Phytotoxin coronatine enhances heat tolerance via maintaining photosynthetic performance in wheat based on Electrophoresis and TOF-MS analysis

Coronatine (COR) is a phytotoxin produced by Pseudomonas syringae. Its structure is similar to Jasmonates, which play a number of diverse roles in plant defense. Both have the COI1 plant receptor, so coronatine can manipulate plant hormone signaling to access nutrients and counteract defense responses. In addition to the hormone system, coronatine affects plant nitrogenous metabolism and chloroplast ultrastructure. In this study, we first examined a typical nitrogen-losing phenotype, and used the polyacrylamide gel approach to demonstrate soluble total protein patterns in a time-course experiment under different temperature conditions. We then employed dimensional gel electrophoresis technology (2-DE) and MALDI-TOF-MS to sequester and identify the sensitive proteins. We found a total of 27 coronatine sensitive proteins, 22 of which were located in the chloroplast and 6 of which were directly involved in photosynthesis. Finally, we measured levels of chlorophyll and photosynthetic performance to reveal the phenotypic effect of these proteins. Taken together, these results demonstrated that coronatine enhanced heat tolerance by regulating nitrogenous metabolism and chloroplast ultrastructure to maintain photosynthetic performance and reduce yield loss under heat stress.

Coronatine inducible proteins under heat stress in wheat leaves. We performed time course experiment to monitor production of soluble total protein. Results showed that coronatine pretreatment could change protein expression patterns. Heat shock proteins were induced by coronatine at an early stage, and disappeared at a later stage. Heat shock proteins reached a maximum concentration at 36 hours after coronatine treatment, and 24 hours delay than control plants (Fig. 2a). Depend on the time course results, we employed dimensional gel electrophoresis technology to identify twenty seven coronatine inducible proteins. Twenty two of these proteins are located in the chloroplast, and six are directly involved in photosynthesis (Fig. 2b,c; Table 2). When heat stress occurred, most of proteins significantly decreased their expression level, while coronatine pretreatment increased the stability of these proteins under heat stress (Figs 2c and 3).
Coronatine inducible proteins regulated nitrogenous metabolism and protein process. Glutamine synthetase (GS) plays an essential role in the metabolism of nitrogen (N) by catalyzing the condensation of glutamate and ammonia to form. GS2 (gi|71362640) is located mainly in the chloroplasts and mitochondria, and involved in nitrite reduction and photorespiration. Coronatine pretreatment increased GS2 expression level under heat stress. Cysteine synthase (gi|585032) and protein disulfide isomerase 2 (gi|13925726) are important in protein processes and structure rebuilding. Coronatine pretreatment significantly increased their expression level under heat stress (Figs 2c and 3).
Coronatine inducible proteins regulated chloroplast ultrastructure and photosynthesis. We found twenty two coronatine sensitive proteins located in the chloroplast, distributed in the chloroplast envelope, chloroplast stroma and chloroplast thylakoid membrane. 30S ribosomal protein S1 (gi|149391139) is involved in the chlorophyll biosynthetic process. OsFTSH2 (gi|75114857) plays a key role in PSII associated light-harvesting complex II catabolic process. CPN-60 alpha (gi|134102) is crucial to chloroplast organization and protein folding. Predicted protein (gi|326523691) is a target of chloroplast and directly involved in photosynthesis. When heat stress occurred, these proteins were unstable and degraded, while coronatine pretreatment increased their stability, and then increased the stability of chloroplast ultrastructure and membrane to maintain a higher photosynthesis under heat stress. (Table 2; Figs 2c and 3).
Coronatine inducible proteins also regulated plant defense and innate immune response. Eighteen of coronatine inducible proteins were response to abiotic stress, and eleven of them were positive regulation of heat stress. Coronatine pretreatment significantly increased the expression level of these eleven proteins under heat stress. Predicted protein (gi|326499830), a negative regulator of defense response, and pathogenesis-related protein 4 (gi|49615737), involved in killing cells of other organs, coronatine pretreatment decreased their expression level under heat stress. There were also several coronatine inducible proteins whose variation looked novel to stress: GST, cp31BHv and Ps16 are innate immune response proteins. Coronatine pretreatment decreased their expression level under heat stress. This result reflected the property of phytotoxin coronatine, it enhanced plant defense in some way, and stimulated plant immune system at the same time. (Table 2; Figs 2c and 3).
Taken together, most of coronatine inducible proteins are important to plant photosynthesis, chloroplast organization, and positive regulation of defense. They were stable under normal conditions. When heat stress occurred, these proteins were unstable and degraded, while coronatine pretreatment increased their stability, and then it increased the stability of chloroplast ultrastructure and membrane to maintain a higher photosynthesis under heat stress.
Coronatine decreased endogenous ABA production under heat stress. Some researches show that coronatine enters plant cells and causes stomata reopening, but the associated mechanisms of this action are still unclear. In our research, we focused on phytohormone ABA, which is closely related to stomata movement. Coronatine had no big difference on ABA production at the control temperature, while under heat stress, coronatine leaded to lower ABA production in both seedlings and flag leaves   (Table 3). This result suggested that coronatine might be involved in stomata movement by regulating ABA levels. Thus, more CO 2 entered plant cells, enhancing photosynthesis at some level. Acceleration of transpiration rate also cooled leaf temperature and facilitated uptake of water and nutrition, reducing the damage of high temperature. Thus, maintaining high relative leaf water content resulted from the balance of physiological process, not from stomata closure.
Coronatine enhanced the capacity of photosynthesis to reduce yield loss under heat stress. We chose both photosynthesis and chlorophyll fluorescence kinetics to reveal the causative mechanism. When heat stress occurred, the photosynthetic rate of coronatine pretreated plants was 20.1% higher than it in control plants, and chlorophyll maximum quantum yield was 15.6% higher than the control (Fig. 4a,b). Mature grain was small and shriveled under heat stress, but coronatine pretreated kernels remained plump, with TKW (Thousand Kernel Weight) 8.2% higher than control (Fig. 4c,d). This result also revealed in field test (Zhou 33 , 2013, Fig. 4e,f). All result suggested that coronatine could reduce yield loss by enhancing photosynthetic performance under heat stress.

Discussion
The structure of coronatine is similar to the phytohormone Jasmonates. Both have the COI1 plant receptor, so coronatine can manipulate plant hormone signaling 16 . During the last 20 years, the number of known plant hormones has grown from five to at least ten. The ubiquitin-proteasome pathway plays a central part in most hormone signaling pathways. The receptors of IAA, JA and Gibberellin are components of the SCF complex that consists of ASK, CUL and RBX 34 , so coronatine can affect the IAA, JA and Gibberellin pathways by the COI1 receptor. Coronatine also promotes stomata reopening through the E3 ligase subunit COI1. The pathway for stomatal closure involves triggering of the salicylic acid (SA) and abscisic acid (ABA) signaling pathways 20,35 . The mechanistic pathway of how coronatine regulates SA, ABA and NO to reopen stomata remains unclear; however, coronatine does affect these pathways. The precursor of the ethylene ACC is similar to CMA, which is the precursor of the coronatine. Some studies report that coronatine treatment can increase the key synthetase of ethylene 1-am inocyclopropane-1-carboxylate synthase, and improve plant production of ethylene 15 . In brief, coronatine can manipulate almost every hormone signaling pathway.
For our study, the plant phytotoxin coronatine showed a concentration effect similar to phytohormone auxin (IAA). High auxin amounts inhibit plant growth, while low auxin amounts can enhance plant growth. Similarly, high coronatine amounts lead to leaf chlorosis 22 , but low coronatine amounts could preserve the green phenotype of wheat leaves under heat stress. The phenotype of nitrogen loss caused us to focus on nitrogenous metabolism, since nitrogen is a key component of protein. Abiotic stresses usually cause protein dysfunction 35 . Maintaining proteins in their functional conformations is particularly important for cell survival under stress. Coronatine sensitive protein disulfide isomerase 2 is involved in protein structure rebuilding. CPN-60 alpha and some heat shock proteins are responsible for protein folding, assembly, translocation and degradation in many cellular processes and can assist in protein refolding under stress conditions. They play a crucial role in protecting plants against stress by reestablishing normal protein conformation. We found twenty two coronatine inducible proteins located in chloroplast, which is distributed in the chloroplast envelope, chloroplast stroma and chloroplast thylakoid membrane. Some proteins are a primary component of chloroplast ultrastructure. The 30S ribosomal protein S1 is involved in chlorophyll biosynthetic process. OsFTSH2 plays a key role in PSII associated light-harvesting complex II catabolic process. Coronatine pretreatment increased their stability, so it increased the stability of chloroplast ultrastructure and membrane to maintain a higher photosynthesis under heat stress. Take together, in physiological metabolism, coronatine mainly affects nitrogenous metabolism via regulating protein processes and chloroplast ultrastructure to maintain photosynthetic performance in wheat (Fig. 5).     Protein extraction. 1 g of wheat leaves was randomly collected and finely ground in liquid nitrogen.

Methods
Protein extraction was performed according to Rinalducci et al. (2011) 37 with some modifications. Three biological replicates were used. The powder was suspended (1 g/mL) in chilled lysis buffer in acetone containing 0.007% DTT and 1% plant protease inhibitor cocktail (Bio-Rad, USA). The mixture was incubated at − 20 °C for at least 1 h, and then centrifuged at 12000 r for 15 minutes before the supernatant was collected.
Twenty-four centimeter IPG strips (Bio-Rad, USA) pH 4-7 were passively rehydrated overnight with 750 μ g of protein in 300 μ l of solution containing 1% carrier ampholyte (Bio-lyte 4-7; Bio-Rad, USA  Table 3. ABA production in wheat seedlings and flag leaves. Each value represents the mean ± SD (n = 10). Bars showing the same latter are not significantly different at P ≤ 0.05 as determined by LSD test. Image analysis. Two dimension gel images were digitized using a flatbed scanner (model Image Scanner-II, GE Healthcare) with a resolution of 300 dpi and 16-bit greyscale pixel depth. Image analysis was carried out with Image Master 7.0, which allows spot detection, background subtraction, and protein spot OD intensity quantification. Spot quantity values were normalized in each gel dividing the raw quantity of each spot by the total quantity of all the spots included in the standard gel. For each protein spot, the average spot quantity value and its variance coefficient in each group was determined. The least significant difference (LSD) test was used to determine significant differences among group means. Protein spots were cut out from coomassie blue stained gels and subjected to trypsin digestion according to Shevchenko 38 with minor modifications. Peptide mixtures were separated using Auto-flex2   (Germany, Brook) system. A sample volume of 1 μ L was loaded by the autosampler. Gene function and location analysis were downloaded from the NCBI and TAIR website. Each protein was classified with respect to its cellular component, biological process, and molecular function. Protein expression pattern analysis was performed using Cluster 3.0. The data were analyzed statistically according to a randomized block design using SAS statistical software. The least significant difference (LSD) was calculated for the significant data at P ≤ 0.05.

RNA extraction, cDNA synthesis and qRT-PCR.
Corresponding genes were double confirmed by quantitative RT-PCR. Total RNAs were prepared from wheat leaves treated with DNase before being subjected to cDNA synthesis using Superscript III reverse transcriptase primed by oligo dT. Quantitative RT-PCR was performed in 96-well plates with an ABI 7500Fast real-time PCR system using the SYBR Green II mix (Applied Biosystems). PCR primers were designed using the Primer Express Software DNAman. Three biological replicates were performed, and the reactions were performed in triplicate for each run. The comparative CT method was used to evaluate the relative quantities of each amplified product in the samples. The threshold cycle (CT) was automatically determined for each reaction by the system according to the default parameters 39 . The specificity of the PCR was determined by dissociation curve analysis of the amplified products using the standard method installed in the system 26 . Primers used for qRT-PCR is listed in Table 4.
Protoplast preparation. Protoplast isolation was based on the protocol for wheat mesophyll protoplasts provided online by J.Sheen's laboratory http://genetics.mgh.harvard.edu/sheenweb/ with some modifications. Coronatine was added in the solution directly.