Signaling pathways involved in microbial indoor air pollutant 3-methyl-1-butanol in the induction of stomatal closure in Arabidopsis.

Indoor air pollution is a global problem and one of the main stress factors that has negative effects on plant and human health. 3-methyl-1-butanol (3MB), an indoor air pollutant, is a microbial volatile organic compound (mVOC) commonly found in damp indoor dwellings. In this study, we reported that 1 mg/L of 3MB can elicit a significant reduction in the stomatal aperture ratio in Arabidopsis and tobacco. Our results also showed that 3MB enhances the reactive oxygen species (ROS) production in guard cells of wild-type Arabidopsis after 24 h exposure. Further investigation of 24 h 3MB fumigation of rbohD, the1-1, mkk1, mkk3, and nced3 mutants revealed that ROS production, cell wall integrity, MAPK kinases cascade, and phytohormone abscisic acid are all involved in the process of 3MB-induced stomatal. Our findings proposed a mechanism by which 3MB regulates stomatal closure in Arabidopsis. Understanding the mechanisms by which microbial indoor air pollutant induces stomatal closure is critical for modulating the intake of harmful gases from indoor environments into leaves. Investigations into how stomata respond to the indoor mVOC 3MB will shed light on the plant's "self-defense" system responding to indoor air pollution.

Uncovering molecular mechanisms involved in microbial volatile compounds-induced stomatal closure in Arabidopsis thaliana.

Microbial volatile compounds (mVCs) may cause stomatal closure to limit pathogen invasion as part of plant innate immune response. However, the mechanisms of mVC-induced stomatal closure remain unclear. In this study, we co-cultured Enterobacter aerogenes with Arabidopsis (Arabidopsis thaliana) seedlings without direct contact to initiate stomatal closure. Experiments using the reactive oxygen species (ROS)-sensitive fluorescent dye, H2DCF-DA, showed that mVCs from E. aerogenes enhanced ROS production in guard cells of wild-type plants. The involvement of ROS in stomatal closure was then demonstrated in an ROS production mutant (rbohD). In addition, we identified two stages of signal transduction during E. aerogenes VC-induced stomatal closure by comparing the response of wild-type Arabidopsis with a panel of mutants. In the early stage (3 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and receptor-like kinase THESEUS1 mutant (the1-1) but not in rbohD, plant hormone-related mutants (nced3, erf4, jar1-1), or MAPK kinase mutants (mkk1 and mkk3). However, in the late stage (24 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and rbohD but not in nced3, erf4, jar1-1, the1-1, mkk1 or mkk3. Taken together, our results suggest that E. aerogenes mVC-induced plant immune responses modulate stomatal closure in Arabidopsis by a multi-phase mechanism.

Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product.

The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-D-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, are reported as the first representative structures of an alkaline α-galactosidase. The structure of AtAkαGal3 comprises three domains: an N-terminal domain with 13 antiparallel ß-strands, a catalytic domain with an (α/ß)8-barrel fold and a C-terminal domain composed of ß-sheets that form two Greek-key motifs. The WW box of the N-terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/ß)8 barrel of the catalytic domain. The C-terminal domain is presumably involved in structural stability. Structures of the D383A mutant in complex with various substrates and products, especially the natural substrate/product stachyose, reveal four complete subsites (-1 to +3) at the catalytic site. A functional loop (residues 329-352) that exists in the alkaline α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-galactosidases, stabilizes the stachyose at the +2 and +3 subsites and extends the catalytic pocket for the transferase mechanism. Considering the similarities in amino-acid sequence, catalytic domain and activity between alkaline α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

Integrating Early Transcriptomic Responses to Rhizotoxins in Rice (Oryza sativa. L.) Reveals Key Regulators and a Potential Early Biomarker of Cadmium Toxicity.

As sessile organisms, plants were constantly challenged with biotic and abiotic stresses. Transcriptional activation of stress-responsive genes is a crucial part of the plant adaptation to environmental changes. Here, early response of rice root to eight rhizotoxic stressors: arsenate, copper, cadmium, mercury, chromate, vanadate, ferulic acid and juglone, was analyzed using published microarray data. There were 539 general stress response (GSR) genes up-regulated under all eight treatments, including genes related to carbohydrate metabolism, phytohormone balance, and cell wall structure. Genes related to transcriptional coactivation showed higher Ka/Ks ratio compared to the other GSR genes. Network analysis discovered complicated interaction within GSR genes and the most connected signaling hubs were WRKY53, WRKY71, and MAPK5. Promoter analysis discovers enriched SCGCGCS cis-element in GSR genes. Moreover, GSR genes tend to be intronless and genes with shorter total intron length were induced in a higher level. Among genes uniquely up-regulated by a single stress, a phosphoenolpyruvate carboxylase kinase (PPCK) was identified as a candidate biomarker for detecting cadmium contamination. Our findings provide insights into the transcriptome dynamics of molecular response of rice to different rhizotoxic stress and also demonstrate potential use of comparative transcriptome analysis in identifying a novel potential early biomarker.

The blue fluorescent protein from Vibrio vulnificus CKM-1 is a useful reporter for plant research.

BACKGROUND: The mBFP is an improved variant of NADPH-dependent blue fluorescent protein that was originally identified from the non-bioluminescent pathogenic bacteria Vibrio vulnificus CKM-1. To explore the application of mBFP in plants, the mBFP gene expression was driven by one of the three promoters, namely, leaf-specific (RbcS), hypoxia-inducible (Adh) or auxin-inducible (DR5) promoters, in different plant tissues such as leaves, roots and flowers under diverse treatments. In addition, the expressed mBFP protein was targeted to five subcellular compartments such as cytosol, endoplasmic reticulum, apoplast, chloroplast and mitochondria, respectively, in plant cells. RESULTS: When the mBFP was transiently expressed in the tobacco leaves and floral tissues of moth orchid, the cytosol and apoplast exhibited brighter blue fluorescence than other compartments. The recombinant mBFP-mS1C fusion protein exhibited enhanced fluorescence intensity that was correlated with more abundant RNA transcripts (1.8 fold) as compared with a control. In the root tips of horizontally grown transgenic Arabidopsis, mBFP could be induced as a reporter under hypoxia condition. Furthermore, the mBFP was localized to the expected subcellular compartments, except that dual targeting was found when the mBFP was fused with the mitochondria-targeting signal peptide. Additionally, the brightness of mBFP blue fluorescence was correlated with NADPH concentration. CONCLUSION: The NADPH-dependent blue fluorescent protein could serve as a useful reporter in plants under aerobic or hypoxic condition. However, to avoid masking the mitochondrial targeting signal, fusing mBFP as a fusion tag in the C-terminal will be better when the mBFP is applied in mitochondria trafficking study. Furthermore, mBFP might have the potential to be further adopted as a NADPH biosensor in plant cells. Future codon optimization of mBFP for plants could significantly enhance its brightness and expand its potential applications.

Alkaline alpha-galactosidase degrades thylakoid membranes in the chloroplast during leaf senescence in rice.

Here, we studied the functional role of a chloroplast alkaline alpha-galactosidase (OsAkalphaGal) in the breakdown of thylakoid membranes during rice (Oryza sativa) leaf senescence. We assayed the enzyme activity of recombinant OsAkalphaGal with different natural substrates and examined the effect of ectopic OsAkalphaGal expression in rice plants. Recombinant OsAkalphaGal showed at least a two-fold greater substrate-binding affinity and a 10-fold greater turnover rate to galactolipid digalactosyl diacylglycerol than the raffinose family of oligosaccharides (verbascose, stachyose, raffinose) and melibiose. The OsAkalphaGal null mutant (osakalphagal) displayed a delayed leaf senescence phenotype. OsAkalphaGal complementation in osakalphagal recovered OsAkalphaGal expression and showed a senescence phenotype similar to that of wild-type plants. Transgenic plants overexpressing OsAkalphaGal (UbiP-OsAkalphaGal) exhibited retarded plant growth and development, and showed a pale-green phenotype coupled with a reduced chlorophyll content to 42% in newly unfolded leaves. UbiP-OsAkalphaGal leaves also showed a 29-fold increase in alkaline alpha-galactosidase activity compared with wild-type leaves. An ultrastructural study of Ubi-OsAkalphaGal chloroplasts in newly unfolded leaves revealed abnormal grana organization. Our findings strongly suggest that OsAkalphaGal is a thylakoid membrane-degrading enzyme involved in the degradation of digalactosyl diacylglycerol during rice leaf senescence.

Early signalling pathways in rice roots under vanadate stress.

Vanadate is beneficial to plant growth at low concentration. However, plant exposure to high concentrations of vanadate has been shown to arrest cell growth and lead to cell death. We are interested in understanding the signalling pathways of rice roots in response to vanadate stress. In this study, we demonstrated that vanadate induced rice root cell death and suppressed root growth. In addition, we found that vanadate induced ROS accumulation, increased lipid peroxidation and elicited a remarkable increase of MAPKs and CDPKs activities in rice roots. In contrast, pre-treatment of rice roots with ROS scavenger (sodium benzoate), serine/threonine protein phosphatase inhibitor (endothall), and CDPK antagonist (W7), reduced the vanadate-induced MAPKs activation. Furthermore, the expression of a MAPK gene (OsMPK3) and four tyrosine phosphatase genes (OsDSP3, OsDSP5, OsDSP6, and OsDSP10) were regulated by vanadate in rice roots. Collectively, these results strongly suggest that ROS, protein phosphatase, and CDPK may function in the vanadate-triggered MAPK signalling pathway cause cell death and retarded growth in rice roots.

A novel alkaline alpha-galactosidase gene is involved in rice leaf senescence.

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG- Osh69 encoding alkaline alpha-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H2O2 and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal alpha-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards alpha-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence.

Molecular characterization of a novel senescence-associated gene SPA15 induced during leaf senescence in sweet potato.

The structure and expression of a novel senescence-associated gene (SPA15) of sweet potato were characterized. The protein coding region of the gene consists of 13 exons encoding 420 amino acids. Apparent homologues of this sweet potato gene are found in a variety of dicot and monocot plants, but not in animals or microorganisms. Examination of the expression patterns of the SPA15 gene in sweet potato reveals that the transcripts of SPA15 are specifically induced in the senescing leaves, and the temporal profile of SPA15 protein accumulation is correlated with that of SPA15 transcripts. Studies on the distribution of SPA15 homologue in rice plants also indicate that SPA15 homologue is up-regulated specifically in senescing rice leaves. Treatment of detached sweet potato leaves with phytohormones including ethylene, methyl jasmonate, salicylic acid and abscisic acid resulted in a high-level induction of SPA15. Immunoelectron microscopic analysis demonstrates that SPA15 is specifically associated with the cell wall. The potential role for SPA15 during leaf senescence is discussed.

Molecular characterization of a senescence-associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet potato.

The structure and expression of a senescence-associated gene (SPG31) encoding a cysteine proteinase precursor of sweet potato have been characterized. The coding region of the gene consists of two exons encoding an enzyme precursor of 341 amino acids with conserved catalytic amino acids of papain. Examination of the expression patterns of the SPG31 gene in sweet potato by Northern blot analyses reveals that the transcripts of SPG31 are specifically induced in the senescing leaves but not in other organs. The differential accumulation of the mature SPG31 protein in the senescing leaves was further identified by two-dimensional electrophoresis of leaf proteins and N-terminal sequencing. This result suggests the important role played by SPG31 in proteolysis and nitrogen remobilization during the leaf senescence process. Furthermore, treatment of mature green leaves with ethylene for 3 d resulted in a high-level induction of SPG31 transcripts. Ethylene-regulated expression of SPG31 is consistent with the presence of a number of putative ethylene-responsive elements in the 899-bp SPG31 promoter region.

Programmed cell death during rice leaf senescence is nonapoptotic.

• The cellular events associated with programmed cell death during leaf senescence in rice (Oryza sativa) plants are reported here. • The cytological sequence of senescence-related changes in rice leaves was studied by transmission electron microscopy, in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and DNA ladder assay. • Cell death in senescing mesophyll cells was marked by depletion of cytoplasm in a tightly controlled manner. However, no apparent morphological feature associated with apoptosis was observed. Nuclear DNA fragmentation was detectable as early as during leaf unfolding and the subsequent developmental and senescent stages. The occurrence of DNA fragmentation correlated well with the size-shift of chromosomal DNA on agarose gel after electrophoresis. However, DNA fragmentation was not accompanied by generation of oligonucleosomal DNA fragments. • These features of cell death occurring during leaf senescence in monocot rice are quite different from features characteristic of apoptosis in animals. The implications of these results for cellular events associated with rice leaf senescence are discussed.