52 research outputs found
Quantitative and functional post-translational modification proteomics reveals that TREPH1 plays a role in plant thigmomorphogenesis
Plants can sense both intracellular and extracellular mechanical forces and
can respond through morphological changes. The signaling components responsible
for mechanotransduction of the touch response are largely unknown. Here, we
performed a high-throughput SILIA (stable isotope labeling in
Arabidopsis)-based quantitative phosphoproteomics analysis to profile changes
in protein phosphorylation resulting from 40 seconds of force stimulation in
Arabidopsis thaliana. Of the 24 touch-responsive phosphopeptides identified,
many were derived from kinases, phosphatases, cytoskeleton proteins, membrane
proteins and ion transporters. TOUCH-REGULATED PHOSPHOPROTEIN1 (TREPH1) and MAP
KINASE KINASE 2 (MKK2) and/or MKK1 became rapidly phosphorylated in
touch-stimulated plants. Both TREPH1 and MKK2 are required for touch-induced
delayed flowering, a major component of thigmomorphogenesis. The treph1-1 and
mkk2 mutants also exhibited defects in touch-inducible gene expression. A
non-phosphorylatable site-specific isoform of TREPH1 (S625A) failed to restore
touch-induced flowering delay of treph1-1, indicating the necessity of S625 for
TREPH1 function and providing evidence consistent with the possible functional
relevance of the touch-regulated TREPH1 phosphorylation. Bioinformatic analysis
and biochemical subcellular fractionation of TREPH1 protein indicate that it is
a soluble protein. Altogether, these findings identify new protein players in
Arabidopsis thigmomorphogenesis regulation, suggesting that protein
phosphorylation may play a critical role in plant force responses
Keeping the rhythm:light/dark cycles during postharvest storage preserve the tissue integrity and nutritional content of leafy plants
Background: The modular body structure of plants enables detached plant organs, such as postharvest fruits and vegetables, to maintain active responsiveness to environmental stimuli, including daily cycles of light and darkness. Twenty-four hour light/darkness cycles entrain plant circadian clock rhythms, which provide advantage to plants. Here, we tested whether green leafy vegetables gain longevity advantage by being stored under light/dark cycles designed to maintain biological rhythms. Results: Light/dark cycles during postharvest storage improved several aspects of plant tissue performance comparable to that provided by refrigeration. Tissue integrity, green coloration, and chlorophyll content were generally enhanced by cycling of light and darkness compared to constant light or darkness during storage. In addition, the levels of the phytonutrient glucosinolates in kale and cabbage remained at higher levels over time when the leaf tissue was stored under light/dark cycles. Conclusions: Maintenance of the daily cycling of light and dark periods during postharvest storage may slow the decline of plant tissues, such as green leafy vegetables, improving not only appearance but also the health value of the crops through the maintenance of chlorophyll and phytochemical content after harvest
XTH31, Encoding an in Vitro XEH/XET-Active Enzyme, Regulates Aluminum Sensitivity by Modulating in Vivo XET Action, Cell Wall Xyloglucan Content, and Aluminum Binding Capacity in Arabidopsis
Xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET) activities, encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes, are involved in cell wall extension by cutting or cutting and rejoining xyloglucan chains, respectively. However, the physiological significance of this biochemical activity remains incompletely understood. Here, we find that an XTH31 T-DNA insertion mutant, xth31, is more Al resistant than the wild type. XTH31 is bound to the plasma membrane and the encoding gene is expressed in the root elongation zone and in nascent leaves, suggesting a role in cell expansion. XTH31 transcript accumulation is strongly downregulated by Al treatment. XTH31 expression in yeast yields a protein with an in vitro XEH:XET activity ratio of >5000:1. xth31 accumulates significantly less Al in the root apex and cell wall, shows remarkably lower in vivo XET action and extractable XET activity, has a lower xyloglucan content, and exhibits slower elongation. An exogenous supply of xyloglucan significantly ameliorates Al toxicity by reducing Al accumulation in the roots, owing to the formation of an Al-xyloglucan complex in the medium, as verified by an obvious change in chemical shift of (27)Al-NMR. Taken together, the data indicate that XTH31 affects Al sensitivity by modulating cell wall xyloglucan content and Al binding capacity
Xyloglucan Endotransglucosylase-Hydrolase17 Interacts with Xyloglucan Endotransglucosylase-Hydrolase31 to Confer Xyloglucan Endotransglucosylase Action and Affect Aluminum Sensitivity in Arabidopsis
Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock
In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic-free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterised. We investigated whether circadian oscillations of [Ca2+] cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage
between CML24 and the circadian oscillator, through pathways involving the circadian
oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).Supported by BBSRC UK research grants BBSRC BB/D010381/1 (A.N.D.), BB/D017904/1 (F.R.) BB/M00113X/1 (H.J.H.) awarded to (A.A.R.W.), Research Studentship (K.H.) and BBSRC Industrial Case (T.H.). A Swiss Science Foundation Award (PBZHP3-123289) and the Isaac Newton Trust Cambridge (M.C.M.R. and S.A.),
678 the National Science Foundation under Grant No. MCB 0817976 (Y-C.T. and J.B.), a Royal Society Grant RG081257 and Corpus Christi College, Cambridge Junior Research Fellowship (M.J.G.), a Cordenadoria de Apoio ao Ensino Superior Brazil 25681 studentship (C.T.H.), IEF Marrie Curie (Project No. 272186) (M.C.M.R.), a Broodbank Fellowship (M.C.M.R.), a Malaysian Government Studentship (N.I.M-H.)
Restriction Site Extension PCR: A Novel Method for High-Throughput Characterization of Tagged DNA Fragments and Genome Walking
BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well
Keeping the rhythm: light/dark cycles during postharvest storage preserve the tissue integrity and nutritional content of leafy plants
Recommended from our members
Genetic analysis of the regulation of TCH gene expression, Final Report
The Arabidopsis TCH genes, originally isolated as a consequence of their upregulation in response to the mechanical stimulus of touch, are also upregulated by a variety of seemingly disparate environmental and hormonal stimuli. To gain insight into the complexities of TCH gene regulation, a number of approaches were taken. Regulatory elements responsible for regulation were identified and characteristics of the regulation were evaluated. Reporter genes were used to monitor expression localization and dynamics. Microarray analyses of genome-wide expression behavior indicated that touch-inducible gene expression is more widespread than generally appreciated. Identification of all touch-regulated genes shed light on the types of cellular processes that may be altered in response to mechanical stress perturbations. Expression of the TCH2 gene, also called CML24, encoding a calmodulin (CaM)-like (CML) protein, was evaluated. CML24 shares over 40% amino acid sequence identity with CaM, has 4 EF hands and undergoes a Ca2+-dependent change in migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs and is induced from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress, in regions undergoing growth, in vascular tissues and various floral organs and in stomata, trichomes and hydathodes. CML24 underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4 and MgCl2. These data present evidence that CML24 encodes a potential Ca2+ sensor that may function to enable responses to ABA, day length and presence of various salts. Further investigation of CML24 function and regulation led to the finding that CML24 has a critical role in nitric oxide regulation. Distinct tilling mutant alleles demonstrated that CML24 can act as a switch in the response to day length perception. Because of potential redundancy with the related CML23 gene, CML23 T-DNA insertion mutants were identified and characterized. Together, CML23 and CML24 impact the autonomous regulatory pathway of the transition to flowering. Nitric oxide levels are elevated in cml23/cml24 double mutants. Therefore, CML23 and CML24 are potential calcium sensors regulate nitric oxide accumulation. In collaboration with Drs. McCann and Carpita, fourier transform infrared spectroscopy (FTIR) was used to assess, verify and classify wall architectural changes that occur as a result of single XTH insertion mutations. Thirty-four homozygous mutant lines of Arabidopsis representing 21 members of the xyloglucan endotransglucosylase/hydrolase gene family provided a set of mutants to characterize. Kohonen networks classified cell wall architectures of xth mutant lines and previously characterized cell wall mutants. The xth mutants were found to have chemical changes in their cell walls not detectable as phenotypic growth and development changes, consistent with the existence of feed-back loops that modify wall composition in response to a life-long deficiency of a cell wall enzyme. To gain insight into the potential physiological relevance of the distinct members of the XTH family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data revealed that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs showed XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes suggesting that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics
- …