The zinc finger protein DHHC09 S-acylates the kinase STRK1 to regulate H2O2 homeostasis and promote salt tolerance in rice.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice.

Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.

The Receptor-Like Cytoplasmic Kinase STRK1 Phosphorylates and Activates CatC, Thereby Regulating H2O2 Homeostasis and Improving Salt Tolerance in Rice.

Salt stress can significantly affect plant growth and agricultural productivity. Receptor-like kinases (RLKs) are believed to play essential roles in plant growth, development, and responses to abiotic stresses. Here, we identify a receptor-like cytoplasmic kinase, salt tolerance receptor-like cytoplasmic kinase 1 (STRK1), from rice (Oryza sativa) that positively regulates salt and oxidative stress tolerance. Our results show that STRK1 anchors and interacts with CatC at the plasma membrane via palmitoylation. CatC is phosphorylated mainly at Tyr-210 and is activated by STRK1. The phosphorylation mimic form CatCY210D exhibits higher catalase activity both in vitro and in planta, and salt stress enhances STRK1-mediated tyrosine phosphorylation on CatC. Compared with wild-type plants, STRK1-overexpressing plants exhibited higher catalase activity and lower accumulation of H2O2 as well as higher tolerance to salt and oxidative stress. Our findings demonstrate that STRK1 improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H2O2 homeostasis. Moreover, overexpression of STRK1 in rice not only improved growth at the seedling stage but also markedly limited the grain yield loss under salt stress conditions. Together, these results offer an opportunity to improve rice grain yield under salt stress.

Overexpression of an NADP(H)-dependent glutamate dehydrogenase gene, TrGDH, from Trichurus improves nitrogen assimilation, growth status and grain weight per plant in rice.

As glutamate dehydrogenases (GDHs) of microorganisms usually have higher affinity for NH4 + than do those of higher plants, it is expected that ectopic expression of these GDHs can improve nitrogen assimilation in higher plants. Here, a novel NADP(H)-GDH gene (TrGDH) was isolated from the fungus Trichurus and introduced into rice (Oryza sativa L.). Investigation of kinetic properties in vitro showed that, compared with the rice GDH (OsGDH4), TrGDH exhibited higher affinity for NH4 + (K m = 1.48 ± 0.11 mM). Measurements of the NH4 + assimilation rate demonstrated that the NADP(H)-GDH activities of TrGDH transgenic lines were significantly higher than those of the controls. Hydroponic experiments revealed that the fresh weight, dry weight and nitrogen content significantly increased in the TrGDH transgenic lines. Field trials further demonstrated that the number of effective panicles, 1,000-grain weight and grain weight per plant of the transgenic lines were significantly higher than those of the controls, especially under low-nitrogen levels. Moreover, glutelin and prolamine were found to be markedly increased in seeds from the transgenic rice plants. These results sufficiently confirm that overexpression of TrGDH in rice can improve the growth status and grain weight per plant by enhancing nitrogen assimilation. Thus, TrGDH is a promising candidate gene for maintaining yields in crop plants via genetic engineering.

Stress signaling in response to polycyclic aromatic hydrocarbon exposure in Arabidopsis thaliana involves a nucleoside diphosphate kinase, NDPK-3.

MAIN CONCLUSION: The study is the first to reveal the proteomic response in plants to a single PAH stress, and indicates that NDPK3 is a positive regulator in the Arabidopsis response to phenanthrene stress. Polycyclic aromatic hydrocarbons (PAHs) are highly carcinogenic pollutants that are byproducts of carbon-based fuel combustion, and tend to persist in the environment for long periods of time. PAHs elicit complex, damaging responses in plants, and prior research at the physiological, biochemical, and transcriptional levels has indicated that reactive oxygen species (ROS) and oxidative stress play major roles in the PAH response. However, the proteomic response has remained largely unexplored. This study hypothesized that the proteomic response in Arabidopsis thaliana to phenanthrene, a model PAH, would include a strong oxidative stress signature, and would provide leads to potential signaling molecules involved. To explore that proteomic signature, we performed 2D-PAGE experiments and identified 30 proteins levels that were significantly altered including catalases (CAT), ascorbate peroxidase (APX), peroxiredoxins (POD), glutathione-S-transferase, and glutathione reductase. Also upregulated was nucleoside diphosphate kinase 3 (NDPK-3), a protein known to have metabolic and stress signaling functions. To address whether NDPK-3 functions upstream of the oxidative stress response, we measured levels of stress-responsive enzymes in NDPK-3 overexpressor, loss-of-function knockout, and wild-type plant lines. In the NDPK-3 overexpressor, the enzyme activities of APX, CAT, POD, as well as superoxide dismutase were all increased compared to wild type; in the NDPK-3 knockout line, these enzymes had reduced activity. This pattern occurred in untreated as well as phenanthrene-treated plants. These data support a model in which NDPK-3 is a positive regulator of the Arabidopsis stress response to PAHs.

Mutational analysis of Arabidopsis PP2CA2 involved in abscisic acid signal transduction.

The homozygous T-DNA mutant of the PP2CA2 gene in Arabidopsis thaliana was identified at DNA and RNA levels. The semi-quantitative RT-PCR analysis showed expression of PP2CA2 was induced by NaCl and ABA. When grown in presence of increasing concentration of exogenous ABA the pp2ca2 mutant showed a significant loss of ABA sensitivity in terms of seed germination, efficiency of post-germination growth and root growth. In presence of all ABA and NaCl concentrations tested the germination percentage of wild-type seeds was lower than that of mutant ppca2 seeds. Furthermore, in the presence of exogenous ABA, the pp2ca2 seeds showed higher germination percentages than wild-type at different stages of development and the pp2ca2 seedlings showed a reduced inhibition of root growth compared with wild-type plants. The above results indicated that the pp2ca2 was an ABA-hyposensitive mutant.

A calcium-dependent protein kinase is involved in plant hormone signal transduction in Arabidopsis.

A number of signal pathways have been found through which abundant calcium-stimulated protein kinase activity in plant is associated with calcium-dependent protein kinases (CDPKs) which act as the calcium sensors mediating numerous responses, including hormone signaling. Basing on previous studies, we made additional functional analysis of the gene AtCPK30 encoding a protein kinase in Arabidopsis. Results of semi-quantitative reverse transcription PCR (RT-PCR) analysis indicated that AtCPK30 was highly expressed in root and induced by ABA, IAA, 2,4-D, GA(3) and 6-BA treatment. The physiological roles of AtCPK30 were studied using a gain-of-function approach. Seedlings of AtCPK30 transgenic lines had longer primary roots than those plants of wild-type at the early stages. Interestingly, when these plants grew on MS lack of Ca(2+) including wild-type and transgenic lines, the roots of transgenic line were more sensitive to calcium, lack of Ca(2+) had less effect on roots of transgenic lines than those of wild-type. Treated with several plant hormones, such as ABA, IAA, GA(3) and 6-BA, the roots of seedlings of transgenic line developed abnormally because they were more sensitive to hormones. Furthermore, NPA relatively less inhibited emergency of lateral roots of transgenic line than those of the wild-type. Green fluorescent protein-CPK30 (GFP-CPK30) fusion protein studies revealed the localization of AtCPK30 to both cell wall and plasma membrane. These results suggest that AtCPK30 acts as the calcium sensor and involved in the hormone-signaling pathways.

Plant architecture and grain yield are regulated by the novel DHHC-type zinc finger protein genes in rice (Oryza sativa L.).

In many plants, architecture and grain yield are affected by both the environment and genetics. In rice, the tiller is a vital factor impacting plant architecture and regulated by many genes. In this study, we cloned a novel DHHC-type zinc finger protein gene Os02g0819100 and its alternative splice variant OsDHHC1 from the cDNA of rice (Oryza sativa L.), which regulate plant architecture by altering the tiller in rice. The tillers increased by about 40% when this type of DHHC-type zinc finger protein gene was over-expressed in Zhong Hua 11 (ZH11) rice plants. Moreover, the grain yield of transgenic rice increased approximately by 10% compared with wild-type ZH11. These findings provide an important genetic engineering approach for increasing rice yields.

Bioconjugated nanoparticle for DNA protection from ultrasound damage.

To research whether poly-L-lysine-starch nanoparticle (PLL-StNP) could protect DNA from ultrasound damage or not, a series experiments were carried out: plasmid DNA-PLL-StNP complexes were treated with ultrasound for diverse times; the electrophoresis result proved that DNA bound to the complexes all the same. To investigate whether the conjugated DNA was completely protected or not, cDNA fragments bound to PLL-StNP were treated with ultrasound, and matched molecular beacons (MBs) were added. The cDNA-MB-PLL-StNP complexes exhibited dramatically increasing fluorescence, and had the same intensity as that of those MBs that were hybridized with free cDNA fragments. After being treated by ultrasound, the pIRGFP plasmid DNA-PLL-StNP complexes were transferred into COS-7 cells mediated by ultrasound. Green fluorescence protein expressed in most of the cells. Those results indicated that PLL-StNP could completely protect DNA from ultrasound damage. Furthermore, the DNA kept the same function as untreated one.

Prediction of regulatory factor X1 binding sites in promoters of RNA-binding proteins genes in mouse brain.

BACKGROUND: There is an increasing interest in the role of regulatory factor X1 (RFX1) and RNA-binding proteins (RBPs) during neural development. However, there are few reports about their interaction.   METHODS: We extracted RNA and performed reverse transcription polymerase chain reaction (RT-PCR) to identify RFX1 expression in imprinting control region (ICR) mouse tissues, analyzed RFX1 domains and motif consensus by comparing public databases on the Internet, tested the motif consensus with affinity-capture and western blotting experiments with mouse brain tissue, and predicted the binding sites of RFX1 in promoter regions of mouse RBPs genes. RESULTS: The expression of RFX1 was higher in embryonic brain compared to embryonic kidney, heart, and liver, and its expression level was relatively stable and higher in mouse embryonic brain than neonatal brain. RFX1 had several domains, including domain A as an activation domain, DBD as a DNA binding domain, domain B and C which played an important role in dimerization, and domain D as dimerization domain. RFX1 had three different profiles motif consensus RFX1M00281, RFX1M00280, and RFX1 (EF-C) M00626. There were 79 RFX1 binding sites at the promoters of 65 of 323 RBPs genes. CONCLUSION: RFX1 as regulatory factor will have putative important regulating role in the expression of RBPs genes during embryonic development of mouse brain.

Over-expression of the AtGA2ox8 gene decreases the biomass accumulation and lignification in rapeseed (Brassica napus L.).

Gibberellin 2-oxidase (GA 2-oxidase) plays very important roles in plant growth and development. In this study, the AtGA2ox8 gene, derived from Arabidopsis (Arabidopsis thaliana), was transformed and over-expressed in rapeseed (Brassica napus L.) to assess the role of AtGA2ox8 in biomass accumulation and lignification in plants. The transgenic plants, identified by resistant selection, polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) analyses, and green fluorescence examination, showed growth retardation, flowering delay, and dwarf stature. The fresh weight and dry weight in transgenic lines were about 21% and 29% lower than those in wild type (WT), respectively, and the fresh to dry weight ratios were higher than that of WT. Quantitative measurements demonstrated that the lignin content in transgenic lines decreased by 10%-20%, and histochemical staining results also showed reduced lignification in transgenic lines. Quantitative real-time PCR analysis indicated that the transcript levels of lignin biosynthetic genes in transgenic lines were markedly decreased and were consistent with the reduced lignification. These results suggest that the reduced biomass accumulation and lignification in the AtGA2ox8 over-expression rapeseed might be due to altered lignin biosynthetic gene expression.

Blue-light-independent activity of Arabidopsis cryptochromes in the regulation of steady-state levels of protein and mRNA expression.

Cryptochromes are blue-light receptors that mediate blue-light inhibition of hypocotyl elongation and blue-light stimulation of floral initiation in Arabidopsis. In addition to their blue-light-dependent functions, cryptochromes are also involved in blue-light-independent regulation of the circadian clock, cotyledon unfolding, and hypocotyl inhibition. However, the molecular mechanism associated with the blue-light-independent function of cryptochromes remains unclear. We reported here a comparative proteomics study of the light regulation of protein expression. We showed that, as expected, the protein expression of many metabolic enzymes changed in response to both blue light and red light. Surprisingly, some light-regulated protein expression changes are impaired in the cry1cry2 mutant in both blue light and red light. This result suggests that, in addition to mediating blue-light-dependent regulation of protein expression, cryptochromes are also involved in the blue-light-independent regulation of gene expression. Consistent with this hypothesis, the cry1cry2 mutant exhibited reduced changes of mRNA expression in response to not only blue light, but also red light, although the cryptochrome effects on the red-light-dependent gene expression changes are generally less pronounced. These results support a hypothesis that, in addition to their blue-light-specific functions, cryptochromes also play roles in the control of gene expression mediated by the red/far-red-light receptor phytochromes.

Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator.

An amperometric biosensor has been developed for the determination of H(2)O(2) in plant samples. Horseradish peroxidase (HRP) is immobilized on a sandwiched nano-Au particle / m-phenylenediamine polymer film by glutaraldehyde cross-linking. The film is formulated on the carbon paste electrode (CPE) blended with ferrocene as an electron transfer mediator. On the low concentration range, the current response is related to the H(2)O(2) concentration linearly from 0 to 8x10(-6) M with a detection limit of 1.3x10(-7) M. On a wider concentration range of 8x10(-6) to 1.4x10(-4) M, the reciprocal of current response is linearly related to the reciprocal of H(2)O(2) concentration. The apparent Michaelis-Menten constant (K(m)(app)) was calculated to be 0.0334 mM. The sensor has been tested by determining H(2)O(2) concentration in plant leaf samples.