Programmed cell death induced by high levels of cytokinin in Arabidopsis cultured cells is mediated by the cytokinin receptor CRE1/AHK4

High levels of cytokinins (CKs) induce programmed cell death (PCD) both in animals and plant cells. High levels of the CK benzylaminopurine (BA) induce PCD in cultured cells of Arabidopsis thaliana by accelerating a senescence process characterized by DNA laddering and expression of a specific senescence marker. In this report, the question has been addressed whether members of the small family of Arabidopsis CK receptors (AHK2, AHK3, CRE1/AHK4) are required for BA-induced PCD. In this respect, suspension cell cultures were produced from selected receptor mutants. Cell growth and proliferation of all receptor mutant and wild-type cell cultures were similar, showing that the CK receptors are not required for these processes in cultured cells. The analysis of CK metabolites instead revealed differences between wild-type and receptor mutant lines, and indicated that all three receptors are redundantly involved in the regulation of the steady-state levels of isopentenyladenine- and trans-zeatin-type CKs. By contrast, the levels of cis-zeatin-type CKs were controlled mainly by AHK2 and AHK3. To study the role of CK receptors in the BA-induced PCD pathway, cultured cells were analysed for their behaviour in the presence of high levels of BA. The results show that CRE1/AHK4, the strongest expressed CK receptor gene of this family in cultured cells, is required for PCD, thus linking this process to the known CK signalling pathway

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T0 generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T1 seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T1 generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use

Proteomic changes associated with expression of a gene (ipt) controlling cytokinin synthesis for improving heat tolerance in a perennial grass species

Cytokinins (CKs) are known to regulate leaf senescence and affect heat tolerance, but mechanisms underlying CK regulation of heat tolerance are not well understood. A comprehensive proteomic study was conducted to identify proteins altered by the expression of the adenine isopentenyl transferase (ipt) gene controlling CK synthesis and associated with heat tolerance in transgenic plants for a C3 perennial grass species, Agrostis stolonifera. Transgenic plants with two different inducible promoters (SAG12 and HSP18) and a null transformant (NT) containing the vector without ipt were exposed to 20 °C (control) or 35 °C (heat stress) in growth chambers. Two-dimensional electrophoresis and mass spectrometry analysis were performed to identify protein changes in leaves and roots in response to ipt expression under heat stress. Transformation with ipt resulted in protein changes in leaves and roots involved in multiple functions, particularly in energy metabolism, protein destination and storage, and stress defence. The abundance levels of six leaf proteins (enolase, oxygen-evolving enhancer protein 2, putative oxygen-evolving complex, Rubisco small subunit, Hsp90, and glycolate oxidase) and nine root proteins (Fd-GOGAT, nucleotide-sugar dehydratase, NAD-dependent isocitrate dehydrogenase, ferredoxin-NADP reductase precursor, putative heterogeneous nuclear ribonucleoprotein A2, ascorbate peroxidase, dDTP-glucose 4–6-dehydratases-like protein, and two unknown proteins) were maintained or increased in at least one ipt transgenic line under heat stress. The diversity of proteins altered in transgenic plants in response to heat stress suggests a regulatory role for CKs in various metabolic pathways associated with heat tolerance in C3 perennial grass species

Treating the acute stroke patient as an emergency: current practices and future opportunities

Developments in acute stroke therapy have followed advances in the understanding of the evolving pathophysiology in both ischaemic stroke and intracerebral haemorrhage (ICH). In ischaemic stroke, rapid reperfusion of the ischaemic penumbra with thrombolysis within 3 h of symptom onset is of proven benefit, but few patients currently receive therapy, mainly due to the short-time window and lack of stroke expertise. In ICH, a recent study indicated that a haemostatic agent can limit ongoing bleeding and improve outcomes when administered within 4 h of stroke onset. These advances in acute stroke therapy underlie the concept that ‘time is brain’ and that urgent intervention can limit cerebral damage. Neuroprotective therapy could offer the prospect of a greater proportion of stroke patients receiving treatment, potentially before imaging and even in the ambulance setting. Virtually all stroke patients would benefit from receiving multidisciplinary care in acute stroke units