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Polygalacturonase (PG) activity in plants can serve as an important index for plant disease. However, the conventional method to detect PG activity is a complex process and requires a skilled technician and expensive analytical equipment. In this study, a paper-based colorimetric sensor was developed based on the principle of the ruthenium red (RR) dye method for easy and simple measurement of PG activity. The proposed paper-based sensor has a three-layer structure for detection of PG activity in samples. The sensor sensitivity was enhanced by optimizing the pH of the sodium acetate buffer used in polygalacturonic acid (PGA)-RR complex formation and the reaction temperature for PG and the PGA-RR complex. Further, for quantitative analysis of PG activity, Delta RGB analysis was conducted to detect color changes in the sensing window of the sensor. Results presented that the linear measurement range of the paper sensor was 0.02-0.1 unit with the limit of detection of 0.02 unit, which showed a similar detection range, but a lower detection limit, compared to the spectrophotometry. Furthermore, PG activity based on culture condition was measured using samples from Sclerotium cepivorum to verify the potential application of the developed paper-based sensor in the field. The measured activity showed no statistically significant difference from the values obtained from the spectrophotometry at 95% confidence level. Therefore, the paper-based colorimetric sensor can be used to predict plant diseases in Allium crops during the stage of pathogen invasion, potentially contributing to the improvement of crop production.

Transduction of glucose (Glc) signaling is critical for plant development, metabolism, and stress responses. However, identifying initial Glc sensing and response stimulating mechanisms in plants has been difficult due to dual functions of glucose as energy sources and signaling component. A basic Helix-Loop-Helix 104 (bHLH104) protein is a homolog of bHLH34 previously isolated from Arabidopsis that functions as a transcriptional activator of Glc and abscisic acid (ABA) responses. In this study, we characterized bHLH104 as a transcription factor that binds to the regulatory region of Arabidopsis Plasma membrane Glc-responsive Regulator (AtPGR) gene. The bHLH104 binds to 5'-AANA-3' element of the promoter region of AtPGR in vitro and represses beta-glucuronidase (GUS) activity in AtPGR promoter-GUS transgenic plants. Genetic approaches show that bHLH104 positively regulates Glc and abscisic acid (ABA) response. These results suggest that bHLH104 is involved in Glc- and ABA-mediated signaling pathway. Taken together, these findings provide evidence that bHLH104 is an important transcription regulator in plant-sensitivity to Glc and ABA signaling.

Proline (Pro) metabolism plays important roles in protein synthesis, redox balance, and abiotic stress response. However, it is not known if cross-talk occurs between proline and brassinosteroid (BR) signaling pathways. Here, an Arabidopsis intergenic enhancer double mutant, namely proline content alterative 41 (pca41), was generated by inserting a T-DNA tag in the Arabidopsis thaliana ring zinc finger 1 (atrzf1 ) mutant background. pca41 had a T-DNA inserted at the site of the gene encoding BES1/BZR1 Homolog 3 (BEH3). pca41 has a drought-insensitive phenotype that is stronger than atrzf1 under osmotic stress, including high Pro accumulation and decreased amounts of reactive oxygen species. Analysis of physiological, genetic, and molecular networks revealed that negative regulation of BEH3 during abiotic stress was linked to the BR signaling pathway. Our data also suggest that AtRZF1, an E3 ubiquitin ligase, might control osmotic stress, abscisic acid, and BR responses in a BEH3-dependent manner. Under darkness, pca41 displays a long hypocotyl phenotype, which is similar to atrzf1 and beh3, suggesting that BEH3 acts in the same pathway as AtRZF1. Overexpression of BEH3 results in an osmotic stress-sensitive phenotype, which is reversed by exogenous BR application. Taken together, our results indicate that AtRZF1 and BEH3 may play important roles in the osmotic stress response via ubiquitination and BR signaling.

Highlight PCA22 negatively regulates the drought response through the modulation of osmolytic components and regulates pollen tube length in Arabidopsis., Proline metabolism is important for environmental responses, plant growth, and development. However, its precise roles in plant abiotic stress tolerance are not well understood. Mutants are valuable for the identification of new genes and for elucidating their roles in physiological mechanisms. We applied a suppressor mutation approach to identify novel genes involved in the regulation of proline metabolism in Arabidopsis. Using the atrzf1 (Arabidopsis thaliana ring zinc finger 1) mutant as a parental line for activation tagging mutagenesis, we selected several mutants with suppressed induction of proline accumulation under dehydration conditions. One of the selected mutants [proline content alterative 22 (pca22)] appeared to have reduced proline contents compared with the atrzf1 mutant under drought stress. Generally, pca22 mutant plants displayed suppressed atrzf1 insensitivity to dehydration and abscisic acid during early seedling growth. Additionally, the pca22 mutant exhibited shorter pollen tube length than wild-type (WT) and atrzf1 plants. Furthermore, PCA22-overexpressing plants were more sensitive to dehydration stress than the WT and RNAi lines. Green fluorescent protein-tagged PCA22 was localized to the cytoplasm of transgenic Arabidopsis cells. Collectively, these results suggest that pca22 acts as dominant suppressor mutant of atrzf1 in the abiotic stress response.

The modulation of glucose (Glc) homeostasis and signaling is crucial for plant growth and development. Nevertheless, the molecular signaling mechanism by which a plant senses a cellular Glc level and coordinates the expression of Glc-responsive genes is still incompletely understood. Previous studies have shown that Arabidopsis thaliana plasma membrane Glc-responsive regulator (AtPGR) is a component of the Glc-responsive pathway. Here, we demonstrated that a transcription factor bHLH34 binds to 5′-GAGA-3′ element of the promoter region of AtPGR in vitro, and activates beta-glucuronidase (GUS) activity upon Glc treatment in AtPGR promoter-GUS transgenic plants. Gain- and loss-of-function analyses suggested that the bHLH34 involved in the responses to not only Glc, but also abscisic acid (ABA) and salinity. These results suggest that bHLH34 functions as a transcription factor in the Glc-mediated stress responsive pathway as well as an activator of AtPGR transcription. Furthermore, genetic experiments revealed that in Glc response, the functions of bHLH34 are different from that of a bHLH104, a homologue of bHLH34. Collectively, our findings indicate that bHLH34 is a positive regulator of Glc, and may affect ABA or salinity response, whereas bHLH104 is a negative regulator and epistatic to bHLH34 in the Glc response.

Abscisic acid (ABA) receptors in plants are thought to be involved in various cellular processes mediated by signal transduction pathways. There are about 14 ABA receptors in Arabidopsis, but only a few have been studied. In this study, we investigated the effect of the disruption and overexpression of an ABA receptor gene, PYL8 (At5g53160) on plant responses to glucose (Glc) and dark-induced leaf senescence. Expression of PYL8 was strongly reduced by Glc treatment. Overexpression of PYL8 in Arabidopsis resulted in significantly reduced seed germination and cotyledon greening under high Glc conditions, while RNAi transgenic lines were more insensitive to Glc stress. Activities of two Glc-responsive genes, Arabidopsis thaliana Hexokinase 1 (AtHXK1) and ABA insensitive 5 (ABI5) were higher in PYL8-overexpressing plants than in the wild-type (WT) plants after Glc treatment, whereas the transcript levels of these genes in RNAi plants decreased. Furthermore, PYL8-overexpressing plants displayed increased yellowing, membrane ion leakage, and reduced chlorophyll content due to dark-induced senescence, and exhibited stronger expression of a group of senescence-inducible genes than did WT. The data show that PYL8 plays essential roles in responses to both Glc and dark-induced senescence in A. thaliana.

Proline (Pro) metabolism in plants is involved in various cellular processes mediated during abiotic stress. However, the Pro-regulatory mechanisms are unclear. We used a suppressor mutation technique to isolate novel genes involved in the regulation of Pro metabolism in Arabidopsis. Using atrzf1 as a parental plant for T-DNA tagging mutagenesis, we identified a suppressor mutant, termed proline content alterative 21 (pca21), that displayed reduced Pro contents compared with the atrzf1 under osmotic stress conditions. Genomic Thermal Asymmetric Interlaced (TAIL)-PCR revealed pca21 harbored an inserted T-DNA in the region of At2g36620 that encodes Ribosomal Protein L24A. In general, the pca21 mutant partially suppressed the insensitivity of atrzf1 to osmotic stress and abscisic acid during seed germination and early seedling stage. Additionally, the pca21 mutant had increased MDA content and lower expression of several Pro biosynthesis-related genes than the atrzf1 mutant during drought condition. These results suggest that pca21 acts as partial suppressor of atrzf1 in the osmotic stress response through the Pro-mediated pathway.

The precise roles of the B-box zinc finger family of transcription factors in plant stress are poorly understood. Functional analysis was performed on AtCOL4, an Arabidopsis thaliana L. CONSTANS-like 4 protein that is a putative novel transcription factor, and which contains a predicted transcriptional activation domain. Analyses of an AtCOL4 promoter-β-glucuronidase (GUS) construct revealed substantial GUS activity in whole seedlings. The expression of AtCOL4 was strongly induced by abscisic acid (ABA), salt, and osmotic stress. Mutation in atcol4 resulted in increased sensitivity to ABA and salt stress during seed germination and the cotyledon greening process. In contrast, AtCOL4-overexpressing plants were less sensitive to ABA and salt stress compared to the wild type. Interestingly, in the presence of ABA or salt stress, the transcript levels of other ABA biosynthesis and stress-related genes were enhanced induction in AtCOL4-overexpressing and WT plants, rather than in the atcol4 mutant. Thus, AtCOL4 is involved in ABA and salt stress response through the ABA-dependent signaling pathway. Taken together, these findings provide compelling evidence that AtCOL4 is an important regulator for plant tolerance to abiotic stress.

Current molecular methods that include PCR have been used to detect norovirus in many food samples. However, the protocols require removing PCR inhibitors and incorporate time-consuming concentration steps to separate virus from analyte for rapid and sensitive detection of norovirus. We developed an immunomagnetic separation (IMS) and a quantum dots (QDs) assay to detect norovirus eluted from fresh lettuce with Tris buffer containing 1% beef extract (pH 9.5). IMS facilitated viral precipitation with a 10-min incubation, whereas virus concentration using polyethylene glycol (PEG) requires more than 3 h and an additional high-speed centrifugation step to precipitate virus before reverse transcription PCR (RT-PCR) analysis. The fluorescence intensity of QDs was detected qualitatively on norovirus dilutions of 10(-1) to 10(-3) in a stool suspension (100 RT-PCR units/ml). The results suggest that a fluorescence assay based on IMS and QDs is valid for detecting norovirus qualitatively according to fluorescent signal intensity within the same virus detection limit produced by IMS-RT-PCR and PEG-RT-PCR.

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.

AtSKIP participated in cytokinin-regulated leaf initiation. Putative phosphorylated AtSKIP (AtSKIP (DD) ) displayed the opposite function in the leaf development from AtSKIP transgenic seedlings.AtSKIP, as a multiple protein, is involved in many physiological processes, such as flowering, cell cycle regulator, photomorphogenesis and stress tolerance. However, the mechanism of AtSKIP in these processes is unclear. Here, we identify one gene, AtSKIP, which is associated with cytokinin-regulated leaf growth process in Arabidopsis. The expression of AtSKIP was regulated by cytokinin. Leaf development in AtSKIP overproduced seedlings was independent of light, but promoted by cytokinin, and phosphorylation of AtSKIP (AtSKIP(DD)) partially interfered with AtSKIP function as a positive regulator in cytokinin signaling, indicative of true leaf formation, and the defects of AtSKIP(DD) in the true leaf formation could be recovered to some extent by the addition of cytokinin. Moreover, different cytokinin-responsive gene Authentic Response Regulator 7 (ARR7) promoter-GUS activity further proved that expression of AtSKIP or AtSKIP(DD) altered endogenous cytokinin signaling in plants. Together, these data indicate that AtSKIP participates in cytokinin-regulated promotion of leaf growth in photomorphogenesis, and that phosphorylation interferes with AtSKIP normal function.