The RNA Polymerase-Associated Factor 1 Complex Is Required for Plant Touch Responses

Journal of Experimental Botany, Vol. 68, No. 3 pp. 499–511, 2017 doi:10.1093/jxb/erw439 Advance Access publication 15 December 2016 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER The RNA Polymerase-Associated Factor 1 Complex Is Required for Plant Touch Responses Gregory S. Jensen1,2, Kateryna Fal3, Olivier Hamant3 and Elizabeth S. Haswell1,* 1 2 3 Department of Biology, Mailbox 1137, Washington University in Saint Louis, Saint Louis, MO 63130, USA Current address: Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, F-69342, Lyon, France Received 1 June 2016; Editorial decision 4 November 2016; Accepted 4 November 2016 Editor: Bruno Moulia, INRA-University Blaise Pascal, France Abstract Thigmomorphogenesis is a stereotypical developmental alteration in the plant body plan that can be induced by repeatedly touching plant organs. To unravel how plants sense and record multiple touch stimuli we performed a novel forward genetic screen based on the development of a shorter stem in response to repetitive touch. The touch insensitive (ths1) mutant identified in this screen is defective in some aspects of shoot and root thigmomorphogenesis. The ths1 mutant is an intermediate loss-of-function allele of VERNALIZATION INDEPENDENCE 3 (VIP3), a previously characterized gene whose product is part of the RNA polymerase II-associated factor 1 (Paf1) complex. The Paf1 complex is found in yeast, plants and animals, and has been implicated in histone modification and RNA processing. Several components of the Paf1 complex are required for reduced stem height in response to touch and normal root slanting and coiling responses. Global levels of histone H3K36 trimethylation are reduced in VIP3 mutants. In addition, THS1/VIP3 is required for wild type histone H3K36 trimethylation at the TOUCH3 (TCH3) and TOUCH4 (TCH4) loci and for rapid touch-induced upregulation of TCH3 and TCH4 transcripts. Thus, an evolutionarily conserved chromatin-modifying complex is required for both short- and long-term responses to mechanical stimulation, providing insight into how plants record mechanical signals for thigmomorphogenesis. Key words: Histone methylation, Paf1 complex, TCH genes, thigmomorphogenesis, touch response, VIP3. Introduction It is firmly established that development in multicellular organisms relies on the local concentration of key biochemical signals such as hormones or growth factors (Wolpert, 1969). However, it is becoming increasingly clear that the temporal pattern of changes in the concentration of these biochemical signals is equally important for the final phenotypic output (Raj and van Oudenaarden, 2008; Lander, 2011; Oates, 2011; Webb et al., 2016). For instance, the intrinsic delays associated with transcription and translation have been proposed to play an instructive role in patterning (Monk, 2003; Gaffney and Monk, 2006). Understanding how the intensity as well as the frequency of a stimulus are measured and recorded is a key challenge for plant biologists, as post-embryonic development is continuously impacted by the environmental conditions in which the plant grows. Abbreviations: Col-0, Columbia-0; FLC, Flowering Locus C; JA, jasmonic acid; LINC, linker of the nucleoskeleton and cytoskeleton; me3, trimethylation; Paf1, RNA polymerase II-associated factor 1; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; qRT-PCR, quantitative reverse-transcriptase PCR; SAM, S-ADENOSYLMETHIONINE SYNTHASE; TBST, Tris-buffered saline with Tween 20; TCH, TOUCHths1, touch insensitive 1; VIP3, VERNALIZATION INDEPENDENCE 3 © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: 500 | Jensen et al. to touch stimulation. The cml24 (tch2) mutation affects root morphology on hard agar and is implicated in microtubule structure and starvation-induced autophagy (Wang et al., 2011; Tsai et al., 2013) but a functional role for TCH genes in thigmomorphogenesis has yet to be established. While these results demonstrate that touch is perceived at the molecular level after a single event, no link between short-term and longterm responses to touch has been identified. In trees, the transcription factor gene ZFP2 is rapidly induced in response to bending (Leblanc-Fournier et al., 2008; Coutand et al., 2009). Interestingly, this induction can be attenuated after multiple bending events, suggesting that plants are able to record the number of mechanical perturbations, albeit through an unknown mechanism (Martin et al., 2010). The growth response is likely to involve the phytohormone jasmonic acid (JA). JA accumulates in the stems of plants after touching of their rosette leaves, and JA biosynthesis and signaling are required for thigmomorphogenesis (Chehab et al., 2012). Conversely, application of the growth regulatory hormone gibberellin or a loss-of-function mutation in the gibberellin-catabolizing enzyme AtGA2ox7 can prevent thigmomorphogenesis (Lange and Lange, 2015). However, these effects are potentially far downstream in the pathway. No real known regulator of thigmomorphogenesis capable of sensing repetition has been identified yet. We therefore performed a novel forward genetic screen designed to identify the key molecular and genetic components that link successive elastic deformations to thigmomorphogenesis. Arabidopsis exhibits several stereotypical thigmomorphogenic responses that require multiple days of touch, including the inhibition of stem elongation, shortened petioles, reduced rosette diameter and delayed transition to flowering (Braam and Davis, 1990; Chehab et al., 2012; Cazzonelli et al., 2014; Lange and Lange, 2015). Here we concentrated on stem thigmomorphogenesis to identify new regulators of the touch response. We screened for plants that did not exhibit shortened stems after repeated touch. The first mutant isolated in this screen identified a known regulator of gene expression, the RNA Polymerase II-associated factor 1 (Paf1) complex, as a key element of thigmomorphogenesis that serves to integrate touch stimuli over time. Materials and methods Touch response assays For Fig. 1 and Fig. 2, plants were grown for one week in long day conditions of 16 hours of light and 8 hours of darkness at 23°C, then transferred to 24 hours of light at 25°C for one more week. These conditions were established during the screen and were replicated in later experiments to reduce variability due to grow (...truncated)