Warm temperature triggers JOX and ST2A-mediated jasmonate catabolism to promote plant growth

Plants respond to warm temperature by increased elongation growth of organs to enhance cooling capacity. Phytohormones, such as auxin and brassinosteroids, regulate this growth process. However, our view on the players involved in warm temperature-mediated growth remains fragmentary. Here, we show that warm temperature leads to an increased expression of JOXs and ST2A, genes controlling jasmonate catabolism. This leads to an elevated 12HSO4-JA level and consequently to a reduced level of bioactive jasmonates. Ultimately this results in more JAZ proteins, which facilitates plant growth under warm temperature conditions. Taken together, understanding the conserved role of jasmonate signalling during thermomorphogenesis contributes to ensuring food security under a changing climate.

C limate change impacts crop productivity and small increases in average temperatures are predicted to lead to large decreases in crop yield 1 . In plants, architectural adaptations to high ambient temperatures (referred to as thermomorphogenesis) allow improved cooling by evaporation to withstand warm temperatures 2 . Also in wheat, shoot elongation is promoted by high ambient temperatures 3 (Supplementary Fig. 1). In Arabidopsis thaliana, phytochromes, such as phytochrome B (phyB), and the basic helix-loop-helix transcription factor PHY-TOCHROME INTERACTING FACTOR 4 (PIF4) are central and required regulators of warm temperature-mediated growth 2 . In addition, plant hormones, such as auxin and brassinosteroids, are critical regulators of thermomorphogenesis 2 . Notably, while jasmonic acid (JA) signaling plays a vital role in various stress responses 4 , including wound response, cold stress, and heat stress, whether and how JA impacts warm temperature-mediated organ growth remains unknown.
JA biosynthesis and signaling pathways are well characterized 5 . The enzyme JASMONATE RESISTANT 1 (JAR1) conjugates JA with isoleucine to yield the bioactive version jasmonoyl-(L)-isoleucine (JA-Ile), which is perceived by a receptor complex including CORONATINE INSENSITIVE 1 (COI1) and a JAS-MONATE ZIM-DOMAIN (JAZ) protein. The JAZ proteins inhibit the functioning of target transcription factors, such as the MYCs, and are subject to 26S proteasome-mediated degradation following ubiquitination by the SCF COI1 E3 ubiquitin ligase complex to modulate JA responses. To control JA signaling, the availability of bioactive JA (mainly JA-Ile) is a critical factor, and thus there are numerous metabolic reactions that convert JA to other active, inactive, or partially active compounds. For example, the three endoplasmic reticulum (ER)-localized cytochrome P450 oxidases CYP94B1, CYP94B3, and CYP94C1 generate the JA derivatives 12-hydroxy JA-Ile (12OH-JA-Ile) and 12-carboxy JA-Ile (12COOH-JA-Ile), the amidohydrolases IAR3 and ILL6 cleave JA-Ile back to free JA and in addition act on 12OH-JA-Ile, defining an indirect biosynthetic route to 12-hydroxy JA (12OH-JA), and the four JASMONATE-INDUCED OXYGENASES (JOXs)/JASMONIC ACID OXIDASES (JAOs) redundantly catalyze the oxidation of JA to 12OH-JA 6 . Subsequently, 12OH-JA can be sulfated by the sulfotransferase ST2A to form 12-hydroxy jasmonoyl sulfate (12HSO 4 -JA) 6,7 .
Here, we report that JAZ proteins are stabilized by a warm temperature that promotes hypocotyl elongation in Arabidopsis. This repression of COI1-mediated JAZ degradation is due to reduced levels of bioactive JA. Furthermore, complementary analyses in wheat indicate that the role of JA in regulating seedling's growth at warm temperature is conserved.

Results and discussion
Our observation of COI1 phosphorylation in wheat seedlings exposed to 34°C for 1 h (Supplementary Fig. 2) prompted us to investigate the role of JA signaling in warm temperaturemediated wheat growth. Wheat seedlings overexpressing Arabidopsis or wheat 12-OXOPHYTODIENOATE REDUCTASE 3 (OPR3), a key enzyme in JA biosynthesis 6 , which display mild and variable elevated 3-oxo-2-(2-(Z)-pentenyl) cyclopentane-1butyric acid (OPC4) and JA levels 8,9 ( Supplementary Fig. 3), or wheat seedlings treated with JA methyl ester (MeJA) exposed to a growth-promoting temperature displayed a significantly shorter second leaf than their respective control plants or treatment (Fig. 1a, b and Supplementary Figs. 4,5). Thus, JA represses warm temperature-mediated wheat seedling growth.
To further explore a possibly conserved role of JA and JA signaling in warm temperature-mediated growth, we then focused on hypocotyl growth in Arabidopsis, which is a hallmark for thermomorphogenesis 2 . Warm temperature-triggered hypocotyl elongation was largely repressed by MeJA treatment at 28°C in a concentration-dependent manner (Fig. 1c, d and Supplementary  Fig. 6a, b). In contrast, the Arabidopsis aos mutant, which lacks JA 10 , displays a slightly longer hypocotyl at 28°C compared to wild type ( Supplementary Fig. 6c, d). In agreement with previous observations 11 , MeJA also leads to reduced Col-0 hypocotyl growth at 21°C, but this is less pronounced than at 28°C (Fig. 1d and Supplementary Fig. 6b). For further analyses, we used 30 µM MeJA, as this resulted in an obviously shorter hypocotyl at 28°C compared to control plants at 21°C. This effect of 30 µM MeJA at 28°C is also reflected in the expression of EXPANSIN-LIKE A 2 (EXLA2), a positive regulator of cell elongation 12 , and EXPANSIN A8 (EXPA8), encoding an auxin-induced cell wall-modifying enzyme involved in turgor-driven cell expansion 13 , which is upregulated at 28°C, but repressed by MeJA at 28°C (Fig. 1e and Supplementary Fig. 7a, b). It should be noted that, in contrast to EXPA8, EXLA2 is not rapidly upregulated by 28°C (Supplementary Fig. 7b). Furthermore, the expression of PIF4 targets INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) and YUC8 14 was (partially) reduced by MeJA at 28°C (Fig. 1f and Supplementary Fig. 7a, b). Interestingly, the expression of HSP70, a transcriptional marker for warm temperature response 15 , is not downregulated by MeJA at 28°C ( Supplementary Fig. 7a), indicating that the overall temperature perception is not affected. Taken together, these results indicate that JA represses warm temperature-mediated organ growth in distinct species.
Next, we checked if the MeJA-mediated repression of hypocotyl length at 28°C required the core JA signaling components, such as COI1, the JAZ proteins, and the MYCs. The hypocotyl of the coi1-21 mutant 16 , the myc2 myc3 myc4 mutant 17 , and lines overexpressing JAZ proteins 18,19 were significantly longer than the wild type at 28°C (Fig. 1g and Supplementary Fig. 8), further indicating that JA signaling exerts repression on growth at a warm temperature. Furthermore, coi1-21 and myc2 myc3 myc4 mutant seedlings were less sensitive to 30 µM MeJA at 28°C (Fig. 1h, i and Supplementary Fig. 9a, b). In contrast, hypocotyl length of coi1-21, myc2 myc3 myc4, and 35S::JAZ1:GUS lines was hardly affected by 30 µM MeJA at 21°C (Supplementary Fig. 10). In addition, lines producing truncated JAZ3 proteins lacking the C-terminal Jas domain that plays a key role in destabilizing JAZ repressors in response to increased JA levels 19 , such as jai3-1 or 35S::JAZ3-Δjas-GFP, were also less sensitive to exogenous 30 µM MeJA application at 28°C ( Fig. 1g and Supplementary Fig. 9c, d). Conversely, the hypocotyl length of the 35S::JAZ3:GFP and 35S:: JAZ1:GUS seedlings, overexpressing wild-type JAZ proteins, was still repressed by 30 µM MeJA at 28°C, comparable to MeJAtreated Col-0 at 28°C (Fig. 1g and Supplementary Fig. 9e). We furthermore observed that in the hypocotyl of the 35S::JAZ1:GUS line 18 , the JAZ1:GUS levels strongly and within a few hours increased at 28°C compared to 21°C, while the JAZ1:GUS expression levels remained unaltered (Fig. 1j, k and Supplementary Figs. 7c, 11a). Similarly, in the hypocotyl of the 35S::Jas9-VENUS line 20 , the Jas9-VENUS levels increased at 28°C compared to 21°C ( Supplementary Fig. 12). Taken together, this indicates that JAZ proteins are stabilized in the hypocotyl at 28°C. Notably, however, the strong JAZ1:GUS signal at 28°C could be fully suppressed by 30 µM MeJA (Fig. 1j, k), indicating that JAZ degradation can be triggered by exogenous MeJA under warm temperature. However, the increased JAZ levels are in contrast to the 28°C-mediated increase in COI1 levels (Supplementary Fig. 11b), which is similar to previous observations of a heat-mediated increase in COI1 levels 21 . Nonetheless, together our data indicate that JA represses thermomorphogenesis and that this requires the JA signaling machinery. Specifically, high levels of JAZ repressor proteins (in coi1 or 35S::JAZΔjas lines) or the absence of MYC-mediated response (in myc2 myc3 myc4) result in a long hypocotyl at 28°C. Our data support the stabilization of JAZ proteins and reduced JA signaling at 28°C, and furthermore suggest that this is likely, not due to an impaired perception of JA at 28°C.
Since degradation of JAZ proteins is dependent on the availability of the bioactive JA, JA-Ile 5 , we speculated that warm temperature affects the availability of bioactive JA. To test this hypothesis, we measured JA-Ile levels in both Arabidopsis and wheat seedlings exposed to high temperature. This indeed revealed that high temperature leads to a clear reduction in JA-Ile levels in Arabidopsis and to a reduction, although less pronounced, in JA-Ile levels in wheat ( Fig. 2a and Supplementary  Fig. 13). However, the expression of several enzymes involved in JA biosynthesis was not affected or even up-regulated at warm temperature ( Supplementary Fig. 14). Furthermore, in Arabidopsis, cis-(+)-12-oxo phytodienoic acid (OPDA) and 10-oxo dinor-phytodienoic acid (dn)-OPDA were not significantly affected and even showed a trend of increase at warm temperature like the expression of the regulatory enzymes (Supplementary Fig. 15a, b). In wheat, however, this trend is less uniform, varies for different compounds, and differs between the varieties ( Supplementary Fig. 15a, c-e). Next, we, therefore, focused on the diverse catabolic processes that control the level of bioactive First, an iar3 ill6 cyp94b1 cyp94b3 cyp94c1 pentuple mutant, which displays increased JA-Ile levels 22 , displayed a slightly shorter hypocotyl at high temperature compared to the wildtype ( Supplementary Fig. 16a). This further supported that reducing JA-Ile is required to promote hypocotyl growth at a warm temperature. However, while IAR3, ILL6, and CYP94 expression levels were upregulated by warm temperature in Arabidopsis (Supplementary Fig. 16b, c), the levels of 12OH-JA-Ile and 12COOH-JA-Ile were not higher at control versus high temperature in Arabidopsis or wheat ( Fig. 2a and Supplementary Fig. 16d) and the iar3 ill6 double or cyp94b1 cyp94b3 cyp94c1 triple mutants did not display a hypocotyl phenotype at warm temperature (Supplementary Fig. 16e). This indicated that this pathway is not the main mechanism to control JA-Ile levels under warm temperature.
Second, with respect to the JOXs and ST2A-mediated catabolic pathway, 12OH-JA levels were below the detection limit in  Samples were harvested at noon. ND, not detected (too low to be detected and considered as 0, and thus replaced with 0 when performing statistical analysis and calculating average). Dots depict 3 biological replicates (white background) and the average (Avg) (gray or red background). An * denotes significant differences (p < 0.05) using a Student's t-test with two-sample equal variance and one-tailed distribution. b, c Relative expression of JOX and ST2A genes in 7-days-old Col-0 wild type shoots at 21 and 28°C collected at dawn (b) and of genes encoding for putative wheat JOX and ST2A orthologs in 10-days-old Chinese Spring shoots at 19 and 29°C collected during the day (c). Graphs depict average (line) of 3 biological replicates (dots). An * denotes significant differences (p < 0.05) using a Student's t-test with two-sample equal variance and one-tailed distribution. The exact p-value (or p < 0.0001) is shown at the top. ns not significant.
Arabidopsis, and 12OH-JA levels were not higher at control versus high temperature in wheat ( Fig. 2a and Supplementary  Fig. 17). However, while 12HSO 4 -JA levels in Arabidopsis were not changed, the 12HSO 4 -JA levels were strongly increased in wheat ( Fig. 2a and Supplementary Fig. 18). In addition, expression of JOX and ST2A, genes encoding for the enzymes involved in regulating the catabolic processes leading to 12HSO 4 -JA, was significantly upregulated at warm temperature in both wheat and Arabidopsis, but-at least in Arabidopsis-this increase in expression is time-of-day-dependent 23,24 (Fig. 2b, c and Supplementary Fig. 19). To explore whether JOX and ST2Amediated catabolism contribute to Arabidopsis thermomorphogenesis, we first used a 35S::JOX1 line as a genetic tool to reduce the levels of JA and JA-Ile at high temperature 25 (Supplementary Fig. 20). We observed a longer hypocotyl of the 35S:: JOX1 line at 28°C compared to the control, while no difference was observed at 21°C (Fig. 3a, b and Supplementary Fig. 21a). Next, to confirm the role of JOX proteins in Arabidopsis thermomorphogenesis, we measured the hypocotyl of jox1, jox2, jox3, jox4 single mutants, and a jox1 jox2 jox3 jox4 (jox-Q) quadruple mutant 25 at 21°C and 28°C. This revealed a significantly shorter hypocotyl and less increased growth for jox1, jox2, jox3, jox4, and jox-Q seedlings compared to the wildtype at 28°C and at warm temperature relative to 21°C, respectively (Fig. 3c, d and Supplementary Fig. 21b). The rather modest phenotype of the jox-Q mutants may be attributed to other catabolic pathways taking over. Indeed, we observed reduced levels of JA and JA-Ile in jox-Q at 28°C, compared to the elevated levels at 21°C (Supplementary Fig. 22). Next, we also explored if ST2A is important for thermomorphogenesis. Indeed, both st2a-1 and st2a-2 displayed a shorter hypocotyl at 28°C compared to the wildtype (Fig. 3e, f and Supplementary Fig. 21c). Finally, we also observed that JOXs and ST2A are required for other thermomorphogenesis phenotypes, such as petiole length and angle 2 ( Supplementary Figs. 21d and 23). The importance of JA and JA-Ile catabolic processes at high temperature is further reflected in the increased sensitivity of jox-Q and iar3 ill6 cyp94b1 cyp94b3 cyp94c1 to MeJA treatment at 28°C (Supplementary Fig. 24). Taken together, this supports that organ growth under warm temperature conditions requires a tight control of available JA-Ile levels, mainly through JOX and ST2Amediated catabolism. It should be noted that although jox and st2a mutants display obvious warm temperature-associated phenotypes, we did not detect 12OH-JA or a change in 12HSO 4 -JA at warm temperature in Arabidopsis to support this. This may indicate that also other regulatory mechanisms, such as glycosylation of 12OH-JA 26 , may be involved in the reduction of JA and JA-Ile levels at warm temperature in Arabidopsis.
In conclusion, warm temperature leads to an increased expression of genes controlling JA catabolism. Subsequent altered levels of bioactive JA-Ile stabilize JAZ proteins, which in turn facilitates plant growth. Similarly, the neighbor competition promotes plant growth by upregulating ST2A that acts to reduce the bioactive JA molecules in a phyB-dependent manner 7 . Additionally, high temperature enhances JA-dependent defense responses in tomato, resulting from increased COI1 levels and wounding-induced bioactive JA levels 21 . Our data add an additional layer of control where bioactive JA levels are tightly controlled at a warm temperature to modulate JAZ protein levels and accommodate warm temperature-mediated growth. The balance between levels of bioactive JA and the amount of COI1 determines-together with JAZ expression levels-JAZ protein levels and thus the response; and this seems to depend on the type of stress and even the combination of stresses. It is important to note that plants have a narrow temperature range for optimal growth and warm temperature-mediated growth promotion, and outside this range-such as extreme cold or heat-JA levels and signaling can be regulated differently leading to different responses. A broader understanding of the conserved role of jasmonate signaling when plants are exposed to low or high

Methods
Plant material and growth conditions. All A. thaliana plants used in this study were in the Col-0 reference accession genetic background and referred to as wild type. The following A. thaliana lines were used: aos (ref. 10 ), coi1-21 (ref. 16 ), myc2 myc3 myc4 (ref. 17 ), jai3-1 (ref. 19 ), jox1-4 single mutants (ref. 25 ), jox-Q (ref. 25 ), st2a-1 and st2a-2 (ref. 7 ), iar3 ill6 (ref. 22 ), cyp94b1 cpy94b3 cpy94c1 (ref. 22 ), iar3 ill6 cyp94b1 cpy94b3 cpy94c1 (ref. 22  sucrose. Seeds were stratified for 3 days at 4°C firstly and then transferred to a growth chamber at 21°C, with continuous light (70-100 μmol m −2 s −1 ) at 50% relative humidity for germination. After germination, seeds on vertical plates were transferred to short-day conditions of 8 h light/16 h dark either at 21°C or 28°C (100 μmol m −2 s −1 photosynthetically active radiation supplied by cool-white, fluorescent tungsten tubes, Osram). Additionally, seeds for JA treatment were sown onto sterilized mesh on medium as mentioned above. After germination, the seeds with mesh were transferred to a medium as mentioned above supplemented with DMSO as mock or 30 µM MeJA (Sigma-Aldrich, CAS: 39924-52-2) for JA treatment at 21°C or 28°C. Plates were scanned at 7 days after germination, unless indicated otherwise. The wheat seeds used in this study were from hexaploid wheat cultivars: Chinese Spring 27 , Sar-60, Tr-3 and Tr-18 (ref. 9 ), KN199, and TaOPR3 OE #3 (ref. 8 ). The seeds were put on wet paper enclosed by plastic wrap and kept at 4°C for 3-4 days, and then transferred to room temperature in the darkness for 1 day for germination. Seeds that germinated uniformly were selected and grown in plastic pots containing soil at the temperatures of interest under 16 h light/8 h dark (100 µmol m -2 s -1 photosynthetically active radiation, supplied by cool-white, fluorescent tungsten tubes, Osram) and 65-75% air humidity. In addition, for JA treatment in wheat plants, 5 ml mock (DMSO) or 200 µM MeJA solution was injected into soil grown with 1 day after germination (DAG) wheat seedlings at the temperatures of interest. The injection was applied every 3 days (three times injection in total). Phenotyping pictures were taken with a Canon digital camera. Measurement was done by using Fiji/ImageJ (https://imagej.net/Fiji). For protein extraction, 7-days-old 35S::GFP-COI1 seedlings were grown at 21°C and 28°C in short-day conditions on ½ MS solid medium. Or, 5-days-old 35S::Jas9-VENUS seedlings were grown at 21°C in short-day conditions on ½ MS solid medium, then 5-days-old seedlings were transferred to ½ MS liquid medium to adapt for 1 day at 21°C at the same conditions. After adapting to the liquid medium, seedlings were transferred to a new liquid medium supplemented with DMSO as a mock for 20 h at 21°C or 28°C. Protein quantification was done by using Fiji/ ImageJ (https://imagej.net/Fiji). For GUS expression, GUS staining and GUS activity, 5-days-old 35S::JAZ1-GUS seedlings were grown onto sterilized mesh at 21°C in the absence or presence of 30 µM MeJA and 28°C in the absence or presence of 30 µM MeJA in short-day conditions on ½ MS solid medium. For short-term treatment, 5-days-old 35S::JAZ1-GUS seedlings were grown onto mesh at 21°C in short-day conditions on ½ MS solid medium, then 5-days-old seedlings with mesh were transferred to ½ MS solid medium as mentioned above supplemented with mock or JA treatment at 28°C for 12 h. Or, 5-days-old Col-0 seedlings were grown onto mesh at 21°C in short-day conditions on ½ MS solid medium, then 5-days-old seedlings with mesh were transferred to ½ MS solid medium as mentioned above supplemented with mock or JA treatment at 21 and 28°C for 12 h.
Gene expression analyses. Shoot samples from 7-days-old or short-term (12 h) treated Arabidopsis seedlings were harvested at dawn or noon. 10-days-old wheat (Chinese Spring) seedlings were collected during the day. Three or six biological replicates were performed for each condition and each tested gene. Total RNA from Arabidopsis shoots or wheat seedlings was extracted and purified with the RNeasy Mini Kit (Qiagen GUS staining assay. 35S::JAZ1-GUS seedlings were fixed for 30 min in ice-cold 90% (v/v) acetone and rinsed with NT buffer (100 mM Tris/50 mM NaCl), incubated in X-Gluc (500 µg ml −1 5-bromo-4-chloro-3-indolyl-β-D-glucuronide) solution (500 µl 100 mM K 3 [Fe(CN) 6 ] + 600 µl X-Gluc + 28.8 ml NT-buffer), and incubated at 37°C in the dark for 6-8 h. The reaction was stopped by rinsing with NT-buffer, then seedlings were mounted in 80% lactic acid on a slide and imaged.
Quantitative GUS activity assay. The GUS activity assay was performed according to a previously published method, with some modifications 28 . For this, 5-days-old 35S::JAZ1-GUS shoot samples were harvested at dawn. Three biological replicates were performed for each condition. Finely ground material from 50 to 100 mg of shoots was homogenized in 500 μl of GUS extraction buffer (100 mM EDTA (pH 8.0), 0.1% SDS, 50 mM sodium phosphate (pH 7.0), 0.1% Triton X-100), adding 10 mM β-mercaptoethanol, 25 μg ml −1 PMSF (ThermoFisher Scientific) just before use. Samples were centrifuged at 20,817×g at 4°C for 10 min and the supernatant (crude extract) was transferred to new pre-cooled 1.5 ml Eppendorf tubes that were kept on ice. Next, 10 μl of crude extract was added to the 1. Confocal microscopy. Five-day-old 35S::Jas9:N7-VENUS seedlings at 21 and 28°C were imaged on a Leica SP8 confocal laser scanning microscope (Leica Microsystems, Germany). Scanner and detectors settings used for one experiment were optimized to avoid saturation and to maximize resolution and kept unchanged throughout the experiment. VENUS was excited using the 514 nm line (Leica SP8). VENUS fluorescence was collected from 520 to 560 nm (using the AOBS of the SP8). RFP was excited using a 561-nm laser diode (SP8). RFP fluorescence was collected from 590 to 680 nm (using the AOBS of the SP8). Images were taken using the ×40 objective, to maximize the number of nuclei being observed.
Statistical analyses and reproducibility. Phenotypic quantification, metabolite measurement, and relative gene expression data were analyzed using either Oneway or Two-way analysis of variance (ANOVA) with Tukey's honest significant difference (HSD) in GraphPad Prism 8. In some cases, data were analyzed using Students' t-test with two-sample equal variance and one-tailed distribution. For box plots, the lower Tukey-based whisker shows the smallest value that is greater than the lower quartile minus 1.5× interquartile range, the upper Tukey-based whisker shows the greatest value that is smaller than the upper quartile plus 1.5× interquartile range and data points outside this range (outliers) are plotted as individual dots.
Phylogenetics. The orthologues of the Arabidopsis genes in other species (Brachypodium, wheat, rice, maize, and barely) were identified using gene trees available at EnsemblPlants. The amino acid sequences of the orthologs were aligned by the "Align by Muscle" function and then the phylogenetic trees were reconstructed using an UPGMA statistical method implemented in MEGA5.05 and then manually edited.

Data availability
All data are available from the corresponding author upon request. Source data are provided with this paper.
Received: 27 January 2021; Accepted: 13 July 2021; Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.