TY - JOUR TI - Comparative transcriptome analysis reveals candidate genes for cold stress response and early flowering in pineapple AU - Yow, Ashley G. AU - Laosuntisuk, Kanjana AU - Young, Roberto A. AU - Doherty, Colleen J. AU - Gillitt, Nicholas AU - Perkins-Veazie, Penelope AU - Jenny Xiang, Qiu-Yun AU - Iorizzo, Massimo T2 - SCIENTIFIC REPORTS AB - Abstract Pineapple originates from tropical regions in South America and is therefore significantly impacted by cold stress. Periodic cold events in the equatorial regions where pineapple is grown may induce early flowering, also known as precocious flowering, resulting in monetary losses due to small fruit size and the need to make multiple passes for harvesting a single field. Currently, pineapple is one of the most important tropical fruits in the world in terms of consumption, and production losses caused by weather can have major impacts on worldwide exportation potential and economics. To further our understanding of and identify mechanisms for low-temperature tolerance in pineapple, and to identify the relationship between low-temperature stress and flowering time, we report here a transcriptomic analysis of two pineapple genotypes in response to low-temperature stress. Using meristem tissue collected from precocious flowering-susceptible MD2 and precocious flowering-tolerant Dole-17, we performed pairwise comparisons and weighted gene co-expression network analysis (WGCNA) to identify cold stress, genotype, and floral organ development-specific modules. Dole-17 had a greater increase in expression of genes that confer cold tolerance. The results suggested that low temperature stress in Dole-17 plants induces transcriptional changes to adapt and maintain homeostasis. Comparative transcriptomic analysis revealed differences in cuticular wax biosynthesis, carbohydrate accumulation, and vernalization-related gene expression between genotypes. Cold stress induced changes in ethylene and abscisic acid-mediated pathways differentially between genotypes, suggesting that MD2 may be more susceptible to hormone-mediated early flowering. The differentially expressed genes and module hub genes identified in this study are potential candidates for engineering cold tolerance in pineapple to develop new varieties capable of maintaining normal reproduction cycles under cold stress. In addition, a total of 461 core genes involved in the development of reproductive tissues in pineapple were also identified in this study. This research provides an important genomic resource for understanding molecular networks underlying cold stress response and how cold stress affects flowering time in pineapple. DA - 2023/11/2/ PY - 2023/11/2/ DO - 10.1038/s41598-023-45722-y VL - 13 IS - 1 SP - SN - 2045-2322 ER - TY - JOUR TI - A normalization method that controls for total RNA abundance affects the identification of differentially expressed genes, revealing bias toward morning-expressed responses AU - Laosuntisuk, Kanjana AU - Vennapusa, Amaranatha AU - Somayanda, Impa M. AU - Leman, Adam R. AU - Jagadish, SV Krishna AU - Doherty, Colleen J. AB - Abstract RNA-Sequencing is widely used to investigate changes in gene expression at the transcription level in plants. Most plant RNA-Seq analysis pipelines base the normalization approaches on the assumption that total transcript levels do not vary between samples. However, this assumption has not been demonstrated. In fact, many common experimental treatments and genetic alterations affect transcription efficiency or RNA stability, resulting in unequal transcript abundance. The addition of synthetic RNA controls is a simple correction that controls for variation in total mRNA levels. However, adding spike-ins appropriately is challenging with complex plant tissue, and carefully considering how they are added is essential to their successful use. We demonstrate that adding external RNA spike-ins as a normalization control produces differences in RNA-Seq analysis compared to traditional normalization methods, even between two times of day in untreated plants. We illustrate the use of RNA spike-ins with 3’ RNA-Seq and present a normalization pipeline that accounts for differences in total transcriptional levels. We evaluate the effect of normalization methods on identifying differentially expressed genes in the context of identifying the effect of the time of day on gene expression and response to chilling stress in sorghum. DA - 2023/10/29/ PY - 2023/10/29/ DO - 10.1101/2023.10.28.564442 UR - https://doi.org/10.1101/2023.10.28.564442 ER - TY - JOUR TI - Structural analysis and functional evaluation of the disordered ß-hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis AU - Dagher, Suzanne F. AU - Vaishnav, Asmita AU - Stanley, Christopher B. AU - Meilleur, Flora AU - Edwards, Brian F. P. AU - Bruno-Barcena, Jose M. T2 - FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY AB - Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1–110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region’s impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%–97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT. DA - 2023/12/14/ PY - 2023/12/14/ DO - 10.3389/fbioe.2023.1291245 VL - 11 SP - SN - 2296-4185 KW - disorder KW - expression KW - kinetics KW - mutagenesis KW - transglycosylation KW - Hamamotoa singularis ER - TY - JOUR TI - SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana AU - Rajabu, Cyprian A. AU - Dallas, Mary M. AU - Chiunga, Evangelista AU - De Leon, Leandro AU - Ateka, Elijah M. AU - Tairo, Fred AU - Ndunguru, Joseph AU - Ascencio-Ibanez, Jose T. AU - Hanley-Bowdoin, Linda T2 - FRONTIERS IN PLANT SCIENCE AB - Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement. DA - 2023/10/17/ PY - 2023/10/17/ DO - 10.3389/fpls.2023.1250105 VL - 14 SP - SN - 1664-462X KW - SEGS-1 KW - begomovirus KW - ACMV KW - Arabidopsis thaliana KW - cassava ER - TY - JOUR TI - Cellulose Acetate-Stabilized Pickering Emulsions: Preparation, Rheology, and Incorporation of Agricultural Active Ingredients AU - Sohail, Mariam AU - Pirzada, Tahira AU - Guenther, Richard AU - Barbieri, Eduardo AU - Sit, Tim AU - Menegatti, Stefano AU - Crook, Nathan AU - Opperman, Charles H. AU - Khan, Saad A. T2 - ACS SUSTAINABLE CHEMISTRY & ENGINEERING AB - We report the use of cellulose acetate (CA) nanoparticles (NPs) to produce oil in water Pickering emulsions. The CA NP can emulsify various oils and form stable emulsions at concentrations as low as 0.5 wt %. Rheological and microscopic analyses show evidence of interconnected NP aggregate networks between droplets. Yield stress measurements display evidence of “double” yielding. We postulate that the presence of the NP aggregates provides a secondary network between droplet clusters resulting in such behavior. We demonstrate the suitability of the emulsions as agriculture formulations by incorporating an agrochemical, abamectin (Abm), and a plant-growth-promoting microbe (PGPM) in the emulsions. Release assays exhibit sustained Abm release, promising higher efficacy at lower usage volumes. Incorporation of nonsporulating PGPM Pseudomonas simiae in the emulsions shows significantly higher microbe viability compared to controls after 70 days of storage. By demonstrating the application of CA NPs as a sustainable Pickering emulsifier, this study introduces the use of CA as a platform technology for the delivery of diverse agriculture cargos. A comprehensive evaluation of the system is articulated in a fundamental microstructure analysis and a demonstration of practical on-site attributes, including shelf-life stability and functional performance, verified through bioassays and plant growth studies. DA - 2023/9/30/ PY - 2023/9/30/ DO - 10.1021/acssuschemeng.3c02428 VL - 11 IS - 42 SP - 15178-15191 SN - 2168-0485 KW - nonsporulating microbes KW - double yielding KW - Pseudomonas simiae KW - microstructure KW - controlledrelease ER - TY - JOUR TI - Multiplex CRISPR editing of wood for sustainable fiber production AU - Sulis, Daniel B. AU - Jiang, Xiao AU - Yang, Chenmin AU - Marques, Barbara M. AU - Matthews, Megan L. AU - Miller, Zachary AU - Lan, Kai AU - Cofre-Vega, Carlos AU - Liu, Baoguang AU - Sun, Runkun AU - Sederoff, Henry AU - Bing, Ryan G. AU - Sun, Xiaoyan AU - Williams, Cranos M. AU - Jameel, Hasan AU - Phillips, Richard AU - Chang, Hou-min AU - Peszlen, Ilona AU - Huang, Yung-Yun AU - Li, Wei AU - Kelly, Robert M. AU - Sederoff, Ronald R. AU - Chiang, Vincent L. AU - Barrangou, Rodolphe AU - Wang, Jack P. T2 - SCIENCE AB - The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits. DA - 2023/7/14/ PY - 2023/7/14/ DO - 10.1126/science.add4514 VL - 381 IS - 6654 SP - 216-+ SN - 1095-9203 ER - TY - JOUR TI - A Ferric-Superoxide Intermediate Initiates P450-Catalyzed Cyclic Dipeptide Dimerization AU - Gering, Hannah E. AU - Li, Xiaojun AU - Tang, Haoyu AU - Swartz, Paul D. AU - Chang, Wei-Chen AU - Makris, Thomas M. T2 - JOURNAL OF THE AMERICAN CHEMICAL SOCIETY AB - The cytochrome P450 (CYP) AspB is involved in the biosynthesis of the diketopiperazine (DKP) aspergilazine A. Tryptophan-linked dimeric DKP alkaloids are a large family of natural products that are found in numerous species and exhibit broad and often potent bioactivity. The proposed mechanisms for C-N bond formation by AspB, and similar C-C bond formations by related CYPs, have invoked the use of a ferryl-intermediate as an oxidant to promote substrate dimerization. Here, the parallel application of steady-state and transient kinetic approaches reveals a very different mechanism that involves a ferric-superoxide species as a primary oxidant to initiate DKP-assembly. Single turnover kinetic isotope effects and a substrate analog suggest the probable nature and site for abstraction. The direct observation of CYP-superoxide reactivity rationalizes the atypical outcome of AspB and reveals a new reaction manifold in heme enzymes. DA - 2023/8/23/ PY - 2023/8/23/ DO - 10.1021/jacs.3c04542 SP - SN - 1520-5126 ER - TY - JOUR TI - Identifying the enzyme responsible for initiating aerobic acetylene metabolism in Rhodococcus rhodochrous ATCC 33258 AU - Bingham, Johnna AU - Hyman, Michael AU - Montoya, Sarah AU - Goshe, Michael AU - Gracieux-Singleton, Cyndell T2 - JOURNAL OF BIOLOGICAL CHEMISTRY DA - 2023/// PY - 2023/// DO - 10.1016/j.jbc.2023.103703 VL - 299 IS - 3 SP - S369-S369 SN - 1083-351X ER - TY - JOUR TI - Genome segment ratios change during whitefly transmission of two bipartite cassava mosaic begomoviruses AU - Kennedy, George G. G. AU - Sharpee, William AU - Jacobson, Alana L. L. AU - Wambugu, Mary AU - Mware, Benard AU - Hanley-Bowdoin, Linda T2 - SCIENTIFIC REPORTS AB - Cassava mosaic disease is caused by a complex of whitefly-transmitted begomoviruses, which often occur in co-infections. These viruses have bipartite genomes consisting of DNA-A and DNA-B that are encapsidated into separate virions. Individual viruses exist in plants and whitefly vectors as populations comprising both genome segments, which can occur at different frequencies. Both segments are required for infection, and must be transmitted for virus spread to occur. Cassava plants infected with African cassava mosaic virus (ACMV) and/or East African cassava mosaic Cameroon virus (EACMCV), in which the ratios of DNA-A:DNA-B titers differed between plants, were used to examine how titers of the segments in a plant relate to their respective probabilities of acquisition by whiteflies and to the titers of each segment acquired and subsequently transmitted by whiteflies. The probabilities of acquiring each segment of ACMV did not reflect their relative titers in the source plant but they did for EACMCV. However, for both viruses, DNA-A:DNA-B ratios acquired by whiteflies differed from those in the source plant and the ratios transmitted by the whitefly did not differ from one - the ratio at which the highest probability of transmitting both segments is expected. DA - 2023/6/21/ PY - 2023/6/21/ DO - 10.1038/s41598-023-37278-8 VL - 13 IS - 1 SP - SN - 2045-2322 ER - TY - JOUR TI - The inhibitor of & kappa;B kinase & beta; (IKK & beta;) phosphorylates I & kappa;B & alpha; twice in a single binding event through a sequential mechanism AU - Stephenson, Anthony A. AU - Taggart, David J. AU - Xu, Guozhou AU - Fowler, Jason D. AU - Wu, Hao AU - Suo, Zucai T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event. Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event. The nuclear factor κB (NF-κB)2 family of transcription factors are evolutionarily conserved master regulators of cell proliferation, innate immunity, inflammation, cell differentiation, and apoptosis (1Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3638) Google Scholar, 2Vallabhapurapu S. Karin M. Regulation and function of NF-kappaB transcription factors in the immune system.Annu. Rev. Immunol. 2009; 27: 693-733Crossref PubMed Scopus (2112) Google Scholar). Dysregulation of NF-κB is associated with many disorders, including cancer, autoimmune disease, neurodegenerative diseases, arthritis, and diabetes (3Karin M. Nuclear factor-kappaB in cancer development and progression.Nature. 2006; 441: 431-436Crossref PubMed Scopus (3042) Google Scholar, 4Courtois G. Gilmore T.D. Mutations in the NF-kappaB signaling pathway: implications for human disease.Oncogene. 2006; 25: 6831-6843Crossref PubMed Scopus (420) Google Scholar, 5Baker R.G. Hayden M.S. Ghosh S. NF-kappaB, inflammation, and metabolic disease.Cell Metab. 2011; 13: 11-22Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). Thus, investigation of the molecular mechanisms of NF-κB activation is important for our understanding of human disease. The mammalian NF-κB family consists of RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2), which form 15 separate homodimeric or heterodimeric complexes (6Oeckinghaus A. Ghosh S. The NF-kappaB family of transcription factors and its regulation.Cold Spring Harb. Perspect. Biol. 2009; 1: a000034Crossref PubMed Scopus (1833) Google Scholar). In resting cells, NF-κB dimers containing RelA, RelB, and/or c-Rel are sequestered in the cytoplasm through interactions with Inhibitor of κB (IκB) proteins IκBα, IκBβ, or IκBε. In contrast, NF-κB dimers containing p100 and p105 are localized to the cytoplasm through a C-terminal inhibitory domain containing multiple ankyrin repeat motifs. In response to various stimuli, NF-kB is activated through one of two separate pathways: the canonical or the noncanonical pathway. Within the canonical pathway, a Ser/Thr-specific IκB kinase (IKK) complex phosphorylates IκB proteins within a conserved DSGXXS destruction box motif, leading to IκB polyubiquitination, 26S proteasome-mediated degradation, and subsequent NF-κB release. Free NF-κB dimers then translocate to the nucleus to regulate transcription. In the noncanonical pathway, the IKK complex phosphorylates p100 to induce proteolytic processing of p100 to the activated NF-κB2 subunit p52, which also localizes to the nucleus. The IKK complex also catalyzes the phosphorylation of several protein substrates within alternative signaling pathways, and this activity is thought to coordinate the functions of the NF-κB pathways with other cellular pathways (reviewed in reference (7Hinz M. Scheidereit C. The IkappaB kinase complex in NF-kappaB regulation and beyond.EMBO Rep. 2014; 15: 46-61Crossref PubMed Scopus (361) Google Scholar)). The IKK complex consists of the nonenzymatic protein NEMO (NF-κB essential modulator, also called IKKγ) and a homodimer or heterodimer of the catalytic subunits IKKα and IKKβ (8Chen Z.J. Parent L. Maniatis T. Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity.Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar, 9DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.Nature. 1997; 388: 548-554Crossref PubMed Scopus (1919) Google Scholar, 10Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation.Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1600) Google Scholar, 11Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. et al.Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar, 12Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. et al.IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.Mol. Cell Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar). Although IKKα and IKKβ share 54% amino acid sequence identity, these kinases possess distinct substrate specificities. For example, IKKα predominantly catalyzes phosphorylation of p100 within the noncanonical pathway (13Xiao G. Fong A. Sun S.C. Induction of p100 processing by NF-kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to p100 and IKKalpha-mediated phosphorylation.J. Biol. Chem. 2004; 279: 30099-30105Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 14Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. et al.Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway.Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1147) Google Scholar). In contrast, IKKβ is primarily responsible for the phosphorylation of IkBα, IκBβ, IκBε within the canonical pathway (15Zandi E. Chen Y. Karin M. Direct phosphorylation of IkappaB by IKKalpha and IKKbeta: Discrimination between free and NF-kappaB-bound substrate.Science. 1998; 281: 1360-1363Crossref PubMed Google Scholar, 16Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Severe liver degeneration in mice lacking the IkappaB kinase 2 gene.Science. 1999; 284: 321-325Crossref PubMed Scopus (860) Google Scholar, 17Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. et al.The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis.J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (826) Google Scholar). Structural studies indicate that IKKβ possesses a trimodular architecture (Fig. 1A) consisting of an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (18Liu S. Misquitta Y.R. Olland A. Johnson M.A. Kelleher K.S. Kriz R. et al.Crystal structure of a human IkappaB kinase beta asymmetric dimer.J. Biol. Chem. 2013; 288: 22758-22767Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 19Xu G. Lo Y.C. Li Q. Napolitano G. Wu X. Jiang X. et al.Crystal structure of inhibitor of kappaB kinase beta.Nature. 2011; 472: 325-330Crossref PubMed Scopus (156) Google Scholar, 20Polley S. Huang D.B. Hauenstein A.V. Fusco A.J. Zhong X. Vu D. et al.A structural basis for IkappaB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.PLoS Biol. 2013; 11e1001581Crossref PubMed Scopus (77) Google Scholar). The KD contains an activation loop with the MEK consensus motif SxxxS (S177 and S181 in human IKKβ, Fig. 1A). This activation loop is essential for the activity of IKKβ as mutational analysis indicates that changing these Ser residues to Ala prevents IKKβ activation, whereas substitution of these critical Ser residues with phosphomimetic Glu residues renders the kinase constitutively active (12Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. et al.IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.Mol. Cell Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 19Xu G. Lo Y.C. Li Q. Napolitano G. Wu X. Jiang X. et al.Crystal structure of inhibitor of kappaB kinase beta.Nature. 2011; 472: 325-330Crossref PubMed Scopus (156) Google Scholar, 21Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. et al.IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.Science. 1997; 278: 860-866Crossref PubMed Scopus (1858) Google Scholar, 22Delhase M. Hayakawa M. Chen Y. Karin M. Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation.Science. 1999; 284: 309-313Crossref PubMed Scopus (754) Google Scholar). Although the phosphorylation of IκB proteins by IKKβ is a critical step within the canonical pathway of NF-κB activation, the molecular mechanism of IKKβ is poorly understood. Here, we used pre–steady-state kinetic analysis to investigate the molecular mechanism of IκBα phosphorylation catalyzed by constitutively active IKKβ. Our data indicate that IKKβ phosphorylates full-length IκBα twice within the conserved DSGXXS destruction box motif (Fig. 1C) during a single binding event. Our study also suggests that IKKβ preferentially phosphorylates IκBα sequentially at Ser32, followed by Ser36. We conclude that these aspects of the kinetic mechanism of IKKβ may be shared among the IKK and IKK-related kinases. The activity of human IKKβ (Fig. 1A) is significantly increased upon phosphorylation of activation loop residues Ser177 and Ser181 within the kinase domain (21Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. et al.IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.Science. 1997; 278: 860-866Crossref PubMed Scopus (1858) Google Scholar, 22Delhase M. Hayakawa M. Chen Y. Karin M. Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation.Science. 1999; 284: 309-313Crossref PubMed Scopus (754) Google Scholar). In order to measure the catalytic rates of activated IKKβ, we chose to utilize a constitutively active IKKβ (S177E, S181E) phosphomimetic amino acid substitution mutation (12Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. et al.IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.Mol. Cell Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 19Xu G. Lo Y.C. Li Q. Napolitano G. Wu X. Jiang X. et al.Crystal structure of inhibitor of kappaB kinase beta.Nature. 2011; 472: 325-330Crossref PubMed Scopus (156) Google Scholar, 21Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. et al.IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.Science. 1997; 278: 860-866Crossref PubMed Scopus (1858) Google Scholar) for our study. Within the canonical pathway of NF-κB activation, IKKβ specifically phosphorylates IκBα at residues Ser32 and Ser36 (Fig. 1, B and C) to induce the proteolytic degradation of IκBα and the subsequent release of NF-κB dimers. Importantly, IκBα is also phosphorylated at other sites in vivo, particularly within the C-terminal PEST domain (Fig. 1B) (23Barroga C.F. Stevenson J.K. Schwarz E.M. Verma I.M. Constitutive phosphorylation of I kappa B alpha by casein kinase II.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7637-7641Crossref PubMed Scopus (140) Google Scholar, 24Lin R. Beauparlant P. Makris C. Meloche S. Hiscott J. Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.Mol. Cell. Biol. 1996; 16: 1401-1409Crossref PubMed Google Scholar). However, unlike phosphorylation of residues within the destruction box motif, phosphorylation of residues within the PEST domain does not specifically target IκBα for ubiquitination and 26S proteasome-mediated degradation (24Lin R. Beauparlant P. Makris C. Meloche S. Hiscott J. Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.Mol. Cell. Biol. 1996; 16: 1401-1409Crossref PubMed Google Scholar). Steady-state kinetic analysis has demonstrated that IKKβ phosphorylates IκBα by using a random sequential mechanism (25Peet G.W. Li J. IkappaB kinases alpha and beta show a random sequential kinetic mechanism and are inhibited by staurosporine and quercetin.J. Biol. Chem. 1999; 274: 32655-32661Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), indicating that IKKβ can bind IκBα and ATP in any order prior to catalysis. However, because IKKβ exhibits autophosphorylation (Fig. 2), we chose to measure the pre–steady-state kinetic parameters of IKKβ-catalyzed phosphorylation of IκBα by first incubating IKKβ with the IκBα protein substrate to generate the IKKβ•IκBα complex and subsequently initiating the reaction by the rapid addition of ATP. To initially characterize the molecular mechanism of IKKβ-catalyzed phosphorylation of full-length IκBα, the ground-state–binding affinity of ATP (Kd) for the IKKβ•IκBα complex and the maximum observed rate (kp) of IKKβ-catalyzed phosphorylation were determined by using pre–steady-state kinetic analysis. To this end, we measured the ATP concentration dependence of the phosphorylation rate of IKKβ under conditions where IKKβ was in a 4-fold molar excess compared to the IκBα substrate to ensure that nearly all of the IκBα substrate was initially bound by the kinase. IKKβ has previously been shown to bind IκBα with an equilibrium dissociation constant (Kd) of 56 nM (26Heilker R. Freuler F. Vanek M. Pulfer R. Kobel T. Peter J. et al.The kinetics of association and phosphorylation of IkappaB isoforms by IkappaB kinase 2 correlate with their cellular regulation in human endothelial cells.Biochemistry. 1999; 38: 6231-6238Crossref PubMed Scopus (23) Google Scholar), and thus, we calculated that >98% of the IκBα substrate was initially bound by IKKβ under our experimental conditions. A preincubated solution of full-length IκBα and IKKβ (S177E, S181E) was rapidly mixed with a solution containing increasing concentrations of [γ-32P]ATP for various times. The reaction products were then separated by SDS-PAGE and phosphorylation of IκBα was quantified by using autoradiography (Fig. 2). A [γ-32P]-labeled, linearized DNA vector was included as a loading control because it was easily separated from both IKKβ and IκBα by SDS-PAGE, and it provided a consistent signal after incubation with IKKβ, IκBα, and ATP (Fig. 2). The amount of phosphorylated IκBα detected at each time point was normalized to the loading control and plotted as a function of reaction time (Fig. 3A). The resulting plots indicated that IKKβ (S177E, S181E) phosphorylated IκBα with an initial, fast exponential phase rate (ke), followed by a significantly slower linear phase rate (kl) and therefore, these data were fit to biphasic Equation 1 (see Experimental procedures). The biphasic nature of the plot can be clearly observed in Figure 4A when longer reaction time points were included in the plot. We hypothesized that the initial exponential rate corresponded to phosphorylation of IκBα at residues Ser32 and Ser36 within the destruction box motif, and the significantly slower linear rate resulted from either the phosphorylation of IκBα at sites outside of the destruction box motif, such as the PEST domain (Fig. 1B), or phosphorylation of IκBα substrate that was not initially bound in a productive complex by IKKβ, or both. The exponential rates of IKKβ-catalyzed phosphorylation of IκBα were then plotted as a function of ATP concentration and fit to hyperbolic Equation 2 (Fig. 3B), yielding a kp of 0.32 ± 0.01 s−1 for the maximum observed rate of IKKβ-catalyzed phosphorylation of full-length IκBα and a Kd of 12 ± 1 μM for ATP binding to the IKKβ•IκBα complex.Figure 4IKKβ phosphorylation of full-length IκBα and full-length IκBα amino acid substitution mutants. A preincubated solution of IKKβ (3 μM) and the indicated full-length IκBα (0.75 μM) was rapidly mixed with a solution containing a [γ-32P]-labeled loading control and [γ-32P]ATP (500 μM) for various times. Phosphorylation of the full-length IκBα substrates was then normalized to the loading control and plotted as arbitrary phosphorylation units (a.u.) as a function of time from (A) 0 to 500 s or (B) 0 to 50 s. The solid lines represent the best fits to biphasic Equation 1 (see Experimental procedures). The exponential phase amplitudes (C), the exponential phase rates (D), and the linear phase rates (E) were separately plotted against WT IκBα and its various mutants. IκB, inhibitor of IκB; IKKβ, IκB Kinase β.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further investigate the molecular mechanism of IKKβ phosphorylation, we sought to test the ability of IKKβ (S177E, S181E) to phosphorylate either Ser32 or Ser36 within the destruction box motif of IκBα (Fig. 1, B and C). To this end, we evaluated the phosphorylation of seven separate full-length IκBα amino acid substitution mutants (Fig. 1C) in which either residue Ser32 or Ser36 were individually changed to (i) Ala to eliminate the phosphorylation site, (ii) Asp to mimic the size and charge of a phosphorylated Ser, or iii) Cys to mimic the size and hydrophilic character of Ser while eliminating the phosphorylation site. An IκBα(S32A, S36A) double alanine substitution mutant was also tested in which both Ser residues within the destruction box motif were eliminated. The amount of phosphorylated IκBα product was plotted as a function of reaction time and fit to biphasic Equation 1 (Fig. 4). Kinetic data derived from the best fit curves are reported in Figure 4, C–E and Table 1.Table 1Kinetic parametersaPhosphorylation of the full-length IκBα substrates by IKKβ (S177E, S181E) were fit to Equation 1, [Product] = A[1 – exp(-ket)] + klt where A is the exponential phase amplitude, ke is the exponential rate and kl is the linear rate (see Experimental procedures). of full-length IκBα phosphorylation by IKKβFull-length IκBα substrateCognate phosphorylation sitesExponential phase amplitude (%)Exponential rate (s-1)Linear rate (s-1)WTS32, S3665 ± 30.28 ± 0.040.0011 ± 0.0002S32AS3627 ± 20.13 ± 0.020.0011 ± 0.0002S32DS3629 ± 10.50 ± 0.090.0012 ± 0.0002S32CS3636 ± 30.18 ± 0.040.0013 ± 0.0003S36AS3224 ± 10.17 ± 0.030.0018 ± 0.0003S36DS3234 ± 30.15 ± 0.050.0012 ± 0.0004S36CS3221 ± 10.17 ± 0.030.0017 ± 0.0003S32A, S36Anone3.8 ± 0.60.048 ± 0.0170.004 ± 0.001a Phosphorylation of the full-length IκBα substrates by IKKβ (S177E, S181E) were fit to Equation 1, [Product] = A[1 – exp(-ket)] + klt where A is the exponential phase amplitude, ke is the exponential rate and kl is the linear rate (see Experimental procedures). Open table in a new tab Comparing the kinetic parameters of IKKβ-catalyzed phosphorylation of wt IκBα and the IκBα amino acid substitution mutants, the amplitude of the exponential phase produced by phosphorylation of each of the IκBα single amino acid substitution mutants was reduced approximately 2-fold when compared to the exponential phase amplitude of wt IκBα (Fig. 4C and Table 1). Additionally, the observed exponential phase amplitude produced by phosphorylation of the IκBα(S32A, S36A) double amino acid substitution mutant was reduced by 17-fold when compared to wt IκBα (Fig. 4C and Table 1). These data indicate that the exponential phase of IKKβ-catalyzed phosphorylation of IκBα corresponds to phosphorylation of IκBα at Ser32 and Ser36. Interestingly, the exponential phase of wt IκBα phosphorylation can be fit to a single rate (Fig. 4), indicating that IKKβ phosphorylated IκBα at Ser32 and Ser36 rapidly, without a significant intervening step. This finding strongly suggests that IKKβ phosphorylated wt IκBα twice, without complete disassociation and reassociation of the IKKβ•IκBα complex. Thus, we concluded that IKKβ phosphorylates IκBα processively within a single binding event. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic substitution mutant was increased by approximately 1.8 fold when compared to the exponential rate of phosphorylation of wt IκBα (Fig. 4D and Table 1). In contrast, the exponential rate of phosphorylation for all of the other IκBα single amino acid substitution mutants, including the IκBα(S36D) phosphomimetic amino acid substitution mutant, was reduced by approximately 1.5-fold when compared to wt IκBα phosphorylation (Fig. 4D and Table 1). These data suggest that if IκBα is phosphorylated at Ser32, the rate at which IKKβ catalyzes the second phosphorylation event at Ser36 is increased, while phosphorylation of IκBα Ser36 does not increase the rate of phosphorylation at Ser32. The linear rate of IKKβ-catalyzed phosphorylation of the IκBα single amino acid substitution mutants was virtually identical (within 2-fold) to the linear rate of IKKβ-catalyzed phosphorylation of wt IκBα (Fig. 4E and Table 1). These results are consistent with the hypothesis that the linear phase of phosphorylation of IκBα and the IκBα amino acid substitution mutants results primarily from phosphorylation of IκBα at sites outside of the destruction box motif (Fig. 1, B and C) by IKKβ. Interestingly, the linear rate of IKKβ-catalyzed phosphorylation of the IκBα(S32A, S36A) double amino acid substitution mutant was 2- to 4-fold faster than for wt IκBα and the IκBα single amino acid substitution mutants (Fig. 4E and Table 1), suggesting that IKKβ-catalyzed phosphorylation of Ser32 and Ser36 may have slowed the linear PEST domain phosphorylation rate in these experiments. Although IKKβ preferentially phosphorylates IκBα within the destruction box motif (Fig. 1, B and C), we predicted that IKKβ is also capable of phosphorylating IκBα at sites within the C-terminal region (residues 55–317), such as within the PEST domain (Fig. 1B). To rule out the possibility that the observed rates of IKKβ-catalyzed phosphorylation of full-length IκBα were significantly influenced by phosphorylation of IκBα residues outside of the destruction box motif, we evaluated the phosphorylation of glutathione-S-transferase (GST)-IκBα(1–54) fusion proteins (Fig. 1, B and C). These GST-IκBα(1–54) protein substrates lack the C-terminal region of IκBα. Thus, we predicted that nonspecific substrate phosphorylation by IKKβ would be reduced when testing these protein substrates. We assessed the IKKβ-catalyzed phosphorylation of GST-IκBα(1–54) bearing the first 54 residues of IκBα, including the destruction box motif, and seven separate amino acid substitution mutations of GST-IκBα(1–54) (Fig. 1, B and C) under pre–steady-state conditions. The amount of phosphorylated IκBα substrate detected was plotted as a function of reaction time (Fig. 5, A and B). In contrast to phosphorylation of the full-length IκBα substrates (Fig. 4, A and B), only a single exponential phase of GST-IκBα(1–54) was observed (Fig. 5, A and B). Therefore, these data were fit to single-exponential Equation 3 (see Experimental procedures). Kinetic data derived from the best fit curves are reported in Figure 5, C and D as well as Table 2. The absence of a clear linear phase was likely due to either a reduction in nonspecific phosphorylation of the GST-IκBα(1–54) fusion protein at sites outside of the destruction box motif when compared to phosphorylation of the full-length IκBα substrates and/or a reduction in the number of initial, unproductive protein complexes. Consistently, the rate of GST-IκBα(1–54)(S32A, S36A) phosphorylation was too slow to be accurately measured (Fig. 5, A and B).Table 2Kinetic parameters of GST-IκBα(1–54)aPhosphorylation of the GST- IκBα(1–54) substrates by IKKβ (S177E, S181E) were fit to Equation 3, [Product] = A[1 – exp(-ket)] where A is the exponential phase amplitude and ke is the exponential rate (see Experimental procedures). phosphorylation by IKKβGST-IκBα(1–54) substrateCognate phosphorylation sitesExponential phase amplitude (%)Exponential rate (s-1)WTS32, S3696 ± 30.29 ± 0.03S32AS3642 ± 30.18 ± 0.04S32DS3659 ± 20.28 ± 0.04S32CS3653 ± 20.20 ± 0.03S36AS3244 ± 20.21 ± 0.03S36DS3248 ± 30.20 ± 0.05S36CS3249 ± 20.18 ± 0.03S32A, S36AbA dash (-) indicates that phosphorylation of GST-IκBα(1–54)(S32A, S36A) by IKKβ was too low to be accurately quantified.none––a Phosphorylation of the GST- IκBα(1–54) substrates by IKKβ (S177E, S181E) were fit to Equation 3, [Product] = A[1 – exp(-ket)] where A is the exponential phase amplitude and ke is the exponential rate (see Experimental procedures).b A dash (-) indicates that phosphorylation of GST-IκBα(1–54)(S32A, S36A) by IKKβ was too low to be accurately quantified. Open table in a new tab It has been previously demonstrated that the C-terminal region (residues 55–317) of IκBα (Fig. 1B) interacts with the ULD and SSD of IKKβ (Fig. 1A) (19Xu G. Lo Y.C. Li Q. Napolitano G. Wu X. Jiang X. et al.Crystal structure of inhibitor of kappaB kinase beta.Nature. 2011; 472: 325-330Crossref PubMed Scopus (156) Google Scholar). Such interaction may help to properly position the N-terminal destruction box motif of IκBα for phosphorylation by the KD of IKKβ (Fig. 6) and thus, influence the molecular mechanism of IKKβ. Interestingly, in agreement with our observations of IKKβ-catalyzed phosphorylation of the full-length IκBα substrates, the amplitude of the exponential phase of GST-IκBα(1–54) wt phosphorylation was approximately 2-fold greater than the exponential phase amplitude of all six of the GST-IκBα(1–54) single amino acid substitution mutants (Fig. 5C and Table 2). Thus, we concluded that interactions between IKKβ and the C-terminal region of IκBα are not required for IKKβ to phosphorylate IκBα twice within a single binding event. Furthermore, the full-length IκBα and the GST-IκBα(1–54) wt substrates were both phosphorylated at similar exponential rates (Figs. 4D and 5D, Tables 1 and 2), suggesting that interactions between IKKβ and the C-terminal domain of IκBα do not significantly alter the overall exponential rate of IKKβ-catalyzed phosphorylation. However, in contrast to the results obtained with the full-length IκBα amino acid substitution mutants (Fig. 4D and Table 1), the GST-IκBα(1–54)(S32D) phosphomimetic amino acid substitution mutant was phosphorylated at a similar rate to the GST-IκBα(1–54) wt substrate, and the remaining GST-IκBα(1–54) single amino acid substitution mutants were phosphorylated at a rate that was only reduced by approximately 1.5-fold when compared to GST-IκBα(1–54) wt (Fig. 5D and Table 2). Thus, substitution of destruction box residues Ser32 or Ser36 of the GST-IκBα(1–54) substrates altered the exponential rate of phosphorylation of these truncated substrates to a lesser extent than substitution of Ser32 or Ser36 within the full-length IκBα substrates. We concluded that although interactions between IKKβ a DA - 2023/1// PY - 2023/1// DO - 10.1016/j.jbc.2022.102796 VL - 299 IS - 1 SP - SN - 1083-351X ER - TY - JOUR TI - Cassava begomovirus species diversity changes during plant vegetative cycles AU - Dye, Anna E. E. AU - Muga, Brenda AU - Mwangi, Jenniffer AU - Hoyer, J. Steen AU - Ly, Vanessa AU - Rosado, Yamilex AU - Sharpee, William AU - Mware, Benard AU - Wambugu, Mary AU - Labadie, Paul AU - Deppong, David AU - Jackai, Louis AU - Jacobson, Alana AU - Kennedy, George AU - Ateka, Elijah AU - Duffy, Siobain AU - Hanley-Bowdoin, Linda AU - Carbone, Ignazio AU - Ascencio-Ibanez, Jose Trinidad T2 - FRONTIERS IN MICROBIOLOGY AB - Cassava is a root crop important for global food security and the third biggest source of calories on the African continent. Cassava production is threatened by Cassava mosaic disease (CMD), which is caused by a complex of single-stranded DNA viruses (family: Geminiviridae , genus: Begomovirus ) that are transmitted by the sweet potato whitefly ( Bemisia tabaci ). Understanding the dynamics of different cassava mosaic begomovirus (CMB) species through time is important for contextualizing disease trends. Cassava plants with CMD symptoms were sampled in Lake Victoria and coastal regions of Kenya before transfer to a greenhouse setting and regular propagation. The field-collected and greenhouse samples were sequenced using Illumina short-read sequencing and analyzed on the Galaxy platform. In the field-collected samples, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Kenya virus (EACMKV), and East African cassava mosaic virus-Uganda variant (EACMV-Ug) were detected in samples from the Lake Victoria region, while EACMV and East African mosaic Zanzibar virus (EACMZV) were found in the coastal region. Many of the field-collected samples had mixed infections of EACMV and another begomovirus. After 3 years of regrowth in the greenhouse, only EACMV-like viruses were detected in all samples. The results suggest that in these samples, EACMV becomes the dominant virus through vegetative propagation in a greenhouse. This differed from whitefly transmission results. Cassava plants were inoculated with ACMV and another EACMV-like virus, East African cassava mosaic Cameroon virus (EACMCV). Only ACMV was transmitted by whiteflies from these plants to recipient plants, as indicated by sequencing reads and copy number data. These results suggest that whitefly transmission and vegetative transmission lead to different outcomes for ACMV and EACMV-like viruses. DA - 2023/5/25/ PY - 2023/5/25/ DO - 10.3389/fmicb.2023.1163566 VL - 14 SP - SN - 1664-302X KW - plant virus KW - Cassava (Manihot esculenta) KW - vegetative (asexual) propagation KW - vector transmission KW - whitefly (Bemisia tabaci) ER - TY - JOUR TI - The Game of Timing: Circadian Rhythms Intersect with Changing Environments AU - Laosuntisuk, Kanjana AU - Elorriaga, Estefania AU - Doherty, Colleen J. T2 - ANNUAL REVIEW OF PLANT BIOLOGY AB - Recurring patterns are an integral part of life on Earth. Through evolution or breeding, plants have acquired systems that coordinate with the cyclic patterns driven by Earth's movement through space. The biosystem responses to these physical rhythms result in biological cycles of daily and seasonal activity that feed back into the physical cycles. Signaling networks to coordinate growth and molecular activities with these persistent cycles have been integrated into plant biochemistry. The plant circadian clock is the coordinator of this complex, multiscale, temporal schedule. However, we have detailed knowledge of the circadian clock components and functions in only a few species under controlled conditions. We are just beginning to understand how the clock functions in real-world conditions. This review examines what we know about the circadian clock in diverse plant species, the challenges with extrapolating data from controlled environments, and the need to anticipate how plants will respond to climate change. DA - 2023/// PY - 2023/// DO - 10.1146/annurev-arplant-070522-065329 VL - 74 SP - 511-538 SN - 1545-2123 KW - circadian KW - rhythms KW - climate change KW - signaling networks KW - environmental adaptation ER - TY - JOUR TI - Neutron Scattering in the Biological Sciences: Techniques And Applications AU - Meilleur, Flora T2 - JOVE-JOURNAL OF VISUALIZED EXPERIMENTS AB - ARTICLES DISCUSSED: Vahdatahar, E., Junius, N., Budayova-Spano, M. Optimization of crystal growth for neutron macromolecular crystallography. Journal of Visualized Experiments. (169), e61685 (2021). Schröder, G. C., Meilleur, F. Neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures. Journal of Visualized Experiments. (166), e61903 (2020). Kelley, E. G., Nguyen, M. H. L., Marquardt, D., Maranville, B. B., Murphy, R. P. Measuring the time-evolution of nanoscale materials with stopped-flow and small-angle neutron scattering. Journal of Visualized Experiments. (174), e62873 (2021). Bilheux, H. Z. et al. Neutron radiography and computed tomography of biological systems at the Oak Ridge National Laboratory's high flux isotope reactor. Journal of Visualized Experiments. (171), e61688 (2021). Stingaciu, L.-R. Study of protein dynamics via neutron spin echo spectroscopy. Journal of Visualized Experiments. (182), e61862 (2022). Kumarage, T., Nguyen, J., Ashkar, R. Neutron spin echo spectroscopy as a unique probe for lipid membrane dynamics and membrane-protein interactions. Journal of Visualized Experiments. (171), e62396 (2021). DA - 2023/1// PY - 2023/1// DO - 10.3791/64806 VL - 1 IS - 191 SP - SN - 1940-087X ER - TY - JOUR TI - Weed-induced changes in the maize root transcriptome reveal transcription factors and physiological processes impacted early in crop-weed interactions AU - Horvath, David P. AU - Doherty, Colleen J. AU - Desai, Jigar AU - Clark, Natalie AU - Anderson, James V AU - Chao, Wun S. T2 - AOB PLANTS AB - Abstract A new paradigm suggests weeds primarily reduce crop yield by altering crop developmental and physiological processes long before the weeds reduce resources through competition. Multiple studies have implicated stress response pathways are activated when crops such as maize are grown in close proximity with weeds during the first 4–8 weeks of growth—the point at which weeds have their greatest impact on subsequent crop yields. To date, these studies have mostly focused on the response of above-ground plant parts and have not examined the early signal transduction processes associated with maize root response to weeds. To investigate the impact of signals from a below-ground competitor on the maize root transcriptome when most vulnerable to weed pressure, a system was designed to expose maize to only below-ground signals. Gene set enrichment analyses identified over-represented ontologies associated with oxidative stress signalling throughout the time of weed exposure, with additional ontologies associated with nitrogen use and transport and abscisic acid (ABA) signalling, and defence responses being enriched at later time points. Enrichment of promoter motifs indicated over-representation of sequences known to bind FAR-RED IMPAIRED RESPONSE 1 (FAR1), several AP2/ERF transcription factors and others. Likewise, co-expression networks were identified using Weighted-Gene Correlation Network Analysis (WGCNA) and Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) algorithms. WGCNA highlighted the potential roles of several transcription factors including a MYB 3r-4, TB1, WRKY65, CONSTANS-like5, ABF3, HOMEOBOX 12, among others. These studies also highlighted the role of several specific proteins involved in ABA signalling as being important for the initiation of the early response of maize to weeds. SC-ION highlighted potential roles for NAC28, LOB37, NAC58 and GATA2 transcription factors, among many others. DA - 2023/6/1/ PY - 2023/6/1/ DO - 10.1093/aobpla/plad013 VL - 15 IS - 3 SP - SN - 2041-2851 KW - plant-plant interaction KW - gene regulation KW - network analysis ER - TY - JOUR TI - Overexpression of a gibberellin 20-oxidase gene in poplar xylem led to an increase in the size of nanocellulose fibrils and improved paper properties AU - Peng, Xiaopeng AU - Tong, Botong AU - Lee, Jongcheol AU - Wang, Kun AU - Yu, Xiaojuan AU - Huang, Xiong AU - Wen, Jialong AU - Makarem, Mohamadamin AU - Pang, Hongying AU - Hinjan, Subin AU - Yan, Xiaojing AU - Yao, Shuangquan AU - Lu, Fachuang AU - Wang, Baichen AU - Peng, Feng AU - Ralph, John AU - Kim, Seong H. AU - Sederoff, Ronald R. AU - Li, Quanzi T2 - CARBOHYDRATE POLYMERS AB - Cellulose, the major component of secondary cell walls, is the most abundant renewable long-chain polymer on earth. Nanocellulose has become a prominent nano-reinforcement agent for polymer matrices in various industries. We report the generation of transgenic hybrid poplar overexpressing the Arabidopsis gibberellin 20-oxidase1 gene driven by a xylem-specific promoter to increase gibberellin (GA) biosynthesis in wood. X-ray diffraction (XRD) and sum frequency generation spectroscopic (SFG) analyses showed that cellulose in transgenic trees was less crystalline, but the crystal size was larger. The nanocellulose fibrils prepared from transgenic wood had an increased size compared to those from wild type. When such fibrils were used as a reinforcing agent in sheet paper preparation, the mechanical strength of the paper was significantly enhanced. Engineering the GA pathway can therefore affect nanocellulose properties, providing a new strategy for expanding nanocellulose applications. DA - 2023/8/15/ PY - 2023/8/15/ DO - 10.1016/j.carbpol.2023.120959 VL - 314 SP - SN - 1879-1344 KW - Nanocellulose microfibril KW - Poplar KW - Gibberellin KW - Pulp ER - TY - JOUR TI - Auxin-cytokinin interplay shapes root functionality under low-temperature stress AU - Tiwari, Manish AU - Kumar, Ritesh AU - Subramanian, Senthil AU - Doherty, Colleen J. AU - Jagadish, S. V. Krishna T2 - TRENDS IN PLANT SCIENCE AB - Low-temperature stress alters root system architecture. In particular, changes in the levels and response to auxin and cytokinin determine the fate of root architecture and function under stress because of their vital roles in regulating root cell division, differentiation, and elongation. An intricate nexus of genes encoding components of auxin and cytokinin biosynthesis, signaling, and transport components operate to counteract stress and facilitate optimum development. We review the role of auxin transport and signaling and its regulation by cytokinin during root development and stem cell maintenance under low-temperature stress. We highlight intricate mechanisms operating in root stem cells to minimize DNA damage by altering phytohormone levels, and discuss a working model for cytokinin in low-temperatures stress response. DA - 2023/4// PY - 2023/4// DO - 10.1016/j.tplants.2022.12.004 VL - 28 IS - 4 SP - 447-459 SN - 1878-4372 ER - TY - JOUR TI - Tizoxanide Antiviral Activity on Dengue Virus Replication AU - Yamamoto, Kristie A. AU - Blackburn, Kevin AU - Goshe, Michael B. AU - Brown, Dennis T. AU - Migoswski, Edimilson AU - Campanhon, Isabele B. AU - Moreira, Monica F. AU - Ferreira, Davis F. AU - Soares, Marcia R. T2 - VIRUSES-BASEL AB - Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections. DA - 2023/3// PY - 2023/3// DO - 10.3390/v15030696 VL - 15 IS - 3 SP - SN - 1999-4915 KW - antiviral KW - tizoxanide KW - dengue virus KW - virucidal activity ER - TY - JOUR TI - Hybrid spatial-temporal Mueller matrix imaging spectropolarimeter for high throughput plant phenotyping AU - Kudenov, Michael W. AU - Krafft, Danny AU - Scarboro, Clifton G. AU - Doherty, Colleen J. AU - Balint-Kurti, Peter T2 - APPLIED OPTICS AB - Many correlations exist between spectral reflectance or transmission with various phenotypic responses from plants. Of interest to us are metabolic characteristics, namely, how the various polarimetric components of plants may correlate to underlying environmental, metabolic, and genotypic differences among different varieties within a given species, as conducted during large field experimental trials. In this paper, we overview a portable Mueller matrix imaging spectropolarimeter, optimized for field use, by combining a temporal and spatial modulation scheme. Key aspects of the design include minimizing the measurement time while maximizing the signal-to-noise ratio by mitigating systematic error. This was achieved while maintaining an imaging capability across multiple measurement wavelengths, spanning the blue to near-infrared spectral region (405-730 nm). To this end, we present our optimization procedure, simulations, and calibration methods. Validation results, which were taken in redundant and non-redundant measurement configurations, indicated that the polarimeter provides average absolute errors of (5.3±2.2)×10-3 and (7.1±3.1)×10-3, respectively. Finally, we provide preliminary field data (depolarization, retardance, and diattenuation) to establish baselines of barren and non-barren Zea maize hybrids (G90 variety), as captured from various leaf and canopy positions during our summer 2022 field experiments. Results indicate that subtle variations in retardance and diattenuation versus leaf canopy position may be present before they are clearly visible in the spectral transmission. DA - 2023/3/10/ PY - 2023/3/10/ DO - 10.1364/AO.483870 VL - 62 IS - 8 SP - 2078-2091 SN - 2155-3165 ER - TY - JOUR TI - Chromosome-level genome assembly of a triploid poplar Populus alba 'Berolinensis' AU - Chen, Song AU - Yu, Yue AU - Wang, Xinyu AU - Wang, Sui AU - Zhang, Tianjiao AU - Zhou, Yan AU - He, Ruihan AU - Meng, Nan AU - Wang, Yiran AU - Liu, Wenxuan AU - Liu, Zhijie AU - Liu, Jinwen AU - Guo, Qiwen AU - Huang, Haijiao AU - Sederoff, Ronald R. AU - Wang, Guohua AU - Qu, Guanzheng AU - Chen, Su T2 - MOLECULAR ECOLOGY RESOURCES AB - Abstract Many recent studies have provided significant insights into polyploid breeding, but limited research has been carried out on trees. The genomic information needed to understand growth and response to abiotic stress in polyploidy trees is largely unknown, but has become critical due to the threats to forests imposed by climate change. Populus alba ‘ Berolinensis ,’ also known “Yinzhong poplar,” is a triploid poplar from northeast China. This hybrid triploid poplar is widely used as a landscape ornamental and in urban forestry owing to its adaptation to adverse environments and faster growth than its parental diploid. It is an artificially synthesized male allotriploid hybrid, with three haploid genomes of P. alba ‘ Berolinensis ’ originating from different poplar species, so it is attractive for studying polyploidy genomic mechanisms in heterosis. In this study, we focused on the allelic genomic interactions in P. alba ‘ Berolinensis ,’ and generated a high‐quality chromosome‐level genome assembly consisting of 19 allelic chromosomes. Its three haploid chromosome sets are polymorphic with an average of 25.73 nucleotide polymorphism sites per kilobase. We found that some stress‐related genes such as RD22 and LEA7 exhibited sequence differences between different haploid genomes. The genome assembly has been deposited in our polyploid genome online analysis website TreeGenomes ( https://www.treegenomes.com ). These polyploid genome‐related resources will provide a critical foundation for the molecular breeding of P. alba ‘ Berolinensis ’ and help us uncover the allopolyploidization effects of heterosis and abiotic stress resistance and traits of polyploidy species in the future. DA - 2023/2/27/ PY - 2023/2/27/ DO - 10.1111/1755-0998.13770 SP - SN - 1755-0998 KW - allele KW - allopolyploid KW - genome KW - Populus alba 'Berolinensis' KW - salinity stress KW - triploid ER - TY - JOUR TI - Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature AU - Tandrup, Tobias AU - Lo Leggio, Leila AU - Meilleur, Flora T2 - ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS AB - Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes which cleave polysaccharides oxidatively and are important in pathogen biology, carbon cycling and biotechnology. The Lentinus similis family AA9 isoform A (LsAA9_A) has been extensively studied as a model system because its activity towards smaller soluble saccharide substrates has allowed detailed structural characterization of its interaction with a variety of substrates by X-ray crystallography at high resolution. Here, the joint X-ray/neutron room-temperature crystallographic structure of carbohydrate-free LsAA9_A in the copper(II) resting state refined against X-ray and neutron data at 2.1 and 2.8 Å resolution, respectively, is presented. The results provide an experimental determination of the protonation states of the copper(II)-coordinating residues and second-shell residues in LsAA9_A, paving the way for future neutron crystallographic studies of LPMO-carbohydrate complexes. DA - 2023/1// PY - 2023/1// DO - 10.1107/S2053230X22011335 VL - 79 SP - 1-7 SN - 2053-230X UR - https://doi.org/10.1107/S2053230X22011335 KW - lytic polysaccharide mono-oxygenases KW - Lentinus similis AA9_A KW - copper metalloenzymes KW - protonation states KW - neutron crystallography ER -