The rice germin-like protein OsGLP1 participates in acclimation to UV-B radiation.

Exposure to ultraviolet B radiation (UV-B) stress can have serious effects on the growth and development of plants. Germin-like proteins (GLPs) may be involved in different abiotic and biotic stress responses in different plants, but little is known about the role of GLPs in UV-B stress response and acclimation in plants. In the present study, knockout of GLP 8-14 (OsGLP1) using the CRISPR/Cas9 system resulted in mutant rice (Oryza sativa L.) plants (herein called glp1) that exhibited UV-B-dependent formation of lesion mimic in leaves. Moreover, glp1 grown under solar radiation (including UV-B) showed decreased plant height and increased leaf angle, but we observed no significant differences in phenotypes between wild-type (WT) plants and glp1 grown under artificial light lacking UV-B. Fv/Fm, Y (II) and the expression of many genes, based on RNA-seq analysis, related to photosynthesis were also only reduced in glp1, but not in WT, after transfer from a growth cabinet illuminated with artificial white light lacking UV-B to growth under natural sunlight. The genes-associated with flavonoid metabolism as well as UV resistance locus 8 (OsUVR8), phytochrome interacting factor-like 15-like (OsPIF3), pyridoxal 5'-phosphate synthase subunit PDX1.2 (OsPDX1.2), deoxyribodipyrimidine photolyase (OsPHR), and deoxyribodipyrimidine photolyase family protein-like (OsPHRL) exhibited lower expression levels, while higher expression levels of mitogen-activated protein kinase 5-like (OsMPK3), mitogen-activated protein kinase 13-like (OsMPK13), and transcription factor MYB4-like (OsMYB4) were observed in glp1 than in WT after transfer from a growth cabinet illuminated with artificial white light to growth under natural sunlight. Therefore, mutations in OsGLP1 resulted in rice plants more sensitive to UV-B and reduced expression of some genes for UV-B protection, suggesting that OsGLP1 is involved in acclimation to UV-B radiation.

Overexpression of OsIAAGLU reveals a role for IAA-glucose conjugation in modulating rice plant architecture.

KEY MESSAGE: OsIAAGLU could catalyze the reaction of IAA with glucose to generate IAA-glucose. Overexpression of OsIAAGLU in rice resulted in altered rice shoot architecture and root gravitropism. The distribution and levels of indole-3-acetic acid (IAA) within plant tissues are well known to play vital roles in plant growth and development. An important mechanism of regulating free IAA levels in monocots is formation of IAA ester conjugates. In this study, a cytosol-localized protein encoded by the rice gene of indole-3-acetic acid glucosyltransferase (OsIAAGLU) was found to catalyze the reaction of free IAA with glucose to generate IAA-glucose. Expression of OsIAAGLU could be induced by IAA and NAA. The number of tillers and leaf angle was significantly increased with a concomitant decrease in plant height and panicle length in the transgenic rice lines overexpressing OsIAAGLU compared to the wild-type (WT) plants. Phenotypes of iaaglu mutants constructed using the CRISPR/Cas9 system had no obvious differences with WT plants. Furthermore, overexpression of OsIAAGLU resulted in reduced sensitivity to IAA/NAA and altered gravitropic response of the roots in the transgenic plants. Free IAA contents in the leaves, root tips, and lamina joint of OsIAAGLU-overexpressing transgenic lines were lower than those of WT plants. These results support that OsIAAGLU could play a regulatory role in IAA homeostasis and rice architecture.

Phytotoxicity testing of diesel-contaminated water using Petunia grandiflora Juss. Mix F1 and Marigold-Nemo Mix (Tagetes patula L.).

Tagetes patula (marigold) and Petunia grandiflora (petunia) have been shown to exhibit potential in phytoremediation of environmental pollutants including heavy metals and textile dyes. To investigate their phytoremediation potential of diesel, it was necessary to evaluate diesel phytotoxicity of these two ornamental plants. Marigold and petunia seeds were incubated, for 10 and 15 days, respectively, in deionised water contaminated with 0 to 4%, v/v, diesel in Petri dishes in a growth room with continuous lighting at 25 °C. It was found that as far as seed germination was concerned, petunia was less sensitive than marigold to 4% diesel in water. In contrast, petunia exhibited poorer seedling root growth than marigold in the presence of diesel contamination. This finding of differential sensitivity of these two ornamental plants to diesel-contaminated water during germination and seedling growth has not been reported before. Therefore, the implications of phytotoxicity evaluation and comparison between different species or genotypes of plants at both seed germination and postgermination seedling growth should both be taken into consideration in screening tolerant plants for phytoremediation.

Influence of toxic diesel fuel on Petunia grandiflora calli and after plant regeneration.

The toxic effects of diesel fuel on whole plants have been reported before, but little is known about the toxic effect of diesel fuel on callus cultures. This knowledge is a pre-requisite for exploring the possibility of using a sub-lethal diesel concentration as an agent for in vitro cell line selection to obtain novel somaclonal variants resistant to diesel toxicity. These novel variants could be useful for the phytoremediation of diesel-contaminated soil. Here, a callus induction medium [Murashige and Skoog medium supplemented with 1.8 µM of naphthlene-1-acetic acid (NAA) and 6.6 µM of 6-benzyladenine (BA)] was found to induce 85% of Petunia grandiflora leaf explants to form light green calli. Since it was not possible to include diesel in aseptic culture, the P. grandiflora calli were exposed to diesel under non-aseptic conditions. It was found that the calli did not exhibit any sign of necrosis immediately after up to 9 min of diesel exposure. The diesel-treated calli were subsequently subcultured successfully on the callus induction medium using the proliferating, non-necrotic cells. Transverse sections of the control and diesel-treated calli after 2 weeks of culture revealed that the control calli exhibited more small meristematic cells while diesel-treated calli exhibited larger, empty-looking parenchyma cells. Moreover, it was possible to induce, though at a low frequency (< 15%), shoot formation in the control calli and those derived from the diesel treatment on the Murashige and Skoog medium supplemented with 1.1 µM of indole-3-acetic acid (IAA) and 13.3 µM of BA. Under glasshouse conditions, the shoots regenerated from the calli derived from the diesel treatment exhibited higher biomass than those from the control calli and P. grandiflora seedlings when grown in a potting mix spiked with 0%, 2% and 7% diesel. Taken together, these results suggest that up to 9 min of diesel exposure of P. grandiflora calli was sub-lethal.

Short-Term Storability of Alginate-Encapsulated Persian Violet Microshoots for Germplasm Exchange.

Microshoots have been widely used for micropropagation. It may be necessary to store microshoots for a short period of time, for example in germplasm exchange needing transport to other research groups. Here, we investigated the short-term storability of alginate-encapsulated Persian violet (Exacum affine Balf. f. ex Regel) microshoots at 4 °C and 25 °C. After storage, the encapsulated microshoots were sown on basal Murashige and Skoog medium for germination and viability determination using tetrazolium chloride staining. The results showed that one or five microshoots encapsulated with a single alginate layer could be stored at 4 °C for up to 30 days, while the percentages of germination and viability of the microshoots encapsulated with two layers of alginate were greatly reduced upon storage. This is the first report on the storability of alginate-encapsulated multiple microshoots, which could be a more efficient way to encapsulate microshoots used for short-term cold storage.

The protective effect of sodium nitroprusside (SNP) treatment on Arabidopsis thaliana seedlings exposed to toxic level of Pb is not linked to avoidance of Pb uptake.

The effects of 100 µM Pb(NO3)2 on 7-day-old Arabidopsis thaliana seedlings grown from seeds pretreated with a nitric oxide donor, sodium nitroprusside (SNP), or a nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were investigated. Physiological stress induced by Pb (reduced root growth) was less evident in seedlings grown from seeds pretreated with SNP. However, SNP pretreatment of seeds did not affect Pb accumulation in the seedlings. Pb exposure caused oxidative stress by elevating hydrogen peroxide (H2O2) and lipid hydroperoxide contents of the seedlings. SNP pretreatment of seeds counteracted Pb toxicity by reducing the H2O2 and lipid hydroperoxide contents of Pb-exposed seedlings. Additionally, Pb-induced rises in antioxidative enzyme activities were reversed by SNP pretreatment of seeds. It was concluded that the ameliorating effects of SNP pretreatment were associated with the release of nitric oxide because cPTIO reversed these effects of SNP pretreatment of seeds and more importantly SNP pretreatment did not trigger an avoidance mechanism.

Improved rapid detection of trypsin isoinhibitors using non-denaturing polyacrylamide gels with immobilised azoalbumin.

INTRODUCTION: Proteinaceous inhibitors of animal trypsin occur naturally as isoforms in seeds and some are of interest as antinutritional or anti-pest agents. OBJECTIVE: To establish a simplified electrophorectic, in-gel method for rapid and direct detection of trypsin isoinhibitors present in crude plant extracts that are particularly suitable for many studies including rapid evaluation of cultivars. METHODOLOGY: Azoalbumin (3%, w/v) is immobilised in 7.5% polyacrylamide gels before electrophoresis under non-denaturing conditions. RESULTS: This improved method eliminates the need for both time-consuming and labourious staining and destaining or renaturation steps. CONCLUSION: Immobilised azoalbumin in polyacrylamide gels, run under non-denaturing electrophoresis conditions, can be used to assist rapid evaluation of trypsin isoinhibitors in numerous crude plant extracts.

Application of Modified Spent Mushroom Compost Biochar (SMCB/Fe) for Nitrate Removal from Aqueous Solution.

The public is already aware that nitrate pollution caused by nutrient runoff from farms is harmful to aquatic life and human health, and there is an urgent need for a product/technology to solve this problem. A biochar adsorbent was synthesized and used to remove nitrate ions from aqueous media based on spent mushroom compost (SMC), pre-treated with iron (III) chloride hexahydrate and pyrolyzed at 600 °C. The surface properties and morphology of SMCB/Fe were investigated using Fourier transform-infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The effect of main parameters such as the adsorbent dosages, pH of the solutions, contact times, and ion concentrations on the efficiency of nitrate removal was investigated. The validity of the experimental method was examined by the isothermal adsorption and kinetic adsorption models. The nitrate sorption kinetics were found to follow the pseudo-second-order model, with a higher determination coefficient (0.99) than the pseudo-first-order (0.86). The results showed that the maximum percentage of nitrate adsorption was achieved at equilibrium pH 5-7, after 120 min of contact time, and with an adsorbent dose of 2 g L-1. The highest nitrate adsorption capacity of the modified adsorbent was 19.88 mg g-1.

Diet factors responsible for the change of the glucose oxidase activity in labial salivary glands of Helicoverpa armigera.

We investigated the change of the glucose oxidase (GOX) activity in labial salivary glands of Helicoverpa armigera larvae fed with the artificial diet or host plant tobacco and the major factors responsible for such a change. Throughout larval development, the labial salivary GOX activities in caterpillars reared on the artificial diet were remarkably higher than those fed with the plant. After fifth-instar plant-fed caterpillars were transferred to the artificial diet, their labial salivary GOX activity increased quickly, which was closely correlated with the time spent feeding on the artificial diet. The total sugar content of the artificial diet was 68 times higher than that of the tobacco leaves. We hypothesized that sugars and secondary metabolites are the possible causes of induction of GOX activity. When fifth-instar caterpillars were fed with tobacco leaves coated with glucose or sucrose, their labial salivary GOX activity was significantly higher than those fed with leaves without sugar coating. Following native PAGE, 1 single band of the labial salivary GOX was observed in all the caterpillars fed with different diets, implying that only the activity of the isoenzyme was changed in response to different diets. Furthermore, the labial salivary GOX activity was determined after caterpillars were fed with artificial diets containing chlorogenic acid, rutin, and quercetin. The results showed that all these phenolic compounds had no effect on the GOX activity. We conclude that sugar in diets was a major factor influencing the labial salivary GOX activity of the larvae. Arch. Insect Biochem. Physiol. 2008.

Minocycline protects photoreceptors from light and oxidative stress in primary bovine retinal cell culture.

PURPOSE: To determine whether minocycline, a compound known to protect the retina against light-induced damage in rodent models, and its structurally related analogues would protect photoreceptor cells in primary bovine retinal cell culture against light and oxidative stress. METHODS: Minocycline and its analogues were tested in primary retinal cell culture to see whether they would inhibit light or oxidative stress-induced cell death. Primary cell cultures composed of photoreceptors, bipolar cells, and glial cells were prepared from bovine retinas. The extent of cell death induced by light or oxidative stress was assessed by using Sytox Green (Invitrogen-Molecular Probes, Eugene, OR) a nucleic acid dye uptake assay. Differential protection of photoreceptor cells from stress were examined using immunocytochemistry. RESULTS: Minocycline and methacycline were cytoprotective against light- or oxidative stress-induced damage of bovine primary photoreceptors in culture with an EC(50) < 10 microM. In contrast, structurally related analogues such as demeclocycline, meclocycline, and doxycycline were phototoxic at >3 to >10 microM. Though demeclocycline was found to be phototoxic, it was cytoprotective (EC(50) = 5 microM) against oxidative stress in the absence of exposure to light. CONCLUSIONS: The protective action of minocycline against light-induced damage in the cell-based assays agrees with earlier reports in animal models and suggests that the in vitro assay using bovine primary retinal cell culture is a suitable model for evaluating compounds for retinal protection. Cellular protection or toxicity produced by structurally related compounds show that minor structural modifications can alter the function of minocycline and lead to potent retinal protective compounds.

Novel potato plants with enhanced cadmium resistance and antioxidative defence generated after in vitro cell line selection.

It is of interest to apply plant tissue culture to generate plants resistant to toxic effects of cadmium (Cd) on plant growth. Callus cultures were initiated from leaf explants of micropropagated potato plantlets (Solanum tuberosum L., cv. Iwa) for in vitro selection comprising 18 different Cd treatments varying in Cd exposure timing and duration. Plantlets regenerated from two different lines of Cd-selected calli, L9 and L11, were found to exhibit enhanced resistance to 218 µM Cd compared to control (source plantlets for leaf explants used to initiate callus cultures for Cd resistance). In response to 218 µM Cd, L11 plantlets had lower levels of lipid peroxidation and hydrogen peroxide than control and L9 plantlets. In addition, antioxidative enzyme activities in L11 were generally higher than control. L11 also had a higher level of proline than control.

Insights into Resistance to Fe Deficiency Stress from a Comparative Study of In Vitro-Selected Novel Fe-Efficient and Fe-Inefficient Potato Plants.

Iron (Fe) deficiency induces chlorosis (IDC) in plants and can result in reduced plant productivity. Therefore, development of Fe-efficient plants is of great interest. To gain a better understanding of the physiology of Fe-efficient plants, putative novel plant variants were regenerated from potato (Solanum tubersosum L. var. 'Iwa') callus cultures selected under Fe deficient or low Fe supply (0-5 µM Fe). Based on visual chlorosis rating (VCR), 23% of callus-derived regenerants were classified as Fe-efficient (EF) and 77% as Fe-inefficient (IFN) plant lines when they were grown under Fe deficiency conditions. Stem height was found to be highly correlated with internodal distance, leaf and root lengths in the EF plant lines grown under Fe deficiency conditions. In addition, compared to the IFN plant lines and control parental biotype, the EF plants including the lines named A1, B2, and B9, exhibited enhanced formation of lateral roots and root hairs as well as increased expression of ferritin (fer3) in the leaf and iron-regulated transporter (irt1) in the root. These morphological adaptations and changes in expression the fer3 and irt1 genes of the selected EF potato lines suggest that they are associated with resistance to low Fe supply stress.

Protective effect of crocin against blue light- and white light-mediated photoreceptor cell death in bovine and primate retinal primary cell culture.

PURPOSE: The present study was performed to investigate the effect of crocin on blue light- and white light-induced rod and cone death in primary retinal cell cultures. METHODS: Primary retinal cell cultures were prepared from primate and bovine retinas. Fifteen-day-old cultures were exposed to blue actinic light or to white fluorescent light for 24 hours. Cultures were treated by the addition of different concentrations of crocin for 24 hours before light exposure or for 8 hours after light exposure. Cultures kept in the dark were used as controls. Green nucleic acid stain assay was used to evaluate cell death. Rods and cones were immunolabeled with specific antibodies and counted. TUNEL labeling was used to detect fragmented DNA in fixed cells after light exposure. RESULTS: Primary retinal cell cultures contained a mixture of retinal cells enriched in photoreceptors, bipolar cells, and Müller cells. Twenty-four-hour exposure to blue and white light induced death in 70% to 80% of the photoreceptors in bovine and primate retinal cell cultures. Crocin protected the photoreceptors against blue light- or white light-mediated damage in a concentration-dependent manner with an EC50 of approximately 30 microM. TUNEL assays confirmed that crocin protected photoreceptors from light damage. CONCLUSIONS: These results show that blue and white light selectively induce rod and cone cell death in an in vitro model. Crocin protects retinal photoreceptors against light-induced cell death.

Divergent biochemical and enzymatic properties of oxalate oxidase isoforms encoded by four similar genes in rice.

The biochemical and enzymatic properties of four highly similar rice oxalate oxidase proteins (OsOxO1-4) were compared after their purification from the leaves of transgenic plants each overexpressing the respective OsOxO1-4 genes. Although alignment of their amino acid sequences has revealed divergence mainly in the signal peptides and they catalyze the same enzymic (oxalate oxidase) reaction, divergence in apparent molecular mass, Km, optimum pH, stability and responses to inhibitors and activators was uncovered by biochemical characterization of the purified OsOxO1-4 proteins. The apparent molecular mass of oligomer OsOxO1 was found to be similar to that of OsOxO3 but lower than the other two. The molecular mass of the subunit of OsOxO1 was lower than that of OsOxO3. The Km value of OsOxO3 was higher than the other three which had similar Km. OsOxO1 and OsOxO4 possessed peak activity at pH 8.5 which was close to that at the optimum pH 4.0. The activity of OsOxO2 at pH 8.5 was only 65% of that at its optimum pH 3.5, while the activity of OsOxO3 did not vary much at pH 6-9 and was also much lower than that at its optimum pH 3. OsOxO2 and OsOxO3 still maintained all their activities after being heated at 70°C for 1h while OsOxO1 and OsOxO4 lost about 30% of their activities. Pyruvate and oxaloacetic acid inhibited the activity of OsOxO3 more strongly than the other three. Interestingly, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-biphosphate related to photosynthetic assimilation of triose phosphate greatly increased the activities of OsOxO3 and OsOxO4. In addition to the differences in the biochemical properties of the four OsOxO proteins, an intriguing finding is that the purified OsOxO1-4 exhibited substrate inhibition, which is a typical of the classical Michaelis-Menten enzyme kinetics exhibited by a majority of other enzymes.

Phospholipase C delta-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells.

BACKGROUND: The expression of the rodent phosphoinositide-specific phospholipase C delta-4 (PLCdelta4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCdelta4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCdelta4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCdelta4 on cell signaling and proliferation in this study. RESULTS: The cDNA for human PLCdelta4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCdelta4 selectively activates protein kinase C-phi and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCdelta4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCdelta4 are not reversible by siRNA. CONCLUSION: Overexpression or dysregulated expression of PLCdelta4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCdelta4 are not reversible, PLCdelta4 itself is not a suitable drug target, but enzymes in pathways activated by PLCdelta4 are potential therapeutic targets for oncogenic intervention.

Relationship between disease resistance and rice oxalate oxidases in transgenic rice.

Differential expression of rice oxalate oxidase genes (OsOxO1-4) in rice leaves (Oryza sativa L.) in response to biotic stress was assayed using RT-PCR. OsOxO4 was induced transiently at 12 h in plants inoculated with the pathogens of bacterial blight and that of the wounding control. Inoculation with the rice blast pathogen induced OsOxO2 expression compared to the mock spray control. Overexpressing OsOxO1 or OsOxO4 in rice resulted in elevated transcript levels of the respective transgene as well as OsOxO3 in leaves compared to that in untransformed wild type (WT). In a line of RNA-i transgenic rice plants (i-12), expression of all four OsOxO genes except that of OsOxO2 was severely inhibited. Oxalate oxidase (OxO, EC 1.2.3.4) activity in plants overexpressing OsOxO1 or OsOxO4 was substantially higher than that in WT and the RNA-i lines. It was found that transgenic rice plants with substantially higher OxO activity were not more resistant to rice blast and bacterial blight than WT. In contrast, some RNA-i lines with less OxO activity seemed to be more resistant to rice blast while some overexpressing lines were more susceptible to rice blast than WT. Therefore, OxO might not be a disease resistance factor in rice.

Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata.

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.

Characterization of activity of a potential food-grade leucine aminopeptidase from kiwifruit.

Aminopeptidase (AP) activity in ripe but firm fruit of Actinidia deliciosa was characterized using L-leucine-p-nitroanilide as a substrate. The enzyme activity was the highest under alkaline conditions and was thermolabile. EDTA, 1,10-phenanthroline, iodoacetamide, and Zn(2+) had inhibitory effect while a low concentration of dithiothreitol (DTT) had stimulatory effect on kiwifruit AP activity. However, DTT was not essential for the enzyme activity. The results obtained indicated that the kiwifruit AP was a thiol-dependent metalloprotease. Its activity was the highest in the seeds, followed by the core and pericarp tissues of the fruit. The elution profile of the AP activity from a DEAE-cellulose column suggested that there were at least two AP isozymes in kiwifruit: one unadsorbed and one adsorbed fractions. It is concluded that useful food-grade aminopeptidases from kiwifruit could be revealed using more specific substrates.

Lead chelation to immobilised Symphytum officinale L. (comfrey) root tannins.

Reported correlations between tannin level and metal accumulation within plant tissues suggest that metal-chelating tannins may help plants to tolerate toxic levels of heavy metal contaminants. This paper supports such correlations using a new method that demonstrated the ability of plant tannins to chelate heavy metals, and showed that the relative levels of tannins in tissues were quantitatively related to lead chelation in vitro. Using this in vitro metal chelation method, we showed that immobilised tannins prepared from lateral roots of Symphytum officinale L., that contained high tannin levels, chelated 3.5 times more lead than those from main roots with lower tannin levels. This trend was confirmed using increasing concentrations of tannins from a single root type, and using purified tannins (tannic acid) from Chinese gallnuts. This study presents a new, simple, and reliable method that demonstrates direct lead-tannin chelation. In relation to phytoremediation, it also suggests that plant roots with more 'built-in' tannins may advantageously accumulate more lead.