Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to ;31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9-and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H 2 O 2 -and a-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondriaenriched fractions prevented H 2 O 2 /clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.

SummaryGSK3/shaggy-like protein kinases have been shown to play diverse roles in development and signal transduction pathways in various organisms. An Arabidopsis homologue of GSK3/shaggy-like kinase, AtGSK1, has been shown to be involved in NaCl stress responses. In order to further clarify the role of AtGSK1 in NaCl stress responses in plants, we generated transgenic Arabidopsis plants that overexpressed AtGSK1 mRNA. These plants showed enhanced resistance to NaCl stress when assayed either as whole plants or by measurement of root growth on NaCl plates. In addition, AtGSK1 transgenic plants in the absence of NaCl stress showed phenotypic changes, such as accumulation of anthocyanin, that were similar to those observed in wild-type plants under NaCl stress. Transgenic plants accumulated 30±50% more Na + than did wild-type plants when subjected to NaCl stress, and Ca 2+ content was increased by 15±30% in the transgenic plants regardless of the NaCl stress level. Northern blotting revealed that AtGSK1 over-expression induced expression of the NaCl stress-responsive genes AtCP1, RD29A and CHS1 in the absence of NaCl stress. In addition, AtCBL1 and AtCP1 were superinduced in the NaCl-stressed transgenic plants. Taken together, these results suggest that AtGSK1 is involved in the signal transduction pathway(s) of NaCl stress responses in Arabidopsis.

SummaryCalpain, a calcium-dependent cysteine protease, plays an essential role in basic cellular processes in animal cells, including cell proliferation, apoptosis, and differentiation. NbDEK encodes the calpain homolog of N. benthamiana. In this study, virus-induced gene silencing (VIGS) of NbDEK resulted in arrested organ development and hyperplasia in all the major plant organs examined. The epidermal layers of the leaves and stems were covered with hyperproliferating cell masses, and stomata and trichome development was severely inhibited. During¯ower development, a single dome-like structure was grown from the¯ower meristem to generate a large cylinder-shaped¯ower lacking any¯oral organs. At the cellular level, cell division was sustained in tissues that were otherwise already differentiated, and cell differentiation was severely hampered. NbDEK is ubiquitously expressed in all the plant tissues examined. In the abnormal organs of the NbDEK VIGS lines, protein levels of D-type cyclins (CycD)2, CycD3, and proliferating cell nuclear antigen (PCNA) were greatly elevated, and transcription of E2F (E2 promoter binding factor), E2F-regulated genes, retinoblastoma (Rb), and KNOTTED1 (KN1)-type homeobox genes was also stimulated. These results suggest that phytocalpain is a key regulator of cell proliferation and differentiation during plant organogenesis, and that it acts partly by controlling the CycD/Rb pathway.

GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stresssensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.