Life Sciences Commons^™

Functional Antagonism Of Wri1 And Tcp20 Modulates Gh3.3 Expression To Maintain Auxin Homeostasis In Roots, Que Kong, Pui Man Low, Audrey R. Q. Lim, Yuzhou Yang, Ling Yuan, Wei Ma

Kentucky Tobacco Research and Development Center Faculty Publications

Auxin is a well-studied phytohormone, vital for diverse plant developmental processes. The GH3 genes are one of the major auxin responsive genes, whose expression changes lead to modulation of plant development and auxin homeostasis. However, the transcriptional regulation of these GH3 genes remains largely unknown. WRI1 is an essential transcriptional regulator governing plant fatty acid biosynthesis. Recently, we identified that the expression of GH3.3 is increased in the roots of wri1-1 mutant. Nevertheless, in this study we found that AtWRI1 did not activate or repress the promoter of GH3.3 (proGH3.3) despite of its binding to proGH3.3. Cross-family …

Arp2/3-Independent Wave/Scar Pathway And Class Xi Myosin Control Sperm Nuclear Migration In Flowering Plants, Mohammad F. Ali, Umma Fatema, Xiongbo Peng, Samuel W. Hacker, Daisuke Maruyama, Meng-Xiang Sun, Tomokazu Kawashima

Plant and Soil Sciences Faculty Publications

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a …

Root Hair Single Cell Type Specific Profiles Of Gene Expression And Alternative Polyadenylation Under Cadmium Stress, Jingyi Cao, Congting Ye, Guijie Hao, Carole Dabney-Smith, Arthur G. Hunt, Qingshun Q. Li

Plant and Soil Sciences Faculty Publications

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root …

Positioning Of The Scrambled Receptor Requires Udp-Glc:Sterol Glucosyltransferase 80b1 In Arabidopsis Roots, Victoria G. Pook, Meera Nair, Kookhui Ryu, James C. Arpin, John Schiefelbein, Kathrin Schrick, Seth Debolt

Horticulture Faculty Publications

The biological function of sterol glucosides (SGs), the most abundant sterol derivatives in higher plants, remains uncertain. In an effort to improve our understanding of these membrane lipids we examined phenotypes exhibited by the roots of Arabidopsis (Arabidopsis thaliana) lines carrying insertions in the UDP-Glc:sterol glucosyltransferase genes, UGT80A2 and UGT80B1. We show that although ugt80A2 mutants exhibit significantly lower levels of total SGs they are morphologically indistinguishable from wild-type plants. In contrast, the roots of ugt80B1 mutants are only deficient in stigmasteryl glucosides but exhibit a significant reduction in root hairs. Sub-cellular investigations reveal that the plasma membrane …

Noncanonical Alternative Polyadenylation Contributes To Gene Regulation In Response To Hypoxia, Laura De Lorenzo, Reed Sorenson, Julia Bailey-Serres, Arthur G. Hunt

Plant and Soil Sciences Faculty Publications

Stresses from various environmental challenges continually confront plants, and their responses are important for growth and survival. One molecular response to such challenges involves the alternative polyadenylation of mRNA. In plants, it is unclear how stress affects the production and fate of alternative mRNA isoforms. Using a genome-scale approach, we show that in Arabidopsis thaliana, hypoxia leads to increases in the number of mRNA isoforms with polyadenylated 3′ ends that map to 5′-untranslated regions (UTRs), introns, and protein-coding regions. RNAs with 3′ ends within protein-coding regions and introns were less stable than mRNAs that end at 3′-UTR poly(A) sites. …

Organelle_Pba, A Pipeline For Assembling Chloroplast And Mitochondrial Genomes From Pacbio Dna Sequencing Data, Aboozar Soorni, David Haak, David Zaitlin, Aureliano Bombarely

Kentucky Tobacco Research and Development Center Faculty Publications

Background: The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present.

Results: We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and …

[14C] Glucose Cell Wall Incorporation Assay For The Estimation Of Cellulose Biosynthesis, Chad Brabham, Jozsef Stork, Seth Debolt

Horticulture Faculty Publications

Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining …

Mechanistic Insights Into Glucan Phosphatase Activity Against Polyglucan Substrates, David A. Meekins, Madushi Raththagala, Kyle D. Auger, Benjamin D. Turner, Diana Santelia, Oliver Kötting, Matthew S. Gentry, Craig W. Vander Kooi

Molecular and Cellular Biochemistry Faculty Publications

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. …

Integration Of Developmental And Environmental Signals Via A Polyadenylation Factor In Arabidopsis, Man Liu, Ruqiang Xu, Carrie Merrill, Liwei Hong, Carol Von Lanken, Arthur G. Hunt, Qingshun Q. Li

Plant and Soil Sciences Faculty Publications

The ability to integrate environmental and developmental signals with physiological responses is critical for plant survival. How this integration is done, particularly through posttranscriptional control of gene expression, is poorly understood. Previously, it was found that the 30 kD subunit of Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF30) is a calmodulin-regulated RNA-binding protein. Here we demonstrated that mutant plants (oxt6) deficient in AtCPSF30 possess a novel range of phenotypes--reduced fertility, reduced lateral root formation, and altered sensitivities to oxidative stress and a number of plant hormones (auxin, cytokinin, gibberellic acid, and ACC). While the wild-type AtCPSF30 (C30G) was …

Mono- And Digalactosyldiacylglycerol Lipids Function Nonredundantly To Regulate Systemic Acquired Resistance In Plants, Qing-Ming Gao, Keshun Yu, Ye Xia, M. B. Shine, Caixia Wang, Duroy Navarre, Aardra Kachroo, Pradeep Kachroo

Plant Pathology Faculty Publications

The plant galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) have been linked to the anti-inflammatory and cancer benefits of a green leafy vegetable diet in humans due to their ability to regulate the levels of free radicals like nitric oxide (NO). Here, we show that DGDG contributes to plant NO as well as salicylic acid biosynthesis and is required for the induction of systemic acquired resistance (SAR). In contrast, MGDG regulates the biosynthesis of the SAR signals azelaic acid (AzA) and glycerol-3-phosphate (G3P) that function downstream of NO. Interestingly, DGDG is also required for AzA-induced SAR, but MGDG is not. Notably, …

A Novel Partitivirus That Confers Hypovirulence On Plant Pathogenic Fungi, Xueqiong Xiao, Jiasen Cheng, Jinghua Tang, Yanping Fu, Daohong Jiang, Timothy S. Baker, Said A. Ghabrial, Jiatao Xie

Plant Pathology Faculty Publications

Members of the family Partitiviridae have bisegmented double-stranded RNA (dsRNA) genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant-pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles, including mitochondria, were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily cotransmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor. S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar …

Induced Transcriptional Profiling Of Phenylpropanoid Pathway Genes Increased Flavonoid And Lignin Content In Arabidopsis Leaves In Response To Microbial Products, Mohammad Babar Ali, David H. Mcnear

Plant and Soil Sciences Faculty Publications

BACKGROUND: The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana.

RESULTS: This study …

Ectopic Expression Of The Phosphomimic Mutant Version Of Arabidopsis Response Regulator 1 Promotes A Constitutive Cytokinin Response Phenotype, Jasmina Kurepa, Yan Li, Sharyn E. Perry, Jan A. Smalle

Plant and Soil Sciences Faculty Publications

Background

Cytokinins control numerous plant developmental processes, including meristem formation and activity, nutrient distribution, senescence timing and responses to both the abiotic and biotic environments. Cytokinin signaling leads to the activation of type-B response regulators (RRBs), Myb-like transcription factors that are activated by the phosphorylation of a conserved aspartate residue in their response receiver domain. Consistent with this, overexpression of RRBs does not substantially alter plant development, but instead leads to cytokinin hypersensitivity.

Results

Here we present comparative analysis of plants overexpressing Arabidopsis RRB 1 (ARR1) or a phosphomimic ARR1^D94E mutant in which the conserved aspartate-94 (D94) is replaced …

An Intergenic Region Shared By At4g35985 And At4g35987 In Arabidopsis Thaliana Is A Tissue Specific And Stress Inducible Bidirectional Promoter Analyzed In Transgenic Arabidopsis And Tobacco Plants, Joydeep Banerjee, Dipak K. Sahoo, Nrisingha Dey, Robert Houtz, Indu B. Maiti

Kentucky Tobacco Research and Development Center Faculty Publications

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and …

Development And Functional Analysis Of Novel Genetic Promoters Using Dna Shuffling, Hybridization And A Combination Thereof, Rajiv Ranjan, Sunita Patro, Bhubaneswar Pradhan, Alok Kumar, Indu B. Maiti, Nrisingha Dey

Kentucky Tobacco Research and Development Center Faculty Publications

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications.

METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most …

Sag101 Forms A Ternary Complex With Eds1 And Pad4 And Is Required For Resistance Signaling Against Turnip Crinkle Virus, Shifeng Zhu, Rae-Dong Jeong, Srivathsa C. Venugopal, Ludmila Lapchyk, Duroy Navarre, Aardra Kachroo, Pradeep Kachroo

Plant Pathology Faculty Publications

EDS1, PAD4, and SAG101 are common regulators of plant immunity against many pathogens. EDS1 interacts with both PAD4 and SAG101 but direct interaction between PAD4 and SAG101 has not been detected, leading to the suggestion that the EDS1-PAD4 and EDS1-SAG101 complexes are distinct. We show that EDS1, PAD4, and SAG101 are present in a single complex in planta. While this complex is preferentially nuclear localized, it can be redirected to the cytoplasm in the presence of an extranuclear form of EDS1. PAD4 and SAG101 can in turn, regulate the subcellular localization of EDS1. We also show that the Arabidopsis genome …

Argonaute10 And Argonaute1 Regulate The Termination Of Floral Stem Cells Through Two Micrornas In Arabidopsis, Lijuan Ji, Xigang Liu, Jun Yan, Wenming Wang, Rae Eden Yumul, Yun Ju Kim, Thanh Theresa Dinh, Jun Liu, Xia Cui, Binglian Zheng, Manu Agarwal, Chunyan Liu, Xiaofeng Cao, Guiliang Tang, Xuemei Chen

Plant and Soil Sciences Faculty Publications

Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, …

Substrates Of The Arabidopsis Thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display And Biopanning, Tingsu Chen, Nihar Nayak, Susmita Maitra Majee, Jonathan Lowenson, Kim R. Schäfermeyer, Alyssa C. Eliopoulos, Taylor D. Lloyd, Randy Dinkins, Sharyn E. Perry, Nancy R. Forsthoefel, Steven G. Clarke, Daniel M. Vernon, Zhaohui Sunny Zhou, Tomas Rejtar, A. Bruce Downie

Horticulture Faculty Publications

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when …

Copy Number Variation Shapes Genome Diversity In Arabidopsis Over Immediate Family Generational Scales, Seth Debolt

Horticulture Faculty Publications

Arabidopsis thaliana is the model plant and is grown worldwide by plant biologists seeking to dissect the molecular underpinning of plant growth and development. Gene copy number variation (CNV) is a common form of genome natural diversity that is currently poorly studied in plants and may have broad implications for model organism research, evolutionary biology, and crop science. Herein, comparative genomic hybridization (CGH) was used to identify and interrogate regions of gene CNV across the A. thaliana genome. A common temperature condition used for growth of A. thaliana in our laboratory and many around the globe is 22 degrees C. …

Enhanced Disease Susceptibility 1 And Salicylic Acid Act Redundantly To Regulate Resistance Gene-Mediated Signaling, Srivathsa C. Venugopal, Rae-Dong Jeong, Mihir Kumar Mandal, Shifeng Zhu, A. C. Chandra-Shekara, Ye Xia, Matthew Hersh, Arnold J. Stromberg, Duroy Navarre, Aardra Kachroo, Pradeep Kachroo

Plant Pathology Faculty Publications

Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or …

Distinctive Interactions Of The Arabidopsis Homolog Of The 30 Kd Subunit Of The Cleavage And Polyadenylation Specificity Factor (Atcpsf30) With Other Polyadenylation Factor Subunits, Suryadevara Rao, Randy D. Dinkins, Arthur G. Hunt

Plant and Soil Sciences Faculty Publications

BACKGROUND: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits.

RESULTS: It was found that AtCPSF30 by itself localizes, not to the nucleus, but to the cytoplasm. AtCPSF30 could be found in the nucleus when co-expressed with AtCPSF160 or AtCPSF73(I), …

Relative Crystallinity Of Plant Biomass: Studies On Assembly, Adaptation And Acclimation, Darby Harris, Seth Debolt

Horticulture Faculty Publications

Plant biomechanical design is central to cell shape, morphogenesis, reproductive performance and protection against environmental and mechanical stress. The cell wall forms the central load bearing support structure for plant design, yet a mechanistic understanding of its synthesis is incomplete. A key tool for studying the structure of cellulose polymorphs has been x-ray diffraction and fourier transform infrared spectroscopy (FTIR). Relative crystallinity index (RCI) is based on the x-ray diffraction characteristics of two signature peaks and we used this technique to probe plant assembly, adaptation and acclimation. Confocal microscopy was used to visualize the dynamics of cellulose synthase in transgenic …

A Polyadenylation Factor Subunit Implicated In Regulating Oxidative Signaling In Arabidopsis Thaliana, Jingxian Zhang, Bala Subrahmanyam Addepalli, Kil-Young Yun, Arthur G. Hunt, Ruqiang Xu, Suryadevara Rao, Qingshun Q. Li, Deane L. Falcone

Kentucky Tobacco Research and Development Center Faculty Publications

BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6.

METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to …

Arabidopsis Mrna Polyadenylation Machinery: Comprehensive Analysis Of Protein-Protein Interactions And Gene Expression Profiling, Arthur G. Hunt, Ruqiang Xu, Balasubrahmanyam Addepalli, Suryadevara Rao, Kevin P. Forbes, Lisa R. Meeks, Denghui Xing, Min Mo, Hongwei Zhao, Amrita Bandyopadhyay, Lavanya Dampanaboina, Amanda Marion, Carol Von Lanken, Qingshun Quinn Li

Plant and Soil Sciences Faculty Publications

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and …

A Novel Endonuclease Activity Associated With The Arabidopsis Ortholog Of The 30-Kda Subunit Of Cleavage And Polyadenylation Specificity Factor, Balasubrahmanyam Addepalli, Arthur G. Hunt

Plant and Soil Sciences Faculty Publications

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [³²P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH …