BackgroundPlant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.ResultsHere, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.ConclusionsWe show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.

Abscisic acid-, stress- and ripening (ASR) -induced proteins are plant-specific proteins whose expression is up-regulated under abiotic stresses or during fruit ripening. In this study, we characterized an ASR protein from plantain to explore its physiological roles under osmotic stress. The expression pattern of MpAsr gene shows that MpAsr gene changed little at the mRNA level, while the MpASR protein accumulates under osmotic treatment. Through bioinformatic-based predictions, circular dichroism spectrometry, and proteolysis and heat-stability assays, we determined that the MpASR protein is an intrinsically unstructured protein in solution. We demonstrated that the hydrophilic MpASR protein could protect L: -lactate dehydrogenase (L: -LDH) from cold-induced aggregation. Furthermore, heterologous expression of MpAsr in Escherichia coli and Arabidopsis enhanced the tolerance of transformants to osmotic stress. Transgenic 35S::MpAsr Arabidopsis seeds had a higher germination frequency than wild-type seeds under unfavorable conditions. At the physiological level, 35S::MpAsr Arabidopsis showed increased soluble sugars and decreased cell membrane damage under osmotic stress. Thus, our results suggest that the MpASR protein may act as an osmoprotectant and water-retaining molecule to help cell adjustment to water deficit caused by osmotic stress.

Asr (abscisic acid, stress, ripening induced) genes are typically upregulated by a wide range of factors, including drought, cold, salt, abscisic acid (ABA) and injury; in addition to plant responses to developmental and environmental signals. We isolated an Asr gene, MpAsr, from a suppression subtractive hybridization (SSH) cDNA library of cold induced plantain (Musa paradisiaca) leaves. MpAsr expression was upregulated in Fusarium oxysporum f. sp. cubense infected plantain leaves, peels and roots, suggesting that MpAsr plays a role in plantain pathogen response. In addition, a 581-bp putative promoter region of MpAsr was isolated via genome walking and cis-elements involved in abiotic stress and pathogen-related responses were detected in this same region. Furthermore, the MpAsr promoter demonstrated positive activity and inducibility in tobacco under F. oxysporum f. sp. cubense infection and ABA, cold, dehydration and high salt concentration treatments. Interestingly, transgenic Arabidopsis plants overexpressing MpAsr exhibited higher drought tolerance, but showed no significant decreased sensitivity to F. oxysporum f. sp. cubense. These results suggest that MpAsr might be involved in plant responses to both abiotic stress and pathogen attack.

Bupleurum scorzonerifolium Willd has been found to have a wide range of immunopharmacologic functions. We isolated an anti-UVB B. scorzonerifolium cell clone and found elevated level of polysaccharides. In this study, we investigated the ability of crude polysaccharide (CP) from the anti-UVB B. scorzonerifolium cell clone to inhibit UVBinduced photodamage using a human skin keratinocyte cell line, HaCaT. Cells were UVB irradiated and then incubated in presence of different concentrations of CP. MTT assay showed that the CP did not induce cytotoxic effect under 10 mg/mL and after UVB irradiation, CP can inhibit UVB-induced HaCaT cell death. Decreased reactive oxygen species and lipid peroxidation and increased superoxide dismutase activity showed that CP can act as a free radical scavenger. Furthermore, CP had a strong protective ability against UVB-induced DNA damage. These effects were compared to the crude polysaccharide (CP 0 ) from normal B. scorzonerifolium callus at concentration of 20 mg/mL. The portion of crude polysaccharide (CP) from the anti-UVB B. scorzonerifolium cell clone was more than 2.5-fold higher than crude polysaccharide (CP 0 ) from normal B. scorzonerifolium callus. Taken together, the protective mechanisms of crude polysaccharide from the anti-UVB B. scorzonerifolium cell clone against UVB-induced photodamage occur by the inhibition of UVB-induced reactive oxygen species production, lipid peroxidation and DNA damage.Keywords Anti-UVB Bupleurum scorzonerifolium cell clone Á Crude polysaccharide Á Oxidative stress Á Photodamage Á UVB

Copper is an essential nutrient, but it is toxic in excess. Here, we cloned and characterized a His-rich low molecular weight dehydrin from Musa paradisiaca, MpDhn12. Analysis by circular dichroism (CD) spectra and a thermal stability assay showed that MpDhn12 is an intrinsically disordered protein, and immobilized-metal affinity chromatography (IMAC) analysis revealed that MpDhn12 can bind Cu(2+) both in vitro and in vivo. Interestingly, MpDhn12 aggregated under excess Cu(2+) conditions, and the aggregation was reversible and impaired by histidine modification with diethylpyrocarbonate (DEPC), while the disordered structure of another dehydrin ERD14 (as a control) was not changed. Furthermore, MpDhn12 could complement the copper-sensitive phenotype of yeast mutant Δsod1. These results together suggested that MpDhn12 may take part in buffering copper levels through chelation and formation of aggregates in excess Cu(2+) conditions. To the best of our knowledge, it is the first report that a dehydrin interchanged between disordered and aggregated state triggered by copper.