Proteomics and functional analyses of <Emphasis Type="Italic">Arabidopsis</Emphasis> nitrilases involved in the defense response to microbial pathogens

Main conclusion

Proteomics and functional analyses of the Arabidopsis – Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.

     

Functional analysis of the <Emphasis Type="Italic">Malus domestica MdHMGR2</Emphasis> gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The first rate-limiting enzyme of the mevalonate pathway during isoprenoid biosynthesis is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In this study, the expression pattern of the MdHMGR2 gene in Malus domestica suggests that MdHMGR2 was expressed in a tissue-specific manner and was significantly induced by ethephon (ETH), indoleacetic acid (IAA), methyl jasmonate (MeJA), and salicylic acid (SA). The MdHMGR2 promoter was isolated, sequenced, and analyzed through bioinformatics tools, and the results suggest the presence of various putative cis-acting elements responsive to different hormones. Activity of β-glucuronidase (GUS) driven by the full length MdHMGR2 promoter and its 5′deletion fragments was detected in transgenic Arabidopsis thaliana. A strong GUS activity was observed in seedlings, roots, newly growing true leaves, anthers, and stigmas in transgenic Arabidopsis containing the full MdHMGR2 promoter. The results indicate that a region from -1050 to -827 was crucial for promoter activity. In addition, the MdHMGR2 promoter was induced in response to ETH, IAA, MeJA, and SA. The analysis suggests that an ethylene-responsive element in the region from -1050 to -1005 was required for the ethylene inducibility.     

Endogenous Salicylic Acid Levels and Signaling Positively Regulate <Emphasis Type="Italic">Arabidopsis</Emphasis> Response to Polyethylene Glycol-Simulated Drought Stress

Salicylic acid (SA) functions in the plant response to drought stress were assessed using SA-altering Arabidopsis mutants, including snc1 (with constitutively high levels of SA) and its nahG-transformed plants (named as snc1/nahG, with a comparable SA level to the wild type), sid2 and transgenic line nahG (both with SA deficiency), and npr1-1 (with SA signaling blockage). The drought stress was simulated by polyethylene glycol (PEG)-6000 treatment. Compared with wild-type (wt) plants, the snc1 plants displayed obvious easing of PEG-induced growth inhibition, leaf water loss, and photosynthesis-related impairment, whereas in nahG, sid2, and npr1-1 mutants the effect was more severe. PEG stress reduced stomatal conductance, to a higher extent in the snc1 line, whereas it was lower in nahG, sid2, and npr1-1 lines as compared with the wt. The snc1 plants accumulated higher levels of H₂O₂ than the other genotypes tested. PEG stress increased activities of superoxide dismutase and peroxidase, but decreased activities of catalase in all lines tested, to a greater extent in snc1 and less in sid2, nahG, and npr1-1 relative to wt. Proline was significantly increased, especially in snc1 line at 6 % and higher PEG stress. Noticeably, the performance of snc1 under PEG stress was dependent on SA levels, as the expression of nahG in snc1 plants did not only significantly reduce SA levels, but largely reversed the above-mentioned parameters, as well as eliminated the drought tolerance. Based on these data, it was concluded that endogenous SA levels and signaling provided a protective role in the Arabidopsis response to PEG-simulated drought.     

ATG5 is required to limit cell death induced by <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> in <Emphasis Type="Italic">Arabidopsis</Emphasis> and may be mediated by the salicylic acid pathway

Autophagy can be regarded as a protection mechanism to restrict programmed cell death (PCD) induced by pathogen infection during plant innate immunity in the early stages. Autophagy related 5 (ATG5) plays an important role in autophagy in Arabidopsis. We investigated the function of ATG5 in Arabidopsis in the hypersensitive response (HR)-PCD elicited by both virulent and avirulent strains of Pseudomonas syringae pv. tomato bacteria DC3000. Results show that ATG5 plays a vital role in limiting HR induced by P. syringae strains and colocalizes with autophagic bodies during the early phase of bacterial infection. In addition, the P. syringae-induced response is mediated by the salicylic acid (SA) signaling pathway. In summary, ATG5 is required for limiting HR-PCD induced in Arabidopsis by P. syringae strains and may be mediated by SA signaling.     

Hormone-Induced Gene Expression During Gravicurvature of <Emphasis Type="Italic">Brassica</Emphasis> Roots

To investigate the spatial and temporal dependence of hormonal regulation during gravitropism, we compared the effects of root cap application of indole-3-acetic acid (IAA) and abscisic acid (ABA) with gene expression changes occurring naturally during gravitropic reaction of Brassica rapa roots. The expression of auxin, ABA, and metabolism-related genes in the tip, elongation zone, and maturation zone varied with time, location, and hormone concentration and characterized polar auxin transport. IAA was transported readily shootward and inhibited growth more than ABA. Expression of PIN3 and IAA5 in the elongation zone showed downregulation on the convex but upregulation on the concave side. Both PIN7 and IAA5 responded near maximally to 10^?8 M IAA within 30 min, suggesting that auxin activates its own transport system. Ubiquitin 1 (UBQ1) responded after a lag time of more than 1 h to IAA. The metabolic control gene Phosphoenolpyruvate carboxylase 1 (PEPC1) was more sensitive to ABA but upregulated by high concentrations of either hormone. The time course and duration of gene activation suggests that ABA is not involved in gravitropic curvature, differential elongation is not simply explained by IAA-induced upregulation, and that reference genes are sensitive to auxin.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Halophilic rhizobacteria from <Emphasis Type="Italic">Distichlis spicata</Emphasis> promote growth and improve salt tolerance in heterologous plant hosts

Rhizobacteria are central components of the plant microbiome and influence root development and function. Desciphering how rhizobacteria contribute to plant performance under adverse environments is a major research challenge. The aims of the present study were to isolate and characterize rhizobacteria from the halophilic grass Distichlis spicata and to test their possible growth promoting and salt protective properties in Arabidopsis thaliana, Cucumis sativus, and Citrullus lanatus. To determine their possible plant growth promoting properties, 38 rhizobacterial isolates were co-cultivated with Arabidopsis seedlings in vitro. Out of these, two halophilic bacteria, LBEndo1 and KBEcto4, were selected following their strong shoot and root biostimulation. 16S rRNA sequencing identified LBEndo1 as Bacillus sp. and KBEcto4 as Pseudomonas lini. Both strains improved growth under standard and saline conditions, which correlated with IAA and siderophore production, as well as phosphate solubilization. Additionally, the KBEcto4 strain expresses the ACC deaminase enzyme (acdS gene), and slightly increases auxin redistribution within Arabidopsis roots expressing an auxin-inducible gene construct. These data reveal the potential of saltgrass (Distichlis spicata) rhizobacteria to promote growth and confer salt tolerance to Arabidopsis and crop plants.     

Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

The benefits of foliar inoculation with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in soybean are explained by an auxin signaling model

Azospirillum sp. is one of the most studied genera of plant growth-promoting rhizobacteria (PGPR). The ability of Azospirillum sp. to promote plant growth has been associated with its ability to produce several phytohormones, such as auxins, gibberellins and cytokinins, but mainly indole-3-acetic acid (IAA). It has been propoosed that the production of IAA explains the positive effects of co-inoculation with Azospirillum sp. on the rhizobia-legume symbiosis. In this study, we constructed an IAA-deficient mutant of A. brasilense Az39 (ipdC ^? ) by using a restriction-free cloning method. We inoculated soybean seeds with 1·10⁶ cfu·seed^?1 of Bradyrhizobium japonicum E109 and co-inoculating leaves at the V3 stage with 1·10⁸ cfu.plant^?1 of A. brasilense Az39 wt or ipdC ^? or inoculated leaves with 20 μg.plant^?1 synthetic IAA. The results confirmed soybean growth promotion as there was increased total plant and root length, aerial and root dry weight, number of nodules on the primary root, and an increase in the symbiosis established with B. japonicum E109. Nodule weight also increased after foliar co-inoculation with the IAA- producer A. brasilense Az39. The exogenous application of IAA decreased aerial and root length, as well as the number of nodules on primary roots in comparison with the Az39 wt strain. These results allow us to propose a biological model of response to foliar co-inoculation of soybean with IAA-producing rhizobacteria. This model clearly shows that both the presence of microorganism as part of the colonization process and the production of IAA in situ are co-responsible, via plant signaling molecules, for the positive effects on plant growth and symbiosis establishment.     

The effect of <Emphasis Type="Italic">UGT1A9</Emphasis>, <Emphasis Type="Italic">CYP2B6</Emphasis> and <Emphasis Type="Italic">CYP2C9</Emphasis> genes polymorphism on individual differences in propofol pharmacokinetics among Polish patients undergoing general anaesthesia

Propofol (2,6-diisopropylphenol) is one of the safest and most commonly used anaesthetic agents for intravenous general anaesthesia. However, in clinical practice, a large inter-individual variability in response to propofol is observed. To limit the risk of adverse effects, pharmacogenetic investigations are recommended. The aim of our study was to verify the impact of genetic changes c.516G>T in the CYP2B6, c.98T>C in the UGT1A9 and c.1075A>C in the CYP2C9 genes on the individual propofol pharmacokinetic profile in the Polish patients undergoing general anaesthesia. Eighty-five patients from the Department of Anaesthesiology and Intensive Therapy, Regional Hospital in Poznan, Poland, anaesthetised with propofol for surgery, were enrolled in the study. We have genotyped CYP2B6, UGT1A9 and CYP2C9 polymorphisms with the use of pyrosequencing. HPLC measurements of propofol plasma concentration were applied for a pharmacokinetic analysis of the anaesthetic. We identified poor (20), intermediate (42) and rapid (23) metabolisers of propofol, which constituted 24%, 49% and 27% of the group, respectively. Homozygotes c.516 T/T in the CYP2B6 gene were statistically more often found in the rapid metabolisers group (p?<?0.05). However, polymorphisms c.98T>C in the UGT1A9 and c.1075A>C in the CYP2C9 genes did not affect the pharmacokinetic profile of propofol. The mean propofol retention time (MRT) correlated with the patient’s body mass index (BMI) (p?<?0.05). From all the analysed changes, only polymorphism c.516G>T in the CYP2B6 gene and BMI affect the metabolism rate of propofol and may play an important role in the optimisation of propofol anaesthesia.     

Contribution of the non-effector members of the HrpL regulon, <Emphasis Type="Italic">iaaL</Emphasis> and <Emphasis Type="Italic">matE</Emphasis>, to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. tomato DC3000 in tomato plants

Background

The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.

Results

A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.

Conclusions

Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

     

Late Embryogenesis Abundant (<Emphasis Type="Italic">LEA</Emphasis>) Gene Family in Maize: Identification,Evolution, and Expression Profiles

Late embryogenesis abundant (LEA) proteins are identified as a large and highly diverse group of polypeptides accumulating in response to cellular dehydration in many organisms. However, there are only very limited reports of this protein family in maize until this study. In the present paper, we identified 32 LEA genes in maize. A total of 83 LEA proteins including 51 members in Arabidopsis and 32 putative members in maize were classified into nine groups. Gene organization and motif compositions of the LEA members are highly conserved in each of the groups, indicative of their functional conservation. The predicted ZmLEA genes were non-random distributed across chromosomes, and transposition event and segmental duplication contributed to the expansion of the LEA gene family in maize. Some abiotic stress-responsive cis-elements were also found in the promoters of ZmLEA genes. Microarray expression analyses revealed different accumulation patterns of ZmLEA family members. Moreover, some members of ZmLEAs were regulated under IAA and some abiotic stresses. This study will provide comprehensive information for maize LEA gene family and may pave the way for deciphering their functions in further studies.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.