bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 1 The plastid-nucleus located DNA/RNA binding protein WHIRLY1 2 regulates microRNA-levels during stress 3 4 Aleksandra Swida-Barteczka2, Anja Krieger-Liszkay3, Wolfgang Bilger1, Ulrike Voigt1, Götz 5 Hensel4, Zofia Szweykowska-Kulinska2, Karin Krupinska1 6 7 1 Institute of Botany, Christian-Albrechts-University, Kiel, Germany 8 2 Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty 9 of Biology, Adam Mickiewicz University in Poznan, Poznan, Poland 10 3 11 Energies Alternatives Saclay, Centre National de la Recherche Scientifique, Université Paris- 12 Sud, Université Paris-Saclay, 91191 Gif-sur-Yvette, France 13 4 14 Germany Institute for Integrative Biology of the Cell, Commissariat à l’Energie Atomique et aux Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland/Gatersleben, 15 16 17 18 In this article a novel mechanism of retrograde signaling by chloroplasts during stress is 19 described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator 20 of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the 21 same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast 22 function and nuclear gene expression (Grabowski et al., 2008; Foyer et al., 2014). In this 23 study the putative involvement of WHIRLY1 in stress dependent retrograde signaling was 24 investigated by comparison of barley (Hordeum vulgare L., cv. Golden Promise) wild-type 25 and transgenic plants with an RNAi-mediated knockdown of WHIRLY1. In contrast to the 26 wild type, the transgenic plants were unable to cope with continuous high light conditions. 27 They were impaired in production of several microRNAs mediating post-transcriptional 28 responses during stress (Kruszka et al., 2012, Sunkar et al., 2012). The results support a 29 central role of WHIRLY1 in retrograde signaling and underpin a so far underestimated role of 30 microRNAs in this process. 31 32 WHIRLY1 belongs to a small plant specific family of DNA/RNA-binding proteins. By 33 immunological methods, WHIRLY1 has been detected in chloroplasts and the nucleus of the 34 same cell (Grabowski et al., 2008). Accordingly, functions of WHIRLY1 were reported for 1 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 35 both compartments. In chloroplasts of barley, WHIRLY1 was shown to be the major 36 compacting protein of nucleoids (Krupinska et al., 2014). Moreover, WHIRLY1 has been 37 found to bind to plastid RNAs (Melonek et al., 2010; Prikryl et al., 2008). In chloroplasts of 38 Arabidopsis thaliana, WHIRLY1 was reported to maintain plastid genome stability (Maréchal 39 et al., 2009). In the nucleus, WHIRLY1 was originally detected as a component of a 40 transcriptional activator of the PR10a gene of potato (Desveaux et al., 2000). Furthermore, it 41 has been found to bind to telomeres (Yoo et al., 2007). 42 Chloroplasts act as sensors of the environmental situation and produce diverse signals 43 informing about the functionality of the photosynthetic apparatus (Pfalz et al., 2012, Kleine 44 and Leister, 2016). These retrograde signals comprise redox changes and reactive oxygen 45 species and regulate gene expression in the nucleus in particular during stress situations 46 (Dietz, 2015). Although in recent years several compounds involved in chloroplast-to-nucleus 47 communication have been identified, the full repertoire of molecular mechanisms adjusting 48 nuclear gene expression to environmental cues remains obscure (Chan et al., 2016). 49 To investigate the impact of WHIRLY1 on stress resistance of barley plants, seedlings of 50 three independent transgenic lines with an RNAi-mediated knockdown of WHIRLY1 (RNAi- 51 W1-1, RNAi-W1-7 and RNAi-W1-9) were grown in continuous light at four different 52 irradiances (50, 120, 200, 350 µmol photons m-2 s-1). Leaves had reduced levels of the 53 WHIRLY1 protein ranging from undetectable traces (RNAi-W1-7) to 10% of the wild-type 54 level (RNAi-W1-1, RNAi-W1-9) (Krupinska et al., 2014). The reduction in length was the 55 same in both lines having 10% the WHILRLY1. Therefore, only the results obtained for line 56 W1-1 besides line W1-7 are presented in Figure 1. The reduction in leaf length occurred 57 irrespective of the irradiance (Fig. 1A) indicating that WHIRLY1 has a general positive effect 58 on growth. 59 Moreover the seedlings of the RNAi-W1 plants showed in contrast to the wild type bleaching 60 and a reduction of the chlorophyll content at 200 µmol photons m-2 s-1 (Fig. 1B). The 61 reduction was more prominent in case of the RNAi-W1-7 line, having the lowest level of 62 WHIRLY1 protein (Krupinska et al., 2014), as compared to the two other lines. 63 Analyses of carotenoids showed that in leaves of the RNAi-W1 plants the ratio of VAZ 64 (V=violaxanthin, A=antheraxanthin, Z=zeaxanthin) pool pigments to chlorophylls was 65 enhanced at irradiances of 200 and 350 µmol photons m-2 s-1 (Fig. 1C). The enhanced ratio of 66 VAZ/chlorophyll in the RNAi-W1 plants coincided with a higher de-epoxidation state of the 2 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 67 VAZ pool (Fig. 1D) indicating synthesis of zeaxanthin from violaxanthin. In line RNAi-W1-7 68 with the most extreme knockdown of WHIRLY1, the alterations were more dramatic than in 69 line RNAi-W1-1. At low light, no differences were detected between wild type and RNAi-W1 70 plants indicating that the alterations in the pigment composition are due to high light stress. 71 Zeaxanthin is known to have the highest antioxidative capacity of the xanthophylls and might 72 protect thylakoid membrane lipids from oxidation (Havaux et al., 2007). Besides its direct 73 effect as ROS scavenger, zeaxanthin plays an important role in non-photochemical quenching 74 dissipating excess energy as heat and avoiding thereby the production of reactive oxygen 75 species (Li et al., 2009). The enhanced de-epoxidation of the xanthophyll cycle pigments in 76 the RNAi-W1 plants compared to the wild type therefore indicates that their photosynthetic 77 apparatus absorbed more light than required for assimilation of carbon. ROS production by 78 thylakoids from RNAi-W1 or from wild-type seedlings grown at 200 µmol photons m-2 s-1 79 was measured by electron paramagnetic spin resonance (EPR). Indirect spin trapping of 80 superoxide/hydrogen peroxide using 4-POBN/ethanol/FeEDTA (Mubarakshina et al., 2010) 3 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 81 showed that RNAi-W1 thylakoids generated in the light about two times larger signals as 82 wild-type thylakoids (Fig. 2A, B). To investigate whether also singlet oxygen production by 83 thylakoids is enhanced in WHIRLY1 deficient chloroplasts, EPR measurements were 84 performed with the specific spin probe TMPD (Krieger-Liszkay et al., 2015). Using TMPD as 85 spin trap, no difference was observed between the wild type and the transgenic lines (Fig. 86 2A). 87 Taken together, analyses of pigments as well as ROS measurements revealed that the 88 WHIRLY1 deficient plants experienced more photooxidative stress than the wild type when 89 grown in continuous high light. This indicates that WHIRLY1, in addition to its positive 90 effect on growth, also promotes stress resistance. 91 Since WHIRLY1 in chloroplasts was shown to bind to RNA as well as to DNA (Melonek et 92 al., 2010) it was obvious to investigate a putative role of WHIRLY1 in controlling the levels 93 of microRNAs which play a central role in the control of plant development as well as in 94 stress responses (Kruszka et al., 2012; Li et al., 2016). For the analysis of microRNAs, 95 primary foliage leaves of wild-type plants and plants of the RNAi-W1-7 line, respectively, 96 grown either at low light (100 µmol photons m-2 s-1) or at high light (350 µmol photons m-2 97 s1), were used. Eight conserved microRNAs reported to be stress responsive in Arabidopsis 98 thaliana (Barciszweska et al., 2015) were selected and their levels were determined by 99 Northern blot analyses as well as by RT-qPCR TaqMan MicroRNA assays. 4 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 100 In wild-type plants the levels of most of these microRNAs were enhanced at high light 101 compared to low light conditions (Supplemental Fig. S1). These findings were confirmed by 102 RT-qPCR TaqMan MicroRNA assays, although the changes were not statistically significant 103 in each case (Fig. 3A). While in Northern blot analyses at least several members of a 104 microRNA family were detected (Supplemental Fig. S1), in RT-qPCR TaqMan MicroRNA 105 assays only specific members of a family were measured. Therefore the results of both 106 approaches are not always directly comparable, e.g. in case of miRNA159. 107 For all microRNAs tested in Northern blot hybridization the levels were reduced in leaves of 108 the two RNAi-W1 lines (RNAi-W1-1 and RNAi-W1-7) (Supplemental Fig. S1). These results 109 were confirmed by RT-qPCR TaqMan MicroRNA assays and were independent of the light 110 conditions (Supplemental Fig. S2A, B). 111 Some mRNA targets for the tested microRNAs are known in several plant species including 112 barley (Supplemental Table 1). Additional missing target mRNAs in barley were identified 113 using the psRNA-Target software (Dai and Zhao, 2011; 114 http://plantgrn.noble.org/psRNATarget/) (Supplemental Material S2). MicroRNAs hvu- 115 miR159a and hvu-miR159b-3p potentially target GAMyb mRNA, and microRNAs named 116 hvu-miR164a, hvu-miR172b-3p, hvu-miR393h and hvu-miR396b-5p target NAC, APETALA, 117 TIR1 and GRF1 mRNAs, respectively. Barley HOX9 and AGO1 mRNAs have been shown 118 experimentally to be targets of microRNA166a and microRNA168-5p, respectively (Kruszka 119 et al., 2014, Pacak et al., 2016). 120 The effects of the selected miRNAs on the levels of targeted mRNAs were tested by qRT- 121 PCR. In primary foliage leaves of wild-type plants grown in high light, the upregulation of 122 microRNAs coincided with a downregulation of targeted mRNAs (Fig. 3B). In contrast, in the 123 RNAi-W1 plants grown in high light most target gene mRNA levels are enhanced compared 124 to the wild type (Fig. 3C) whereas at low light the levels of target gene mRNAs are similar 125 between the wild type and RNAi-W1-7 plants (Supplemental Fig. S3). 126 The results indicate that high light induced signals from chloroplasts stimulate a WHIRLY1 127 dependent downregulation of the level of mRNAs targeted by the tested microRNAs being 128 upregulated in the wild type. In contrast to the wild type, plants of the RNAi-W1-7 line did 129 neither show a light-induced increase in microRNAs nor a decrease in the mRNA levels of 130 their target genes. This indicates that the WHIRLY1 deficient plants can’t respond to stress 131 and thereby suffer from a higher ROS production. 5 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 132 In the transgenic plants grown either in low light or in high light, the levels of targeted 133 mRNAs did not show essential differences (Fig. 3D). The only exception is NAC 134 transcription factor mRNA ( GenBank: AK356223.1) that is downregulated in W1-7 high and 135 low light grown plants despite the low level of its potential cognate microRNA164a. The 136 reason for this result remains unclear. NAC transcription factors comprise one of the largest 137 gene families and are involved in the regulation of plant development, senescence and 138 response to various stresses. Their activities can be regulated at different levels (transcription 139 efficiency, alternative splicing, posttranslational regulation) that possibly might affect the 140 final level of NAC mRNAs (Shao et al., 2015). 141 WHIRLY1 has been proposed to move from the chloroplast to the nucleus in response to 142 environmental cues such as high light intensity (Foyer et al., 2014). In this study it has been 143 demonstrated that the repertoire of the plants’ responses towards high light involves a 144 WHIRLY1 dependent increase in the levels of diverse nuclear microRNAs. As WHIRLY1 6 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 145 can bind to RNA it might be a general factor influencing the biogenesis and/or stability of 146 microRNAs. The observed phenomenon might be caused either by direct binding of 147 WHIRLY1 to the nuclear microRNAs and/or its architectural impact on nuclear chromatin as 148 observed in chloroplasts (Krupinska et al., 2014). To elucidate the specific role of WHIRLY1 149 in the regulation of the levels of microRNAs and targeted mRNAs during retrograde signaling 150 further detailed studies are required. 151 152 Acknowledgements 153 We would like to thank Artur Jarmolowski (AMU Poznan, Poland) for fruitful discussions. 154 Susanne Braun an Jens Hermann (CAU Kiel, Germany) are thanked for technical assistance. 155 We thank the German Research Foundation (DFG: Kr1350/7, Kr1350/9), National Science 156 Centre Poland (NSC UMO-2016/23/B/NZ9/00862), and the KNOW RNA Research Centre in 157 Poznan 01/KNOW2/2014 for financial support. 158 159 160 161 162 163 164 FIGURE LEGENDS 165 166 Figure 1. Characterization of WHIRLY1 knockdown lines at the seedling stage. Seedlings 167 were exposed to continuous irradiation at 50, 120, 200 or 350 µmol photons m s for 7 days. 168 Lengths of the primary leaves (cm) are indicated (A). Pigment extracts from the wild type 169 (WT) and the RNAi-W1 lines (W1-1, W1-7, W1-9) were compared by HPLC for the content 170 of chlorophylls/leaf area (B), the ratio of xanthophyll cycle pigments (VAZ) to chlorophyll 171 (C) and the de-epoxidation state of VAZ (D). De-epoxidation state was calculated as 172 (Z+0.5A)/(V+A+Z). All data are means of three samples, error bars denote standard 173 deviation. The results obtained for lines RNAi-W1-1 and RNAi-W1-9 are rather similar. Only 174 the results of RNAi-W1-1 are therefore shown. -2 -1 175 176 Figure 2. ROS production by thylakoids from wild type and RNAi-W1-1 as well as RNAi- 177 W1-7 lines. Thylakoids were prepared from seedlings grown in continuous light of 200 µmol 7 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 178 photons m-1s-1 . Superoxide/Hydrogen peroxide levels were measured by spin trapping EPR 179 using 4-POBN/EtOH/FeEDTA as spintrap and singlet oxygen by the spin probe TEMPD- 180 HCl (for experimental details, see Krieger-Liszkay et al., 2015). Thylakoids were illuminated 181 for two minutes with red light (500 µmol quanta m-2s-1) in the presence of the chemicals. Left: 182 representative spectra, right: EPR signal sizes (4-POBN/EtOH/FeEDTA) were normalized to 183 the signal obtained in wild-type thylakoids (mean ± SD, n=6). 184 185 Figure 3. RT-qPCR analysis of microRNAs and target genes expression in wild type (WT) 186 and transgenic RNAi -W1-7 plants exposed to either low (LL) or high light (HL). (A) In WT 187 plants exposed to high light the levels of microRNAs were enhanced. Results are presented as 188 fold change and results for WT plants grown in low light are treated as 1. (B) In the wild type 189 plants high light lead to a downregulation of the levels of target mRNAs. (C) Levels of most 190 target mRNAs were enhanced in high light treated W1-7 plants when compared to the wild 191 type. (D) Target mRNAs expression stayed mostly unchanged when RNAi-W1-7 plants 192 exposed to low and high light are compared. Error bars indicate SD (n=3), and the asterisk 193 indicates a significant difference between the sample and control (t test, *P≤0.05, **P≤0.01, 194 ***P≤0.001). 195 196 8 bioRxiv preprint doi: https://doi.org/10.1101/197202. this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. 197 SUPPLEMENTAL DATA 198 199 Supplemental Figure S1. Northern blot analysis of microRNA levels in low light (LL) and 200 high light (HL) in wild type (WT) and WHIRLY1 deficient barley plants (RNAi-W1-1 and 201 RNAi-W1-7). 202 203 Supplemental Figure S2. RT-qPCR analysis of microRNAs in wild type (WT) and 204 transgenic RNAi -W1-7 plants exposed to either low (LL) or high light (HL). 205 206 Supplemental Figure S3. RT-qPCR analysis of target genes expression in wild type (WT) 207 and transgenic RNAi -W1-7 plants exposed to either low light (LL). 208 209 Supplemental Table 1. List of microRNAs, their sequences, NCBI GEO accession numbers 210 of barley Next Generation Sequencing results and references. 211 212 Supplemental Table 2. List of microRNA sequences, TaqMan™ MicroRNA assays and 213 Northern probes used in the study. 214 215 Supplemental Table 3. Primer sequences used in the RT-qPCR of target mRNA levels. 216 217 Supplemental Material and Methods S1. 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