No abstract

The cyanide-resistant alternative oxidase (AOX) is a homodimeric protein whose activity can be regulated by the oxidation/reduction state and by a-keto acids. To further clarify the role of AOX in the skunk cabbage, Symplocarpus renifolius, we have performed expression and functional analyses of the encoding gene. Among the various tissues in the skunk cabbage, SrAOX transcripts were found to be specifically expressed in the thermogenic spadix. Moreover, our data demonstrate that the SrAOX protein exists as a non-covalently associated dimer in the thermogenic spadix, and is more sensitive to pyruvate than to other carboxylic acids. Our results suggest that the pyruvatemediated modification of SrAOX activity plays a significant role in thermoregulation in the skunk cabbage.

Plastids, found in plants and some parasites, are of endosymbiotic origin. The best-characterized plastid is the plant cell chloroplast. Plastids provide essential metabolic and signaling functions, such as the photosynthetic process in chloroplasts. However, the role of plastids is not limited to production of metabolites. Plastids affect numerous aspects of plant growth and development through biogenesis, varying functional states and metabolic activities. Examples include, but are not limited to, embryogenesis, leaf development, gravitropism, temperature response and plant-microbe interactions. In this review, we summarize the versatile roles of plastids in plant growth and development.

Sex-dependent thermogenesis during reproductive organ development in the inflorescence is a characteristic feature of some of the protogynous arum species. One such plant, skunk cabbage (Symplocarpus renifolius), can produce massive heat during the female stage but not during the subsequent male stage in which the stamen completes development, the anthers dehisce, and pollen is released. Unlike other thermogenic species, skunk cabbage belongs to the bisexual flower group. Although recent studies have identified the spadix as the thermogenic organ, it remains unclear how individual tissues or intracellular structures are involved in thermogenesis. In this study, reproductive organ development and organelle biogenesis were examined during the transition from the female to the male stage. During the female stage, the stamens exhibit extensive structural changes including changes in organelle structure and density. They accumulate high levels of mitochondrial proteins, including possible thermogenic factors, alternative oxidase, and uncoupling protein. By contrast, the petals and pistils do not undergo extensive changes during the female stage. However, they contain a larger number of mitochondria than during the male stage in which they develop large cytoplasmic vacuoles. Comparison between female and male spadices suggests that mitochondrial number rather than their level of activity correlates with thermogenesis. Their spadices, even in the male, contain a larger amount of mitochondria that had greater oxygen consumption, compared with non-thermogenic plants. Taken together, our data suggest that the extensive maturation process in stamens produces massive heat through increased metabolic activities. The possible mechanisms by which petal and pistil metabolism may affect thermogenesis are also discussed.

In mammalian brown adipose tissue, uncoupling protein 1 (UCP1), an integral inner mitochondrial membrane protein, triggers a proton leak and converts the energy generated by the resulting electron flow into heat. Although the recent finding of plant UCPs in non-thermogenic tissues has questioned their involvement in thermogenesis, there are few studies of plant UCPs in thermogenic tissues. Therefore, in this work, two cloned UCP cDNAs, SrUCPA and SrUCPB, isolated from the thermogenic spadix of skunk cabbage, were analysed. SrUCPA, not SrUCPB, was identified as the major uncoupling protein, and it was found to be integrated into the inner mitochondrial membrane. Topological analyses indicate that the 1st and 2nd intra-matrix loops are sensitive to trypsin treatment, but the 3rd intra-matrix loop is resistant to it. Using spadix mitochondria, the uncoupling activity of SrUCPA was examined. Although SrUCPA transcripts were constitutively expressed in various tissues irrespective of thermogenic stage, the SrUCPA protein was detected only in the thermogenic tissue or stage. On the other hand, both gene and protein expression for another heat-generating protein, SrAOX, were increased specifically in the thermogenic tissue or stage. Quantitative immunoblot analysis revealed that SrUCPA was an abundant protein in spadix mitochondria, accounting for about 3% of the total mitochondrial protein in the spadix. The results suggest that specific co-expression of SrUCPA and SrAOX protein in the thermogenic tissue or stage, as well as the high expression of SrUCPA protein in spadix mitochondria, may play a role in thermogenesis of skunk cabbage.

Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM) of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

No abstract

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.