Hirotada Fukushige

Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence

In this work, the role of jasmonic acid (JA) in leaf senescence is examined. Exogenous application of JA caused premature senescence in attached and detached leaves in wild-type Arabidopsis but failed to induce precocious senescence of JA-insensitive mutantcoi1 plants, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence. JA levels in senescing leaves are 4-fold higher than in non-senescing ones. Concurrent with the increase in JA level in senescing leaves, genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway are differentially activated during leaf senescence in Arabidopsis, except for allene oxide synthase, which is constitutively and highly expressed throughout leaf development. Arabidopsis lipoxygenase 1 (cytoplasmic) expression is greatly increased but lipoxygenase 2 (plastidial) expression is sharply reduced during leaf senescence. Similarly,AOC1 (allene oxide cyclase 1),AOC2, and AOC3 are all up-regulated, whereas AOC4 is down-regulated with the progression of leaf senescence. The transcript levels of 12-oxo-PDA reductase 1 and 12-oxo-PDA reductase 3 also increase in senescing leaves, as does PED1 (encoding a 3-keto-acyl-thiolase for β-oxidation). This represents the first report, to our knowledge, of an increase in JA levels and expression of oxylipin genes during leaf senescence, and indicates that JA may play a role in the senescence program.

Activation of a Stress-Responsive Mitogen-Activated Protein Kinase Cascade Induces the Biosynthesis of Ethylene in Plants

Plants under stress from both biotic and abiotic sources produce increased levels of ethylene, which is perceived by ethylene receptors and triggers cellular responses further downstream. Protein phosphorylation and dephosphorylation were implicated in the regulation of ethylene induction by stresses based on studies using protein kinase and phosphatase inhibitors. However, the kinase(s) involved remains to be determined. Using a conditional gain-of-function transgenic system, we demonstrate that the activation of SIPK, a tobacco mitogen-activated protein kinase (MAPK), by NtMEK2DD, an active mutant of the upstream kinase of SIPK, resulted in a dramatic increase in ethylene production. The increase in ethylene after the activation of SIPK coincided with a dramatic increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, which was followed by the activation of a subgroup of ACS and ACC oxidase (ACO) genes, suggesting that either the activation of unidentified ACS(s) or post-transcriptional regulation is involved. Infection with Tobacco mosaic virus (TMV), which is known to activate the SIPK cascade and induce ethylene biosynthesis, also induced the same ACSs and ACOs. After ethylene production in NtMEK2DD plants, strong activation of ETHYLENE-RESPONSE FACTOR (ERF) genes was observed, similar to the effect in NN tobacco plants infected with TMV. In contrast to previous reports, no major increase in jasmonic acid (JA) and methyl jasmonate (MJ) was detected after the activation of SIPK/WIPK in NtMEK2DD transgenic plants. These results suggest that the induction of ethylene but not JA/MJ is involved in plant defense responses mediated by the NtMEK2-SIPK/WIPK pathway.

Regulation of oxylipin synthesis

Two very common groups of oxylipins formed in plants involve the conversion of fatty acid hydroperoxides, such as hydroperoxy-octadecatrienoic acid, into further metabolites by allene oxide synthase and hydroperoxide lyase. Both of these oxylipin branch pathways appear to be ubiquitous or nearly so in plants, but the relative activities of these two branches vary among plant species. In most plants examined, including Arabidopsis, product formation from either of these pathways is minimal until elicited by wounding or some other means, upon which products from both pathways, such as jasmonic acid and C(6) aldehydes and alcohols, can increase by orders of magnitude. In some plant species such as Artemisia and Jasminum spp. oxylipin product formation is heavily skewed towards allene oxide synthase products. Others such as watermelon (Citrullus lanatus) produce 10-fold higher amounts or more of hydroperoxide lyase than allene oxide synthase products. Arabidopsis and tobacco are intermediate between these extremes. Artemisia and Jasminum are also unusual in that they do not require wounding or other types of induction for high oxylipin product formation. Release of non-esterified fatty acids appears to be correlated with oxylipin formation, but phospholipase A(2) appears not to be involved with oxylipin production, at least in the case of Artemisia leaves.

A gain-of-function mutation in Msl10 triggers cell death and wound-induced hyperaccumulation of jasmonic acid in Arabidopsis

Jasmonates (JAs) are rapidly induced after wounding and act as key regulators for wound induced signaling pathway. However, what perceives the wound signal and how that triggers JA biosynthesis remains poorly understood. To identify components involved in Arabidopsis wound and JA signaling pathway, we screened for mutants with abnormal expression of a luciferase reporter, which is under the control of a wound-responsive promoter of an ethylene response factor (ERF) transcription factor gene, RAP2.6 (Related to APetala 2.6). The rea1 (RAP2.6 expresser in shoot apex) mutant constitutively expressed the RAP2.6-LUC reporter gene in young leaves. Along with the typical JA phenotypes including shorter petioles, loss of apical dominance, accumulation of anthocyanin pigments and constitutive expression of JA response gene, rea1 plants also displayed cell death and accumulated high levels of JA in response to wounding. The phenotype of rea1 mutant is caused by a gain-of-function mutation in the C-terminus of a mechanosensitive ion channel MscS-like 10 (MSL10). MSL10 is localized in the plasma membrane and is expressed predominantly in root tip, shoot apex and vascular tissues. These results suggest that MSL10 is involved in the wound-triggered early signal transduction pathway and possibly in regulating the positive feedback synthesis of JA.

Nondestructive DNA Extraction Techniques for Soybean (Glycine Max) Seeds

ABSTRACT While maintaining high germination rates, genotyping single seeds are useful in breeding and genetics. Nondestructive single-seed genomic DNA extraction protocols using 12 mg cotyledon tissue with a modified cetyl trimethyl ammonium bromide (CTAB) technique and a commercial seed DNA extraction kit using 1 mg cotyledon tissue were developed for dry soybean seeds and cross-verified with leaf DNA analysis. The DNA extracted by these methods was sufficient for polymerase chain reaction (PCR), and the results were reproducible for several endogenous genes and transgenes in D-myo-inositol 3-phosphate synthase (MIPS) mutant seeds (GM-lpa-TW1). This single-seed DNA-analysis method is amenable to rapid high-throughput genotype screening and verifying the genetic purity of seed stocks.

Hydroperoxide Lyase and Leaf Aldehyde Formation can be Greatly Increased in Leaves

2(E)-hexenal, leaf aldehyde, is used as flavor to impart a green character and freshness in foods and beverages. Leaf aldehyde is formed by the hydroperoxide lyase (HL) branch pathway of oxylipin pathway (Zimmerman and Coudron, 1979; Hatanaka et al., 1987; Vick and Zimmerman, 1987b). Linolenic acid (18:3), a major fatty acid in leaf lipids, is first oxidized by lipoxygenase (LOX) to produce 13-hydroperoxy 18:3 (13-HPOT). 13-HPOT is then cleaved by HL to produce the C6 aldehyde, 3(Z)-hexenal and a 12- carbon oxo acid. 3(Z)-hexenal is then isomerized to leaf aldehyde or reduced to 3(Z)-hexenol, leaf alcohol (Fig. 1). Though this compound can been synthesized chemically, there are increasing demands for natural flavors as well as natural chemicals for fungicides and pesticides as more and more consumers are interested in natural products, organic production and ‘green’ industries (Whitehead et al., 1995). Also a more efficient plant-based production system will be more competitive with petroleum-based production making use from such a renewable source more economically attractive. Currently commercial production of natural leaf aldehyde is achieved from watermelon leaves because of the high yield of this compound from this source. This involves making large-scale homogenates of watermelon leaves with added linolenic acid or hydrolyzed linseed oil (Chou and Chin, 1994; Holtz et al. 2001). Hildebrand et al. (1990 and 1991) and Whitehead et al. (1995) proposed the usage of soybean LOX to produce 13-HPOT in combination with plant materials to generate C6 aldehydes. Leaf aldehyde is currently valued as much as

Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant.

100 per kg and the market for such compounds has increased > 70% in the past 10 years. This research reports the increased formation of leaf aldehyde from plant leaves by enhanced expression of the key enzyme involved in its formation and the usage of plant leaves as a factory for this compound. Open image in new window Figure1 HL branch of the oxylipin pathway IF = Isomerization Factor, ADH = Alcohol Dehydrogenase