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Vacuolar H

The basal level of the plant defense hormone jasmonate (JA) in unstressed leaves is low, but wounding causes its near instantaneous increase. How JA biosynthesis is initiated is uncertain, but the lipolysis step that generates fatty acid precursors is generally considered to be the first step. Here, we used a series of physiological, pharmacological, genetic, and kinetic analyses of gene expression and hormone profiling to demonstrate that the early spiking of JA upon wounding does not depend on the expression of JA biosynthetic genes in Arabidopsis (Arabidopsis thaliana). Using a transgenic system, we showed how decoupling the responses to wounding and JA prevents the perpetual synthesis of JA in wounded leaves. We then used DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) as a model wound-responsive lipase to demonstrate that although its transient expression in leaves can elicit JA biosynthesis to a low level, an additional level of activation is triggered by wounding, which causes massive accumulation of JA. This wound-triggered boosting effect of DAD1-mediated JA synthesis can happen directly in damaged leaves or indirectly in undamaged remote leaves by the systemically transmitted wound signal. Finally, protein stability of DAD1 was influenced by wounding, α-linolenic acid, and mutation in its catalytic site. Together, the data support mechanisms that are independent of gene transcription and translation to initiate the rapid JA burst in wounded leaves and demonstrate how transient expression of the lipase can be used to reveal changes occurring at the level of activity and stability of the key lipolytic step.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a “fly-casting mechanism.” We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light–dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.

Hydrogen sulfide (H2S) serves as an important gaseous signaling molecule that is involved in intra- and intercellular signal transduction in plant–environment interactions. In plants, H2S is formed in sulfate/cysteine reduction pathways. The activation of endogenous H2S and its exogenous application has been found to be highly effective in ameliorating a wide variety of stress conditions in plants. The H2S interferes with the cellular redox regulatory network and prevents the degradation of proteins from oxidative stress via post-translational modifications (PTMs). H2S-mediated persulfidation allows the rapid response of proteins in signaling networks to environmental stimuli. In addition, regulatory crosstalk of H2S with other gaseous signals and plant growth regulators enable the activation of multiple signaling cascades that drive cellular adaptation. In this review, we summarize and discuss the current understanding of the molecular mechanisms of H2S-induced cellular adjustments and the interactions between H2S and various signaling pathways in plants, emphasizing the recent progress in our understanding of the effects of H2S on the PTMs of proteins. We also discuss future directions that would advance our understanding of H2S interactions to ultimately mitigate the impacts of environmental stresses in the plants.

Active molecules uncovered through chemical genetics studies have provided unique molecular genetic tools with which to study specific life processes. Different strategies have been developed to study the modes of action of these small molecules, especially for the target identification, including affinity chromatography (for target identification) and genetic/genomic methods. In this chapter we describe the protocols for a conventional forward genetics screening against seed germination inhibitors to study their working mechanism in model plant Arabidopsis. Such methods have been applied to study small molecules germostatin and triplin, the copper ion-binding small molecule.

A new synthetic auxin AAL1 with new structure was identified. Different from known auxins, it has weak effects. By AAL1, we found specific amino acids could restore the effects of auxin with similar structure. Auxin, one of the most important phytohormones, plays crucial roles in plant growth, development and environmental response. Although many critical regulators have been identified in auxin signaling pathway, some factors, especially those with weak fine-tuning roles, are still yet to be discovered. Through chemical genetic screenings, we identified a small molecule, Auxin Activity Like 1 (AAL1), which can effectively inhibit dark-grown Arabidopsis thaliana seedlings. Genetic screening identified AAL1 resistant mutants are also hyposensitive to indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). AAL1 resistant mutants such as shy2-3c and ecr1-2 are well characterized as mutants in auxin signaling pathway. Genetic studies showed that AAL1 functions through auxin receptor Transport Inhibitor Response1 (TIR1) and its functions depend on auxin influx and efflux carriers. Compared with known auxins, AAL1 exhibits relatively weak effects on plant growth, with 20 µM and 50 µM IC50 (half growth inhibition chemical concentration) in root and hypocotyl growth respectively. Interestingly, we found the inhibitory effects of AAL1 and IAA could be partially restored by tyrosine and tryptophan respectively, suggesting some amino acids can also affect auxin signaling pathway in a moderate manner. Taken together, our results demonstrate that AAL1 acts through auxin signaling pathway, and AAL1, as a weak auxin activity analog, provides us a tool to study weak genetic interactions in auxin pathway.

Active molecules uncovered through chemical genetics studies have provided unique molecular genetic tools with which to study specific life processes. Different strategies have been developed to study the modes of action of these small molecules, especially for the target identification, including affinity chromatography (for target identification) and genetic/genomic methods. In this chapter we describe the protocols for a conventional forward genetics screening against seed germination inhibitors to study their working mechanism in model plant Arabidopsis. Such methods have been applied to study small molecules germostatin and triplin, the copper ion-binding small molecule.

Seed development is an important process of reproductive development and consists of embryo and endosperm development; both comprise several key processes. To determine and investigate the functions of the dynamic DNA methylome during seed development, we profiled the DNA methylation genome wide in a series of developmental stages of rice (Oryza sativa) embryo and endosperm by methylcytosine immunoprecipitation followed by Illumina sequencing. The results showed that embryo is hypermethylated predominantly around non-transposable element (TE) genes, short DNA-TEs, and short interspersed TEs compared with endosperm, and non-TE genes have the most diverse methylation status across seed development. In addition, lowly expressed genes are significantly enriched in hypermethylated genes, but not vice versa, confirming the crucial role of DNA methylation in suppressing gene transcription. Further analysis revealed the significantly decreased methylation at early developing stages (from 2 to 3 d after pollination), indicating a predominant role of demethylation during early endosperm development and that genes with a consistent negative correlation between DNA methylation change and expression change may be potentially directly regulated by DNA methylation. Interestingly, comparative analysis of the DNA methylation profiles revealed that both rice indica and japonica subspecies showed robust fluctuant profiles of DNA methylation levels in embryo and endosperm across seed development, with the highest methylation level at 6 d after pollination (2 d after pollination of endosperm in japonica as well), indicating that a complex and finely controlled methylation pattern is closely associated with seed development regulation. The systemic characterization of the dynamic DNA methylome in developing rice seeds will help us understand the effects and mechanism of epigenetic regulation in seed development.

Abscisic acid (ABA), the most important stress-induced phytohormone, regulates seed dormancy, germination, plant senescence, and the abiotic stress response. ABA signaling is repressed by group A type 2C protein phosphatases (PP2Cs), and then ABA binds to its receptor of the ACTIN RESISTANCE1 (PYR1), PYR1-LIKE (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family, which, in turn, inhibits PP2Cs and activates downstream ABA signaling. The agonist/antagonist of ABA receptors have the potential to reveal the ABA signaling machinery and to become lead compounds for agrochemicals; however, until now, no broad-spectrum antagonists of ABA receptors blocking all PYR/PYL-PP2C interactions have been identified. Here, using chemical genetics screenings, we identified ABA ANTAGONIST1 (AA1), the first broad-spectrum antagonist of ABA receptors in Arabidopsis (Arabidopsis thaliana). Physiological analyses revealed that AA1 is sufficiently active to block ABA signaling. AA1 interfered with all the PYR/PYL-HAB1 interactions, and the diminished PYR/PYL-HAB1 interactions, in turn, restored the activity of HAB1. AA1 binds to all 13 members. Molecular dockings, the non-AA1-bound PYL2 variant, and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2. Using AA1, we tested the genetic relationships of ABA receptors with other core components of ABA signaling, demonstrating that AA1 is a powerful tool with which to sidestep this genetic redundancy of PYR/PYLs. In addition, the application of AA1 delays leaf senescence. Thus, our study developed an efficient broad-spectrum antagonist of ABA receptors and demonstrated that plant senescence can be chemically controlled through AA1, with a simple and easy-to-synthesize structure, allowing its availability and utility as a chemical probe synthesized in large quantities, indicating its potential application in agriculture.

Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in Arabidopsis thaliana. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown Arabidopsis seedlings through ethylene signaling pathway. ran1-1 and ran1-2 are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of ran1-1 and ran1-2. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in Arabidopsis, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human., Author summary Copper ions are cofactors of protein functions, and their disorder is closely related to many human diseases which drive to develop new copper chelator related drugs. In plants, copper ions are essential for ethylene receptors, but many details are unclear. Researchers need novel specific copper chelators to study these questions. Here, by using plant chemical genetics, we identified a novel chemical triplin, which could activate ethylene signaling pathway by chelating copper ions essential for ethylene receptors. Ethylene resistance mutant etr1-1 and ein2 were resistant to triplin, and copper transporter mutants ran1-1 and ran1-2 were hypersensitive to triplin. Using triplin as a molecular genetics tool, we showed copper chaperone ATX1 acts the upstream of RAN1 which transports copper ions to ethylene receptors. We provide a sample that metabolism could be studied by combining chemical genetics and known signaling pathway. Moreover, triplin could chelate copper ions effectively and specifically in Arabidopsis, and could be a useful tool to study copper ion transport in plant, and valuable for designing copper chelator related drug.

Seed dormancy and germination are the most important adaptive traits of seed plants, which control the germination in a proper space and time. Internal genetic factors together with environmental cues govern seed dormancy and germination. Abscisic acid (ABA), a key phytohormone induces seed dormancy and inhibits seed germination through its molecular genetic signaling network responding the seed inherent physiological and environmental factors. Recently, auxin has been shown to be another phytohormone that induces seed dormancy. We have recently shown that germonstatin (GS), a small synthetic molecule identified by high through-put chemical genetic screenings, inhibits seed germination through up-regulating auxin signaling and inducing auxin biosynthesis. GERMOSTATIN RESISTANCE LOCUS 1 (GSR1) encodes a plant homeodomain (PHD) finger protein and is responsible for GS seed germination inhibition. Its knockdown mutant gsr1 displays decreased dormancy. In this report, we show that GS is not an ABA analog and provided 2 other GS-resistant mutants related to the chemical's function in seed germination inhibition other than gsr1, suggesting that GS may have pleiotropic effects through targeting different pathway governing seed germination.