Regulation of CYP94B1 by WRKY33 controls apoplastic barrier formation in the roots leading to salt tolerance

Salinity is an environmental stress that causes decline in crop yield. Avicennia officinalis and other mangroves have adaptations such as ultrafiltration at the roots aided by apoplastic cell-wall barriers to thrive in saline conditions. We studied a Cytochrome P450 gene, AoCYP94B1 from A. officinalis and its Arabidopsis ortholog AtCYP94B1 that are involved in apoplastic barrier formation, and are induced by 30 minutes of salt treatment in the roots. Heterologous expression of AoCYP94B1 in atcyp94b1 Arabidopsis mutant and wild-type rice conferred increased NaCl tolerance to seedlings by enhancing root suberin deposition. Histochemical staining and GC-MS/MS quantification of suberin precursors confirmed the role of CYP94B1 in suberin biosynthesis. Using chromatin immunoprecipitation, yeast one-hybrid and luciferase assays, we identified AtWRKY33 as the upstream regulator of AtCYP94B1 in Arabidopsis. In addition, atwrky33 mutants exhibited reduced suberin and salt sensitive phenotypes, which were rescued by expressing 35S::AtCYP94B1 in atwrky33 mutant. This further confirms that the regulation of AtCYP94B1 by AtWRKY33 is part of the salt tolerance mechanism, and our findings can help in generating salt tolerant crops. One sentence summary AtWRKY33 transcription factor regulates AtCYP94B1 to increase plant salt tolerance by enhanced suberin deposition in the endodermal cells of Arabidopsis roots


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Salinity is a major environmental stress factor that leads to reduced crop productivity. The 50 progressive increase in soil salinization exacerbates the already damaging effect of steady 51 reduction in the area of arable land worldwide (Parida and Das, 2005;Agarwal et al., 2014). 52 Na + is the major toxic ion found in high saline soils, which imparts osmotic as well as ionic 53 stresses. It is imperative to limit the entry of excess Na + into plant cells in order to maintain 54 proper ion homeostasis, and normal metabolism. Mangroves have evolved various adaptive 55 strategies to flourish under high saline conditions. One of the important adaptations exhibited 56 by most plants, and to a greater extent by mangroves, is ultrafiltration at the roots by the 57 presence of apoplastic barriers in the roots (Scholander, 1968). In an earlier study, we have 58 shown that a salt secretor mangrove, A. officinalis restricts 90-95 % salt at the roots due to the 59 presence of enhanced apoplastic barriers (Krishnamurthy et al., 2014). 60 The main apoplastic diffusion barriers in roots are: epidermis, which is the outermost layer of 61 young roots, endodermis surrounding the vasculature of young roots and peridermis which 62 replaces both epidermis and endodermis in the older roots upon secondary thickening 63 (Nawrath et al., 2013;Wunderling et al., 2018). These apoplastic barriers mainly consisting 64 of Casparian strips (CSs) and suberin lamellae (SL) block the apoplastic and coupled 65 transcellular leakage of ions and water into the xylem, which is the major path of Na + uptake 66 (Yeo et al., 1987;Ma and Peterson, 2003;Krishnamurthy et al., 2011;Kronzucker and Britto, 67 2011;Schreiber and Franke, 2011;Andersen et al., 2015;Barberon et al., 2016). While CSs 68 are formed as radial wall thickenings, SL are secondary wall thickenings on the inner face of 69 primary cell-walls (Schreiber et al., 1999;Naseer et al., 2012). Chemically, CSs are made up 70 of mainly lignin and SL are made up of suberin and/or lignin depositions (Schreiber et al., 71 1999;Naseer et al., 2012). Together, these barriers function in biotic and abiotic stress 72 responses (Enstone et al., 2003;Krishnamurthy et al., 2009;Chen et al., 2011;Schreiber and 73 Franke, 2011;Ranathunge et al., 2011a). Suberin is a biopolymer consisting of aliphatic and 74 aromatic domains, with the aliphatic domain contributing mainly to its barrier properties 75 (Kolattukudy, 1984;Schreiber et al., 1999;Ranathunge and Schreiber, 2011b). Suberin 76 biosynthesis is a complex pathway involving elongases, hydroxylases and peroxidases 77 (Bernards et al., 2004;Franke et al., 2005;Hofer et al., 2008;Franke et al., 2009).
Several cytochrome P450 genes in the CYP94B subfamily such as, AoCYP94B1 and 114 AoCYP94B3 were identified in our earlier transcriptomic study of A. officinalis roots 115 (Krishnamurthy et al., 2017). Since some reports (Benveniste et al., 2006) suggest a role for 116 this subfamily genes in ω-hydroxylation, an important step in suberin biosynthesis, we chose 117 AoCYP94B1 for further characterization. A phylogenetic tree was constructed based on the 118 derived amino acid sequence of AoCYP94B1 with other members of this subfamily 119 (Supplemental Figure S1A). Rice OsCYP94B3 and Arabidopsis AtCYP94B1 were among 120 the homologs that share high level of sequence similarity with AoCYP94B1. AoCYP94B1 121 showed 60 % identity and 74 % similarity with AtCYP94B1, and 60 % identity and 71 % 122 similarity with OsCYP94B3. The Cytochrome P450 cysteine heme-iron ligand signature 123 motif was conserved across various plant species (Supplemental Figure S1B). 124 In A. officinalis seedlings without salt treatment, the AoCYP94B1 transcripts were 125 constitutively expressed in all tissues, but higher level of expression was observed in the 126 leaves and stems compared to roots ( Figure 1A). The transcript levels in the roots increased showed survival (with more green and healthy leaves) after recovery growth, only 33% of 160 atcyp94b1 plants could survive the treatment ( Figure 3B). There was no significant difference 161 in the leaf area among the genotypes, although a reduction in effective leaf area could be seen 162 due to curling up of leaves upon salt treatment in all the genotypes ( Figure 3C). Other growth 163 parameters such as chlorophyll content and FW/DW were measured and found to be 164 generally reduced in all the genotypes upon salt treatment. While atcyp94b1 mutants showed 165 3-and 4.5-fold reduction in chlorophyll content and FW/DW ratio respectively, these 166 reductions occurred to a lesser extent (~1.5-fold) in WT and 35S::AoCYP94B1 lines ( Figure   167 3D, E). These observations suggest that introduction of 35S::AoCYP94B1 into atcyp94b1 168 mutant rescues its salt sensitive phenotype even in the older plants. 169 We measured the total Na + and K + ion contents in the leaves and roots of WT, mutant and 170 35S::AoCYP94B1 plants under untreated and salt-treated (100 mM NaCl for 2 days) 171 conditions in order to understand the ion accumulation and distribution. There were no 172 differences in the ion contents among the WT and transgenic lines in the absence of salt 173 treatment. Upon NaCl treatment, the amount of Na + increased from 1 to 38 mg/g DW in the 7 leaves of atcyp94b1 mutants, while in the 35S::AoCYP94B1 leaves, the amount was 175 significantly lower (6, 8 and 7 mg/g DW in lines 1, 2 and 3, respectively) ( Figure 3F). No 176 significant differences were observed in the Na + and K + concentrations within the roots of the 177 different genotypes tested ( Figure 3F, G). These data indicate that the 35S::AoCYP94B1 lines 178 efficiently control endogenous Na + accumulation.  192 In order to gain further insights into the underlying molecular mode of action, we chose to 193 work with the Arabidopsis ortholog. This would permit more detailed biochemical and 194 molecular genetic analyses to be performed, which would not be feasible with Avicennia, a 195 perennial tree species that is not amenable to genetic transformation. mannitol treatment, as an alternate abiotic stress, did not cause significant changes to the 205 seedling root growth in any of these genotypes (Supplemental Figure S3).

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In view of the suggested role for CYP94B family genes in suberin biosynthesis, combined 207 with our observation of reduced Na + accumulation in the shoots of 35S::AoCYP94B1 lines, 208 we carried out GC-MS/MS quantification of several aliphatic components of the root suberin 209 monomers in WT, atcyp94b1 mutant, pAtCYP94B1::AtCYP94B1 complementation lines and 210 35S::AoCYP94B1 heterologous expression lines. The atcyp94b1 mutant showed a significant 211 reduction in the amount of ω-hydroxy acids and α, ω-dicarboxylic acid compared to the other 212 genotypes tested ( Figure 5E). No significant differences in the amounts of p-coumaric acid, 213 C-18 octadecanoic acid and C-22 docosanol was found between the genotypes tested. While 214 atcyp94b1 mutant showed ~50 % reduction in the amounts of alcohols (C-18 octadecanol and 215 C-20 eicosanol), ω-hydroxy acids (C-16 and C-22) and C-16 α, ω-dicarboxylic acid ( Figure   216 5E), the amounts were restored to the WT levels in pAtCYP94B1::AtCYP94B1 217 complementation lines. The increase seen in 35S::AoCYP94B1 lines was higher compared to 218 that of the WT, which could be either due to the strength of 35S promoter or because 219 AoCYP94B1 functions much more efficiently. Similarly, significantly higher amounts of C-220 16 ω-hydroxy acid and C-16 α, ω-dicarboxylic acid were present in 35S::AoCYP94B1 line 221 compared to WT indicating a role for CYP94B1 in their biosynthesis. All the standards 222 quantified for this study are shown in the chromatogram in Figure 5F. that AtCYP94B1 has a critical role in the formation of SL as the apoplastic barrier leading to 250 salt tolerance, but also that AoCYP94B1 could function similar to its Arabidopsis ortholog.

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Therefore, the use of AtCYP94B1 for further understanding of its molecular regulatory 252 mechanism can be justified.   Figure 7H). While AtWRKY6 did not show any better growth 293 compared to the control, there was very weak interaction with AtWRKY9 ( Figure 7H).

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Additionally, luciferase assay using atwrky33 Arabidopsis mutant protoplasts was carried out 295 to check the in vivo transcriptional activation of AtCYP94B1 promoter by AtWRKY33.

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Protoplasts transfected with pAtCYP94B1::LUC along with 35S::AtWRKY33 showed ~3-fold 297 higher luminescence compared to the ones transfected with the control, pAtCYP94B1::LUC 298 ( Figure 7I). The mutant pAtCYP94B1::LUC (with two WRKY binding sites mutated) showed 299 only ~1.5-fold higher luminescence compared to the control indicating that the mutation in 300 the TF binding sites indeed affects the promoter activity. Collectively, these results show that 301 AtWRKY33 TF acts as the upstream regulator of AtCYP94B1 gene.  previously. The potential to learn from such adaptive mechanisms to devise strategies for 326 crop improvement has been highlighted, but that is yet to be accomplished. The present study 327 represents a successful example of discovering and applying such mechanistic knowledge.

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To understand the role of CYP94B1 in salt stress response, we have used three plant species 329 (A. officinalis, Arabidopsis and rice) of varying ages. Our earlier transcriptomic study 330 involving A. officinalis which led to the identification of AoCYP94B1, was carried out using 331 2-month-old seedlings treated with 500 mM NaCl. Therefore, similar conditions were used 332 for A. officinalis in the current study. Experiments in Arabidopsis were carried out using the 333 young seedlings (one-week-old) and older (4-week-old) plants in order to understand their 334 response to salt in two developmental stages. While 50 mM NaCl was used for most of the 335 studies with younger seedlings as this did not damage the roots, 100 mM NaCl was used to 336 challenge the older plants. Similarly, two developmental stages (one-week-old and 4-week-337 old) of rice plants were used for our studies.   In silico analysis 491 The NCBI database was used as a search engine for nucleotide and protein sequences. Chlorophyll concentrations were determined spectrophotometrically using 100 mg FW of 517 untreated and recovered (from NaCl treatment) leaf material ground in 2 ml of acetone 80%

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(v/v). After complete extraction, the mixture was filtered and the volume adjusted to 5 ml 519 with cold acetone. The absorbance of the extract was read at 663 and 645 nm and pigment 520 concentrations were calculated as described previously (Arnon, 1949). The data represented 521 are mean ± SD of 4 biological replicates each with single plants.  where an endodermal cell length is clearly more than twice its width). The roots were divided 570 into various zones ( Figure 6A) such as undifferentiated zone (young part of the root with no 571 CS and SL), non-suberized zone (only CS, no SL) and suberized zone (patchy and continuous 572 SL). For FDA transport assay, seedlings were incubated for 1 min in 0.5 x MS FDA (5 µg ml -573 1 ), rinsed, and observed using a confocal laser scanning microscope (FV3000, Olympus).  Figure S6B) by site directed mutagenesis and the mutant promoter was cloned into pGreen II-610 0800-LUC vector and used as an additional control. The luciferase assay was carried out 611 using the Dual-Luciferase ® Reporter Assay System (Promega) following the manufacturer's 612 instructions. The luminescence was measured using the GloMax discover (Promega). Firefly 613 luciferase activity was normalized to Renilla luciferase activity. Data shown were taken from 614 four independent biological replicates each with three technical replicates.      AtCYP94B1 promoter fragment with mutated WRKY binding sites was used as additional 778 control. Firefly luciferase activity was normalized to Renilla luciferase activity and plotted.

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Data represent mean ± SD of four independent biological replicates each with three technical 780 replicates. Asterisks indicate statistically significant differences (**=P< 0.01) as measured by 781 Student's t-test between the control and the test.  Photographs were taken at the end of one week after germination. Scale bar=10 mm.        Chromatin immunoprecipitation (ChIP)-qPCR of HA-tagged AtWRKY33 in Arabidopsis protoplasts, AtWRKY6 and AtWRKY9 were used as negative controls. Fold change in the enrichment of promoter fragments compared to no protein control are plotted. qRT-PCR data represent means ± SD from 3 biological replicates each with 3 technical replicates. Asterisks indicate statistically significant differences (**=P< 0.01) between no protein control and AtWRKY33 as measured by Student's t-test. (H) Yeast one-hybrid assay showing regulation of AtCYP94B1 by AtWRKY33. AtWRKY6 and AtWRKY9 were used as additional controls. The representative growth status of yeast cells is shown on SD/-Leu agar medium with or without 100 ng of aureobasidin A. Numbers on the top of each photograph indicate relative densities of the cells 4 days post-inoculation. (I) Luciferase assay was carried out using the mesophyll protoplasts obtained from the leaves of 4-week-old atwrky33 mutants. The pAtCYP94B1::LUC was used as the control and 35S::AtWRKY33 was used as the test. AtCYP94B1 promoter fragment with mutated WRKY binding sites was used as additional control. Firefly luciferase activity was normalized to Renilla luciferase activity and plotted. Data represent mean ± SD of four independent biological replicates each with three technical replicates. Asterisks indicate statistically significant differences (**=P< 0.01) as measured by Student's t-test between the control and the test.