Differential response of phenylpropanoid pathway as linked to hormonal change in two Brassica napus cultivars contrasting drought tolerance.

Oilseed rape (Brassica napus L.) is a significant agro-economic crop with a wide range of uses. Drought is the most frequent unfavourable environmental stressor restraining its growth and development worldwide. This study was conducted to characterize the drought-responsive phenylpropanoid pathway and its link to hormonal changes in two cultivars, drought-resistant "Saturnin" and drought-susceptible "Mosa." Drought susceptibility in cv. Mosa was confirmed by its lower water use efficiency and higher lipid peroxidation levels with reactive oxygen species (ROS) accumulation. In cv. Saturnin, higher salicylic acid (SA) levels and expression of dehydration-responsive element binding 2 (DREB2) and non-expressor of pathogenesis-related gene 1 (NPR1) led to an upregulation of production of anthocyanin pigment 1 (PAP1) and phenylpropanoid pathway-related gene (CHS, F5H and COMT1) expression, increasing hydroxycinnamic acid and flavonoid compound concentrations. However, in cv. Mosa, higher increases in the activity of lignifying enzymes (polyphenol oxidase, coniferyl alcohol peroxidase, syringaldazine peroxidase, guaiacol peroxidase) and expression of the lignin synthesis-related gene cinnamyl alcohol dehydrogenase 2 (CAD2) were found along with greater increases in abscisic acid (ABA) levels and upregulation of ABA-responsive element binding 2 (AREB2) and basic helix-loop-helix transcription factor MYC2. These results indicate that drought-induced SA-mediated activation of the hydroxycinnamic acid and flavonoid pathways contributes to drought resistance, whereas ABA-mediated lignification contributes to drought susceptibility.

Target-Based Physiological Modulations and Chloroplast Proteome Reveals a Drought Resilient Rootstock in Okra (Abelmoschus esculentus) Genotypes.

As climate changes increase, drought stress is becoming a problem for all major horticultural crops; among them is okra (Abelmoschus esculentus). Despite its superior resilience to heat stress and high nutritional content, it is still underutilized in contrast to other vegetable crops. Moreover, the drought-resistant and drought-sensitive genotypes of okra are also not well known and require further exploration to improve their productivity. To investigate this in more detail, we performed comparative physiological and large-scale chloroplast proteomics on drought-stressed genotypes of okra. We evaluated four major genotypes of okra, viz., NS7774, NS7772, Green Gold, and OH3312 for drought resilient rootstock. The physiological modulations demonstrated a significant change by 50-76% in biomass, net-photosynthetic machinery, water transport, and absorption both in early and late stages of drought stress compared to well-watered crops in all genotypes. Maximum oxidative damage due to drought stress was observed for the genotypes NS7772, Green Gold and OH3312 as depicted by H2O2 and O2- determination. Greater oxidative stress was correlated to lesser antioxidant activity and expression of antioxidant enzymes, such as catalase and ascorbate peroxidase under stress in okra genotypes. The overall photosynthetic pigments, such as total chlorophyll, and total carotenoid content, were also decreased, and stomatal guard cells were disrupted and appeared closed compared to the control for the above three mentioned genotypes, except NS7774. A subsequent tissue-specific proteome analysis of chloroplasts and thylakoids analyzed by BN-PAGE (blue native polyacrylamide gel electrophoresis) revealed either over or under expression of specific proteins, such as ATPase, PSI, PSII core dimer, PSII monomer and ATP synthase. The expression of multiprotein complex proteins, including PSII-core dimer and PSII-core monomer, was slightly higher for the genotype NS7774 when compared to three other genotypes for both 5 and 10 days of drought stress. Further identification of specific proteins obtained in second dimension BN-PAGE provided descriptive detail of seven proteins involved in drought resistance across all genotypes. The identified proteins are majorly involved in photosynthesis under drought stress, suggesting NS7774 as a drought tolerant genotype. Further, the proteomic results were confirmed using Immunoblot by selecting specific protein such as PsaA. Overall, from our physiological modulations and chloroplast proteomics in all genotypes, we summarized NS7774 as a resilient rootstock and the other three genotypes (NS7772, OH3312, and Green Gold) as sensitive ones.

Microtubule Dynamics Plays a Vital Role in Plant Adaptation and Tolerance to Salt Stress.

Although recent studies suggest that the plant cytoskeleton is associated with plant stress responses, such as salt, cold, and drought, the molecular mechanism underlying microtubule function in plant salt stress response remains unclear. We performed a comparative proteomic analysis between control suspension-cultured cells (A0) and salt-adapted cells (A120) established from Arabidopsis root callus to investigate plant adaptation mechanisms to long-term salt stress. We identified 50 differentially expressed proteins (45 up- and 5 down-regulated proteins) in A120 cells compared with A0 cells. Gene ontology enrichment and protein network analyses indicated that differentially expressed proteins in A120 cells were strongly associated with cell structure-associated clusters, including cytoskeleton and cell wall biogenesis. Gene expression analysis revealed that expressions of cytoskeleton-related genes, such as FBA8, TUB3, TUB4, TUB7, TUB9, and ACT7, and a cell wall biogenesis-related gene, CCoAOMT1, were induced in salt-adapted A120 cells. Moreover, the loss-of-function mutant of Arabidopsis TUB9 gene, tub9, showed a hypersensitive phenotype to salt stress. Consistent overexpression of Arabidopsis TUB9 gene in rice transgenic plants enhanced tolerance to salt stress. Our results suggest that microtubules play crucial roles in plant adaptation and tolerance to salt stress. The modulation of microtubule-related gene expression can be an effective strategy for developing salt-tolerant crops.

Proteomic analyses of the interaction between the plant-growth promoting rhizobacterium Paenibacillus polymyxa E681 and Arabidopsis thaliana.

Plant growth-promoting rhizobacteria (PGPR) facilitate the plant growth and enhance their induced systemic resistance (ISR) against a variety of environmental stresses. In this study, we carried out integrative analyses on the proteome, transcriptome, and metabolome to investigate Arabidopsis root and shoot responses to the well-known PGPR strain Paenibacillus polymyxa (P. polymyxa) E681. Shoot fresh and root dry weights were increased, whereas root length was decreased by treatment with P. polymyxa E681. 2DE approach in conjunction with MALDI-TOF/TOF analysis revealed a total of 41 (17 spots in root, 24 spots in shoot) that were differentially expressed in response to P. polymyxa E681. Biological process- and molecular function-based bioinformatics analysis resulted in their classification into seven different protein groups. Of these, 36 proteins including amino acid metabolism, antioxidant, defense and stress response, photosynthesis, and plant hormone-related proteins were up-regulated, whereas five proteins including three carbohydrate metabolism- and one amino acid metabolism-related, and one unknown protein were down-regulated, respectively. A good correlation was observed between protein and transcript abundances for the 12 differentially expressed proteins during interactions as determined by qPCR analysis. Metabolite analysis using LC-MS/MS revealed highly increased levels of tryptophan, indole-3-acetonitrile (IAN), indole-3-acetic acid (IAA), and camalexin in the treated plants. Arabidopsis plant inoculated P. polymyxa E681 also showed resistance to Botrytis cinerea infection. Taken together these results suggest that P. polymyxa E681 may promote plant growth by induced metabolism and activation of defense-related proteins against fungal pathogen.

Comparative investigation of seed coats of brown- versus yellow-colored soybean seeds using an integrated proteomics and metabolomics approach.

Seed coat color is an important attribute determining consumption of soybean seeds. Soybean cultivar Mallikong (M) has yellow seed coat while its naturally mutated cultivar Mallikong mutant (MM), has brown colored seed coat. We used integrated proteomics and metabolomics approach to investigate the differences between seed coats of M and MM during different stages of seed development (4, 5, and 6 weeks after flowering). 2DE profiling of total seed coat proteins from three stages showed 178 differentially expressed spots between M and MM of which 172 were identified by MALDI-TOF/TOF. Of these, 62 were upregulated and 105 were downregulated in MM compared with M, while five spots were detected only in MM. Proteins involved in primary metabolism showed downregulation in MM suggesting energy in MM might be utilized for proanthocyanidin biosynthesis via secondary metabolic pathways that leads to the development of brown seed coat color. Besides, downregulation of two isoforms of isoflavone reductase indicated reduced isoflavones in seed coat of MM that was confirmed by quantitative estimation of total and individual isoflavones using HPLC. We propose that low isoflavones level in MM may offer a high substrate for proanthocyanidin production that results in the development of brown seed coat in MM.

Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium.

A NAC transcription factor and SNI1 cooperatively suppress basal pathogen resistance in Arabidopsis thaliana.

Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the in planta function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in Arabidopsis. A T-DNA insertional mutant (cbnac1) displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae DC3000 (PstDC3000), whereas resistance was reduced in transgenic CBNAC overexpression lines. The observed changes in disease resistance were correlated with alterations in pathogenesis-related protein 1 (PR1) gene expression. CBNAC bound directly to the PR1 promoter. SNI1 (suppressor of nonexpressor of PR genes1, inducible 1) was identified as a CBNAC-binding protein. Basal resistance to PstDC3000 and derepression of PR1 expression was greater in the cbnac1 sni1 double mutant than in either cbnac1 or sni1 mutants. SNI1 enhanced binding of CBNAC to its cognate PR1 promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense.

Circadian-based approach for improving physiological, phytochemical and chloroplast proteome in Spinacia oleracea under salinity stress and light emitting diodes.

Salt stress is a recognized annihilating abiotic stress that has a significant impact on agricultural and horticulture crop productivity. Plant development faces three distinct dangers as a result of salt stress: oxidative stress, osmotic stress, and ionic toxicity. It has been shown that plants can forecast diurnal patterns using the circadian clock; moreover, they can manage their defensive mechanism for the detoxification of reactive oxygen species (ROS). Circadian rhythmicity in gene expression assembles transcription and translation feedback networks to govern plant shape, physiology, cellular and molecular activities. Both external and internal variables influence the systemic rhythm via input routes. The Malav Jyoti (MJ) and Delhi Green (DG) genotypes of spinach (Spinacia oleracea) were grown in the plant growth chamber. The chamber had an optimized temperature of 25 °C and humidity of 65% containing light emitting diode (LED) having Red: Blue: white (one side) and White fluorescent (other side) under salinity stress. The samples were collected on the basis of 4 h intervals of circadian hours (0 h, 4 h, 8 h and 12 h) during Day-10 and Day-20 of salt treatments. Under salt stress, the circadian and light-emitting diode-based strategy had a substantial influence on spinach's anti-oxidative responses, stomatal movement, CO2 assimilation, PS-I and II efficiency, phytochrome pigment efficiency, and photosynthesis. Based on the findings of the free radical scavenging enzyme tests, the photoperiodic hours for the proteome analysis were set to 11 am and 3 pm on Day-20. When compared to white fluorescent, this study found that LED has the capacity to influence the entrainment cues of the circadian clock in the cultivation of salt-sensitive spinach genotypes. According to our findings, changing the cellular scavenging mechanism and chloroplast proteome has increased the survival rate of spinach genotypes under LED when compared to white fluorescent.

Determination of the variations in levels of phenolic compounds in soybean (Glycine max Merr.) sprouts infected by anthracnose (Colletotrichum gloeosporioides).

BACKGROUND: Soybean sprouts (Kongnamool) are one of the most popular and nutritive traditional vegetables in East Asia. Anthracnose caused by Colletotrichum gloeosporioides is one of the most serious diseases of soybean sprouts. In order to obtain basic information for breeding and/or selecting soybean genotypes with increased natural defense against anthracnose, phenolic compounds were profiled for healthy and infected soybean (Glycine max Merr.) sprouts by using high-performance liquid chromatography coupled with tandem mass spectrometry. RESULTS: Tryptophan and eight phenolic compounds (daidzin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, genistein and coumestrol) were determined from healthy and inoculated sprouts. Total identified phenolic content was 40.02 ± 0.03 mg kg⁻¹, 99.4% of which was isoflavones. CONCLUSION: The monitoring suggested that de novo induced glycitein appeared to act as a phytoalexin in the defence mechanism of the soybean sprouts against C. gloeosporioides, and constitutively formed seven phenolic components that functioned as phytoanticipins in the diseased soybean sprouts.

Drought-Induced Xylem Sulfate Activates the ABA-Mediated Regulation of Sulfate Assimilation and Glutathione Redox in Brassica napus Leaves.

Drought intensity modifies the assimilatory pathway of glutathione (GSH) synthesis. Abscisic acid (ABA) is a representative signaling hormone involved in regulating plant stress responses. This study aimed to investigate an interactive regulation of sulfate and/or ABA in GSH metabolism and redox. The drought-responsive alterations in sulfate assimilation and GSH-based redox reactions were assessed relative to ABA responses on the time-course of drought intensity. Drought-responsive H2O2 concentrations were divided into two distinct phases-an initial 4 days of no change (Ψw ≥ -0.49 MPa) and a phase of higher accumulation during the late phase of the drought (days 10-14; Ψw ≤ -1.34 MPa). During the early phase of the drought, GSH/GSSG redox state turned to the slightly reduced state with a transient increase in GSH, resulting from a strong activation of H2O2 scavenging enzymes, ascorbate peroxidase (APOX) and glutathione reductase (GR). The late phase of the drought was characterized by a decrease in GSH due to cysteine accumulation, shifting GSH- and NADPH-based redox states to higher oxidization, increasing sulfate and ABA in xylem, and causing ABA accumulation in leaves. Regression analysis revealed that sulfate in xylem sap was positively correlated with H2O2 concentrations and ABA was closely related to decreases in the GSH pool and the oxidation of GSH catalyzed by glutathione peroxidase (GPOX). These results indicate that drought-induced oxidation proceeds through the suppression of GSH synthesis and further GSH oxidation in a sulfate-activated ABA-dependent manner.

H2O2-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves.

Drought alters the level of endogenous reactive oxygen species (ROS) and hormonal status, which are both involved in the regulation of stress responses. To investigate the interplay between ROS and hormones in proline metabolism, rapeseed (Brassica napus L.) plants were exposed to drought or exogenous H2O2 (Exo-H2O2) treatment for 10 days. During the first 5 days, the enhanced H2O2 concentrations in drought treatment were associated with the activation of superoxide dismutase (SOD) and NADPH oxidase, with enhanced ABA and SA levels, while that in Exo-H2O2 treatment was mainly associated with SA-responsive POX. During the latter 5 days, ABA-dependent ROS accumulation was predominant with an upregulated oxidative signal-inducible gene (OXI1) and MAPK6, leading to the activation of ABA synthesis and the signaling genes (NCED3 and MYC2). During the first 5 days, the enhanced levels of P5C and proline were concomitant with SA-dependent NDR1-mediated signaling in both drought and Exo-H2O2 treatments. In the latter 5 days of drought treatment, a distinct enhancement in P5CR and ProDH expression led to higher proline accumulation compared to Exo-H2O2 treatment. These results indicate that SA-mediated P5C synthesis is highly activated under lower endogenous H2O2 levels, and ABA-mediated OXI1-dependent proline accumulation mainly occurs with an increasing ROS level, leading to ProDH activation as a hypersensitive response to ROS and proline overproduction under severe stress.

Dimethylthiourea Alleviates Drought Stress by Suppressing Hydrogen Peroxide-Dependent Abscisic Acid-Mediated Oxidative Responses in an Antagonistic Interaction with Salicylic Acid in Brassica napus Leaves.

In plants, prolonged drought induces oxidative stress, leading to a loss of reducing potential in redox components. Abscisic acid (ABA) is a representative hormonal signal regulating stress responses. This study aimed to investigate the physiological significance of dimethylthiourea (DMTU, an H2O2 scavenger) in the hormonal regulation of the antioxidant system and redox control in rapeseed (Brassica napus L.) leaves under drought stress. Drought treatment for 10 days provoked oxidative stress, as evidenced by the increase in O2•- and H2O2 concentrations, and lipid peroxidation levels, and a decrease in leaf water potential. Drought-induced oxidative responses were significantly alleviated by DMTU treatment. The accumulation of O2•- and H2O2 in drought-treated plants coincided with the enhanced expression of the NADPH oxidase and Cu/Zn-SOD genes, leading to an up-regulation in oxidative signal-inducible 1 (OXI1) and mitogen-activated protein kinase 6 (MAPK6), with a concomitant increase in ABA levels and the up-regulation of ABA-related genes. DMTU treatment under drought largely suppressed the drought-responsive up-regulation of these genes by depressing ABA responses through an antagonistic interaction with salicylic acid (SA). DMTU treatment also alleviated the drought-induced loss of reducing potential in GSH- and NADPH-based redox by the enhanced expression of glutathione reductase 1 (GR1) and up-regulation of oxidoreductase genes (TRXh5 and GRXC9). These results indicate that DMTU effectively alleviates drought-induced oxidative responses by suppressing ABA-mediated oxidative burst signaling in an antagonistic regulation of SA.

Proteomic analysis of placentas from cloned cat embryos identifies a set of differentially expressed proteins related to oxidative damage, senescence and apoptosis.

Production of cloned mammals by somatic cell nuclear transfer is associated with functional and structural abnormalities of placentation and with abnormal fetal development. A proteomic analysis was performed in domestic cats (Felis catus) to compare cloned term placentas (CTP) obtained from cesarean section (CS) to control placentas obtained from CS or vaginal delivery. The expression of 20 proteins was altered in CTP (p<0.05) compared to control placentas. The two control groups showed that the method of delivery, vaginal delivery or CS, did not affect protein expression (p>0.05). A total of 13 proteins were up-regulated in CTP, including apoptosis-related cathepsin D (CD), annexin A1 and heat shock protein 27 (HSP 27), and seven proteins were down-regulated in CTP, including prohibitin (PHB). The expression of PHB and CD was confirmed by Western blotting and immunofluorescence staining. The abnormal expression of PHB and CD correlated with the generation of reactive oxygen species, leading to decreased mitochondrial membrane potential and telomeric DNA, which are associated with cellular senescence and apoptosis. In summary, a specific pattern of abnormal protein expression is associated with the impaired development and functions of cloned placentas and hence with decreased fetal viability. Strategies aimed at restoring normal placental protein expression may increase the efficiency of somatic cell nuclear transfer and transgenic cat production and help restore endangered species.

Systemic transplantation of human adipose-derived stem cells stimulates bone repair by promoting osteoblast and osteoclast function.

Systemic transplantation of adipose-derived stem cells (ASCs) is emerging as a novel therapeutic option for functional recovery of diverse damaged tissues. This study investigated the effects of systemic transplantation of human ASCs (hASCs) on bone repair. We found that hASCs secrete various bone cell-activating factors, including hepatocyte growth factor and extracellular matrix proteins. Systemic transplantation of hASCs into ovariectomized mice induced an increased number of both osteoblasts and osteoclasts in bone tissue and thereby prevented bone loss. We also observed that conditioned medium from hASCs is capable of stimulating proliferation and differentiation of osteoblasts via Smad/extracellular signal-regulated kinase (ERK)/JNK (c-jun NH(2) -terminal kinase) activation as well as survival and differentiation of osteoclasts via ERK/JNK/p38 activation in vitro. Overall, our findings suggest that paracrine factors secreted from hASCs improve bone repair and that hASCs can be a valuable tool for use in osteoporosis therapy.

Arabidopsis MAP kinase phosphatase 1 is phosphorylated and activated by its substrate AtMPK6.

Arabidopsis MAP kinase phosphatase 1 (AtMKP1) is a member of the mitogen-activated protein kinase (MPK) phosphatase family, which negatively regulates AtMPKs. We have previously shown that AtMKP1 is regulated by calmodulin (CaM). Here, we examined the phosphorylation of AtMKP1 by its substrate AtMPK6. Intriguingly, AtMKP1 was phosphorylated by AtMPK6, one of AtMKP1 substrates. Four phosphorylation sites were identified by phosphoamino acid analysis, TiO(2) chromatography and mass spectrometric analysis. Site-directed mutation of these residues in AtMKP1 abolished the phosphorylation by AtMPK6. In addition, AtMKP1 interacted with AtMPK6 as demonstrated by the yeast two-hybrid system. Finally, the phosphatase activity of AtMKP1 increased approximately twofold following phosphorylation by AtMPK6. By in-gel kinase assays, we showed that AtMKP1 could be rapidly phosphorylated by AtMPK6 in plants. Our results suggest that the catalytic activity of AtMKP1 in plants can be regulated not only by Ca(2+)/CaM, but also by its physiological substrate, AtMPK6.

Ethephon-Induced Ethylene Enhances Starch Degradation and Sucrose Transport with an Interactive Abscisic Acid-Mediated Manner in Mature Leaves of Oilseed rape (Brassica napus L.).

The leaf senescence process is characterized by the degradation of macromolecules in mature leaves and the remobilization of degradation products via phloem transport. The phytohormone ethylene mediates leaf senescence. This study aimed to investigate the ethephon-induced ethylene effects on starch degradation and sucrose remobilization through their interactive regulation with other hormones. Ethephon (2-chloroethylphosphonic acid) was used as an ethylene-generating agent. Endogenous hormonal status, carbohydrate compounds, starch degradation-related gene expression, sucrose transporter gene expression, and phloem sucrose loading were compared between the ethephon-treated plants and controls. Foliar ethephon spray enhanced the endogenous ethylene concentration and accelerated leaf senescence, as evidenced by reduced chlorophyll content and enhanced expression of the senescence-related gene SAG12. Ethephon-enhanced ethylene prominently enhanced the endogenous abscisic acid (ABA) level. accompanied with upregulation of ABA synthesis gene 9-cis-epoxycarotenoid dioxygenase (NCED3), ABA receptor gene pyrabactin resistance 1 (PYR1), and ABA signaling genes sucrose non-fermenting 1 (Snf1)-related protein kinase 2 (SnRK2), ABA-responsive element binding 2 (AREB2), and basic-helix-loop-helix (bHLH) transcription factor (MYC2).) Ethephon treatment decreased starch content by enhancing expression of the starch degradation-related genes α-amylase 3 (AMY3) and ß-amylase 1 (BAM1), resulting in an increase in sucrose content in phloem exudates with enhanced expression of sucrose transporters, SUT1, SUT4, and SWEET11. These results suggest that a synergistic interaction between ethylene and ABA might account for sucrose accumulation, mainly due to starch degradation in mature leaves and sucrose phloem loading in the ethephon-induced senescent leaves.

Interactive Regulation of Hormone and Resistance Gene in Proline Metabolism Is Involved in Effector-Triggered Immunity or Disease Susceptibility in the Xanthomonas campestris pv. campestris-Brassica napus Pathosystem.

To characterize cultivar variations in hormonal regulation of the transition between pattern-triggered immunity (PTI) and effector-triggered immunity or susceptibility (ETI or ETS), the responses of resistance (R-) genes, hydrogen peroxide, and proline metabolism in two Brassica napus cultivars to contrasting disease susceptibility (resistant cv. Capitol vs. susceptible cv. Mosa) were interpreted as being linked to those of endogenous hormonal levels and signaling genes based on a time course of disease symptom development. Disease symptoms caused by the Xanthomonas campestris pv. campestris (Xcc) infections were much more developed in cv. Mosa than in cv. Capitol, as shown by an earlier appearance (at 3 days postinoculation [3 DPI]) and larger V-shaped necrosis lesions (at 9-15 DPI) in cv. Mosa. The cultivar variations in the R-genes, hormone status, and proline metabolism were found in two different phases (early [0-3 DPI] and later [9-15 DPI]). In the early phase, Xcc significantly upregulated PTI-related cytoplasmic kinase (Botrytis-induced kinase-1 [BIK1]) expression (+6.3-fold) with salicylic acid (SA) accumulation in cv. Capitol, while relatively less (+2.6-fold) with highly increased jasmonic acid (JA) level in cv. Mosa. The Xcc-responsive proline accumulation in both cultivars was similar to upregulated expression of proline synthesis-related genes (P5CS2 and P5CR). During the later phase in cv. Capitol, Xcc-responsive upregulation of ZAR1 (a coiled-coil-nucleotide binding site-leucine-rich repeat [CC-NB-LRR-type R-gene]) was concomitant with a gradual increase in JA levels without additional proline accumulation. However, in cv. Mosa, upregulation of TAO1 (a toll/interleukin-1 receptor-nucleotide binding site-leucine-rich repeat [TIR-NB-LRR-type R-gene]) was consistent with an increase in SA and abscisic acid (ABA) levels and resulted in an antagonistic depression of JA, which led to a proline accumulation. These results indicate that Xcc-induced BIK1- and ZAR1-mediated JA signaling interactions provide resistance and confirm ETI, whereas BIK1- and TAO1-enhanced SA- and/or ABA-mediated proline accumulation is associated with disease susceptibility (ETS).

Drought Intensity-Responsive Salicylic Acid and Abscisic Acid Crosstalk with the Sugar Signaling and Metabolic Pathway in Brassica napus.

The aim of this study was to characterize hormonal crosstalk with the sugar signaling and metabolic pathway based on a time course analysis of drought intensity. Drought intensity-responsive changes in the assimilation of newly fixed carbon (C) into soluble sugar, the content of sugar and starch, and expression of genes involved in carbohydrate metabolism were interpreted as being linked to endogenous abscisic acid (ABA) and salicylic acid (SA) levels and their signaling genes. The ABA and SA levels in the drought-stressed leaves increased together during the early drought period (days 0-6), and additional ABA accumulation occurred with depressed SA during the late period (days 6-14). Although drought treatment decreased the assimilation of newly fixed C into soluble sugar, representing a 59.9%, 33.1%, and 62.9% reduction in 13C-glucose, 13C-fructose, and 13C-sucrose on day 14, respectively, the drought-responsive accumulation of soluble sugars was significant. During the early period, the drought-responsive accumulation of hexose and sucrose was concurrent with the upregulated expression of hexokinase 1 (HXK1), which, in turn, occurred parallel to the upregulation of ABA synthesis gene 9-sis-epoxycarotenoid dioxygenase (NCED3) and SA-related genes (isochorismate synthase 1 (ICS1) and non-expressor of pathogenesis-related gene (NPR1)). During the late period, hexose accumulation, sucrose phloem loading, and starch degradation were dominant, with a highly enhanced expression of the starch degradation-related genes ß-amylase 1 (BAM1) and α-amylase 3 (AMY3), which were concomitant with the parallel enhancement of sucrose non-fermenting-1 (Snf1)-related protein kinase 2 (SnRK2).2 and ABA-responsive element binding 2 (AREB2) expression in an ABA-dependent manner. These results indicate that the drought-responsive accumulation of sugars (especially SA-mediated sucrose accumulation) is part of the acclamatory process during the early period. Conversely, ABA-responsive hexose accumulation and sucrose phloem loading represent severe drought symptoms during the late drought period.

Label-Free Quantitative Proteomics Analysis in Susceptible and Resistant Brassica napus Cultivars Infected with Xanthomonas campestris pv. campestris.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is the main disease of cruciferous vegetables. To characterize the resistance mechanism in the Brassica napus-Xcc pathosystem, Xcc-responsive proteins in susceptible (cv. Mosa) and resistant (cv. Capitol) cultivars were investigated using gel-free quantitative proteomics and analysis of gene expression. This allowed us to identify 158 and 163 differentially expressed proteins following Xcc infection in cv. Mosa and cv. Capitol, respectively, and to classify them into five major categories including antioxidative systems, proteolysis, photosynthesis, redox, and innate immunity. All proteins involved in protein degradation such as the protease complex, proteasome subunits, and ATP-dependent Clp protease proteolytic subunits, were upregulated only in cv. Mosa, in which higher hydrogen peroxide accumulation concurred with upregulated superoxide dismutase. In cv. Capitol, photosystem II (PS II)-related proteins were downregulated (excepting PS II 22 kDa), whereas the PS I proteins, ATP synthase, and ferredoxin-NADP+ reductase, were upregulated. For redox-related proteins, upregulation of thioredoxin, 2-cys peroxiredoxin, and glutathione S-transferase occurred in cv. Capitol, consistent with higher NADH-, ascorbate-, and glutathione-based reducing potential, whereas the proteins involved in the C2 oxidative cycle and glycolysis were highly activated in cv. Mosa. Most innate immunity-related proteins, including zinc finger domain (ZFD)-containing protein, glycine-rich RNA-binding protein (GRP) and mitochondrial outer membrane porin, were highly enhanced in cv. Capitol, concomitant with enhanced expression of ZFD and GRP genes. Distinguishable differences in the protein profile between the two cultivars deserves higher importance for breeding programs and understanding of disease resistance in the B. napus-Xcc pathosystem.

Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles.

Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.