Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon

Liu, Derui; Zehfroosh, Nina; Hancock, Brandon L.; Hines, Kevin; Fang, Wenjuan; Kilfoil, Maria; Learned-Miller, Erik; Sanguinet, Karen A.; Goldner, Lori S.; Baskin, Tobias I.

doi:10.1038/s41598-017-14988-4

Title: Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon

Journal Article · 08 November 2017 · Scientific Reports

DOI: https://doi.org/10.1038/s41598-017-14988-4 · OSTI ID:1507735

^[1]; Zehfroosh, Nina ^[1]; Hancock, Brandon L. ^[1]; Hines, Kevin ^[1]; Fang, Wenjuan ^[1]; Kilfoil, Maria ^[1]; Learned-Miller, Erik ^[1]; Sanguinet, Karen A. ^[1]; Goldner, Lori S. ^[1]; Baskin, Tobias I. ^[1]

Univ. of Massachusetts, Amherst, MA (United States)

The mechanism of cellulose synthesis has been studied by characterizing the motility of cellulose synthase complexes tagged with a fluorescent protein; however, this approach has been used exclusively on the hypocotyl of Arabidopsis thaliana. Here we characterize cellulose synthase motility in the model grass, Brachypodium distachyon. We generated lines in which mEGFP is fused N-terminal to BdCESA3 or BdCESA6 and which grew indistinguishably from the wild type (Bd21-3) and had dense fluorescent puncta at or near the plasma membrane. Measured with a particle tracking algorithm, the average speed of GFP-BdCESA3 particles in the mesocotyl was 164 ± 78 nm min^-1 (error gives standard deviation [SD], n = 1451 particles). Mean speed in the root appeared similar. For comparison, average speed in the A. thaliana hypocotyl expressing GFP-AtCESA6 was 184 ± 86 nm min^-1 (n = 2755). For B. distachyon, we quantified root diameter and elongation rate in response to inhibitors of cellulose (dichlorobenylnitrile; DCB), microtubules (oryzalin), or actin (latrunculin B). Neither oryzalin nor latrunculin affected the speed of CESA complexes; whereas, DCB reduced average speed by about 50% in B. distachyon and by about 35% in A. thaliana. Evidently, between these species, CESA motility is well conserved.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Univ. of Massachusetts, Amherst, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division; USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB)

Grant/Contract Number:: FG02-03ER15421

OSTI ID:: 1507735

Alternate ID(s):: OSTI ID: 2483315

Journal Information:: Scientific Reports, Vol. 7, Issue 1; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (71)

In Vitro Versus in Vivo Cellulose Microfibrils from Plant Primary Wall Synthases: Structural Differences Lai-Kee-Him, Joséphine; Chanzy, Henri; Müller, Martin Journal of Biological Chemistry, Vol. 277, Issue 40 https://doi.org/10.1074/jbc.M203530200	journal	July 2002
Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments Gutierrez, Ryan; Lindeboom, Jelmer J.; Paredez, Alex R. Nature Cell Biology, Vol. 11, Issue 7 https://doi.org/10.1038/ncb1886	journal	June 2009
Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments Gutierrez, Ryan; Lindeboom, Jelmer J.; Paredez, Alex R. Nature Cell Biology, Vol. 11, Issue 7 https://doi.org/10.1038/ncb1886	journal	June 2009
The roles of the cytoskeleton during cellulose deposition at the secondary cell wall Wightman, Raymond; Turner, Simon R. The Plant Journal, Vol. 54, Issue 5 https://doi.org/10.1111/j.1365-313X.2008.03444.x	journal	June 2008
The Cell Biology of Cellulose Synthesis McFarlane, Heather E.; Döring, Anett; Persson, Staffan Annual Review of Plant Biology, Vol. 65, Issue 1 https://doi.org/10.1146/annurev-arplant-050213-040240	journal	April 2014
A Set of Modular Binary Vectors for Transformation of Cereals Himmelbach, Axel; Zierold, Uwe; Hensel, Götz Plant Physiology, Vol. 145, Issue 4 https://doi.org/10.1104/pp.107.111575	journal	November 2007
Brachypodium distachyon as a Genetic Model System Kellogg, Elizabeth A. Annual Review of Genetics, Vol. 49, Issue 1 https://doi.org/10.1146/annurev-genet-112414-055135	journal	November 2015
Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth Vidali, L.; van Gisbergen, P. A. C.; Guerin, C. Proceedings of the National Academy of Sciences, Vol. 106, Issue 32 https://doi.org/10.1073/pnas.0901170106	journal	July 2009
Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth Vidali, L.; van Gisbergen, P. A. C.; Guerin, C. Proceedings of the National Academy of Sciences, Vol. 106, Issue 32 https://doi.org/10.1073/pnas.0901170106	journal	July 2009
Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase Harris, D. M.; Corbin, K.; Wang, T. Proceedings of the National Academy of Sciences, Vol. 109, Issue 11 https://doi.org/10.1073/pnas.1200352109	journal	February 2012
Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase Chen, S.; Ehrhardt, D. W.; Somerville, C. R. Proceedings of the National Academy of Sciences, Vol. 107, Issue 40 https://doi.org/10.1073/pnas.1012348107	journal	September 2010
A Molecular Description of Cellulose Biosynthesis McNamara, Joshua T.; Morgan, Jacob L. W.; Zimmer, Jochen Annual Review of Biochemistry, Vol. 84, Issue 1 https://doi.org/10.1146/annurev-biochem-060614-033930	journal	June 2015
Brachypodium: a promising hub between model species and cereals Girin, Thomas; David, Laure C.; Chardin, Camille Journal of Experimental Botany, Vol. 65, Issue 19 https://doi.org/10.1093/jxb/eru376	journal	September 2014
Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen Plant Physiology, Vol. 170, Issue 1 https://doi.org/10.1104/pp.15.01395	journal	November 2015
Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase Harris, D. M.; Corbin, K.; Wang, T. Proceedings of the National Academy of Sciences, Vol. 109, Issue 11 https://doi.org/10.1073/pnas.1200352109	journal	February 2012
Quinclorac does not inhibit cellulose (cell wall) biosynthesis in sensitive barnyard grass and maize roots Tresch, Stefan; Grossmann, Klaus Pesticide Biochemistry and Physiology, Vol. 75, Issue 3 https://doi.org/10.1016/S0048-3575(03)00013-0	journal	March 2003
Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase Chen, S.; Ehrhardt, D. W.; Somerville, C. R. Proceedings of the National Academy of Sciences, Vol. 107, Issue 40 https://doi.org/10.1073/pnas.1012348107	journal	September 2010
Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement DeBolt, S.; Gutierrez, R.; Ehrhardt, D. W. Proceedings of the National Academy of Sciences, Vol. 104, Issue 14 https://doi.org/10.1073/pnas.0700789104	journal	March 2007
Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes Li, S.; Lei, L.; Somerville, C. R. Proceedings of the National Academy of Sciences, Vol. 109, Issue 1 https://doi.org/10.1073/pnas.1118560109	journal	December 2011
Pausing of Golgi Bodies on Microtubules Regulates Secretion of Cellulose Synthase Complexes in Arabidopsis Crowell, E. F.; Bischoff, V.; Desprez, T. The Plant Cell, Vol. 21, Issue 4, p. 1141-1154 https://doi.org/10.1105/tpc.108.065334	journal	April 2009
The trafficking of the cellulose synthase complex in higher plants Bashline, Logan; Li, Shundai; Gu, Ying Annals of Botany, Vol. 114, Issue 6 https://doi.org/10.1093/aob/mcu040	journal	March 2014
A Set of Modular Binary Vectors for Transformation of Cereals Himmelbach, Axel; Zierold, Uwe; Hensel, Götz Plant Physiology, Vol. 145, Issue 4 https://doi.org/10.1104/pp.107.111575	journal	November 2007
3,7-Dichloroquinolinecarboxylic Acid Inhibits Cell-Wall Biosynthesis in Maize Roots Koo, S. J.; Neal, J. C.; DiTomaso, J. M. Plant Physiology, Vol. 112, Issue 3 https://doi.org/10.1104/pp.112.3.1383	journal	November 1996
Thaxtomin A affects CESA-complex density, expression of cell wall genes, cell wall composition, and causes ectopic lignification in Arabidopsis thaliana seedlings Bischoff, Volker; Cookson, Sarah Jane; Wu, Shuang Journal of Experimental Botany, Vol. 60, Issue 3 https://doi.org/10.1093/jxb/ern344	journal	March 2009
Precise Nanometer Localization Analysis for Individual Fluorescent Probes Thompson, Russell E.; Larson, Daniel R.; Webb, Watt W. Biophysical Journal, Vol. 82, Issue 5 https://doi.org/10.1016/S0006-3495(02)75618-X	journal	May 2002
Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes Li, S.; Lei, L.; Somerville, C. R. Proceedings of the National Academy of Sciences, Vol. 109, Issue 1 https://doi.org/10.1073/pnas.1118560109	journal	December 2011
Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen Plant Physiology, Vol. 170, Issue 1 https://doi.org/10.1104/pp.15.01395	journal	November 2015
Brachypodium: a promising hub between model species and cereals Girin, Thomas; David, Laure C.; Chardin, Camille Journal of Experimental Botany, Vol. 65, Issue 19 https://doi.org/10.1093/jxb/eru376	journal	September 2014
Plant cellulose synthesis: CESA proteins crossing kingdoms Kumar, Manoj; Turner, Simon Phytochemistry, Vol. 112 https://doi.org/10.1016/j.phytochem.2014.07.009	journal	April 2015
Thaxtomin A affects CESA-complex density, expression of cell wall genes, cell wall composition, and causes ectopic lignification in Arabidopsis thaliana seedlings Bischoff, Volker; Cookson, Sarah Jane; Wu, Shuang Journal of Experimental Botany, Vol. 60, Issue 3 https://doi.org/10.1093/jxb/ern344	journal	March 2009
Brachypodium distachyon and Setaria viridis : Model Genetic Systems for the Grasses Brutnell, Thomas P.; Bennetzen, Jeffrey L.; Vogel, John P. Annual Review of Plant Biology, Vol. 66, Issue 1 https://doi.org/10.1146/annurev-arplant-042811-105528	journal	April 2015
Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro Cifuentes, Carolina; Bulone, Vincent; Emons, Anne Mie C. Journal of Integrative Plant Biology, Vol. 52, Issue 2 https://doi.org/10.1111/j.1744-7909.2010.00919.x	journal	February 2010
Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E. Plant Physiology, Vol. 162, Issue 2 https://doi.org/10.1104/pp.113.215277	journal	April 2013
NIH Image to ImageJ: 25 years of image analysis Schneider, Caroline A.; Rasband, Wayne S.; Eliceiri, Kevin W. Nature Methods, Vol. 9, Issue 7 https://doi.org/10.1038/nmeth.2089	journal	June 2012
Microrheology of Microtubule Solutions and Actin-Microtubule Composite Networks Pelletier, Vincent; Gal, Naama; Fournier, Paul Physical Review Letters, Vol. 102, Issue 18 https://doi.org/10.1103/physrevlett.102.188303	journal	May 2009
Perturbation of Brachypodium distachyon CELLULOSE SYNTHASE A4 or 7 results in abnormal cell walls Handakumbura, Pubudu P.; Matos, Dominick A.; Osmont, Karen S. BMC Plant Biology, Vol. 13, Issue 1 https://doi.org/10.1186/1471-2229-13-131	journal	January 2013
Perturbation of Brachypodium distachyon CELLULOSE SYNTHASE A4 or 7 results in abnormal cell walls Handakumbura, Pubudu P.; Matos, Dominick A.; Osmont, Karen S. BMC Plant Biology, Vol. 13, Issue 1 https://doi.org/10.1186/1471-2229-13-131	journal	January 2013
Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR Hong, Shin-Young; Seo, Pil; Yang, Moon-Sik BMC Plant Biology, Vol. 8, Issue 1 https://doi.org/10.1186/1471-2229-8-112	journal	January 2008
Visualization of cellulose synthases in Arabidopsis secondary cell walls Watanabe, Y.; Meents, M. J.; McDonnell, L. M. Science, Vol. 350, Issue 6257 https://doi.org/10.1126/science.aac7446	journal	October 2015
Visualization of Cellulose Synthase Demonstrates Functional Association with Microtubules Paredez, A. R. Science, Vol. 312, Issue 5779 https://doi.org/10.1126/science.1126551	journal	June 2006
Drilling accurate nanopores for biosensors by energetic multi-wall carbon nanotubes: a molecular dynamics investigation Li, Changsheng; Wang, Zilin; Ma, Lei Journal of Molecular Modeling, Vol. 28, Issue 10 https://doi.org/10.1007/s00894-022-05276-8	journal	September 2022
Visualization of Cellulose Synthase Demonstrates Functional Association with Microtubules Paredez, A. R. Science, Vol. 312, Issue 5779 https://doi.org/10.1126/science.1126551	journal	June 2006
Making parallel lines meet: Transferring information from microtubules to extracellular matrix Baskin, Tobias I.; Gu, Ying Cell Adhesion & Migration, Vol. 6, Issue 5 https://doi.org/10.4161/cam.21121	journal	September 2012
Genetic Evidence That Cellulose Synthase Activity Influences Microtubule Cortical Array Organization Paredez, Alexander R.; Persson, Staffan; Ehrhardt, David W. Plant Physiology, Vol. 147, Issue 4 https://doi.org/10.1104/pp.108.120196	journal	June 2008
Phytochrome Regulation of Cellulose Synthesis in Arabidopsis Bischoff, Volker; Desprez, Thierry; Mouille, Gregory Current Biology, Vol. 21, Issue 21 https://doi.org/10.1016/j.cub.2011.09.026	journal	November 2011
Auxin, actin and growth of the Arabidopsis thaliana primary root: Auxin and actin interaction Rahman, Abidur; Bannigan, Alex; Sulaman, Waheeda The Plant Journal, Vol. 50, Issue 3 https://doi.org/10.1111/j.1365-313X.2007.03068.x	journal	April 2007
A Mechanism for Sustained Cellulose Synthesis during Salt Stress Endler, Anne; Kesten, Christopher; Schneider, René Cell, Vol. 162, Issue 6 https://doi.org/10.1016/j.cell.2015.08.028	journal	September 2015
Erratum to “Quinclorac does not inhibit cellulose (cell wall) biosynthesis in sensitive barnyard grass and maize roots” [Pestic. Biochem. Physiol. 75 (2003) 73–78] Tresch, Stefan; Grossmann, Klaus Pesticide Biochemistry and Physiology, Vol. 76, Issue 2 https://doi.org/10.1016/s0048-3575(03)00064-6	journal	June 2003
Visualization of cellulose synthases in Arabidopsis secondary cell walls Watanabe, Y.; Meents, M. J.; McDonnell, L. M. Science, Vol. 350, Issue 6257 https://doi.org/10.1126/science.aac7446	journal	October 2015
Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana Desprez, T.; Juraniec, M.; Crowell, E. F. Proceedings of the National Academy of Sciences, Vol. 104, Issue 39 https://doi.org/10.1073/pnas.0706569104	journal	September 2007
Stimulation of radial expansion in arabidopsis roots by inhibitors of actomyosin and vesicle secretion but not by various inhibitors of metabolism Baskin, TobiasI.; Bivens, NathanJ. Planta, Vol. 197, Issue 3 https://doi.org/10.1007/BF00196673	journal	October 1995
3,7-Dichloroquinolinecarboxylic Acid Inhibits Cell-Wall Biosynthesis in Maize Roots Koo, S. J.; Neal, J. C.; DiTomaso, J. M. Plant Physiology, Vol. 112, Issue 3 https://doi.org/10.1104/pp.112.3.1383	journal	November 1996
Biosynthesis of (1→3)-β- d -glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana : Biosynthesis of (1→3)-β- Him, Joséphine L. K.; Pelosi, Ludovic; Chanzy, Henri European Journal of Biochemistry, Vol. 268, Issue 17 https://doi.org/10.1046/j.1432-1327.2001.02382.x	journal	September 2001
The Fragile Fiber1 Kinesin Contributes to Cortical Microtubule-Mediated Trafficking of Cell Wall Components Zhu, Chuanmei; Ganguly, Anindya; Baskin, Tobias I. Plant Physiology, Vol. 167, Issue 3 https://doi.org/10.1104/pp.114.251462	journal	February 2015
The Fragile Fiber1 Kinesin Contributes to Cortical Microtubule-Mediated Trafficking of Cell Wall Components Zhu, Chuanmei; Ganguly, Anindya; Baskin, Tobias I. Plant Physiology, Vol. 167, Issue 3 https://doi.org/10.1104/pp.114.251462	journal	February 2015
Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E. Plant Physiology, Vol. 162, Issue 2 https://doi.org/10.1104/pp.113.215277	journal	April 2013
Nonmotile Cellulose Synthase Subunits Repeatedly Accumulate within Localized Regions at the Plasma Membrane in Arabidopsis Hypocotyl Cells following 2,6-Dichlorobenzonitrile Treatment DeBolt, Seth; Gutierrez, Ryan; Ehrhardt, David W. Plant Physiology, Vol. 145, Issue 2 https://doi.org/10.1104/pp.107.104703	journal	October 2007
Herbicides that inhibit cellulose biosynthesis Sabba, Robert P.; Vaughn, Kevin C. Weed Science, Vol. 47, Issue 6 https://doi.org/10.1017/S004317450009144X	journal	December 1999
Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro Cifuentes, Carolina; Bulone, Vincent; Emons, Anne Mie C. Journal of Integrative Plant Biology, Vol. 52, Issue 2 https://doi.org/10.1111/j.1744-7909.2010.00919.x	journal	February 2010
The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes Fujita, Miki; Himmelspach, Regina; Ward, Juliet Plant Physiology, Vol. 162, Issue 1 https://doi.org/10.1104/pp.112.211565	journal	March 2013
On the alignment of cellulose microfibrils by cortical microtubules: A review and a model Baskin, Tobias I. Protoplasma, Vol. 215, Issue 1-4 https://doi.org/10.1007/BF01280311	journal	March 2001
Phytochrome Regulation of Cellulose Synthesis in Arabidopsis Bischoff, Volker; Desprez, Thierry; Mouille, Gregory Current Biology, Vol. 21, Issue 21 https://doi.org/10.1016/j.cub.2011.09.026	journal	November 2011
Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex Konopka, Catherine A.; Bednarek, Sebastian Y. The Plant Journal, Vol. 53, Issue 1 https://doi.org/10.1111/j.1365-313X.2007.03306.x	journal	January 2008
Brachypodium distachyon and Setaria viridis : Model Genetic Systems for the Grasses Brutnell, Thomas P.; Bennetzen, Jeffrey L.; Vogel, John P. Annual Review of Plant Biology, Vol. 66, Issue 1 https://doi.org/10.1146/annurev-arplant-042811-105528	journal	April 2015
Making parallel lines meet: Transferring information from microtubules to extracellular matrix Baskin, Tobias I.; Gu, Ying Cell Adhesion & Migration, Vol. 6, Issue 5 https://doi.org/10.4161/cam.21121	journal	September 2012
High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3 Vogel, John; Hill, Theresa Plant Cell Reports, Vol. 27, Issue 3 https://doi.org/10.1007/s00299-007-0472-y	journal	November 2007
Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement DeBolt, S.; Gutierrez, R.; Ehrhardt, D. W. Proceedings of the National Academy of Sciences, Vol. 104, Issue 14 https://doi.org/10.1073/pnas.0700789104	journal	March 2007
Distribution fields for tracking Sevilla-Lara, L.; Learned-Miller, E. 2012 IEEE Conference on Computer Vision and Pattern Recognition (CVPR) https://doi.org/10.1109/CVPR.2012.6247891	conference	June 2012
A Mechanism for Sustained Cellulose Synthesis during Salt Stress Endler, Anne; Kesten, Christopher; Schneider, René Cell, Vol. 162, Issue 6 https://doi.org/10.1016/j.cell.2015.08.028	journal	September 2015
The Cell Biology of Cellulose Synthesis McFarlane, Heather E.; Döring, Anett; Persson, Staffan Annual Review of Plant Biology, Vol. 65, Issue 1 https://doi.org/10.1146/annurev-arplant-050213-040240	journal	April 2014
Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR Hong, Shin-Young; Seo, Pil; Yang, Moon-Sik BMC Plant Biology, Vol. 8, Issue 1 https://doi.org/10.1186/1471-2229-8-112	journal	January 2008

Cited By (3)

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens Tran, Mai L.; McCarthy, Thomas W.; Sun, Hao Scientific Reports, Vol. 8, Issue 1 https://doi.org/10.1038/s41598-017-18994-4	journal	January 2018
Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens Tran, Mai L.; McCarthy, Thomas W.; Sun, Hao Scientific Reports, Vol. 8, Issue 1 https://doi.org/10.1038/s41598-017-18994-4	journal	January 2018
Biochemical and physiological flexibility accompanies reduced cellulose biosynthesis in Brachypodium cesa1S830N Brabham, Chad; Singh, Abhishek; Stork, Jozsef AoB PLANTS, Vol. 11, Issue 5 https://doi.org/10.1093/aobpla/plz041	journal	July 2019