SlJAZ10 and SlJAZ11 mediate dark-induced leaf senescence and regeneration

Abstract

During evolutionary adaptation, the mechanisms for self-regulation are established between the normal growth and development of plants and environmental stress. The phytohormone jasmonate (JA) is a key tie of plant defence and development, and JASMONATE-ZIM DOMAIN (JAZ) repressor proteins are key components in JA signalling pathways. Here, we show that JAZ expression was affected by leaf senescence from the transcriptomic data. Further investigation revealed that SlJAZ10 and SlJAZ11 positively regulate leaf senescence and that SlJAZ11 can also promote plant regeneration. Moreover, we reveal that the SlJAV1-SlWRKY51 (JW) complex could suppress JA biosynthesis under normal growth conditions. Immediately after injury, SlJAZ10 and SlJAZ11 can regulate the activity of the JW complex through the effects of electrical signals and Ca2+ waves, which in turn affect JA biosynthesis, causing a difference in the regeneration phenotype between SlJAZ10-OE and SlJAZ11-OE transgenic plants. In addition, SlRbcs-3B could maintain the protein stability of SlJAZ11 to protect it from degradation. Together, SlJAZ10 and SlJAZ11 not only act as repressors of JA signalling to leaf senescence, but also regulate plant regeneration through coordinated electrical signals, Ca2+ waves, hormones and transcriptional regulation. Our study provides critical insights into the mechanisms by which SlJAZ11 can induce regeneration.

Fig 1. Expression of SlJAZ10 and SlJAZ11 is induced after dark treatment.

(a) Distribution of genes upregulated or downregulated by dark treatment in the wild-type. (b) Expression of representative SlJAZ genes during aging. The fold-change in the average expression (log2 scale) of each gene is shown. WT samples were taken 8 days after dark treatment. Three independent biological samples were used. (c) The relative expression level of SlJAZ10 and SlJAZ11 during aging. (d) Phylogenetic analysis of JAZ proteins in tomato. (e) Amino acid sequence alignment of SlJAZ10 and SlJAZ11 homologues. (f) sljaz10^ko (CRISPR/Cas9-SlJAZ10) and sljaz11^ko (CRISPR/Cas9-SlJAZ11) alleles identified from the T1 mutants of tomato. (g) The SlJAZ10-overexpressing (OE) and SlJAZ11-overexpressing (OE) lines. The tissue examined was mature leaf.

Fig 2. Physiological analyses of the mature leaves in tomato wild-type (WT), knockdown lines and overexpression lines after 8 days dark treatment.

(a) Mature leaves of the wild type, knock-out, and overexpression lines under dark treatment, incubated with wetted filter paper in dark conditions for 8d, and photographed. (b) Cell death was detected using the Tunel assay. Representative images show Tunel staining results (blue, DAPI; green, Tunel). (c-f) Contents of chlorophyll, MDA, ROS and GSH in the leaves of the WT, sljaz10, SlJAZ10-OE, sljaz11 and SlJAZ11-OE after dark treatment. (g-n) Relative expression of eight genes related to the above physiological phenotypes. All data are means (±SE) of three independent biological replicates. *P<0.01 (significant difference between mutants and WT according to Student^`s t-test).

Fig 3. SlJAZ10 and SlJAZ11 regulate JA and IAA signaling in dark-induced senescence.

(a) Mature leaves of the wild type and transgenic lines under dark treatment, incubated with wetted filter paper in dark conditions for 14d, and photographed. (b) The rooting rate of the wild type and transgenic lines under 14d dark treatment. *P<0.01 (significant difference between mutants and WT according to Student^`s t-test). (c) Expression of JA-responsive related genes in the RNA-seq experiments. The fold-change in the average expression (log2 scale) of each gene is shown. (d) Expression of IAA-responsive related genes in the RNA-seq experiments. The fold-change in the average expression (log2 scale) of each gene is shown. (e) JA content and IAA content of senescent leaves. (f) Heatmaps of hormone metabolism. The heat maps represent the log2 fold changes of DEGs related to hormone metabolisms.

Fig 4. The JJW (SlJAZ10-SlJAV1-SlWRKY51 or SlJAZ10-SlJAV1-SlWRKY51).

(a) Y2H assay shows that SlJAV1 interacts with SlJAZ10 and SlJAZ11. (b) Subcellular localization of SlJAZ10 and SlJAZ11 in the epidermal cells of N. benthamiana leaves. Bars = 40μm. (c) BiFC assay confirms the interactions among SlJAZ10, SlJAZ11, SlJAV1 and SlWRKY51 in N. benthamiana leaves. (d) Y1H assay shows the transcriptional binding activity of SlJAV1 and SlWRKY51 with SlAOC promoter. (e) Transient transcriptional activation assays show that SlJAZ10 and SlJAZ11 have transcriptional repression activity. (f) SlJAZ10-SlJAV1-SlWRKY51 and SlJAZ11-SlJAV1-SlWRKY51complex effectively suppresses the expression of SlAOC_Pro-LUC in N. benthamiana transient expression assay. Relative ratio of LUC/REN are means ± SEM (n≥6); *p < 0. and *p < 0.05; Student’s t test. (g) Relative expression of the luciferase gene (LUC) driven by the 130bp upstream fragment of the SlAOC promoter, with the W-box intacted or mutated.

Fig 5. Changes in electrical signals, calcium signals and hormone transports during injury-induced regeneration.

(a) Typical WASPs in wild type (WT), sljaz10, SlJAZ10-OE sljaz11 and SlJAZ11-OE after wounding leaves. (b) Confocal image of [Ca²⁺]_cyt in WT, sljaz10, SlJAZ10-OE sljaz11 and SlJAZ11-OE after wounding leaves. (c) Relative contents of JA and IAA at both ends of the wounding leaves. (d) Heatmap of auxin transport unigenes at both ends of the wounding leaves.

Fig 6. Ca²⁺ and SlRbcs-3B inactivates JJW Complex.

(a) Ca²⁺ alter transcriptional repression of SlJAZ10-SlJAV1-SlWRKY51 and SlJAZ11-SlJAV1-SlWRKY51 complex on the SlAOC_Pro-LUC expression. Relative ratio of LUC/REN are means ± SEM (n≥6); *p < 0.01; Student’s t test. (b) Y2H assay shows that SlRbcs-3B interacts with SlJAZ10 and SlJAZ11. (c) Subcellular localization of SlRbcs-3B in the epidermal cells of N. benthamiana leaves. Bars = 40μm. (d) BiFC assay confirms the interactions among SlJAZ10, SlJAZ11, and SlRbcs-3B in N. benthamiana leaves. (e) The interaction of His-SlJAZ10 and His-SlJAZ11 with GST-SlRbcs-3B detected by the pull-down assay. GST-SlRbcs-3B was used as bait, and pull-down of His-SlJAZ10 and His-SlJAZ11 was detected by anti-His antibody. (f) SlRbcs-3B alters transcriptional repression of SlJAZ11-SlJAV1-SlWRKY51 complex on the SlAOC_Pro-LUC expression. Relative ratio of LUC/REN are means ± SEM (n≥6); *p < 0; Student’s t test. (g) qRT-PCR analysis of transcript levels of SlJAV1, SlWRKY51 and SlRbcs-3B in dark-treatment leaves of WT, sljaz10, SlJAZ10-OE, sljaz11 and SlJAZ11-OE plants. Data are means ± SEM (n≥3); *p < 0.01; Student’s t test. (h) SlRbcs-3B promotes maintenance the stabilization of SlJAZ11 proteins in vivo. Proteins were extracted form N. benthamiana leaves transiently expressing SlJAZ11-GFP or SlRbcs-3B-Flag alone. Extracts containing SlRbcs-3B-Flag were incubated with SlJAZ11-GFP or GFP extracts for different times. Degradation of SlJAZ11-GFP was detected by anti-GFP antibody. An equal amount of SlRbcs-3B-Flag or Flag was detected by anti-Flag antibody. The equal amount of protein stained by CBB was used as a loading control. (i) A simplified model for injury-triggered JA biosynthesis in plant defense.