudomonas palustris in an apo form or in complex with a 2-chloro-1,3-thiazole-5-carboxyl-AMP or 2-methylthiazole-5-carboxyl-AMP intermediate were determined at resolutions of 1.57 Å, 1.7 Å, and 2.13 Å, respectively, which reinforces its capacity in production of unusual CoA starters. STS exhibits broad substrate promiscuity effectively affording structurally diverse polyketide products. Seven novel products showed desired cytotoxicity against a panel of cancer cell lines (A549, HCT116, Cal27). With the treatment of two selected compounds, the cancer cells underwent cell apoptosis in a dose-dependent manner. The precursor-directed biosynthesis alongside structure-guided enzyme engineering greatly expands the pharmaceutical repertoire of lead compounds with promising/enhanced biological activities.

Marimuthu Anandharaj, Yu-Ju Lin, Rizwana Parveen Rani, Eswar Kumar Nadendla, Meng-Chiao Ho, Chieh-Chen Huang, Jan-Fang Cheng, Jui-Jen Chang, and Wen-Hsiung Li* (2020) Constructing a yeast to express the largest cellulosome complex on the cell surface Proceedings of the National Academy of Sciences of the United States of America, 2020, 201916529; DOI: 10.1073/pnas.1916529117

Sustainable utilization of cellulosic biomasses to produce valuable compounds is an ideal approach but hydrolysis of recalcitrant cellulose is complex and time-consuming. Several cellulolytic bacteria produced multi-enzyme complexes called ‘cellulosomes’ that efficiently degrade the cellulose. Hence, we engineered the yeast Kluyveromyces marxianus to express the ‘largest cellulolytic complex’, which can accommodate up to 63 enzymes, on its cell-surface. To achieve this, we have engineered the cellulosomal genes including Scaffoldin protein (CipA), anchoring protein (OlpB) and cellulolytic enzymes into K. marxianus genome. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid swollen cellulose (PASC), respectively, which are higher than any previously constructed yeast cellulosome. In future, this superb cellulosome complex may also be used for the synthesis of various biopharmaceutical products (e.g., astaxanthin and morphine), which involve multiple enzymatic conversion steps.

Chih-Cheng Lin, Ya-Ting Chao, Wan-Chieh Chen, Hsiu-Yin Ho, Mei-Yi Chou, Ya-Ru Li, Yu-Lin Wu, Hung-An Yang, Hsiang Hsieh, Choun-Sea Lin, Fu-Hui Wu, Shu-Jen Chou, Hao-Chung Jen, Yung-Hsiang Huang, Deli Irene, Wen-Jin Wu, Jian-Li Wu, Daniel J. Gibbs, Meng-Chiao Ho*, and Ming-Che Shih* (2019) Regulatory cascade involving transcriptional and N-end rule pathways in rice under submergence. Proc. Natl. Acad. Sci. (2019) 116(8): 3300-3309. (https://doi.org/10.1073/pnas.1818507116)

The rice SUB1A-1 gene, which encodes a group VII ethylene response factor (ERFVII) plays a pivotal role in rice survival under flooding stress, as well as other abiotic stresses. In Arabidopsis, five ERFVII factors play roles in regulating hypoxic responses. A characteristic feature of Arabidopsis ERFVIIs is a destabilizing N-terminus, which functions as an N-degron that targets them for degradation via the oxygen-dependent N-end rule pathway of proteolysis, but permits their stabilization during hypoxia for hypoxia-responsive signaling. Despite having the canonical N-degron sequence, SUB1A-1 is not under N-end rule regulation, suggesting a distinct hypoxia signaling pathway in rice during submergence. Herein we show that two other rice ERFVIIs, ERF66 and ERF67, are directly transcriptionally up-regulated by SUB1A-1 under submergence. In contrast to SUB1A-1, ERF66 and ERF67 are substrates of the N-end rule pathway, which are stabilized under hypoxia and may be responsible for triggering a stronger transcriptional response to promote submergence survival. In support of this, overexpression ERF66 or ERF67 leads to activation of anaerobic survival genes and enhanced submergence tolerance. Furthermore, using structural and protein-interaction analyses, we show that the C-terminus of SUB1A-1 prevents its degradation via the N-end rule and directly interacts with the SUB1A-1 N-terminus, which may explain enhanced stability of SUB1A-1 despite bearing an N-degron sequence. In summary, our results suggest that SUB1A-1, ERF66, and ERF67 form a regulatory cascade involving transcriptional and N-end rule control, which allows rice to distinguish flooding from other SUB1A-1-regulated stresses.

The proposed model of the regulatory cascade of SUB1A-1, ERF66, and ERF67 that involves transcriptional and N-end rule pathways in response to submergence stress in submergence-tolerant rice cultivars.

Reena Sharma, Kuo-Chen Yeh* (2019). The dual benefit of a dominant mutation in Arabidopsis IRON DEFICIENCY TOLERANT1 for iron biofortification and heavy metal phytoremediation. Plant Biotechnology Journal https://doi.org/10.1111/pbi.13285

One of the goals of biofortification is to generate iron‐enriched crops to combat growth and developmental defects especially iron (Fe) deficiency anemia. Genetic engineering is an alternative approach for Fe biofortification but so far strategies to increase Fe content have only encompassed a few genes with limited success. In our study, we demonstrate that the Ethyl methanesulfonate (EMS) mutant, iron deficiency tolerant1 (idt1) can accumulate 4‐7 times higher amounts of Fe than the wild type in roots, shoots and seeds, and exhibits the metal tolerance and iron accumulation (Metina) phenotype in Arabidopsis. The C to T transition mutation resulting in substitution of alanine to valine at amino acid position 320 of bHLH34 (designated as IDT1A320V) in a conserved motif among mono‐ and dicots was found to be responsible for a dominant phenotype that possesses constitutive activation of the Fe regulatory pathway. Overexpression of IDT1A320V in Arabidopsis and tobacco led to the Metina phenotype; a phenotype that has escalated specificity towards optimizing Fe homeostasis and may be useful in Fe‐biofortification. Knowledge of the high tolerance and accumulation of heavy metals of this mutant can aid the development of tools for phytoremediation of contaminants.

Bou-Yun Lin, Chueh-Ju Shih, Hsin-Yu Hsieh, Hsiu-Chen Chen, Shih-Long Tu (2019) Phytochrome coordinates with a hnRNP to regulate alternative splicing via an exonic splicing silencer. Plant Physiol pp.00289.2019

Phytochromes play vital roles among plant growth and development in order to sense and optimize the gene expression at different level according to the changing environment. In addition to transcription factors, which directly regulate gene expression, accumulating evidence has showed that pre-mRNA splicing is also necessary for the regulation after exposing to light. Dr. Tu led his research group and discovered that in moss Physcomitrella patens, the red-light photoreceptor interacts with a splicing regulator PphnRNP-F1 in a red light dependent manner. Moreover, the interaction between phytochrome and PphnRNP-F1 might increase the protein level of the splicing regulator, PphnRNP-F1. Furthermore, the PphnRNP-F1 could be further recruited to the pre-mRNA by the cis-regulatory element located on the exonic region. Dr. Tu’s finding provides a novel phytochrome-mediated signaling pathway for pre-mRNA splicing regulation.

Maria Karmella Apaya, Jeng-Yuan Shiau, Guo-Shiou Liao, Yu-Jen Liang, Chia-Wei Chen, Hsin-Chou Yang, Chi-Hong Chu, Jyh-Cherng Yu and Lie-Fen Shyur (2019). Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer. J Exp Clin Cancer Res. 38(1):187.

Current prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Avenues for molecular stratification and personalized therapy for TNBC patients are thus needed. Oxylipins, a class of bioactive lipids derived from the dietary fatty acids by the action of CYP450, cyclooxygenases and lipoxygenases may offer new insights towards this direction. In this study, we examined the global oxylipin metabolite profiles of normal mammary tissues and breast cancer tumors and show the importance of epoxyeicosatrienoic acids (EETs), an oxylipin derived from the action of CYP epoxyenases on arachidonic acid, in relation to hormone receptor subtype, metastasis status and prospective clinical outcomes. We used integrative genomics, proteomics and metabolomics data from public cohorts, i.e., The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data sets, and actual patient specimens. In vitro functional assays using a panel of breast cancer cell lines were also carried out to further confirm the roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Our findings uncover a new set of vulnerabilities and associated biomarkers within the TNBC lipid metabolite-protein-gene network and provide a clinically-relevant roadmap for the development of personalized intervention strategies for TNBC patients.

Chueh-Ju Shih, Hsiang-Wen Chen, Hsin-Yu Hsieh, Yung-Hua Lai1 Fang-Yi Chiu, Yu-Rong Chen and Shih-Long Tu (2019) Heterogeneous Nuclear Ribonucleoprotein H1 Coordinates with Phytochrome and the U1 snRNP Complex to Regulate Alternative Splicing in Physcomitrella patens The Plant Cell, Vol. 31: 2510–2524

Alternative splicing is of importance to organisms, producing various protein isoforms in response to environmental changes. A research team led by Dr. Tu discovered that in moss Physcomitrella patens, the red-light photoreceptor interacts with the splicing factor PphnRNP-H1 in a red light dependent manner. Furthermore, PphnRNP-H1 interacts with PpPRP39-1, one of spliceosome components which functions in pre-mRNA splicing. Our results suggest that phytochromes target the early step of spliceosome assembly via a cascade of protein–protein interactions to control pre-mRNA splicing.

Chih-Cheng Chien, Mei-Yi Chou, Chun-Yi Chen, and Ming-Che Shih. (2019) Analysis of genetic diversity of Xanthomonas oryzae pv. oryzae populations in Taiwan. Sci. Rep. 9, 316. doi:10.1038/s41598-018-36575-x.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major rice disease. In Taiwan, the tropical indica type of Oryza sativa originally grown in this area is mix-cultivated with the temperate japonica type of O. sativa, and this might have led to adaptive changes of both rice host and Xoo isolates. In order to better understand how Xoo adapts to this unique environment, we collected and analyzed fifty-one Xoo isolates in Taiwan. Three different genetic marker systems consistently identified five groups. Among these groups, two of them had unique sequences in the last acquired ten spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) region, and the other two had sequences that were similar to the Japanese isolate MAFF311018 and the Philippines isolate PXO563, respectively. The genomes of two Taiwanese isolates with unique CRISPR sequence features, XF89b and XM9, were further completely sequenced. Comparison of the genome sequences suggested that XF89b is phylogenetically close to MAFF311018, and XM9 is close to PXO563. Here, documentation of the diversity of groups of Xoo in Taiwan provides evidence of the populations from different sources and hitherto missing information regarding distribution of Xoo populations in East Asia

Punsaldulam Dashnyam, Ramesh Mudududdla, Tung-Ju Hsieh, Ting-Chien Lin, Hsien-Ya Lin, Peng-Yuan Chen, Chia-Yi Hsu, and Chun-Hung Lin. (2018) β-Glucuronidases of opportunistic bacteria are the major contributors to xenobiotic-induced toxicity in the gut. Sci. Rep. 8:16372, doi:10.1038/s41598-018-34678-z.

Gut bacterial β-D-glucuronidases (GUSs) catalyze the removal of glucuronic acid from liver-produced β-D-glucuronides. These reactions can have deleterious consequences when they reverse xenobiotic metabolism. The human gut contains hundreds of GUSs of variable sequences and structures. To understand how any particular bacterial GUS(s) contributes to global GUS activity and affects human health, the individual substrate preference(s) must be known. Herein, we report that representative GUSs vary in their ability to produce various xenobiotics from their respective glucuronides. To attempt to explain the distinct substrate preference, we solved the structure of a bacterial GUS complexed with coumarin-3-β-D-glucuronide. Comparisons of this structure with other GUS structures identified differences in loop 3 (or the α2-helix loop) and loop 5 at the aglycone-binding site, where differences in their conformations, hydrophobicities and flexibilities appear to underlie the distinct substrate preference(s) of the GUSs. Additional sequence, structural and functional analysis indicated that several groups of functionally related gut bacterial GUSs exist. Our results pinpoint opportunistic gut bacterial GUSs as those that cause xenobiotic-induced toxicity. We propose a structure-activity relationship that should allow both the prediction of the functional roles of GUSs and the design of selective inhibitors.

Huei-Hsuan Tsai, Jorge Rodriguez-Celma, Ping Lan, Yu-Ching Wu, Isabel Cristina Vélez-Bermúdez and Wolfgang Schmidt. (2018) Scopoletin 8-Hydroxylase-Mediated Fraxetin Production is Crucial for Iron Mobilization. Plant physiol. 2018 Mar 20. doi: 10.1104/pp.18.00178.

As a key edaphic factor, soil pH has a strong impact on the availability of mineral nutrients and the distribution of species in natural plant communities. Iron solubility decreases dramatically with increasing pH, excluding so-called calcifuge (‘chalk-fleeing’) species from carbonate-rich, alkaline soils due to their inability to acquire sufficient Fe under such conditions. The question as to the traits that allow calcicole plants to thrive on neutral or alkaline soil has puzzled plant biologists for more than two centuries. In collaboration with the metabolomics core facility at IPMB, Wolfgang Schmidt’s group showed that scopoletin 8-hydroxylase (S8H; At3g12900) participates in Fe acquisition by mediating an as yet undocumented step in the biosynthesis of fraxetin (7,8-dihydroxy-6-methoxycoumarin), a coumarin derived from the scopoletin pathway. Fraxetin exhibited Fe-reducing properties in vitro with higher rates being observed at neutral relative to acidic pH. In natural Arabidopsis accessions differing in their performance on media containing immobilized Fe, the amount of secreted fraxetin was highly correlated with growth, Fe and chlorophyll content, indicating that fraxetin secretion is a decisive factor for calcicole-calcifuge behavior of plants.

Van Cam Nguyen, Yuki Nakamura and Kazue Kanehara (2019) Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana. Plant J https://doi.org/10.1111/tpj.14338

Endoplasmic reticulum (ER) stress is caused by a variety of factors including abiotic stresses and biotic stresses, which lead to a disturbance of cellular homeostasis for protein biogenesis and glycerolipid biosynthesis in the ER. The current study provides conceptual advances in understanding the circumvention mechanism of ER stress by the desaturation of ER membrane lipid. The relationship between the polyunsaturated fatty acid metabolism and stress adaptation is clarified by investigating biological functions of FATTY ACID DESATURASE 2 (FAD2) which converts oleic acid (18:1) to linoleic acid (18:2) in the acyl groups of ER-localized phospholipids. Our results indicate the importance of hydrophobic tails in the lipid bilayer, which advances the understanding of membrane lipid function in the stress response in plants and beyond. (Nguyen et al, 2019, The Plant Journal. https://onlinelibrary.wiley.com/doi/10.1111/tpj.14338)

Min May Wong, Govinal Badiger Bhaskara, Tuan-Nan Wen, Wen-Dar Lin, Thao Thi Nguyen, Geeng Loo Chong, and Paul E. Verslues* (2019). Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook Like10 phosphorylation required for stress growth regulation. Proceedings of the National Academy of Sciences of the United States of America. 116(6): 2354-2363. doi: 10.1073/pnas.1819971116

The mechanisms plants use to detect and signal abiotic stress are not well understood but have major implications for plant productivity during periods of drought and other environmental stresses. Clade A Protein Phosphatases 2Cs (PP2Cs) are part of the core signaling pathway of the stress hormone Abscisic Acid which regulates many plant stress responses. In previous research, the Verslues laboratory noted that the Clade A PP2C Highly ABA-Induced 1 (HAI1) has especially prominent effects on drought phenotypes such as growth and proline accumulation (Bhaskara et al., 2012). To investigate how HAI1 affects drought response, phosphoproteomic analysis of the hai1-2 mutant was conducted for plants under unstressed conditions or after low water potential (drought) treatment (Wong et al., 2019 PNAS). This identified more than 100 phosphopeptides of increased abundance in hai1-2, indicating that phosphorylation status of these sites could be regulated by HAI1 directly of via HAI1-regulated kinases. Further experiments on At-Hook Like 10, a nuclear localized DNA-binding protein of unclear function, demonstrated that it could be directly dephosphorylated by HAI1. The S314 phosphorylation site identified in our proteomics analysis was crucial for AHL10 regulation of growth and gene expression during low water potential stress and was also required for AHL10 complexes to form nuclear foci. By identifying HAI1-affected phosphoproteins and functionally important AHL10 phosphorylation site, these results elucidate HAI1 and AHL10 function and also demonstrate a mechanism plants use to balance maximal growth versus robust response to environmental stress.

Shang Jye Leong, Wen-Chien Lu and Tzyy-Jen Chiou (2018) Phosphite-mediated suppression of anthocyanin accumulation regulated by mitochondrial ATP synthesis and sugars in Arabidopsis. Plant and Cell Physiology 59(6): 1158–1169.

Despite the essential role of phosphate (Pi) in plant growth and development, how plants sense and signal the change of Pi supply to adjust its uptake and utilization is not yet well understood. Pi itself has been proposed to be a signaling molecule that regulates Pi starvation responses (PSRs) because phosphite (Phi), a non-metabolized Pi analog, suppresses several PSRs. In this study, we identified a phosphite-insensitive1 (phi1) mutant which retained anthocyanin, a visible PSR, in Phi-containing but Pi-deficient medium. phi1 mutants were impaired in the gene encoding an FAd subunit of mitochondrial F1Fo-ATP synthase and showed a reduced mitochondrial ATP level in roots, growth hypersensitivity to oligomycin and an increased mitochondrial membrane potential, suggesting that this gene has a crucial role in mitochondrial ATP synthesis. phi1 mutants accumulated a high level of sugars in shoots, which may account for the increased accumulation of anthocyanin and starch in Phi-containing conditions. Gene expression analysis showed that a subset of genes involved in carbohydrate metabolism in phi1 was misregulated in response to Phi. The majority of genes were repressed by Pi starvation and, unlike wild-type plants, their repression in phi1 was not affected by the addition of Phi. Our findings show that defective mitochondrial ATP synthesis results in sugar accumulation, leading to alteration of Phi-mediated suppression of PSRs. This study reinforces the role of sugars, and also reveals a cross-talk among ATP, sugars and Pi/Phi molecules in mediating PSRs.

Chien-Yun Lee, Chu-Cheng Lin, Yi-Liang Liu, Guang-Yaw Liu, Jyung-Hurng Liu, Hui-Chih Hung*. (2017) Molecular interplay between the dimer interface and the substrate-binding site of human peptidylarginine deiminase 4. Scientific Reports 7: 42662.

Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should bea dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis.

Chien-Yun Lee, Dongxue Wang, Mathias Wilhelm, Daniel P Zolg, Tobias Schmidt, Karsten Schnatbaum, Ulf Reimer, Fredrik Pontén, Mathias Uhlén, Hannes Hahne, Bernhard Kuster. (2018) Mining the human tissue proteome for protein citrullination. Molecular & cellular proteomics 17(7):1378-1391.

Citrullination is a posttranslational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations, and while the modification has been linked to several diseases, including rheumatoid arthritis (RA) and cancer, its physiological or pathophysiological roles remain largely unclear. In part, this is owing to limitations in available methodology to robustly enrich, detect, and localize the modification. As a result, only a few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass-spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra, which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused ith deamidation, we synthetized ~2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins.

Anh H. Ngo, Ying-Chen Lin, Yu-chi Liu, Katharina Gutbrod, Helga Peisker, Peter Dörmann and Yuki Nakamura* (2018) A pair of non-specific phospholipases C, NPC2 and NPC6, is involved in gametophyte development and glycerolipid metabolism in Arabidopsis. New Phytologist. 2018 April 14. doi: 10.1111/nph.15147.

Plant reproduction is a key to propagate species and rich harvest of agricultural product. A research team led by Dr. Nakamura discovered in Arabidopsis thaliana that successful gametogenesis requires a pair of non-specific phospholipase C (NPC), NPC2 and NPC6, a unique family of lipase found only in seed plants and certain bacterial species. Due to its potentially toxic activity to non-specifically degrades the cellular membrane lipids, this type of enzyme was thought to function only under certain stress conditions. Our finding on the function of NPC2 and NPC6, two last uncharacterized NPC family member, shed light on critical function of phospholipid catabolism in plant reproductive development. This work was conducted in collaboration with Prof. Peter Dörmann (University of Bonn, Germany).

Po-Yuan Shih, Shu-Jen Chou, Caroline Müller, Barbara Ann Halkier, Rosalia Deeken, and Erh-Min Lai* (2018) Differential Roles of Glucosinolates and Camalexin at Different Stages of Agrobacterium-Mediated Transformation. Molecular Plant Pathology 2018 Mar 2. doi: 10.1111/mpp.12672.

Agrobacterium tumefaciens is a plant pathogen, which causes crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. At the different transformation stages, many plant genes were known to regulate the transient and stable transformation. However, how plant secondary metabolites involved in this transformation process are still unknown. Erh-Min Lai’s group at the Institute of Plant and Microbial Biology studied the roles of the plant secondary metabolites, glucosinolates and camalexin, at the different stages of Agrobacterium transformation (Shih et al., 2018, Molecular Plant Pathology). Based on the Arabidopsis mutant studies, metabolite profiling and exogenous metabolite applications, they found that the indole glucosinolate (iGS) hydrolysis serves as the first layer to defend Agrobacterium infection at the early stage, and then camalexin accumulation plays an inhibitory role for tumor formation at the later stage. In addition, some aliphatic glucosinolate hydrolysis products (aGS-ITC) were shown to have various effects to control transient transformation efficiency on Arabidopsis seedlings. This study revealed the differential roles of glucosinolates and camalexin at different stages of Agrobacterium-mediated transformation and provided new insights into crown gall disease control and improvement of plant transformation.

Yueh Cho, Chao-Yuan Yu, Yuki Nakamura and Kazue Kanehara* (2017) Arabidopsis dolichol kinase AtDOK1 is involved in flowering time control – Journal of Experimental Botany. DOI: 10.1093/jxb/erx095

Dolichols are a class of isoprenoids that play crucial roles for protein glycosylation because an oligosaccharide is assembled on a lipid carrier, dolichol diphsophate. Dr. Kazue Kanehara’s group previously identified DOLICHOL KINASE 1 (AtDOK1) that catalyzes the last step of dolichol phosphate biosynthesis in Arabidopsis [Plant J. (2015) 81, 292-303]. In this study, Dr. Kanehara group demonstrated that AtDOK1 was involved in flowing time control by use of a microRNA-mediated gene suppression technique because knockout of AtDOK1 causes lethality. Indeed, AtDOK1 was highly expressed in emerging shoot apical meristems as well as inflorescence and floral meristems as shown in the figure. Furthermore, a subcellular localization study indicated that AtDOK1 was localized at the endoplasmic reticulum (ER) in Arabidopsis. Dr. Kanehara group proposes the ER-localized catalytically active AtDOK1 is highly expressed in the meristems and possesses important roles in the control of flowering time, possibly by post-transcriptional regulations.

Chun-Ming Lai, Kun-Yang Lin, Shih-Han Kao, Yi-Ning Chen, Fu Huang, and Hwei-Jan Hsu* (2017) Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion. The Journal of Cell Biology 10.1083

Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. We report that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. We also report that Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell-cell adhesion by negative regulation of E-cadherin expression. Our results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation.

Yao-Pin Lin, Meng-Chen Wu, and Yee-yung Charng* Identification of Chlorophyll Dephytylase Involved in Chlorophyll Turnover, The Plant Cell, 2016, 28, 2974-2990

In photosynthesis, chlorophylls absorb light energy, which is transformed into chemical energy by the multi-protein-pigment complex called photosystem. The photosystem, composing of chlorophylls and proteins, tends to be damaged during the process of photosynthesis. The plant cells must repair the damaged photosystem to maintain the photosynthetic efficiency, causing the bound chlorophylls to turnover. Previous studies suggest that chlorophylls in damaged photosystem would be salvaged by two successive steps: 1. Dephytylation of chlorophylls; 2. Rephytylation of the dephytylated chlorophylls (or chlorophyllides). The latter is catalyzed by chlorophyll synthase, but the enzyme involved in the first reaction has been enigmatic. More than a century, chlorophyllase has been the only enzyme known to hydrolyze chlorophyll into chlorophyllide in vitro. However, the role of chlorophyllase in chlorophyll catabolism remains controversial. In an effort in cloning the mutant gene responsible for a heat sensitive phenotype in Arabidopsis, we identified a novel chloroplast protein capable of hydrolyzing chlorophylls and named it chlorophyll dephytylase1 (CLD1). The results from genetic and biochemical experiments suggested that CLD1 is involved in the first step of the chlorophyll salvage cycle. Plants lacking CLD1 significantly reduced the photosynthetic efficiency and viability under prolonged heat stress, underscoring the importance of the chlorophyll salvage cycle for plant thermotolerance. The discovery of CLD1 fills the gap in chlorophyll metabolism and facilitates further studies in the understanding of photosystem repair and its regulation.

Yueh Cho and Kazue Kanehara*(2017) Endoplasmic reticulum stress response in Arabidopsis roots – Frontier in Plant Science. 8: 144.

Roots are the frontier of plant body to perceive underground environmental change. ER stress response represents circumvention of cellular stress caused by various environmental changes; however, a limited number of studies are available on ER stress responses in roots. Here, we report the tunicamycin (TM) -induced ER stress response in Arabidopsis roots by monitoring expression patterns of BiP3, a representative marker for the response. Roots promptly responded to the TM-induced ER stress through induction of the similar sets of ER stress-responsive genes. However, not all cells responded uniformly to the TM-induced ER stress in roots, as BiP3 was highly expressed in root tips, an outer layer in elongation zone, and an inner layer in mature zone of roots. We suggest that ER stress response in roots has tissue specificity.

Ying-Chen Lin, Koichi Kobayashi, Chun-Hsien Hung, Hajime Wada, and Yuki Nakamura* (2016) Arabidopsis PHOSPHADYLGLCEROPHOSPHATE PHOSPHATASE1 (PGPP1) Involved in Phosphatidylglycerol Biosynthesis and Photosynthetic Function. Plant J. DOI: 10.1111/tpj.13311 http://onlinelibrary.wiley.com/doi/10.1111/tpj.13311/full

Phosphatidylglycerol (PG) is an indispensible lipid constituent of photosynthetic membrane, whose function is essential in photosynthetic activity. In higher plants, the biological function of the last step of PG biosynthesis remains elusive because an enzyme catalyzing this reaction step, namely, phosphatidylglycerophosphate phosphatase (PGPP), has been a missing piece in the entire glycerolipid metabolic map. In this study, Dr. Nakamura’s team reported AtPGPP1 encoding a PGPP in Arabidopsis thaliana. Heterologous expression of AtPGPP1 in yeast Δgep4 complemented growth defect phenotype and PG-producing activity, suggesting that AtPGPP1 encodes functional PGPP. The knockout mutant of AtPGPP1, pgpp1-1, showed pale-green phenotype with reduced PG and chlorophyll contents, defective development of chloroplast, as well as impaired photosynthetic activity. This study identified that AtPGPP1 is a primary plastidic PGPP required for PG biosynthesis and photosynthetic function in Arabidopsis.

Tzu-Yin Liu, Teng-Kuei Huang, Shu-Yi Yang, Yu-Ting Hong, Sheng-Min Huang, Fu-Nien Wang, Su-Fen Chiang, Shang-Yueh Tsai, Wen-Chien Lu and Tzyy-Jen Chiou* (2016)Identification of plant vacuolar transporters mediating phosphate storage Nature Communications 7:11095

Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. Here we demonstrate that the SPX-MFS proteins, designated as Phosphate Transporter 5 family (PHT5), also named Vacuolar Phosphate Transporter (VPT), function as vacuolar Pi transporters. Based on 31P-magnetic resonance spectroscopy analysis, Arabidopsis pht5;1 loss-of-function mutants accumulate less Pi and exhibit a lower vacuolar-to-cytoplasmic Pi ratio than controls. Conversely, overexpression of PHT5 leads to massive Pi sequestration into vacuoles and altered regulation of Pi starvation-responsive genes. Furthermore, we show that heterologous expression of OsSPX-MFS1, the rice PHT5 homolog, OsSPX-MFS1 mediates Pi influx to yeast vacuoles. Our findings uncover a group of Pi transporters in vacuolar membranes that regulate the cytoplasmic Pi homeostasis required for the fitness of plant growth.

Devanand D. Bondage, Jer-Sheng Lin, Lay-Sun Ma, Chih-Horng Kuo and Erh-Min Lai* (2016) VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex- Proceedings of the National Academy of Sciences of the United States of America: doi:10.1073/pnas.1600428113.

Type VI secretion system (T6SS) is a molecular weapon used by many Gram-negative bacteria to inject effectors into eukaryotic host cells or competing prokaryotes for bacterial survival and fitness. T6SS involves multiple strategies for effector delivery via fusion or interaction of effectors to structural components of the phage tail-like structure. However, the identity of the molecular components and mechanisms that governs the delivery of type VI effectors remains limited. The research team (Bondage et al.) at the Institute of Plant and Microbial Biology studied the delivery mechanism of antibacterial type VI effectors, namely Tde1 and Tde2 DNase toxins, in Agrobacterium tumefaciens. They identified the protein components named as spike (VgrG1 and VgrG2 protein), adaptor/chaperone (Tap-1 and Atu3641), and a sharp tip (PAAR) are required for delivery of cognate Tde toxin. Importantly, the divergent C-terminus of VgrG1 and VgrG2 spike proteins governs the Tde toxin delivery specificity. In-depth genetic and biochemical evidence demonstrated that VgrG1 C-terminus is the molecular determinant required for interacting with a specific adaptor/chaperone protein and PAAR tip protein to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. Further genome-wide survey discovered the widespread presence of these genetic modules in various Proteobacteria, suggesting a conserved mechanism in type VI effector delivery.

Hsing-Yi Cho, Tuan-Nan Wen, Ying-Tsui Wang and Ming-Che Shih* (2016) Quantitative Phosphoproteomics of Protein Kinase SnRK1 regulated protein phosphorylation in Arabidopsis under Submergence - Journal of Experimental Botany. 67(9): 2745-60.

SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of plant stress and energy starvation signaling pathways. We observed in this study that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1K48M) had lower tolerance to submergence than the wild-type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signaling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3 h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We further showed that the energy conservation was disrupted in SnRK1.1K48M, mpk6 and PTP1S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signaling factors related to submergence.

Ding Hua Lee, Yu-Hsin Kao, Jia-Chi Ku, Chien-Yu Lin, Robert Meeley, Ya-Shiun Jan, Chung-Ju Rachel Wang* (2015) The axial element protein DESYNAPTIC2 mediates meiotic double-strand break formation and synaptonemal complex assembly in maize. The Plant Cell, 27(9), 2516-2529.

During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient, but tight connection between homologous chromosomes. We showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize. The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RAD51 foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super resolution structured illumination microscopy showed that the DSY2 protein is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASY1. In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZYP1 is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2, but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis.

Bayanmunkh Baatar, Pei-Wen Chiang, Denis Yu Rogozin, Yu-Ting Wu, Ching-Hung Tseng, Cheng-Yu Yang, Hsiu-Hui Chiu, Bolormaa Oyuntsetseg, Andrey G. Degermendzhy, Sen-Lin Tang . (2016) Bacterial Communities of Three Saline Meromictic Lakes in Central Asia. PLOS ONE. DOI: 10.1371/journal.pone.0150847

Meromictic lakes are unique ecosystems with water profiles strongly stratified chemically and incompletely mixed over multi-year intervals. Approximately 200 saline meromictic lakes have been discovered, corresponding to <1% of all known lakes. The water columns of these lakes are commonly stratified into two major zones, the mixolimnion (i.e., upper, oxygenic water) and monimolimnion (i.e., bottom, anoxic water), which are frequently separated by a chemocline (transition zone). Stratification generally results from stable gradients in physiochemical factors in these lakes. The goals of this study were to characterize and compare bacterial communities throughout the water column and determine associations between environmental parameters and bacterial populations among individual layers of these meromictic lakes. Bacterial community composition and biodiversity of the three lakes were determined by 454-pyrosequencing the V1/V2 hyper-variable regions of the 16S rRNA gene. There were clear similarities and differences in bacterial community structure among lakes, as well as unique bacterial profiles in distinct water layers. Furthermore, associations between bacterial community and environmental parameters were explored. Finally, this was the first report to characterize the bacterial community and diversity in Lake Oigon.

Frank DiMaio†, Chun-Chieh Chen†, , Xiong Yu, Brandon Frenz, Yau-Heiu Hsu*, Na-Sheng Lin* and Edward H. Egelman* (2015) The molecular basis for flexibility in the flexible filamentous plant viruses. Nature Structural & Molecular Biology, Volume 22, pp. 642-644.

Viruses that are responsible for more than half the viral crop damage in the world, but have also been viewed as potentially useful for biotechnology, such as in vaccines or recombinant protein production in plants. Structural studies of these viruses date from 1941, but unfortunately no atomic model has been possible due to the fact that the viruses cannot be crystallized and have proven to be too flexible for high-resolution x-ray fiber diffraction or electron cryo-microscopy (cryo-EM). In contrast, tobacco mosaic virus (TMV), the first virus to be discovered, is a rigid filamentous plant virus that has been a model system in structural biology and virology and atomic models of TMV have been produced by both x-ray diffraction and cryo-EM. We have taken advantage of new advances in direct electron detectors to solve the cryo-EM structure of bamboo mosaic virus (BaMV) at 5.6 Å resolution, which when combined with a crystal structure of a large fragment from a homolog (papaya mosaic virus, PapMV) allows for a full atomic model to be built. It has been widely assumed that the flexible filamentous plant viruses are right-handed, like TMV, but we show that the RNA and protein subunits follow a left-handed path. While the rigid TMV is assembled from compact subunits, BaMV contains a compact core with extended N- and C-terminal portions that make contacts with surrounding subunits in both the same and adjacent helical turns. The flexible links between the core and the extensions allow the virion to bend and twist while still maintaining its structural integrity. The new finding will help the understanding BaMV, a flexible filamentous virus, in its CP interaction with viral RNA, its virion assembly, its structural stability, the development of viral vectors and can be potentially useful for biotechnology, such as in vaccines, biomaterials for drug delivery or imaging, or recombinant protein production in plants.

Yao-Pin Lin, Tsung-yuan Lee, Ayumi Tanaka, and Yee-yung Charng* (2014) Analysis of an Arabidopsis heat-sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover. The Plant Journal, DOI: 10.1111/tpj.12611

Chlorophylls, the most abundant pigments in the photosynthetic apparatus, are constantly turned over as a result of the degradation and replacement of the damage-prone reaction center D1 protein of photosystem II. Results from isotope labeling experiments suggest that chlorophylls are recycled by reutilization of chlorophyllide and phytol, but the underlying mechanism is unclear. In this study, by characterization of a heat-sensitive Arabidopsis mutant we provide evidence of a salvage pathway for chlorophyllide a. A missense mutation in CHLOROPHYLL SYNTHASE (CHLG) was identified and confirmed to be responsible for a light-dependent, heat-induced cotyledon bleaching phenotype. Following heat treatment, mutant (chlg-1) but not wild-type seedlings accumulated a substantial level of chlorophyllide a, which resulted in a surge of phototoxic singlet oxygen. Immunoblot analysis suggested that the mutation destabilized the chlorophyll synthase proteins and caused a conditional blockage of esterification of chlorophyllide a after heat stress. Accumulation of chlorophyllide a after heat treatment occurred during recovery in the dark in the light-grown but not the etiolated seedlings, suggesting that the accumulated chlorophyllides were not derived from de novo biosynthesis but from de-esterification of the existing chlorophylls. Further analysis of the triple mutant harboring the CHLG mutant allele and null mutations of CHLOROPHYLLASE1 (CLH1) and CLH2 indicated that the known chlorophyllases are not responsible for the accumulation of chlorophyllide a in chlg-1. Taken together, our results show that chlorophyll synthase acts in a salvage pathway for chlorophyll biosynthesis by re-esterifying the chlorophyllide a produced during chlorophyll turnover.

Wei-Yi Lin, Teng-Kuei Huang, Tzyy-Jen Chiou* (2013) NITROGEN LIMITATION ADAPTATION, a Target of MicroRNA827, Mediates Degradation of Plasma Membrane–Localized Phosphate Transporters to Maintain Phosphate Homeostasis in Arabidopsis. The Plant Cell, DOI 10.1105/tpc.113.116012

Members of the Arabidopsis thaliana PHOSPHATE TRANSPORTER1 (PHT1) family are key players in acquisition of Pi from the rhizosphere, and their regulation is indispensable for the maintenance of cellular Pi homeostasis. Here, we reveal posttranslational regulation of Pi transport through modulation of degradation of PHT1 proteins by the RING-type ubiquitin E3 ligase, NITROGEN LIMITATION ADAPTATION (NLA). Loss of function of NLA caused high Pi accumulation resulting from increases in the levels of several PHT1s at the protein rather than the transcript level. Evidence of decreased endocytosis and ubiquitination of PHT1s in nla mutants and interaction between NLA and PHT1s in the plasma membranes suggests that NLA directs the ubiquitination of plasma membrane–localized PHT1s, which triggers clathrin-dependent endocytosis followed by endosomal sorting to vacuoles. Furthermore, different subcellular localization of NLA and PHOSPHATE2 (PHO2; a ubiquitin E2 conjugase) and the synergistic effect of the accumulation of PHT1s and Pi in nla pho2 mutants suggest that they function independently but cooperatively to regulate PHT1 protein amounts. Intriguingly, NLA and PHO2 are the targets of two Pi starvation-induced microRNAs, miR827 and miR399, respectively. Therefore, our findings uncover modulation of Pi transport activity in response to Pi availability through the integration of a microRNA-mediated posttranscriptional pathway and a ubiquitin-mediated posttranslational regulatory pathway.

Yu-Hsin Hsieh and Sheng-Yang Wang. Lucidone from Lindera erythrocarpa suppresses adipogenesis in 3T3-L1 cells and attenuates obesity and consequent metabolic disorders in high-fat diet C57BL/6 mice. Phytomedicine 20:394-400.

The incidence of obesity has increased dramatically worldwide over the last few decades because of the life style changes. Obesity is associated with an increased risk for the development of a spectrum of diseases such as heart disease and type 2 diabetes that reduce quality of life but raise mortality. Meanwhile, the cost of drugs for obesity-related diseases is often high because it is a lifetime drug expenditure. Medicinal plants and the derived phytocompounds have been used worldwide because plant metabolites have been proven to confer various bioactivities, and are thus considered an abundant resource for new drug development. We selected specific phytocompounds that exhibit a potential on regulating the differentiation and maturation of adipocytes. Furthermore, we characterized the in vivo effect of the phytocompound in mice with high-fat diet induced obesity by observing the changes of physiological and metabolic variables. The data here reveals the potential of our target compound as a nutraceutical for preventing obesity and the consequent metabolic disorders that we often confront with under unhealthy eating habits.

Pablo Bolaños-Villegas, Xiaohui Yang, Huei-Jing Wang, Chien-Ta Juan, Min-Hsiang Chuang, Christopher A. Makaroff and Guang-Yuh Jauh. (2013). Arabidopsis CHROMOSOME TRANSMISSION FIDELITY 7 (AtCTF7/ECO1) is required for DNA repair, mitosis and meiosis. Plant Journal doi: 10.1111/tpj.12261.

In baker’s yeast inactivation of acetyltransferase Eco1 typically disrupts chromosome segregation and homologous recombination-dependent DNA repair in dividing cells, ultimately resulting in lethality. We report here the isolation and detailed characterization of two homozygous T-DNA insertion mutants for the Arabidopsis thaliana Eco1 homolog, CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1), called ctf7-1 and ctf7-2. Mutants exhibited dwarfism, poor anther development and sterility. Analysis of somatic tissues by flow cytometry, scanning electron microscopy and quantitative real-time PCR identified defects in DNA repair and cell division, including an increase in the area of leaf epidermal cells, an increase in DNA content and the upregulation of genes involved in DNA repair such as PARP2. Analysis of meiocytes identified changes in chromosome morphology and defective segregation (shown in the picture above, scale bar: 10 µm). The abundance of chromosomal-bound cohesion subunits was also reduced. Transcript levels for several meiotic genes, including the recombinase DMC1 and the S-phase licensing factor CDC45 were elevated in mutant anthers. Taken together these results demonstrate that Arabidopsis CTF7/ECO1 plays important roles in the preservation of genome integrity and meiosis. This article was collaboration between NCHU, Academia Sinica and Miami University in the United States.

M.Nagaraj kumar, Wann-Neng Jane and Paul Verslues (2013) Role of the putative osmosensor Arabidopsis Histidine kinase 1 in dehydration avoidance and low-water-potential response. Plant Physiology 161: 942-953

The molecular basis of plant osmosensing remains unknown. Arabidopsis Histidine Kinase1 (AHK1) can complement the osmosensitivity of yeast mutants lacking SLN-SHO osmosensors and has been proposed to act as a plant osmosensor. We found that ahk1 mutants in either the Nos-0 or Col-0 background had increased stomatal density and stomatal index consistent with greater transpirational water loss. However, growth of ahk1 mutants was not more sensitive to controlled moderate low water potentials (ψw) or to salt stress. Also, ahk1 mutants had increased, rather than reduced, solute accumulation across a range of low ψw severities. ahk1 mutants had reduced low ψw induction of P5CS1 and NCED3, which encode rate limiting enzymes in proline and ABA synthesis, respectively. However, neither proline nor ABA accumulation was reduced in ahk1mutants at low ψw. P5CS1 protein level was not reduced in ahk1 mutants. This indicated that proline accumulation was regulated in part by post transcriptional control of P5CS1 that was not affected by AHK1. Expression of AHK1 itself was reduced by low ψw, in contrast to previous reports. These results define a role of AHK1 in controlling stomatal density and transcription of stress responsive genes. These phenotypes may be mediated in part by reduced ABA sensitivity. More rapid transpiration and water depletion can also explain previously reported sensitivity of ahk1 to uncontrolled soil drying. The unimpaired growth, ABA, proline and solute accumulation of ahk1 mutants at low ψw suggest that AHK1 may not be the main plant osmosensor required for low ψw tolerance.

Sundaravelpandian K, Chandrika NNP, Schmidt W (2013) PFT1, a transcriptional Mediator complex subunit, controls root hair differentiation through reactive oxygen species (ROS) distribution in Arabidopsis. New Phytologist Volume 197, Issue 1

Transcriptional Mediators, central co-regulators of transcription, have been identified as large protein complexes in eukaryotes. They form a bridge between RNA polymerase II and transcription factors to initiate transcription. The Mediator complex plays important roles in several developmental processes in yeast, Drosophila, mice and humans. However, the function of Mediator subunits in plant development has only recently begun to be explored. In an attempt to identify signaling mechanisms involved in root hair morphogenesis, we screened Arabidopsis Mediator subunit mutants for root hair phenotype. We found that the Mediator subunit PFT1 controls the distribution of reactive oxygen species along the roots, a process that is critical for root hair differentiation and elongation. Transcriptional profiling of pft1 roots revealed that PFT1 activates class III peroxidases-mediated hydrogen peroxide formation to maintain the balance between hydrogen peroxide and superoxide during root hair formation. Our findings revealed a novel mechanism in which the Mediator controls ROS homeostasis by regulating the transcriptional machinery.

Working model of PHYTOCHROME AND FLOWERING TIME1 (PFT1)-regulated root hair differentiation. Mediator complex subunit PFT1 (blue) interacts with activators and RNA polymerase II (RNA Pol II) to initiate transcription. Possibly, PFT1 positively regulates class III peroxidases which produce hydrogen peroxide (H2O2) in the elongation zone (red). Superoxide (O2•─ ) is produced by NADPH oxidases in the meristematic zone (black). The distribution of both H2O2 and O2•─ reaches a threshold concentration, which acts as a signal that determines the differentiation of root hair cells. This signal could be H2O2. GTFs, general transcription factors.

Yi-Fang Chiu, Yung-Han Chen, Mercedes Roncel, Preston L. Dilbeck, Jine-Yung Huang, Shyue-Chu Ke, José M. Ortega, Robert L. Burnap, Hsiu-An Chu* (2013) Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b559 in photosystem II. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1827:507–519.

Cytochrome (Cyt) b559 is one of the essential proteins in photosystem II reaction center (PSII). Despite the recent progress in understanding the structure and function of PSII, the exact function of Cyt b559 remains elusive. We conducted spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of Cyt b559 in PSII. The results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b559 are important to the structure and redox properties of Cyt b559. In addition, the blue light-induced nonphotochemical quenching was significantly perturbed in Cyt b559 mutant cells. Furthermore, UPLC-APCI-QTOF mass spectrometry results showed that the plastoquinone pool was more reduced in Cyt b559 mutant cells in the dark. Our new findings support the important function of Cyt b559 in protection of PSII under photoinhibition conditions in vivo.

Govinal Badiger Bhaskara, Thao Thi Nguyen, Paul E. Verslues (2012). Unique drought resistance functions of the highly ABA-induced Clade A Protein phosphatase 2Cs. Plant Physiology 160: 379-395

Plant drought responses are regulated by ABA signaling and also by less studied ABA –independent signaling mechanisms. The ABA signaling pathway consists of inhibition of Clade A protein phosphatase 2C (PP2Cs) activity by PYL/RCAR-ABA receptors. These ABA signaling PP2Cs function in negative regulation of classical ABA-sensitivity phenotypes such as ABA-hypersensitive seed germination and ABA induced stomatal closure. However, physiological functions of Highly ABA-induced Clade A PP2Cs (HAI PP2Cs) have remained unclear despite the fact that they are closely related to ABA signaling Clade A PP2Cs. Also, regulation of drought related phenotypes such as proline and osmoregulatory solute accumulation remain unknown.
We found that the HAI PP2Cs act as negative regulators of proline and osmoregulatory solute accumulation. These phenotypes are ABA independent or less ABA dependent drought responses. Knockout mutants of HAI PP2Cs had less or no effect on ABA hypersensitive phenotypes regulated by other Clade A PP2Cs. The HAI PP2Cs also had limited interactions with PYLs which differed from other Clade A PP2Cs. The HAI PP2Cs were highly induced during low water potential stress when their PYL interactors were strongly repressed. This provides further indication that the HAI PP2Cs are active during low water potential and act to attenuate certain key stress responses. Overall, this study demonstrated a new level of functional differentiation among Clade A PP2Cs with the HAI PP2Cs controlling the distinct set of drought responses.

Nien-Chen Huang, Wann-Neng Jane, Jychian Chen, and Tien-Shin Yu (2012) Systemic floral inhibition by a florigen paralog in Arabidopsis. Plant Journal doi: 10.1111/j.1365-313X.2012.05076.x

Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote-control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti-florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS (ATC), an Arabidopsis FT paralog, may act in a non-cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short-day induced floral inhibitor. Cell type-specific expression showed that companion cells and apex expressing ATC are sufficient to inhibit floral initiation. Histochemical analysis showed the promoter activity of ATC mainly in vasculature but under the detection limit in apex, which suggests that ATC may moves from the vasculature to the apex to regulate flowering. Consistent with this notion, Arabidopsis seedling-grafting experiments demonstrated that ATC moved over long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and regulate the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT paralogs to systemically regulate floral initiation.

Munkhtsetseg Tsednee, Yit-Wai Mak, Yet-Ran Chen, and Kuo- Chen Yeh* (2012) A sensitive LC-ESI-Q-TOF-MS method reveals novel phytosiderophores and phytosiderophore–iron complexes in barley. New Phytologist 195: 951-961.

Under Fe deficiency, plant roots secrete specific compounds such as organic acids and phytosiderophores into the rhizosphere to increase Fe bioavailability by changing the soil pH or by binding to ferric Fe. Phytosiderophores are non-proteinogenic amino acids. The biosynthesis and secretion of phytosiderophores are specific to graminaceous plants. The acquisition of Fe from soil by grasses by phytosiderophores and a chelation-based mechanism is called strategy II, whereas non-grass plants use a reduction-based mechanism termed strategy I. The direct analysis of phytosiderophores and their metal complexes is critical to understand the biological functions of different phytosiderophores. Here we report on a rapid and highly sensitive LC-ESI-Q-TOF-MS method for the direct and simultaneous determination of free phytosiderophores and their ferric complexes in plants. With the developed platform, we identify two novel phytosiderophores, AVA and HAVA in barley.

Ming-Jung Liu, Szu-Hsien Wu, Ho-Ming Chen and Shu-Hsing Wu (2012) Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis; Molecular Systems Biology 8: 566

Environmental 'light' has a vital role in regulating plant growth and development. Transcriptomic profiling has been widely used to examine how light regulatesmRNA levels on a genome-wide scale, but the global role of translational regulation in the response to light is unknown. Through a transcriptomic comparison of steady-state and polysome-bound mRNAs, we reveal a clear impact of translational control on thousands of genes, in addition to transcriptomic changes, during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, which possibly contributes to the enhanced translation efficiency. We also reveal that mRNAs regulated at the translational level share characteristics of longer half-lives and shorter cDNA length, and that transcripts with a cis-element, TAGGGTTT, in their 50 untranslated region have higher translatability. We report a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions for mechanistic studies of light-triggered translational enhancement in Arabidopsis.

Shu-Yi Yin, Chien-Yu Wang and Ning-Sun Yang (2011) Interleukin-4 Enhances Trafficking and Functional Activities of GM-CSF-Stimulated Mouse Myeloid-Derived Dendritic Cells at Late Differentiation Stage. Experimental Cell Research 317(15): 2210–2221

Mouse bone marrow-derived dendritic cells (BMDCs) are being employed as an important model for translational research into the development of DC-based therapeutics. For such use, the localization and specialized mobility of injected BMDCs within specific immune tissues are known to define their immunity and usefulness in vivo. In this study, we demonstrate that IL-4, a key driving factor for in vitro propagation and differentiation of BMDCs, when added during a late culture stage can enhance the in vivo trafficking activity of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced BMDCs. It suggests that the temporal control of IL-4 stimulation during the in vitro generation of DCs drastically affects the DC trafficking efficiency in vivo. With this modification of IL-4 stimulation, we also show that much less cytokine was needed to generate BMDCs with high purity and yield that secrete a high level of cytokines and possess a good capacity to induce proliferation of allogeneic CD4+ T cells, as compared to the conventional method that uses a continuous supplement of GM-CSF and IL-4 throughout cultivation. These results provide us with an important know-how for differentiation of BMDCs from myeloid stem cells, and for use of other immune cells in related medical or stem cell applications.

Kuan-Ju Lu, Nien-Chen Huang, Yu-Shan Liu, Chung-An Lu and Tien-Shin Yu (2012) Long-distance movement of Arabidopsis FLOWERING LOCUS T RNA participates in systemic floral regulation. RNA Biology Volume 9, Issue 5

The finding of mRNA acting as a systemic information molecule is one of the most exciting discoveries in recent plant biology. However, evidence demonstrating the functional significance of non-cell autonomous RNA remains limited. Recent analyses of Arabidopsis and rice revealed FLOWERING LOCUS T (FT) protein as a systemic florigenic signal. However, whether the FT RNA also participates in systemic floral regulation remains controversial. By using Arabidopsis cleft-grafting experiments, we showed that the RNA of Arabidopsis FT undergoes long-distance movement from the stock to the scion apex in both FT transformants and non-transformants. In addition, the sequences of FT RNA are sufficient to target a cell-autonomous RNA for long-distance movement. Therefore, FT RNA is a bona fide non-cell autonomous RNA. To examine the systemic action of FT RNA, we uncoupled the movement of FT RNA from protein by fusing FT with RED FLUORESCENT PROTEIN (RFP). When RFP-FT protein was retained in companion cells, the detection of RFP-FT RNA correlates with floral promotion in the scion. Further depletion of the translocated RFP-FT RNA by RNAi or artificial miRNA against FT delayed the floral promotion, indicating that the translocated FT RNA acts as a part of the systemic floral signaling. Our results indicate that both FT RNA and protein move long distance and act redundantly to integrate the photoperiodic signals.

RFP-FT mRNA trafficked in seedling grafting from stock to scion and promoter flowering of ft-10. (A) RT-PCR analysis of RFP-FT mRNA movement in seedling grafting. RFP-Cterm-For and FT-IR1-Rev were used in PCR to distinguish RFP-FT from endogenous transcripts. IMPORTIN-α (IMP-α) was used as the loading control. The gel was stained with SYBR green for visulization. (B-C) Image on the 28th day-after-grafting of ft-10 scion on PSUC2-RFP-FT stock (B) and ft-10 self-grafting control (C). Note that flower was already formed in (B) but still no flower in (C). (D) Flowering time of different seedling grafting combinations. SC: scion; ST: stock. IMPORTIN-α (IMP-α) was used as the loading control.

Wai-Leng Lee, Tuan-Nan Wen, Jeng-Yuan Shiau, Lie-Fen Shyur (2010) Differential Proteomic Profiling Identifies Novel Molecular Targets of Paclitaxel and Phytoagent Deoxyelephantopin against Mammary Adenocarcinoma Cells. Journal of Proteome Research 9(1): 237-253

A major germacranolide sesquiterpene lactone, deoxyelephantopin, identified from Elephantopus scaber L. (known as "Didancao" in Chinese medicine) showed significant antitumor growth and antimetastatic effect on murine mammary adenocarcinoma TS/A cells in vitro and in vivo in mice. Deoxyelephantopin exhibited a superior effect to that of the paclitaxel in prolonging median survival time of tumor-bearing animals in our recent study. To investigate the molecular mechanisms underlying the difference in efficacy between deoxyelephantopin and paclitaxel, we used 2-D DIGE and LC-ESI-MS/MS to profile proteins differentially expressed in the nucleus and cytoplasm of TS/A cells and used the MetaCore database to determine the functional protein networks affected by both treatments. Deoxyelephantopin and paclitaxel treatment produced regulation of molecules involved in proteolysis and calcium ion transport, suggesting the possible effects of both drugs on proteasome and endoplasmic reticulum machinery in TS/A cells. Western blot analysis of marker proteins (e.g., PDI, GRP78, TXND5, caspase-12, caspase-3 and PARP) further verified that induction of endoplasmic reticulum stress was associated with apoptosis induced by both deoxyelephantopin and paclitaxel, but only deoxyelephantopin inhibited proteasomal proteolysis in TS/A cells. The novel effects on targeting ER machinery and suppressing proteasome activity suggest the great potential of deoxyelephantopin for mammary cancer therapy.

Ma, Lay-Sun, Lin, Jer-Sheng, and Lai, Erh-Min* (2009) An IcmF Family Protein ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL, and Its Walker A Motif is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens J. Bacteriol 191: 4316-4329.

Intracellular multiplication F (IcmF)-family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. In this study, we characterized the IcmF-family protein, ImpL and its interacting partner, another T6SS essential component, ImpK. A combination of β-lactamase-/GFP- fusion and biochemical fractionation analyses revealed that both ImpL and ImpK are integral inner membrane proteins with their N-terminal domains facing the cytoplasm and the C-terminal domains exposed to the periplasm. Protein-protein interaction and comprehensive yeast two-hybrid assays dissecting ImpL-ImpK interaction domains suggested that ImpL interacts with ImpK via the N-terminal cytoplasmic domains of the proteins. We also showed the importance of ImpL nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. impLM mutants with substitutions or deletion in the Walker A motif failed to complement the impLM deletion mutant for Hcp secretion, which provided evidence that ImpLM may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. In conclusion, ImpL interacts with ImpK, and its Walker A motif is required for its function in mediating Hcp secretion from A. tumefaciens.

Agrobacterium tumefaciens type VI secretion inner membrane protein complex. (A) The topology and subcellular localization of ImpL and ImpK. Both proteins are integral inner membrane proteins with their N-terminal domains face to cytosol. (B). ImpL interacts with ImpK in E. coli co-purification. (C). The impLM mutants with substitutions or deletion in the Walker A motif failed to complement the impLM deletion mutant for Hcp secretion.

Li Y-H, Chen H-C, Gong H-Y, Hu S-Y, Li Y-W, Lin G-W, Lin C-C, Liu W, Wu J-L. (2010) Progranulin A-mediated MET Signaling Is Essential for Liver Morphogenesis in Zebrafish. Journal of Biological Chemistry 285(52):41001-09.

The mechanism that regulates embryonic liver morphogene- sis remains elusive. Progranulin (PGRN) is postulated to play a critical role in regulating pathological liver growth. Neverthe- less, the exact regulatory mechanism of PGRN in relation to its functional role in embryonic liver development remains to be elucidated. In our study, the knockdown of progranulin A (GrnA), an orthologue of mammalian PGRN, using antisense morpholinos resulted in impaired liver morphogenesis in ze- brafish (Danio rerio). The vital role of GrnA in hepatic out- growth and not in liver bud formation was further confirmed using whole-mount in situ hybridization markers. In addition, a GrnA deficiency was also found to be associated with the de- regulation of MET-related genes in the neonatal liver using a microarray analysis. In contrast, the decrease in liver size that was observed in grnA morphants was avoided when ectopic MET expression was produced by co-injecting met mRNA and grnA morpholinos. This phenomenon suggests that GrnA might play a role in liver growth regulation via MET signaling. Furthermore, our study has shown that GrnA positively modu- lates hepatic MET expression both in vivo and in vitro. There- fore, our data have indicated that GrnA plays a vital role in embryonic liver morphogenesis in zebrafish. As a result, a novel link between PGRN and MET signaling is proposed.

Ya-Ling Lin, Shu-Chiun Sung, Hwang-Long Tsai, Ting-Ting Yu, Ramalingam Radjacommare, Raju Usharani, Antony S. Fatimababy, Hsia-Yin Lin, Ya-Ying Wang, and Hongyong Fu* (2011) The defective proteasome but not substrate recognition function is responsible for the null phenotypes of the Arabidopsis proteasome subunit RPN10. Plant Cell 23: 2754–2773.

Ubiquitin/26S proteasome-mediated proteolysis is a vital regulatory mechanism in almost all aspects of plant growth and development. Ubiquitylated protein substrates are recognized by distinct pathways mediated by multiple and evolutionarily conserved ubiquitin receptors. To dissect the complexity and functional roles of the distinct substrate recognition pathways in Arabidopsis, we characterized the major ubiquitin receptors, including the proteasome subunits RPN10 and RPN13 and the UBL and UBA domain-containing factors such as RAD23, DSK2, DDI1, and NUB1, using protein-protein interaction and genetic approaches. This study shows that the major ubiquitin receptors involved in targeting ubiquitylated proteins for proteasome-mediated proteolysis are functionally redundant in Arabidopsis. Interestingly, in addition to playing a redundant role in substrate recognition, the Arabidopsis proteasome subunit

Proteasome stabilization but not substrate recognition is critical for in vivo function of Arabidopsis proteasome subunit RPN10. All major RPN10 null mutant phenotypes examined, including reduced growth rate, late flowering (A), altered leaf morphology (B), defective gamete function (C), sterility (D), increased floral organ size (E), and reduced two-capped proteasome abundance (F), can be rescued by a substrate recognition defective mutant (u123).

Shanmugam, V, Tsednee, M and Yeh, K,C (2011). ZINC TOLERANCE INDUCED BY IRON 1 reveals the importance of glutathione in the cross-homeostasis between zinc and iron in Arabidopsis thaliana. Plant Journal. 69 (6): 1006-1017.

Zinc is an essential micronutrient for plants, but it is toxic in excess concentrations. In Arabidopsis, additional iron (Fe) can increase Zn tolerance. We isolated a mutant, zinc tolerance induced by iron 1, designated zir1, with a defect in Fe-mediated Zn tolerance. Using map-based cloning and genetic complementation, we identified that zir1 has a mutation of glutamate to lysine at position 385 on γ-glutamylcysteine synthetase (GSH1), the enzyme involved in glutathione biosynthesis. The zir1 mutant contains only 15% of the wild-type glutathione level. Blocking glutathione biosynthesis in wild-type plants by a specific inhibitor of GSH1, buthionine sulfoximine, resulted in loss of Fe-mediated Zn tolerance, which provides further evidence that glutathione plays an essential role in Fe-mediated Zn tolerance. Two glutathione-deficient mutant alleles of GSH1, pad2-1 and cad2-1, which contain 22% and 39%, respectively, of the wild-type glutathione level, revealed that a minimal glutathione level between 22% and 39% of the wild-type level is required for Fe-mediated Zn tolerance. Under excess Zn and Fe, the recovery of shoot Fe contents in pad2-1 and cad2-1 was lower than that of the wild type. However, the phytochelatin-deficient mutant cad1-3 showed normal Fe-mediated Zn tolerance. These results indicate a specific role of glutathione in Fe-mediated Zn tolerance. The induced accumulation of glutathione in response to excess Zn and Fe suggests that glutathione plays a specific role in Fe-mediated Zn tolerance in Arabidopsis. We conclude that glutathione is required for the cross-homeostasis between Zn and Fe in Arabidopsis.