Role of Tomato Lipoxygenase D in Wound-Induced Jasmonate Biosynthesis and Plant Immunity to Insect Herbivores

In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs) in tomato (Solanum lycopersicum) provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA). The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8) mutant, which was isolated as a suppressor of (pro)systemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera) and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against insects and pathogens.     

Wound and insect‐induced jasmonate accumulation in carnivorous Drosera capensis: two sides of the same coin

Carnivorous sundew plants catch and digest insect prey for their own nutrition. The sundew species Drosera capensis shows a pronounced leaf bending reaction upon prey capture in order to form an ‘outer stomach’. This formation is triggered by jasmonates, phytohormones typically involved in defence reactions against herbivory and wounding. Whether jasmonates still have this function in D. capensis in addition to mediating the leaf bending reaction was investigated here. Wounded, insect prey‐fed and insect‐derived oral secretion‐treated leaves of D. capensis were analysed for jasmonates (jasmonic acid, JA; jasmonic acid‐isoleucine conjugate, JA‐Ile) using LC‐MS/MS. Prey‐induced jasmonate accumulation in D. capensis leaves was persistent, and showed high levels of JA and JA‐Ile (575 and 55.7 pmol·g·FW^?1, respectively), whereas wounding induced a transient increase of JA (maximum 500 pmol·g·FW^?1) and only low (3.1 pmol·g·FW^?1) accumulation of JA‐Ile. Herbivory, mimicked with a combined treatment of wounding plus oral secretion (W+OS) obtained from Spodoptera littoralis larvae induced both JA (4000 pmol·g·FW^?1) and JA‐Ile (25 pmol·g·FW^?1) accumulation, with kinetics similar to prey treatment. Only prey and W+OS, but not wounding alone or OS, induced leaf bending. The results indicate that both mechanical and chemical stimuli trigger JA and JA‐Ile synthesis. Differences in kinetics and induced jasmonate levels suggest different sensing and signalling events upon injury and insect‐dependent challenge. Thus, in Drosera, jasmonates are still part of the response to wounding. Jasmonates are also employed in insect‐induced reactions, including responses to herbivory and carnivory.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

Microbes in Helicoverpa armigera oral secretions contribute to increased senescence around plant wounds

1. Plants have long been exposed to insect herbivore attack. Crucial to the plant's ability to defend itself is its ability to identify specific signals associated with attacking insects. Distinctive chemical cues, such as those associated with chewing insect oral secretions (OS), activate targeted defence responses to chewing insect herbivores. 2. Herbivore-associated cues can be complicated by the fact that many herbivores form associations with microbes that produce their own specific signals, which may induce alternative defence processes. 3. Here we report that OS of the global pest, the cotton bollworm (Helicoverpa armigera), induce senescence around wounds in Brachypodium distachyon leaves. Crude OS activate greater levels of senescence than OS with reduced microbial abundance or mechanical wounding alone. Nonetheless, plants closed mechanical wounds more rapidly when treated with H. armigera OS regardless of the microbial component. 4. This study concludes that H. armigera OS can activate senescence and wound closure in plant tissues and that microbes within OS have an important role in shaping plant-herbivore interactions through additional increases in senescence.     

Characterization of four defense-related genes up-regulated in root nodules of Casuarina glauca

Actinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a root-nodule symbiosis with N₂-fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus non-inoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question “defense, development or both” remains open.     

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

By cultivating tipburn-susceptible plants in modified Hoagland’s medium containing of gradient exogenous calcium (Ca²⁺), we have shown that Ca²⁺ deficiency is one of the main causes of tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effect of endogenous plant Ca²⁺ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca²⁺ transporter genes that function in cytosolic Ca²⁺ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA β-glucoside (SAG) in tipburn-susceptible plants was ～3-fold higher than it was in resistant plants following Ca²⁺ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca²⁺ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage.     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

Induced plant defense via volatile production is dependent on rhizobial symbiosis

Nitrogen-fixing rhizobia can substantially influence plant–herbivore interactions by altering plant chemical composition and food quality. However, the effects of rhizobia on plant volatiles, which serve as indirect and direct defenses against arthropod herbivores and as signals in defense-associated plant–plant and within-plant signaling, are still unstudied. We measured the release of jasmonic acid (JA)-induced volatiles of rhizobia-colonized and rhizobia-free lima bean plants (Fabaceae: Phaseolus lunatus L.) and tested effects of their respective bouquets of volatile organic compounds (VOCs) on a specialist insect herbivore (Mexican bean beetle; Coccinellidae: Epilachna varivestis Mulsant) in olfactometer choice trials. In a further experiment, we showed that VOC induction by JA reflects the plant responses to mechanical wounding and insect herbivory. Following induction with JA, rhizobia-colonized plants released significantly higher amounts of the shikimic acid-derived compounds, whereas the emission of compounds produced via the octadecanoid, mevalonate and non-mevalonate pathways was reduced. These changes affected the choice behavior of beetles as the preference of non-induced plants was much more pronounced for plants that were colonized by rhizobia. We showed that indole likely represents the causing agent for the observed repellent effects of jasmonic acid-induced VOCs of rhizobia-colonized lima bean plants. Our study demonstrates a rhizobia-triggered efficacy of induced plant defense via volatiles. Due to these findings, we interpret rhizobia as an integral part of legume defenses against herbivores.     

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

Background

Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca²⁺-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca²⁺ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca²⁺ influx mediated by mechanosensitive Ca²⁺ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.

Results

We here identified and investigated OsMCA1, the sole rice homolog of putative Ca²⁺-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca²⁺ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca²⁺ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca²⁺-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.

Conclusions

We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca²⁺-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca²⁺ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.     

Adipokinetic hormone exerts its anti-oxidative effects using a conserved signal-transduction mechanism involving both PKC and cAMP by mobilizing extra- and intracellular Ca stores

The involvement of members of the adipokinetic hormone (AKH) family in regulation of response to oxidative stress (OS) has been reported recently. However, despite these neuropeptides being the best studied family of insect hormones, their precise signaling pathways in their OS responsive role remain to be elucidated. In this study, we have used an in vitro assay to determine the importance of extra and intra-cellular Ca^2 + stores as well as the involvement of protein kinase C (PKC) and cyclic adenosine 3′,5′-monophosphate (cAMP) pathways by which AKH exerts its anti-oxidative effects. Lipid peroxidation product (4-HNE) was significantly enhanced and membrane fluidity reduced in microsomal fractions of isolated brains (CNS) of Pyrrhocoris apterus when treated with hydrogen peroxide (H₂O₂), whereas these biomarkers of OS were reduced to control levels when H₂O₂ was co-treated with Pyrap-AKH. The effects of mitigation of OS in isolated CNS by AKH were negated when these treatments were conducted in the presence of Ca^2 + channel inhibitors (CdCl₂ and thapsigargin). Presence of either bisindolylmaliemide or chelyrythrine chloride (inhibitors of PKC) in the incubating medium also compromised the anti-oxidative function of AKH. However, supplementing the medium with either phorbol myristate acetate (PMA, an activator of PKC) or forskolin (an activator of cAMP) restored the protective effects of exogenous AKH treatment by reducing 4-HNE levels and increasing membrane fluidity to control levels. Taken together, our results strongly implicate the importance of both PKC and cAMP pathways in AKHs' anti-oxidative action by mobilizing both extra and intra-cellular stores of Ca^2 +.     

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions

Calcium (Ca²⁺) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca²⁺ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca²⁺ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca²⁺-buffering protein, is able to bind Ca²⁺ reversibly, it can serve as a mobile store of easily releasable Ca²⁺ (so called an exchangeable Ca²⁺) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca²⁺ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca²⁺ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca²⁺ storage and mobilization during sexual reproduction of angiosperms.     

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca²⁺-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca²⁺ in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca²⁺-permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca²⁺ and protein interactions.