MYB30 orchestrates systemic reactive oxygen signaling and plant 5 acclimation 6

27 Systemic acquired acclimation (SAA) is a key biological process essential for plant survival 28 under conditions of abiotic stress. SAA was recently shown to be controlled by a rapid systemic 29 signaling mechanism termed the reactive oxygen species (ROS) wave in Arabidopsis 30 ( Arabidopsis thaliana ). MYB30 is a key transcriptional regulator mediating many different 31 biological processes. MYB30 was found to act downstream of the ROS wave in systemic tissues 32 of Arabidopsis in response to local high light (HL) stress treatment. However, the function of 33 MYB30 in systemic signaling and SAA is unknown. To determine the relationship between 34 MYB30, the ROS wave, and systemic acclimation in Arabidopsis, the SAA response to HL 35 stress of myb30 mutants and wild-type plants was determined. Although myb30 plants were 36 found to display enhanced rates of ROS wave propagation and their local tissues acclimated to 37 the HL stress, they were deficient in SAA to HL stress. Compared to wild type, the systemic 38 transcriptomic response of myb30 plants was also deficient, lacking in the expression of over 39 3,500 transcripts. A putative set of 150 core transcripts directly associated with MYB30 function 40 during HL stress was determined. Our study identifies MYB30 as a key regulator that links 41 systemic ROS signaling with systemic transcriptomic responses, SAA, and plant acclimation to 42 HL stress. In addition, it demonstrates that plant acclimation and systemic ROS signaling are 43 interlinked, and that the lack of systemic acclimation drives systemic ROS signaling to occur at 44 faster rates, suggesting a feedback mechanism (potentially involving MYB30) between these two 45 processes. 46 47 48 50


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Although we previously identified MYB30 as a core ROS wave-associated transcript 84 (Zandalinas et al., 2019), its function in the regulation of the ROS wave and/or SAA was not 85 experimentally defined. In the current study, we used a genetic approach to study the role of 86 MYB30 in the ROS wave and SAA responses of Arabidopsis to HL stress.

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To determine the involvement of MYB30 in the systemic response of Arabidopsis to HL stress, 89 two independent T-DNA insertion mutants of MYB30 (myb30-1 and myb30-2) were studied 90 (Fig. 1A). Wild-type (WT) and mutant plants were exposed to a local HL stress treatment and  (Fichman et al., 2019;Zandalinas et al., 2019). In contrast to a local treatment of HL stress, the 96 rate of systemic ROS accumulation in the myb30-1 and myb30-2 mutants was similar to WT in 97 response to a local treatment of wounding or heat stress (Supplemental Fig. S2), suggesting that 98 the role of MYB30 could be specific to HL stress. To determine the relationship between 99 MYB30, the ROS wave, and plant acclimation, we compared the SAA of WT plants to that of 100 the myb30 mutants. It was shown earlier that SAA to HL stress is dependent on ROS wave 101 signaling (Suzuki et al., 2013;Devireddy et al., 2018;Zandalinas et al., 2019Zandalinas et al., , 2020. Wild-type 102 plants, showing normal levels of the ROS wave, displayed local and systemic acclimation to HL 103 stress following a 10 min application of HL stress to a single local leaf (Fig. 1C). In contrast, 104 following the same treatment, myb30 mutants displayed local acclimation, but were deficient in 105 SAA (Fig. 1C). These findings revealed a surprising effect in the myb30 mutants in which the 106 ROS wave and SAA were uncoupled. Although the myb30 mutants displayed a stronger and 107 faster ROS wave response, this response was not accompanied by SAA, suggesting that MYB30 108 may be required to couple these two processes. 109 To determine the underlying cause of the decoupling between the ROS wave and SAA in the 110 myb30 mutants, transcriptomic analysis was performed. Changes in transcript steady-state levels 111 in WT and the myb30-1 ( Fig. 1A; Li et al., 2009;Liao et al., 2017) Tables S1-S9). Similar to Zandalinas et al., 114 (2019Zandalinas et al., 114 ( , 2020, large sets of transcripts responded within this time frame in the local and systemic 115 leaves of WT plants to the local application of light stress (Fig. 2, Supplemental Fig. S4). 116 Comparing these sets of transcripts between WT and the myb30-1 mutant revealed that the 117 systemic response of WT to HL stress that included over 4,200 upregulated transcripts, was 118 much more comprehensive than that of the myb30-1 mutant that included over 1,400 upregulated 119 transcripts ( Fig. 2A). Similar differences were found between WT and myb30 for downregulated 120 transcripts in systemic leaves in response to the same treatment ( Fig. 2A; Supplemental Fig. S4).  Tables S1, S2). Interestingly, although 136 MYB30 is a single transcriptional regulator, these transcripts could be segregated into three 137 similar size clusters, representing fast and transiently responsive transcripts, fast responsive 138 transcripts that are altered in abundance but thereafter are maintained at their new level, and 139 slower response transcripts (Fig. 2B). This finding suggests that MYB30 could function during 140 early stages of the systemic response and that its disruption affects multiple responses that span 141 different groups of transcripts and pathways. In support of such a central role of MYB30 is also 142 the sheer number of transcripts affected by the lack of MYB30 (over 5,000 transcripts that are 143 altered in abundance in WT but not in myb30-1; Fig. 2A; Supplemental Tables S1, S2). MYB30-6 dependent upregulated transcripts include a high representation of protein transport, proteolysis, 145 ubiquitination, and stress-response pathways; while MYB30-dependent downregulated 146 transcripts include mostly translation related pathways (Fig. 2B). Comparing the MYB30-147 dependent transcripts identified in this study (Supplemental Tables S1, S2) with groups of 148 transcripts associated with different abiotic stresses (drought, cold, heat, high light, salt, and 149 ozone), response to wounding, pathogen, and hormones, different types of ROS, or 150 overexpression of MYB30 in the presence or absence of ROS (Mabuchi et al., 2018;Zandalinas 151 et al., 2019;Willems et al., 2016) revealed that they contain a high proportion of high light-, 152 wounding-, H 2 O 2 -, MYB30-associated, and ABA-response transcripts (Supplemental Table S3,  Table S4). Because the ROS wave is enhanced in myb30 mutants but 165 SAA does not occur, it is possible that these transcripts are required for SAA. Another possibility 166 is that these MYB30-dependent transcripts are involved in suppressing the ROS wave and their 167 absence enables the ROS wave to propagate faster. Two of the ROS wave-associated transcripts 168 induced in systemic leaves of WT, but not myb30, GATA8, and the GDSL esterase/lipase, were 169 previously shown to be essential for SAA to HL stress in Arabidopsis (Zandalinas et al., 2019).

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In addition, the promoters of these genes contain putative MYB binding motifs, suggesting that 171 they could be under the control of MYB30 (Supplemental Tables S5-S7 and text below). We 172 therefore tested whether mutants of these genes display a ROS wave phenotype similar to the 173 myb30 mutants. As shown in Figure 3, compared to WT, the gata8-1, gata8-2, gdsl-1, and gdsl-2 174 mutants displayed an enhanced ROS wave phenotype, similar to the myb30 mutants ( Fig. 1 Table S5). Next, a screen for the following MYB cis-binding elements,  Table S6) was conducted to   on the other, balancing these two responses and resulting in tolerance to excess light at the whole 226 plant level. In its absence, the coupling of these two processes is lost and acclimation does not 227 occur even though the ROS wave occurs at high levels ( Fig. 1). MYB30 could also be required 228 for acclimation, without directly impacting the ROS wave, and in its absence the ROS wave 229 could be turned on even faster and stronger in an attempt to compensate for the lack of SAA 230 (Fig. 4C).

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The two plausible scenarios described above could be reflected in the transcriptomic analysis  Table S1), suggesting that MYB30 could, at least partially, 249 regulate systemic light responses via PIF4 (Fig. 4C). In addition to regulating light responses, 250 PIF4 was also found by another recent study to regulate ROS homeostasis (Sun et al., 2020).  Table S9). This finding 262 supports previous findings and suggests that MYB30 has a vast array of overlapping and non-263 overlapping functions in cells that may or may not be linked to ROS, HL stress, and/or SAA  Supplemental Table S10. PCR cycles included 5 min denaturation at 95°C, 5 min 285 annealing at 60°C, and 1 min elongation at 72°C. The cycles were repeated 30 times and then 286 followed by 5 min of 72°C. PCR products were separated on a 1.2% Tris-Acetic acid-EDTA 287 agarose gel with 0.5µg/ml ethidium bromide and visualized using Uvidoc HD6 gel  Leaf injury following light stress was measured using the electrolyte leakage assay, as previously 325 described (Suzuki et al., 2013;Devireddy et al., 2018;Zandalinas et al., 2019). In short, a single 326 leaf from a 4-week-old plant was illuminated with 1,700 µmol photons s -1 m -2 for 45 min and    14 In addition, a list of MYB30-associated genes was obtained using the TF2Network prediction 384 tool (Kullkarni et al., 2016).

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The RNA-seq data analyzed in this study were deposited in the Gene Expression Omnibus 387 (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE141916). Quantitative 388 data of the described transcripts is provided in Supplemental Tables S1 and S2.

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The following supplemental materials are available.