Higher plant cells contain several distinct organelles that play vital roles in cellular physiology. During proliferation and differentiation of the cells, the organelles often undergo dynamic changes. The biogenesis of new organelles may occur, existing organelles may undergo a transformation of function, while other organelles may degenerate. Because the dynamic transformation of organellar function (differentiation of organelles) is responsible for flexibility of differentiation events in higher plant cells, the elucidation of regulatory mechanisms underlying organelle transformation are currently studied in this division.

I. Reversible transformation of plant peroxisomes

Dramatic metabolic changes which underlie the shift from heterotrophic to autotrophic growth occur in greening of seed germination. Accompanying these metabolic changes, many constitutive organelles are functionally transformed. Etioplasts differentiate into chloroplasts and mitochondria acquire the ability to oxidize glycine. Glyoxysomes which are microbodies engaged in the degradation of reserve oil via b-oxidation and the glyoxylate cycle, are transformed into leaf peroxisomes that function in several crucial steps of photorespiration. After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse transition of leaf peroxisomes to glyoxysomes occurs during senescence. The functional transformation between glyoxysomes and leaf peroxisomes is controlled by gene expression, alternative splicing, protein translocation and protein deg-radation. We now engage in proteomic and transcriptomic analyses of the reversible peroxisomal transition in Arabidopsis cotyledons.

II. Transcriptomics and proteomics of plant peroxisomes

Enzymes localized in plant peroxisomes are synthesized in the cytosol, and function after their post-translational transport into peroxisomes. Almost all of the peroxisomal matrix proteins are known to contain one of two targeting signals (PTS1 and PTS2) within the molecules. PTS1 is a unique tripeptide sequence found in carboxyl terminus of the mature proteins. The permissible combinations of amino acids for PTS1 in plant cells are [C/A/S/P]-[K/R]-[I/L/M]. In contrast, PTS2 is involved in a cleavable amino terminal presequence of peroxisomal proteins that are synthesized as precursor protein with larger molecular mass. PTS2 consists of a consensus sequence [R]-[L/Q/I]-X5-[H]-[L].

We identified 256 gene candidates of PTS1- and PTS2-containing proteins and other 30 genes of non-PTS-containing proteins from Arabidopsis genome. Custom-made DNA microarray covering all these genes was used to investigate expression profiles of the peroxisomal genes in various organs. Statistical analyses revealed that the peroxisomal genes could be divided into five groups in terms of their transcription. One group showed ubiquitous expression in all organs examined, while the other four were classified as showing organ-specific expression in seedlings, cotyledons, roots and in both cotyledons and leaves. These data proposed more detailed description of differentiation of plant peroxisomes (Fig. 1).

Fig. 1 Novel nomenclature of plant peroxisomes identified by peroxisomal gene-specific transcriptomics.

In parallel, we made two-dimensional protein map of glyoxysomes and leaf peroxisomes isolated from Arabidopsis. Peptide MS fingerprinting analyses allowed us to identify novel proteins exists in either glyoxysomes or leaf peroxisomes. Some of these proteins contain no obvious PTS1 and PTS2. Of these, we characterized GPK1 as a novel protein kinase in glyoxysomes.

III. Involvement of the same dynamin molecule in peroxisomal and mitochondrial division in higher plants

To better understand peroxisome biogenesis, we mutagenized seeds of transgenic Arabidopsis, GFP-PTS1, in which peroxisomes with normal size and number can be visualized with GFP, and screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants) based on the different pattern of GFP. The apm mutants were classified into four classes. These were mutants with (1) long peroxisomes, (2) giant peroxisomes, (3) GFP fluorescence in the cytosol as well as in peroxisomes, and (4) other distributions of GFP.

Fig. 2 Phenotype of apm1 mutant and subcellular localization of APM1/DRP3A protein. (A) and (B) show GFP-labelled peroxisomes in GFP-PTS1 as a parent plant (A) and apm1 mutant (B). (C) and (D) represent electron microscopic observation of leaf cells in GFP-PTS1 (C) and apm1 mutants (D). Mt: mitochondrion, Ch: chloroplast. (E) and (F) show the co-localization of DRP3A protein with peroxisomes (E) and mitochondria (F). Spherical spots in green indicate DRP3A proteins. Red signals show peroxisomes (E) and mitochondria (F), respectively. Arrow heads represent the sites of interaction with DRP3A proteins. Each bar indicates 50 µm for (A) and (B), 1 µm for (C) and (D), and 10 µm for (E) and (F).

In one of these mutants, apm1, the peroxisomes are long and reduced in number, apparently as a result of inhibition of division (Fig. 2). APM1 gene encodes DRP3A (Dynamin related protein 3A). Interestingly, mutations in APM1/ADL2A also caused aberrant morphology of mitochondria. The growth of Arabidopsis, which requires the cooperation of various organelles including peroxisomes and mitochondria, is repressed in apm1, indicating that the changes of morphology of peroxisomes and mitochondria reduce the efficiency of metabolism in these organelles. These findings indicate that the same dynamin molecule is involved in peroxisomal and mitochondrial division in higher plants.

IV. ER derived organelles for transport of proteins to vacuoles.

Precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors from endoplasmic reticulum (ER) to protein storage vacuoles in maturing pumpkin seeds. PAC vesicles had diameters of 300 to 400 nm, are derived from ER and contained an electron-dense core of storage proteins. PV72, a type I integral membrane protein was found on the membrane of the PAC vesicles. PV72 has been shown to bind to the precursor of pumpkin 2S albumin, implying that PV72 functions as a vacuolar sorting receptor (VSR) for storage proteins in pumpkin seeds. Arabidopsis has seven homologue of PV72. The homologue closely related to PV72 was designated AtVSR1. We used a reverse-genetic approach to explore the function of AtVSR1. Two T-DNA insertion mutants (atvsr1-1 and atvsr1-2) missort storage proteins by secreting them from cells, and abnormally accumulate the precursors of storage proteins, together with the mature forms of these proteins in the seeds. These finding demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.

ER bodies are another ER-derived compartment specific to the Brassicaceae, including Arabidopsis. ER bodies are rod-shaped structures (5 µm long and 0.5 µm wide) that is surrounded by ribosomes. ER bodies can be visualized in transgenic plants of Arabidopsis (GFP-h) expressing green fluorescent protein fused with an ER retention signal (GFP-HDEL). ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. nai1 is an Arabidopsis mutant in which ER bodies were hardly detected in whole plants. Analysis of the nai1 mutant reveales that a b-glucosidase with an ER-retention signal (KDEL), called PYK10, is the main component of ER bodies. The putative biological function of PYK10 and the inducibility of ER bodies in rosette leaves by wound stress suggest that the ER body functions in the defense against herbivores.

V. Maturation of vacuolar/lysosomal proteins by vacuolar processing enzyme in animal and plant cells.

Vacuolar processing enzyme (VPE) belongs to the cysteine protease family C13. This family is found in various eukaryote organisms including higher plants and animals. VPE was originally identified as an enzyme responsible for the processing and maturation of seed storage proteins in plants. VPE exhibit substrate specificity toward an asparagine residue, the amino acid well conserved at the P1 position in the processing sites of various vacuolar/lysosomal proteins. Plant VPE homologues were separated to two subfamilies: one seed type and the other vegetative type. We have identified one seed type (bVPE) and two vegetative type (aVPE and gVPE) VPE genes from Arabidopsis. We isolated six Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins. All mutants had a defect in the bVPE gene, indicating that bVPE is involved in the processing of storage proteins in vivo. We further generated various mutants lacking different VPE isoforms: aVPE, bVPE and/or gVPE. avpe-1/bvpe-3/gvpe-1 triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to bvpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (avpe-1 and gvpe-1) and double (avpe-1/gvpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative type VPEs, aVPE and gVPE, compensates for the deficiency in bVPE in bvpe mutant seeds.

To explore the physiological function of VPE in mammals, we generated and characterized VPE-deficient mice. VPE was abundantly expressed in kidney and localized in the late endosomes of the proximal tuble cells. Disruption of the VPE gene led to the enlargement of lysosomes in these cells in an age-dependent manner, which suggests that the materials to be degraded are being accumulated within the lysosomal compartments. The processing of the lysosomal proteases, cathepsins B, H, and L, from the single-chain forms into the two-chain forms was completely defected in the deficient mice. Thus, the VPE deficiency caused the accumulation of macromolecules in the lysosomes, highlighting a pivotal role of VPE in the endosomeal/lysosomal degradation system.

VI. Role of molecular chaperones on cell differentiation.

Molecular chaperones are cellular proteins that function in the folding and assembly of certain other polypeptides into oligomeric structures but that are not, themselves, components of the final oligomeric structure. To clarify the roles of molecular chaperones on cell differentiation, we have purified and characterized chaperonin and Hsp70s and analyzed their roles in the translocation of proteins into chloroplasts.
Previously, we also characterized a mitochondrial co-chaperonin (Cpn10), chloroplast co-chaperonins (Cpn20 and Cpn10) and a small heat shock protein from Arabidopsis. In 2003, we started to characterize HSP90s. Their evolutional and functional characterization is now under experiments.