Induced Jasmonate Signaling Leads to Contrasting Effects on Root Damage and Herbivore Performance

Induced defenses play a key role in plant resistance against leaf feeders. However, very little is known about the signals that are involved in defending plants against root feeders and how they are influenced by abiotic factors. We investigated these aspects for the interaction between rice (Oryza sativa) and two root-feeding insects: the generalist cucumber beetle (Diabrotica balteata) and the more specialized rice water weevil (Lissorhoptrus oryzophilus). Rice plants responded to root attack by increasing the production of jasmonic acid (JA) and abscisic acid, whereas in contrast to in herbivore-attacked leaves, salicylic acid and ethylene levels remained unchanged. The JA response was decoupled from flooding and remained constant over different soil moisture levels. Exogenous application of methyl JA to the roots markedly decreased the performance of both root herbivores, whereas abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid did not have any effect. JA-deficient antisense 13-lipoxygenase (asLOX) and mutant allene oxide cyclase hebiba plants lost more root biomass under attack from both root herbivores. Surprisingly, herbivore weight gain was decreased markedly in asLOX but not hebiba mutant plants, despite the higher root biomass removal. This effect was correlated with a herbivore-induced reduction of sucrose pools in asLOX roots. Taken together, our experiments show that jasmonates are induced signals that protect rice roots from herbivores under varying abiotic conditions and that boosting jasmonate responses can strongly enhance rice resistance against root pests. Furthermore, we show that a rice 13-lipoxygenase regulates root primary metabolites and specifically improves root herbivore growth.

Benzoxazinoid Metabolites Regulate Innate Immunity against Aphids and Fungi in Maize

Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. The first step in BX biosynthesis converts indole-3-glycerol phosphate into indole. In maize (Zea mays), this reaction is catalyzed by either BENZOXAZINELESS1 (BX1) or INDOLE GLYCEROL PHOSPHATE LYASE (IGL). The Bx1 gene is under developmental control and is mainly responsible for BX production, whereas the Igl gene is inducible by stress signals, such as wounding, herbivory, or jasmonates. To determine the role of BXs in defense against aphids and fungi, we compared basal resistance between Bx1 wild-type and bx1 mutant lines in the igl mutant background, thereby preventing BX production from IGL. Compared to Bx1 wild-type plants, BX-deficient bx1 mutant plants allowed better development of the cereal aphid Rhopalosiphum padi, and were affected in penetration resistance against the fungus Setosphaeria turtica. At stages preceding major tissue disruption, R. padi and S. turtica elicited increased accumulation of DIMBOA-glucoside, DIMBOA, and 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucoside (HDMBOA-glc), which was most pronounced in apoplastic leaf extracts. Treatment with the defense elicitor chitosan similarly enhanced apoplastic accumulation of DIMBOA and HDMBOA-glc, but repressed transcription of genes controlling BX biosynthesis downstream of BX1. This repression was also obtained after treatment with the BX precursor indole and DIMBOA, but not with HDMBOA-glc. Furthermore, BX-deficient bx1 mutant lines deposited less chitosan-induced callose than Bx1 wild-type lines, whereas apoplast infiltration with DIMBOA, but not HDMBOA-glc, mimicked chitosan-induced callose. Hence, DIMBOA functions as a defense regulatory signal in maize innate immunity, which acts in addition to its well-characterized activity as a biocidal defense metabolite.

The Chloroplast-Localized Phospholipases D a4 and a5 Regulate Herbivore-Induced Direct and Indirect Defenses in Rice

The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDα4 and OsPLDα5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDα4 and -α5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.

Chlorophyll Breakdown in Senescent Arabidopsis Leaves. Characterization of Chlorophyll Catabolites and of Chlorophyll Catabolic Enzymes Involved in the Degreening Reaction

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide andO-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. UsingLemna minor, we analyzed the effects of omission of CO2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5′-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO2, APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO2 also recovered both enzyme activities, with OAS again influenced only APR.35SO4 2− feeding showed that treatment in air without CO2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.

Increasing the Glutathione Content in a Chilling-Sensitive Maize Genotype Using Safeners Increased Protection against Chilling-Induced Injury

With the aim of analyzing their protective function against chilling-induced injury, the pools of glutathione and its precursors, cysteine (Cys) and gamma -glutamyl-Cys, were increased in the chilling-sensitive maize (Zea mays) inbred line Penjalinan using a combination of two herbicide safeners. Compared with the controls, the greatest increase in the pool size of the three thiols was detected in the shoots and roots when both safeners were applied at a concentration of 5 muM. This combination increased the relative protection from chilling from 50% to 75%. It is interesting that this increase in the total glutathione (TG) level was accompanied by a rise in glutathione reductase (GR; EC 1.6.4.2) activity. When the most effective safener combination was applied simultaneously with increasing concentrations of buthionine sulfoximine, a specific inhibitor of glutathione synthesis, the total gamma -glutamyl-Cys and TG contents and GR activity were decreased to very low levels and relative protection was lowered from 75% to 44%. During chilling, the ratio of reduced to oxidized thiols first decreased independently of the treatments, but increased again to the initial value in safener-treated seedlings after 7 d at 5 degreesC. Taking all results together resulted in a linear relationship between TG and GR and a biphasic relationship between relative protection and GR or TG, thus demonstrating the relevance of the glutathione levels in protecting maize against chilling-induced injury.

The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity

The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling.

A physiological and behavioral mechanism for leaf-herbivore induced systemic root resistance

Indirect plant-mediated interactions between herbivores are important drivers of community composition in terrestrial ecosystems. Among the most striking examples are the strong indirect interactions between spatially separated leaf- and root-feeding insects sharing a host plant. Although leaf feeders generally reduce the performance of root herbivores, little is known about the underlying systemic changes in root physiology and the associated behavioral responses of the root feeders. We investigated the consequences of maize (Zea mays) leaf infestation by Spodoptera littoralis caterpillars for the root-feeding larvae of the beetle Diabrotica virgifera virgifera, a major pest of maize. D. virgifera strongly avoided leaf-infested plants by recognizing systemic changes in soluble root components. The avoidance response occurred within 12 h and was induced by real and mimicked herbivory, but not wounding alone. Roots of leaf-infested plants showed altered patterns in soluble free and soluble conjugated phenolic acids. Biochemical inhibition and genetic manipulation of phenolic acid biosynthesis led to a complete disappearance of the avoidance response of D. virgifera. Furthermore, bioactivity-guided fractionation revealed a direct link between the avoidance response of D. virgifera and changes in soluble conjugated phenolic acids in the roots of leaf-attacked plants. Our study provides a physiological mechanism for a behavioral pattern that explains the negative effect of leaf attack on a root-feeding insect. Furthermore, it opens up the possibility to control D. virgifera in the field by genetically mimicking leaf herbivore-induced changes in root phenylpropanoid patterns.

Assimilatory Sulfate Reduction in C3, C3-C4, and C4 Species of Flaveria

The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C4 monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C4 photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C3, C3-C4, C4-like, and C4 species of the dicot genusFlaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C4-like and C4 species than in C3 and C3-C4 species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C4 speciesFlaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C4 plants and therefore is neither a prerequisite nor a consequence of C4photosynthesis.

Limited Correlation between Expansin Gene Expression and Elongation Growth Rate

The aim of this work was to study the role of the cell wall protein expansin in elongation growth. Expansins increase cell wall extensibility in vitro and are thought to be involved in cell elongation. Here, we studied the regulation of two tomato (Lycopersicon esculentum cv Moneymaker) expansin genes,LeExp2 and LeExp18, in rapidly expanding tissues. LeExp2 was strongly expressed in the elongation zone of hypocotyls and in the faster growing stem part during gravitropic stimulation. LeExp18 expression did not correlate with elongation growth. Exogenous application of hormones showed a substantial auxin-stimulation of LeExp2 mRNA in etiolated hypocotyls and a weaker auxin-stimulation ofLeExp18 mRNA in stem tissue. Analysis of transcript accumulation revealed higher levels of LeExp2 andLeExp18 in light-treated, slow-growing tissue than in dark-treated, rapidly elongating tissue. Expansin protein levels and cell wall extension activities were similar in light- and dark-grown hypocotyl extracts. The results show a strong correlation between expansin gene expression and growth rate, but this correlation is not absolute. We conclude that elongation growth is likely to be controlled by expansin acting in concert with other factors that may limit growth under some physiological conditions.

Regulation of Sulfate Assimilation by Nitrogen in Arabidopsis

Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5′-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mM each of NO3 −, NH4 +, or glutamine (Gln), or 1 mM O-acetylserine (OAS). 35SO4 2− feeding showed that after addition of NH4 +, Gln, or OAS to nitrogen-starved plants, incorporation of 35S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.

Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12–24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 μmol g−1 fresh weight h−1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

Hypersensitivity of an Arabidopsis Sugar Signaling Mutant toward Exogenous Proline Application

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Δ1-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutantrsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS andP5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Δ1-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.