Cloning and characterisation of JAZ gene family in Hevea brasiliensis

Mechanical wounding or treatment with exogenous jasmonates (JA) induces differentiation of the laticifer in Hevea brasiliensis. JA is a key signal for latex biosynthesis and wounding response in the rubber tree. Identification of JAZ (jasmonate ZIM‐domain) family of proteins that repress JA responses has facilitated rapid progress in understanding how this lipid‐derived hormone controls gene expression and related physiological processes in plants. In this work, the full‐length cDNAs of six JAZ genes were cloned from H. brasiliensis (termed HbJAZ). These HbJAZ have different lengths and sequence diversity, but all of them contain Jas and ZIM domains, and two of them contain an ERF‐associated amphiphilic repression (EAR) motif in the N‐terminal. Real‐time RT‐PCR analyses revealed that HbJAZ have different expression patterns and tissue specificity. Four HbJAZ were up‐regulated, one was down‐regulated, while two were less effected by rubber tapping treatment, suggesting that they might play distinct roles in the wounding response. A yeast two‐hybrid assay revealed that HbJAZ proteins interact with each other to form homologous or heterogeneous dimer complexes, indicating that the HbJAZ proteins may expand their function through diverse JAZ–JAZ interactions. This work lays a foundation for identification of the JA signalling pathway and molecular mechanisms of latex biosynthesis in rubber trees.     

外源茉莉酸甲酯通过调节相关基因表达诱导光温敏雄性不育小麦BS366离体花药开裂

植物育性与花药开裂性状相关. 茉莉酮酸甲酯(methyl jasmonic acid, MeJA)是一种植物激素,广泛参与植物的胁迫应答和生长发育过程. 植物体内存在6类基因所编码的酶共同维持茉莉酮酸(JA)代谢途径的畅通,并与植物花药开裂有关. 本研究以小麦光温敏不育系BS366为材料,于抽穗期—开花期进行穗部离体48 h的 MeJA外源处理. 分别于0、1、3、6、12、24和48 h时间点收集花药并进行RNA提取. 通过半定量RT-PCR分析发现,在内参基因甘油醛-3-磷酸脱氢酶(glyceraldehyde-3- phosphate dehydrogenase, GAPDH)表达基本一致的情况下,BS366的JA代谢途径中参与调控的6种基因均存在不同程度的诱导表达. 经MeJA 的48 h处理后的离体花药开裂程度明显增大. 本研究获得了BS366离体环境下JA代谢途径基因经MeJA诱导后表达水平的概况,并为诱导BS366花药开裂及人工调控育性,达到高效制种及繁种的目的提供了初步的理论依据.     

Molecular Cloning and Characterization of Rubber Biosynthetic Genes from <Emphasis Type="Italic">Taraxacum koksaghyz</Emphasis>

Rubber biosynthesis requires the action of specific enzymes known as cis-prenyltransferases (CPTs). These enzymes are responsible for the sequential addition of isopentenyl pyrophosphate units to the growing polyisoprene chain, a biochemical reaction thought to be stimulated by the presence of small rubber particle proteins (SRPPs). We have cloned, characterized, and analyzed the expression of three CPT genes (TkCPT1–3) and five SRPP genes (TkSRPP1–5) from the rubber-producing plant Taraxacum koksaghyz. The deduced TkCPT amino acid sequences showed significant levels of sequence identity with Hevea brasiliensis CPTs. We also found no obvious differences between SRPPs from T. koksaghyz, another rubber producer, and a non-rubber plant. The roles of the individual TkCPTs and TkSRRPs in rubber biosynthesis are discussed.     

Effects of light on production of camptothecin and expression of key enzyme genes in seedlings of <Emphasis Type="Italic">Camptotheca acuminate</Emphasis> Decne

Camptotheca acuminata (C. acuminata) is utilized in preparation of drugs and as constituent in functional foods of China due to high camptothecin (CPT) content in different plant parts. Light intensity is one of the most critical factors which affect plant growth and secondary metabolites. Pot experiment was conducted to study the effect of light intensity (i.e., 100 % irradiance (control), 75 % irradiance, 50 % irradiance and 25 % irradiance) on contents of CPT, activity of enzymes and genes expression related to CPT biosynthesis of C. acuminata seedlings. The study examined total leaf biomass, CPT content, activities of tryptophan synthase (TSB) and tryptophan decarboxylase (TDC), and relative expression of TSB, TDC1, and TDC2 genes. Plants grown in 75 % irradiance possessed the greatest leaf biomass compared with 100 % light irradiance. Highest values of CPT contents were found after 60 days in plants grown in 50 % irradiance, followed by 25, 75 % and full sunlight. Furthermore, activities of TSB, TDC and relative expression of genes of TSB, TDC1, and TDC2, were significantly increased after 60 days of 50 % irradiance compared with full sunlight. Irradiance of 50 % up-regulated the expression of CPT biosynthesis-related genes and induced CPT biosynthesis. In addition to that lower or higher irradiance inhibited the expression of CPT biosynthesis-related genes and CPT biosynthesis. It is concluded that manipulating light intensity can be an effective means to achieve highest CPT yield in medicinal plants.     

The Response of Physiological Characteristics,Expression of OSC Genes,and Accumulation of Triterpenoids in <Emphasis Type="Italic">Betula platyphylla</Emphasis> Sukto MeJA and SA Treatment

The pentacyclic triterpenoids from birch (Betula platyphylla suk) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we explored the effects of spraying 3-year-old white birch with different concentration of methyl jasmonate (MeJA) and salicylic acid (SA) on the expression of key genes in triterpenoid biosynthesis pathways and on the accumulation and physiological characteristics of triterpenoids in birch saplings. The results showed that spraying different concentration of MeJA and SA could obviously promote accumulation of total triterpenoids in 3-year-old white birch. The triterpenoid content in the stem bark was increased by 46.11 %, reaching 81.86 mg/g, after 1 day of treatment with 1 mmol·L^?1 MeJA (MJ2), and by 45.07 %, reaching 91.4 mg/g, after 14 days of treatment with 5 mmol·L^?1 SA (SA1). In addition, MeJA and SA treatment increased the contents of chlorophyll a and b, antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as photosynthetic performance, and affected the content of soluble sugar and soluble protein in birch leaf. Fluorescence quantitative polymerase chain reaction (qPCR) results showed that MeJA and SA treatment deferentially enhanced the key gene expression of cycloartenol synthase (BPX and BPX2), lupeol synthase (BPW) and beta-amyrin synthase (BPY) in triterpenoid synthesis pathway in birch bark and leaves. The results showed that MeJA and SA induced triterpenoid synthesis of birch plant is closely related with not only the expression of key genes of triterpenoid synthesis pathway but also photosynthesis, anti-stress response and physiological indexes, suggesting that regulation of triterpenoid synthesis of birch by MeJA and SA may involve in more complex mechanisms at physiological and molecular levels.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Jasmonate-dependent plant defenses mediate soybean thrips and soybean aphid performance on soybean

Insect herbivores from different feeding guilds induce different signaling pathways in plants. In this study, we examined the effects of salicylic acid (SA)- and jasmonic acid (JA)-mediated defenses on performance of insect herbivores from two different feeding guilds: cell-content feeders, soybean thrips and phloem feeders, soybean aphids. We used a combination of RT-qPCR analysis and elicitor-induced plant resistance to determine induction of SA and JA signaling pathways and the impact on herbivore performance. In the early interaction between the host plant and the two herbivores, SA and JA signaling seems to occur simultaneously. But overall, soybean thrips induced JA-related marker genes, whereas soybean aphids increased SA and ABA-related marker genes over a 24-h period. Populations of both soybean thrips and soybean aphids were reduced (47 and 25 %, respectively) in methyl jasmonate (MeJA)-pretreated soybean plants. SA treatment has no effect on either herbivore performance. A combination pretreatment of SA and MeJA did not impact soybean thrips population but reduced soybean aphid numbers which was comparable with MeJA treatment. Our data suggest that SA–JA antagonism could be responsible for the effect of hormone pretreatment on thrips performance, but not on aphid performance. By linking plant defense gene expression and elicitor-induced resistance, we were able to pinpoint the role for JA signaling pathway in resistance to two herbivores from different feeding guilds.     

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.     

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree)

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches—resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9–194.2 kDa and in isoelectric point values of 4.0–11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.     

Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.     

Overexpression of a chrysanthemum transcription factor gene <Emphasis Type="Italic">DgNAC1</Emphasis> improves drought tolerance in chrysanthemum

Phenolic acids and tanshinones are two groups of pharmaceutical components present in Salvia miltiorrhiza Bunge. Methyl jasmonate (MeJA) has been reported to influence the accumulation of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. However, there is currently a lack of information regarding the comparison of how these two groups of bioactive compounds in S. miltiorrhiza respond to MeJA under the same conditions. In the present study, the effect of 100 µM MeJA on the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza hairy roots was investigated. The results showed that MeJA dramatically induced the accumulation of five different phenolic acids, especially rosmarinic acid and salvianolic acid B, which reached their highest contents at day 3 (20.3 mg/g DW, 1.5-fold of control) and day 6 (47.49 mg/g DW, 2.5-fold of control), respectively. The total production of phenolic acids was induced by as much as 3.3-fold of the control (day 9 after treatment), reaching 357.5 mg/L at day 6. However, tanshinone I was almost unaffected by MeJA treatment, and the accumulation of tanshinone IIA was inhibited. Furthermore, cryptotanshinone and dihydrotanshinone I were moderately induced by MeJA. The gene expression results indicated that MeJA probably induced the whole pathways, especially the tyrosine-derived pathway and the methylerythritol phosphate pathway, and finally resulted in the increased production of these metabolites. This study will help us to further understand how the different biosynthetic mechanisms of phenolic acids and tanshinones respond to MeJA and provide a reference for the future selection of elicitors for application to improving the production of targeted compounds.