Pyk2 downstream of G12/13 pathways regulates platelet shape change through RhoA/p160ROCK.

Rho/Rho-kinase downstream of G12/13 plays an important role in the regulation of calcium-independent platelet shape change. We have previously shown that proline-rich tyrosine kinase 2 (Pyk2) is activated downstream of G12/13 pathways. In this study, we evaluated the role of Pyk2 in G12/13-induced platelet shape change. We used low concentrations of YFLLRNP, a heptapeptide binding to protease-activated receptor 1 (PAR1), or PAR4-activating peptide AYPGKF in the presence of Gαq inhibitor YM254890 to selectively stimulate G12/13 pathways. We found that G12/13-induced platelet shape change was completely inhibited in the presence of Pyk2 inhibitors AG17 and TAT-Pyk2-CT, suggesting an important role of Pyk2 in platelet shape change. In addition, AYPGKF-induced shape change in Gq -/- platelets was completely inhibited in the presence of AG17 or RhoA/p160ROCK inhibitor Y27632, confirming the role of Pyk2 in RhoA-dependent shape change. Furthermore, AYPGKF-induced platelet aggregation and dense granule secretion were inhibited by blocking Pyk2 or RhoA. Finally, G12/13-induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation was inhibited by AG17, confirming that Pyk2 regulates RhoA/p160ROCK activation in platelets. These results demonstrate that Pyk2 downstream of G12/13 pathways regulates platelet shape change as well as platelet aggregation and dense granule secretion through the regulation of RhoA/p160ROCK.

Hymenobacter jeollabukensis sp. nov., isolated from soil.

A Gram-stain-negative, non-motile, rod-shaped, aerobic bacterial strain, designated 1-3-3-8T, was isolated from soil and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 1-3-3-8T belongs to the family Cytophagaceae of phylum Bacteroidetes and is most closely related to Hymenobacter paludis KBP-30T (96.8% similarity), Hymenobacter ocellatus Myx2105T (96.8%), Hymenobacter coalescens WW84T (95.6%), and Hymenobacter deserti ZLB-3T (95.4%). The G + C content of the genomic DNA of strain 1-3-3-8T was 63.6 mol%. The isolate contained C15:0 iso (28.4%), summed feature 4 (C17:1 anteiso B/C17:1 iso I; 18.9%), and C15:0 anteiso (17.6%) as major fatty acids, MK-7 as the predominant respiratory quinone, and sym-homospermidine as the predominant polyamine. The major polar lipids were phosphatidylethanolamine and an unidentified lipid. The phenotypic and chemotaxonomic data supported the affiliation of strain 1-3-3-8T with the genus Hymenobacter. The DNA-DNA relatedness between strain 1-3-3-8T and H. paludis KCTC 32237T and H. ocellatus DSM 11117T were 24.5 and 27.4% respectively, clearly showing that the isolate is not related to them at the species level. Overall, the novel strain could be differentiated from its phylogenetic neighbors on the basis of several phenotypic, genotypic, and chemotaxonomic features. Therefore, strain 1-3-3-8T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter jeollabukensis sp. nov. has been proposed. The type strain is 1-3-3-8T (= KCTC 52741T = JCM 32192T).

Hymenobacter terrigena sp. nov., isolated from soil.

A Gram-stain-negative, non-motile, non-spore-forming, rodshaped, aerobic bacterial strain, designated S1-2-2-5T, was isolated from the Jeollabuk-do province, Republic of Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-2-5T belonged to the family Cytophagaceae in phylum Bacteroidetes, and was most closely related to Hymenobacter terrae DG7AT (98.2%), Hymenobacter rubidus DG7BT (98.0%), Hymenobacter soli PB17T (97.7%), Hymenobacter daeguensis 16F3Y-2T (97.2%) and Hymenobacter saemangeumensis GSR0100T (97.0%). The G + C content of the genomic DNA of strain S1-2-2-5T was 59.4 mol%. The detection of menaquinone MK-7 as the predominant respiratory quinone, a fatty acid profile with summed feature 3 (C16:1ω7c/C16:1ω6c; 32.0%), C15:0 iso (19.0%), and C15:0 anteiso (15.0%) as the major components, and a polar lipid profile with phosphatidylethanolamine as the major component supported the affiliation of strain S1-2-2-5T to the genus Hymenobacter. The DNA-DNA relatedness between strain S1-2-2-5T and H. terrae KCTC 32554T, H. rubidus KCTC 32553T, H. soli KCTC 12607T, H. daeguensis KCTC 52537T, and H. saemangeumensis KACC 16452T were 49.5, 48.2, 34.1, 28.1, and 31.8% respectively, clearly showing that the isolate is not related to them at the species level. Strain S1-2-2-5T could be clearly differentiated from its closest neighbors on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain S1-2-2-5T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter terrigena sp. nov. is proposed. The type strain is S1-2-2-5T (= KCTC 52737T = JCM 32195T).

Synergistic Effect of Nano-Sliver with Sucrose on Extending Vase Life of the Carnation cv. Edun.

We investigated the effects of sucrose and nano-silver (NAg) on extending the vase life of cut carnation flowers "Edun". Sucrose (pulse treatment) suppressed ethylene production by downregulating the genes that code for its biosynthesis. Relative to the control, however, sucrose significantly promoted xylem blockage on cut stem surfaces and reduced relative fresh weight, antioxidant activity, and cysteine proteinase inhibitor gene (DcCPi) expression. Consequently, the sucrose-treated flowers had shorter vase lives than the control. In contrast, NAg suppressed ethylene production in the petal, prevented xylem blockage in the cut stem surface, and improved all the aforementioned parameters. Therefore, NAg increased flower longevity. The most effective treatment in terms of longevity extension and parameter improvement, however, was the combination of NAg and sucrose. These results suggest that sucrose can suppress ethylene production but does not necessarily extend the vase life of the flower cultivar. The role of NAg in increasing cut carnation longevity is mainly to inhibit xylem blockage rather than suppress ethylene production, and the combined effect of NAg and sucrose is most effective at prolonging cut carnation vase life, likely due to their synergetic effects on multiple modes of action.

Role of Nano-silver and the Bacterial Strain Enterobacter cloacae in Increasing Vase Life of Cut Carnation 'Omea'.

We investigated the role of nano-silver (NAg) and the bacterial strain Enterobacter cloacae in increasing the vase life of cut carnation flowers 'Omea.' NAg treatment extended vase life of the flowers by increasing relative fresh weight, antioxidant activities, and expression level of the cysteine proteinase inhibitor gene (DcCPi), and by suppressing bacterial blockage in stem segments, ethylene production and expression of ethylene biosynthesis genes and DcCP1 gene, compared with the control. Out of all the treatments, administration of 25 mg L-1 NAg gave the best results for all the analyzed parameters. Interestingly, application of E. cloacae also extended the vase life of cut flowers by 3 days in comparison with control flowers, and overall, showed better results than the control for all the analyzed parameters. Taken together, these results demonstrate the positive role of NAg and E. cloacae in increasing the longevity of cut carnation flowers, and indicate that this effect is brought about through multiple modes of action.

Overexpression of snapdragon Delila (Del) gene in tobacco enhances anthocyanin accumulation and abiotic stress tolerance.

BACKGROUND: Rosea1 (Ros1) and Delila (Del) co-expression controls anthocyanin accumulation in snapdragon flowers, while their overexpression in tomato strongly induces anthocyanin accumulation. However, little data exist on how Del expression alone influences anthocyanin accumulation. RESULTS: In tobacco (Nicotiana tabacum 'Xanthi'), Del expression enhanced leaf and flower anthocyanin production through regulating NtCHS, NtCHI, NtF3H, NtDFR, and NtANS transcript levels. Transgenic lines displayed different anthocyanin colors (e.g., pale red: T0-P, red: T0-R, and strong red: T0-S), resulting from varying levels of biosynthetic gene transcripts. Under salt stress, the T2 generation had higher total polyphenol content, radical (DPPH, ABTS) scavenging activities, antioxidant-related gene expression, as well as overall greater salt and drought tolerance than wild type (WT). CONCLUSION: We propose that Del overexpression elevates transcript levels of anthocyanin biosynthetic and antioxidant-related genes, leading to enhanced anthocyanin production and antioxidant activity. The resultant increase of anthocyanin and antioxidant activity improves abiotic stress tolerance.

MYB transcription factor isolated from Raphanus sativus enhances anthocyanin accumulation in chrysanthemum cultivars.

A MYB transcription factor gene, RsMYB1, from radish was introduced into the chrysanthemum cultivars 'Peach ND', 'Peach Red', and 'Vivid Scarlet' under the control of the cauliflower mosaic virus 35S promoter. Presence of RsMYB1 in transgenic lines was confirmed using polymerase chain reaction (PCR). Results of reverse-transcription-PCR analysis revealed that the expression of RsMYB1 was stable in all transgenic lines and could enhance the expression levels of three key biosynthetic genes (F3H, DFR, and ANS) involved in anthocyanin production. Accordingly, anthocyanin content was significantly higher in transgenic lines than in control of all the cultivars, although the increasement was not visually observed in any of the transgenic lines. Therefore, these results demonstrate that RsMYB1 has potential to enhance anthocyanin content in the chrysanthemums.

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum.

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1 mgL(-1) of 6-benzyladenine (BA) and 2 mgL(-1) of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.

Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato.

Deregulated expression of an Arabidopsis H⁺/Ca²âº antiporter (sCAX1) in agricultural crops increases total calcium (Ca²âº) but may result in yield losses due to Ca²âº deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²âº binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²âº content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²âº transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²âº within the plant matrix.

Expression of an Arabidopsis Ca2+/H+ antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation.

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100µM CdCl(2) for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation.

Expression of an Arabidopsis CAX2 variant in potato tubers increases calcium levels with no accumulation of manganese.

Previously, we made a chimeric Arabidopsis thaliana vacuolar transporter CAX2B [a variant of N-terminus truncated form of CAX2 (sCAX2) containing the "B" domain from CAX1] that has enhanced calcium (Ca(2+)) substrate specificity and lost the manganese (Mn(2+)) transport capability of sCAX2. Here, we demonstrate that potato (Solanum tuberosum L.) tubers expressing the CAX2B contain 50-65% more calcium (Ca(2+)) than wild-type tubers. Moreover, expression of CAX2B in potatoes did not show any significant increase of the four metals tested, particularly manganese (Mn(2+)). The CAX2B-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through the successive generations and showed no deleterious alternations on plant growth and development. These results demonstrate the enhanced substrate specificity of CAX2B in potato. Therefore, CAX2B can be a valuable tool for Ca(2+) nutrient enrichment of potatoes with reduced accumulation of undesirable metals.

Genetic manipulation for enhancing calcium content in potato tuber.

Increased calcium (Ca) in potatoes may increase the production rate by enhancing tuber quality and storability. Additionally, increased Ca levels in important agricultural crops may help ameliorate the incidence of osteoporosis. However, the capacity to alter Ca levels in potato tubers through genetic manipulations has not been previously addressed. Here we demonstrate that potato tubers expressing the Arabidopsis H+/Ca2+ transporter sCAX1 (N-terminal autoinhibitory domain truncated version of CAtion eXchanger 1) contain up to 3-fold more Ca than wild-type tubers. The increased Ca appears to be distributed throughout the tuber. The sCAX1-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through successive generations. The expression of sCAX1 does not appear to alter potato growth and development. Furthermore, increased Ca levels in sCAX1-expressing tubers do not appear to alter tuber morphology or yield. Given the preponderance of potato consumption worldwide, these transgenic plants may be a means of marginally increasing Ca intake levels in the population. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of potatoes.