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Arabidopsis 56–Amino Acid Serine Palmitoyltransferase- Interacting Proteins Stimulate Sphingolipid Synthesis, Are Essential, And Affect Mycotoxin Sensitivity, Athen N. Kimberlin, Saurav Majumder, Gongshe Han, Ming Chen, Rebecca E. Cahoon, Julie M. Stone, Teresa M. Dunn, Edgar B. Cahoon

Center for Plant Science Innovation: Faculty and Staff Publications

Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB1 and LCB2 subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56–amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB1/LCB2 heterodimer by >100-fold through physical interaction with LCB1/LCB2. ssSPTa transcripts were …

Dynamic Expression Of Imprinted Genes Associates With Maternally Controlled Nutrient Allocation During Maize Endosperm Development, Mingming Xin, Ruolin Yang, Guosheng Li, Hao Chen, John Laurie, Chuang Ma, Dongfang Wang, Yingyin Yao, Brian A. Larkins, Qixin Sun, Ramin Yadegari, Xiangfeng Wang, Zhongfu Ni

Center for Plant Science Innovation: Faculty and Staff Publications

In angiosperms, the endosperm provides nutrients for embryogenesis and seed germination and is the primary tissue where gene imprinting occurs. To identify the imprintome of early developing maize (Zea mays) endosperm, we performed highthroughput transcriptome sequencing of whole kernels at 0, 3, and 5 d after pollination (DAP) and endosperms at 7, 10, and 15 DAP, using B73 byMo17reciprocal crosses.Weobserved gradually increased expression of paternal transcripts in 3- and 5-DAP kernels. In 7-DAP endosperm, the majority of the genes tested reached a 2:1 maternal versus paternal ratio, suggesting that paternal genes are nearly fully activated by 7 DAP. …

Plant Micrornas Display Differential 3' Truncation And Tailing Modifications That Are Argonaute1 Dependent And Conserved Across Species, Jixian Zhai, Yuanyuan Zhao, Stacey A. Simon, Sheng Huang, Katherine Petsch, Siwaret Arikit, Manoj Pillay, Lijuan Ji, Meng Xie, Xiaofeng Cao, Bin Yu, Marja Timmermans, Bing Yang, Xuemei Chen, Blake C. Meyers

Center for Plant Science Innovation: Faculty and Staff Publications

Plant small RNAs are 3' methylated by the methyltransferase HUA1 ENHANCER1 (HEN1). In plant hen1 mutants, 3' modifications of small RNAs, including oligo-uridylation (tailing), are associated with accelerated degradation of microRNAs (miRNAs). By sequencing small RNAs of the wild type and hen1 mutants from Arabidopsis thaliana, rice (Oryza sativa), and maize (Zea mays), we found 39 truncation prior to tailing is widespread in these mutants. Moreover, the patterns of miRNA truncation and tailing differ substantially among miRNA families but are conserved across species. The same patterns are also observable in wild-type libraries from a broad …

Pseudomonas Hopu1 Modulates Plant Immune Receptor Levels By Blocking The Interaction Of Their Mrnas With Grp7, Valerie Nicaise, Anna Joe, Byeong-Ryool Jeong, Christin Korneli, Freddy Boutrot, Isa Westedt, Dorothee Staiger, James R. Alfano, Cyril Zipfel

Center for Plant Science Innovation: Faculty and Staff Publications

Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III-secreted effector HopU1 is a mono-ADP-ribosyltransferase required for full virulence on Arabidopsis thaliana. HopU1 targets several RNA-binding proteins including GRP7, whose role in immunity is still unclear. Here, we show that GRP7 associates with translational components, as well as with the pattern recognition receptors FLS2 and EFR. Moreover, GRP7 binds specifically FLS2 and EFR transcripts in vivo through its RNA recognition motif. HopU1 does not affect the protein–protein associations between GRP7, FLS2 and translational components. Instead, HopU1 blocks the interaction between GRP7 and FLS2 and …

Small Interfering Rna–Mediated Translation Repression Alters Ribosome Sensitivity To Inhibition By Cycloheximide In Chlamydomonas Reinhardtii, Xinrong Ma, Eun-Jeong Kim, Insun Kook, Fangrui Ma, Adam Voshall, Etsuko N. Moriyama, Heriberto D. Cerutti

Center for Plant Science Innovation: Faculty and Staff Publications

Small RNAs (sRNAs; approximately 20 to 30 nucleotides in length) play important roles in gene regulation as well as in defense responses against transposons and viruses in eukaryotes. Their biogenesis and modes of action have attracted great attention in recent years. However, many aspects of sRNA function, such as the mechanism(s) of translation repression at postinitiation steps, remain poorly characterized. In the unicellular green alga Chlamydomonas reinhardtii, sRNAs derived from genomeintegrated inverted repeat transgenes, perfectly complementary to the 39 untranslated region of a target transcript, can inhibit protein synthesis without or with only minimal mRNA destabilization. Here, we report …

Comparative Studies Of Differential Gene Calling Using Rna-Seq Data, Ximeng Zheng, Etsuko N. Moriyama

Center for Plant Science Innovation: Faculty and Staff Publications

Background: With its massive amount of data, gene-expression profiling by RNA-Seq has many advantanges compared with microarray experiments. RNA-Seq analysis, however, is fundamentally different from microarray data analysis. Techniques developed for analyzing microarray data thus cannot be directly applicable for the digital gene expression data. Several statistical methods have been developed for identifying differentially expressed genes specifically from RNA-Seq data over the past few years.
Results: In this study, we examined the performance of differential gene-calling methods using RNA-Seq data in practical situations. We focused on two representative methods: one parametric method, DESeq, and one nonparametric method, NOISeq. …

Camelina Seed Transcriptome: A Tool For Meal And Oil Improvement And Translational Research, Huu T. Nguyen, Jillian E. Silva, Ram Podicheti, Jason Macrander, Wenyu Yang, Tara J. Nazarenus, Jeong-Won Nam, Jan G. Jaworski, Chaofu Lu, Brian E. Scheffler, Keithanne Mockaitis, Edgar B. Cahoon

Center for Plant Science Innovation: Faculty and Staff Publications

Camelina (Camelina sativa), a Brassicaceae oilseed, has received recent interest as a biofuel crop and production platform for industrial oils. Limiting wider production of camelina for these uses is the need to improve the quality and content of the seed protein-rich meal and oil, which is enriched in oxidatively unstable polyunsaturated fatty acids that are deleterious for biodiesel. To identify candidate genes for meal and oil quality improvement, a transcriptome reference was built from 2047 Sanger ESTs and more than 2 million 454-derived sequence reads, representing genes expressed in developing camelina seeds. The transcriptome of approximately 60K transcripts …

Functional Modeling Identifies Paralogous Solanesyl-Diphosphate Synthases That Assemble The Side Chain Of Plastoquinone-9 In Plastids, Anna Block, Rikard Fristedt, Sara Rogers, Jyothi Kumar, Brian Barnes, Joshua Barnes, Christian Elowsky, Yashitola Wamboldt, Sally Ann Mackenzie, Kevin Redding, Sabeeha S. Merchant, Gilles J. Basset

Center for Plant Science Innovation: Faculty and Staff Publications

Background: Plastid isoforms of solanesyl-diphosphate synthase catalyze the elongation of the prenyl side chain of plastoquinone-9.

Results: Corresponding mutants display lower levels of plastoquinone-9 and plastochromanol-8 and display intact levels of vitamin E.

Conclusion: Plastochromanol-8 originates from a subfraction of non-photoactive plastoquinol-9 and is not essential for seed longevity.

Significance: Viable plastoquinone-9 mutants are invaluable tools for understanding plastid metabolism.

Mutational Analyses Of A Fork Head Associated Domain Protein, Dawdle, In Arabidopsis Thaliana, Lakshmi Ayiloor Narayanan, Dipaloke Mukherjee, Shuxin Zhang, Bin Yu, David Chevalier

Center for Plant Science Innovation: Faculty and Staff Publications

DAWDLE (DDL) gene encodes a protein that contains an N-terminal arginine-rich domain and a C-terminal Fork Head Associated (FHA) domain in Arabidopsis thaliana. DDL protein is believed to function in microRNA biogenesis by mediating the recruitment of pri-microRNA to DICER-LIKE 1 and also stabilizing the microRNA. The aim of this study was to conduct a structure-function analysis to identify the regions in DDL that are of functional significance. Targeted Induced Local Lesions in Genome screen was performed in the Columbia erecta-105 background of Arabidopsis resulting in the identification of eight point mutations spanning DDL. The mutants were characterized by …

Complementarity To An Mirna Seed Region Is Sufficient To Induce Moderate Repression Of A Target Transcript In The Unicellular Green Alga Chlamydomonas Reinhardtii, Tomohito Yamasaki, Adam Voshall, Eun-Jeong Kim, Etsuko N. Moriyama, Heriberto Cerutti, Takeshi Ohama

Center for Plant Science Innovation: Faculty and Staff Publications

MicroRNAs (miRNAs) are 20–24 nt noncoding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, , which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, …

A Thraustochytrid Diacylglycerol Acyltransferase 2 With Broad Substrate Specificity Strongly Increases Oleic Acid Content In Engineered Arabidopsis Thaliana Seeds, Chunyu Zhang, Umidjon Iskandarov, Elliott T. Klotz, Robyn L. Stevens, Rebecca E. Cahoon, Tara J. Nazarenus, Suzette L. Pereira, Edgar B. Cahoon

Center for Plant Science Innovation: Faculty and Staff Publications

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol …

A Novel Function Prediction Approach Using Protein Overlap Networks, Shide Liang, Dandan Zheng, Daron M. Standley, Huarong Guo, Chi Zhang

Center for Plant Science Innovation: Faculty and Staff Publications

Background: Construction of a reliable network remains the bottleneck for network-based protein function prediction. We built an artificial network model called protein overlap network (PON) for the entire genome of yeast, fly, worm, and human, respectively. Each node of the network represents a protein, and two proteins are connected if they share a domain according to InterPro database.
Results: The function of a protein can be predicted by counting the occurrence frequency of GO (gene ontology) terms associated with domains of direct neighbors. The average success rate and coverage were 34.3% and 43.9%, respectively, for the test genomes, …

Pseudomonas Syringae Pv. Syringae Uses Proteasome Inhibitor Syringolin A To Colonize From Wound Infection Sites, Johana C. Misas-Villamil, Izabella Kolodziejek, Emerson Crabill, Farnusch Kaschani, Sherry Niessen, Takayuki Shindo, Markus Kaiser, James R. Alfano, Renier A. L. Van De Hoorn

Center for Plant Science Innovation: Faculty and Staff Publications

Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a …

Cdc5, A Dna Binding Protein, Positively Regulates Posttranscriptional Processing And/Or Transcription Of Primary Microrna Transcripts, Shuxin Zhang, Meng Xie, Guodong Ren, Bin Yu

Center for Plant Science Innovation: Faculty and Staff Publications

CDC5 is a MYB-related protein that exists in plants, animals, and fungi. In Arabidopsis, CDC5 regulates both growth and immunity through unknown mechanisms. Here, we show that CDC5 from Arabidopsis positively regulates the accumulation of microRNAs (miRNAs), which control many biological processes including development and adaptations to environments in plants. CDC5 interacts with both the promoters of genes encoding miRNAs (MIR) and the DNA-dependent RNA polymerase II. As a consequence, lack of CDC5 reduces the occupancy of polymerase II at MIR promoters, as well as MIR promoter activities. In addition, CDC5 is associated with the DICER–LIKE1 complex, …

Fuzzy Clustering Of Cpp Family In Plants With Evolution And Interaction Analyses, Tao Lu, Yongchao Dou, Chi Zhang

Center for Plant Science Innovation: Faculty and Staff Publications

Background: Transcription factors have been studied intensively because they play an important role in gene expression regulation. However, the transcription factors in the CPP family (cystein-rich polycomb-like protein), compared with other transcription factor families, have not received sufficient attention, despite their wide prevalence in a broad spectrum of species, from plants to animals. The total number of known CPP transcription factors in plants is 111 from 16 plants, but only 2 of them have been studied so far, namely TSO1 and CPP1 in Arabidopsis thaliana and soybean, respectively.
Methods: In this work, to study their functions, we applied …

Comparative Analyses Of Two Geraniaceae Transcriptomes Using Next-Generation Sequencing, Jinxin Zhang, Tracey A. Ruhlman, Jeffrey P. Mower, Robert K. Jansen

Center for Plant Science Innovation: Faculty and Staff Publications

Background: Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum.

Results: Normalized cDNA libraries of G. maderense and …

Arabidopsis 56–Amino Acid Serine Palmitoyltransferase- Interacting Proteins Stimulate Sphingolipid Synthesis, Are Essential, And Affect Mycotoxin Sensitivity, Athen N. Kimberlin, Saurav Majumder, Gongshe Han, Ming Chen, Rebecca E. Cahoon, Julie M. Stone, Teresa M. Dunn, Edgar B. Cahoon

Center for Plant Science Innovation: Faculty and Staff Publications

Maintenance of sphingolipid homeostasis is critical for cell growth and programmed cell death (PCD). Serine palmitoyltransferase (SPT), composed of LCB₁ and LCB₂ subunits, catalyzes the primary regulatory point for sphingolipid synthesis. Small subunits of SPT (ssSPT) that strongly stimulate SPT activity have been identified in mammals, but the role of ssSPT in eukaryotic cells is unclear. Candidate Arabidopsis thaliana ssSPTs, ssSPTa and ssSPTb, were identified and characterized. Expression of these 56–amino acid polypeptides in a Saccharomyces cerevisiae SPT null mutant stimulated SPT activity from the Arabidopsis LCB₁/LCB₂ heterodimer by >100-fold through physical interaction with LCB …