Abstract
The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events.
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Andreeva Z, Ho AYY, Barthet MM, Potocky M, Bezvoda R, Zarsky V, Marc J (2009) Phospholipase D family interactions with the cytoskeleton: isoform delta promotes plasma membrane anchoring of cortical microtubules. Funct Plant Biol 36(7):600–612. https://doi.org/10.1071/FP09024
Bargmann BO, Laxalt AM, ter Riet B, van Schooten B, Merquiol E, Testerink C, Haring MA, Bartels D, Munnik T (2009) Multiple PLDs required for high salinity and water deficit tolerance in plants. Plant Cell Physiol 50(1):78–89. https://doi.org/10.1093/pcp/pcn173
Cai G (2010) Assembly and disassembly of plant microtubules: tubulin modifications and binding to MAPs. J Exp Bot 61(3):623–626. https://doi.org/10.1093/jxb/erp395
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x
Dhonukshe P, Laxalt AM, Goedhart J, Gadella TW, Munnik T (2003) Phospholipase d activation correlates with microtubule reorganization in living plant cells. Plant Cell 15(11):2666–2679. https://doi.org/10.1105/tpc.014977
Dixit R, Cyr RJ (2004) The cortical microtubule array: from dynamics to organization. Plant Cell 16(10):2546–2552. https://doi.org/10.1105/tpc.104.161030
Gardiner JC, Harper JD, Weerakoon ND, Collings DA, Ritchie S, Gilroy S, Cyr RJ, Marc J (2001) A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane. Plant Cell 13(9):2143–2158. https://doi.org/10.1105/tpc.13.9.2143
Golldack D, Li C, Mohan H, Probst N (2014) Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci 5:151
Granger CL, Cyr RJ (2001) Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD. Protoplasma 216(3-4):201–214. https://doi.org/10.1007/BF02673872
Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42(6):819–832. https://doi.org/10.1023/A:1006496308160
Ho AYY, Day DA, Brown MH, Marc J (2009) Arabidopsis phospholipase D as an initiator of cytoskeleton-mediated signalling to fundamental cellular processes. Funct Plant Biol 36(2):190–198. https://doi.org/10.1071/FP08222
Hong Y, Zhao J, Guo L, Kim SC, Deng X, Wang G, Zhang G, Li M, Wang X (2016) Plant phospholipases D and C and their diverse functions in stress responses. Prog Lipid Res 62:55–74. https://doi.org/10.1016/j.plipres.2016.01.002
Huesmann C, Reiner T, Hoefle C, Preuss J, Jurca ME, Domoki M, Feher A, Huckelhoven R (2012) Barley ROP binding kinase1 is involved in microtubule organization and in basal penetration resistance to the barley powdery mildew fungus. Plant Physiol 159:311–320
Johansson ON, Fahlberg P, Karimi E, Nilsson AK, Ellerstrom M, Andersson MX (2014) Redundancy among phospholipase D isoforms in resistance triggered by recognition of the Pseudomonas syringae effector AvrRpm1 in Arabidopsis thaliana. Front Plant Sci 5(639). https://doi.org/10.3389/fpls.2014.00639
Katagiri T, Takahashi S, Shinozaki K (2001) Involvement of a novel Arabidopsis phospholipase D, AtPLD-delta, in dehydration-inducible accumulation of phosphatidic acid in stress signalling. Plant J 26:595–605
Li JF, Park E, von Arnim AG, and Nebenführ A (2009) The FAST technique: a simplified Agrobacteriumbased transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6–21
Marc J, Sharkey DE, Durso NA, Zhang M, Cyr RJ (1996) Isolation of a 90-kD microtubule-associated protein from tobacco membranes. Plant Cell 8(11):2127–2138. https://doi.org/10.1105/tpc.8.11.2127
Marc J, Granger CL, Brincat J, Fisher DD, Kao T (1998) A GFP–MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10:1927–1939
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int Rev Cytol 132:1–30
Pinosa F, Buhot N, Kwaaitaal M, Fahlberg P, Thordal-Christensen H, Ellerstrom M, Andersson MX (2013) Arabidopsis phospholipase D delta is involved in basal defense and nonhost resistance to powdery mildew fungi. Plant Physiol 163:896–906
RCoreTeam (2013) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna
Sugimoto K, Williamson RE, Wasteneys GO (2000) New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis. Plant Physiol 124(4):1493–1506. https://doi.org/10.1104/pp.124.4.1493
Voight B, Timmers ACJ, Šamaj J, Müller J, Baluška F, Menzel D (2005) GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton. Eur J Cell Biol 84:494–507
Wang C, Wang X (2001) A novel phospholipase D of Arabidopsis that is activated by oleic acid and associated with the plasma membrane. Plant Physiol 127(3):1102–1112. https://doi.org/10.1104/pp.010444
Wang C, Li J, Yuan M (2007) Salt tolerance requires cortical microtubule reorganization in Arabidopsis. Plant Cell Physiol 48:1534–1547
Wang C, Zhang L, Yuan M, Ge Y, Liu Y, Fan J, Ruan Y, Cui Z, Tong S, Zhang S (2010) The microfilament cytoskeleton plays a vital role in salt and osmotic stress tolerance in Arabidopsis. Plant Biol 12:70–78
Wang S, Kurepa J, Hashimoto T, Smalle JA (2011) Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1. Plant Cell 23:3412–3427
West G, Inze D, Beemster GT (2004) Cell cycle modulation in the response of the primary root of Arabidopsis to salt stress. Plant Physiol 135:1050–1058
Zhang Q, Lin F, Mao T, Nie J, Yan M, Yuan M, Zhang W (2012) Phosphatidic acid regulates microtubule organization by interacting with MAP65-1 in response to salt stress in Arabidopsis. Plant Cell 24(11):4555–4576. https://doi.org/10.1105/tpc.112.104182
Zhang Q, Song P, Qu Y, Wang P, Jia Q, Guo L, Zhang C, Mao T, Yuan M, Wang X, Zhang W (2017) Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis. Plant Cell Environ 40:2220–2235 Article accepted
Acknowledgements
We thank Jan Petrášek for microscopy and further guidance and to Jiří Kubásek for the help with data processing.
Funding
This work was supported by the Czech Science Foundation Grant No. 14-09685S and by the Ministry of Education, Youth and Sports of the Czech Republic, projects NPU I, LO1417, and LM2015062. Microscope Zeiss 880 and LSM5DUO: IEB Imaging Facility is supported by OPPK—Operational Program Prague Competitiveness CZ.2.16/3.1.00/21519.
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Fig. S1
Subcellular localization of GFP-PLDδ pattern. a-d – dotted GFP-PLDδ pattern (a, c, d) in root cells of seven-day-old Arabidopsis thaliana stably transformed with 35S::GFP-PLDδ construct, stained with FM 4-64 dye (b, c, d). All pictures represents the same optical section of x,y plane (a, b, c) and y, z plane (d) imaging of GFP-PLDδ layer (a, c, d) and FM 4-64 stained plasma membrane (b, c, d). (c, d) merged a and b images. e-h - dotted and linear GFP-PLDδ pattern (e, g, h) in cotyledon pavement cells of nine-day-old Arabidopsis thaliana stably transformed with 35S::GFP-PLDδ construct. Microtubules (f, g, h) visualized 72 h after transient transformation with vector carrying 35S::RFP-MBD construct. Pictures display maximal intensity projection of twelve optical sections. Plane x,y (e, f, g) and y, z (h). (g, h) merged e and f images. i-j - linear GFP-PLDδ pattern (i, j) in hypocotyl cells of twelve-day-old Arabidopsis thaliana stably transformed with 35S::GFP-PLDδ construct. Pictures display maximal intensity projection of five optical sections. Plane x,y (i) and x, z (j). All pictures were taken with confocal laser scanning microscope Zeiss 880. Scale bars = 10 μm. (GIF 4092 kb)
Fig. S2
GFP-PLDδ linear pattern after treatment with 200 nM latrunculin B. a-c – linear GFP-PLDδ pattern in hypocotyl of twelve-day-old Arabidopsis thaliana stably transformed with 35S::GFP-PLDδ construct. Seedlings without treatment (a) or 18 min (b) and 34 min (c) after treatment with 200 nM latrunculin B. d-f - visualised actin filaments in leaf cells of ten-day-old Arabidopsis stably transformed with 35S::GFP-fABD2. Seedlings without treatment (d) or 15 min (e) and 21 min (f) after treatment with 200 nM latrunculin B. Single optical sections observed with confocal laser scanning microscope Zeiss 880. Scale bars = 10 μm. (GIF 4092 kb) (GIF 4736 kb)
Fig. S3
Differences in root length of Arabidopsis thaliana (Col-0) wild-type, Atpldδ and GFP-PLDδ exposed to salt stress – line chart. Five-day-old seedlings of wild type (WT), Atpldδ mutant (pldδ) and plants overexpressing 35S::GFP-PLDδ construct (GFP-PLDδ) were transferred to agar nutrient media containing 150 (b) or 200 mM (c) NaCl. Seedlings transferred to normal medium were used as control (ctrl - a). The root length was measured every day till day eleven. Values shown are averages ± SE (n = 15) from two independent experiments, with high significance. Scale bars = 10 μm. (GIF 119 kb)
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Angelini, J., Vosolsobě, S., Skůpa, P. et al. Phospholipase Dδ assists to cortical microtubule recovery after salt stress. Protoplasma 255, 1195–1204 (2018). https://doi.org/10.1007/s00709-018-1204-6
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DOI: https://doi.org/10.1007/s00709-018-1204-6