Chang-Hyo Goh

The Arabidopsis Dynamin-Like Proteins ADL1C and ADL1E Play a Critical Role in Mitochondrial Morphogenesis

Dynamin-related proteins are high molecular weight GTP binding proteins and have been implicated in various biological processes. Here, we report the functional characterization of two dynamin homologs in Arabidopsis, Arabidopsis dynamin-like 1C (ADL1C) and Arabidopsis dynamin-like 1E (ADL1E). ADL1C and ADL1E show a high degree of amino acid sequence similarity with members of the dynamin family. However, both proteins lack the C-terminal Pro-rich domain and the pleckstrin homology domain. Expression of the dominant-negative mutant ADL1C[K48E] in protoplasts obtained from leaf cells caused abnormal mitochondrial elongation. Also, a T-DNA insertion mutation at the ADL1E gene caused abnormal mitochondrial elongation that was rescued by the transient expression of ADL1C and ADL1E in protoplasts. In immunohistochemistry and in vivo targeting experiments in Arabidopsis protoplasts, ADL1C and ADL1E appeared as numerous speckles and the two proteins colocalized. These speckles were partially colocalized with F1-ATPase-γ:RFP, a mitochondrial marker, and ADL2b localized at the tip of mitochondria. These results suggest that ADL1C and ADL1E may play a critical role in mitochondrial fission in plant cells.

Loss of cytosolic fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth retardations in rice (Oryza sativa)

During photosynthesis, triose-phosphates (trioseP) exported from the chloroplast to the cytosol are converted to sucrose via cytosolic fructose-1,6-bisphosphatase (cFBPase). Expression analysis in rice suggests that OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day. The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation with reduced chlorophyll content, which results in plant death. Analysis of primary carbon metabolites revealed both significantly reduced levels of sucrose, glucose, fructose and starch in leaves of these mutants, and a high accumulation of sucrose to starch in leaves of rice plants. In the oscfbp1 mutants, products of glycolysis and the TCA cycle were significantly increased. A partitioning experiment of (14)C-labelled photoassimilates revealed altered carbon distributions including a slight increase in the insoluble fraction representing transitory starch, a significant decrease in the neutral fraction corresponding to soluble sugars and a high accumulation of phosphorylated intermediates and carboxylic acid fractions in the oscfbp1 mutants. These results indicate that the impaired synthesis of sucrose in rice cannot be sufficiently compensated for by the transitory starch-mediated pathways that have been found to facilitate plant growth in the equivalent Arabidopsis mutants.

Regulation of Dark-Induced Stomatal Closure in Arabidopsis Dynamin-Like Protein 1E (adl1e) Mutant Leaves

Arabidopsis thaliana dynamin-like protein 1E (ADL1E) is known to regulate mitochondrial elongation. The adl1e mutant has no morphological phenotype, and the growth and photosynthetic activity of the mutant are similar to those of the wild type. Leaf O2 uptake, which is supported by mitochondrial activity in the dark, is increased 1.7-fold by mutation in adl1e gene. The ATP content in the dark of guard and mesophyll cell protoplasts (GCPs and MCPs, respectively) was 2.5- to 4-fold higher in GCPs of the mutant and the wild type, and increased upon the addition of glucose in both genotypes. Oligomycin, an inhibitor of mitochondrial ATPase, suppressed ATP synthesis in both GCPs and MCPS isolated from adl1e plants, indicating that mutant had higher mitochondrial activity. The stomatal apertures of mutant and wild-type plants were then analyzed in vitro. In the light, the stomata of both genotypes showed similar patterns of opening. However, in the dark response, the stomata of the adl1e mutant closed faster than did those of the wild type. Oligomycin severely inhibited dark-induced stomatal closure in both cell types. The results suggest that stomatal closure in the dark is governed by cytosolic ATP concentration, which is stimulated by mitochondrial activity.