Chih Wen Sun

Leaf-Specific Upregulation of Chloroplast Translocon Genes by a CCT Motif-Containing Protein, CIA2

Chloroplasts are a major destination of protein traffic within leaf cells. Protein import into chloroplasts is mediated by a set of translocon complexes at the chloroplast envelope. Current data indicate that the expression of translocon genes is regulated in a tissue-specific manner, possibly to accommodate the higher import demand of chloroplasts in leaves and the lower demand of plastids in other tissues. We have designed a transgene-based positive screen to isolate mutants disrupted in protein import into plastids. The first locus we isolated, CIA2, encodes a protein containing a motif conserved within the CCT family of transcription factors. Biochemical analysis indicates that CIA2 is responsible for specific upregulation of the translocon genes atToc33 and atToc75 in leaves. Identification of CIA2 provides new insights into the tissue-specific regulation of translocon gene expression.

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.

Characterization of Arabidopsis Glutamine Phosphoribosyl Pyrophosphate Amidotransferase-Deficient Mutants

Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.

CIA2 Coordinately Up-Regulates Protein Import and Synthesis in Leaf Chloroplasts

Plastid biogenesis and maintenance depend on the coordinated assembly of proteins imported from the cytosol with proteins translated within plastids. Chloroplasts in leaf cells have a greater need for protein import and protein synthesis than plastids in other organs due to the large amount of proteins required for photosynthesis. We previously reported that the Arabidopsis (Arabidopsis thaliana) transcription factor CIA2 specifically up-regulates leaf expression of genes encoding protein translocons Toc33 and Toc75, which are essential for protein import into chloroplasts. Protein import efficiency was therefore reduced in cia2 mutant chloroplasts. To further understand the function of CIA2, gene expression profiles of the wild type and a cia2 mutant were compared by microarray analysis. Interestingly, in addition to genes encoding protein translocon components, other genes down-regulated in cia2 almost exclusively encode chloroplast ribosomal proteins. Isolated cia2 mutant chloroplasts showed reduced translation efficiency and steady-state accumulation of plastid-encoded proteins. When CIA2 was ectopically expressed in roots, expression of both the protein translocon and ribosomal protein genes increased. Further analyses in vivo revealed that CIA2 up-regulated these genes by binding directly to their promoter regions. We propose that CIA2 is an important factor responsible for fulfilling the higher protein demands of leaf chloroplasts by coordinately increasing both protein import and protein translation efficiencies.

A model for the evolution of polyubiquitin genes from the study of Arabidopsis thaliana ecotypes

Polyubiquitin genes encode the highly conserved 76-amino acid protein ubiquitin that is covalently attached to substrate proteins targeting most for degradation. Polyubiquitin genes are characterized by the presence of tandem repeats of the 228 bp that encode a ubiquitin monomer. Five polyubiquitin genes UBQ3, UBQ4, UBQ10, UBQ11, and UBQ14, previously isolated from Arabidopsis thaliana ecotype Columbia [10] encode identical mature ubiquitin proteins, but differ in synonymous substitutions, nature of amino acids terminating the open reading frame, and in the number of ubiquitin repeats. The presence of these five genes in nine other Arabidopsis ecotypes was verified by polymerase chain reaction (PCR). Size differences in UBQ3 and UBQ11 amplified products from several ecotypes were observed, suggesting that alleles differ in ubiquitin repeat number. DNA sequence of UBQ11 alleles from each size class (ecotypes Be-0, Ler, and Rld-0) verified that PCR product size differences resulted from changes in the number of ubiquitin repeats. Nucleotide sequence between two UBQ11 alleles containing the same number of repeats was identical. Transcript size differences for UBQ3 and UBQ11 mRNAs between ecotypes Columbia and Landsberg indicated that repeat number changes did not inactivate these genes. Nucleotide sequence comparisons between UBQ11 repeats from different ecotypes suggest that first repeats are related to each other and last repeats are related to each other. We hypothesize that changes in UBQ11 ubiquitin repeat number occurred via the contraction and/or expansion of specific internal repeats or portions thereof by misalignment of alleles and recombination, most likely via unequal crossing-over events.

Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and microarray analysis

To gain a better understanding of gene expression in bamboo (Bambusa edulis Murno), we have used a combination of suppressive subtractive hybridization (SSH), microarray hybridization analysis, sequencing, and bioinformatics to identify bamboo genes differentially expressed in a bamboo albino mutant. Ten expressed sequence tags (ESTs) were found to be differentially expressed; these were isolated and sequenced. RT-PCR analysis of these ESTs supported the results of the microarray analysis. Six ESTs that were nucleus-encoded exhibited differential expression patterns in the green wild-type bamboo relative to the albino mutant. These genes (exception being the Rubisco small subunit) were non-photosynthesis-related genes. The development of a specific SSH cDNA library in which most of the chloroplast-encoded or photosynthesis-related genes had been subtracted proved to be useful for studying the function of non-photosynthesis-related genes in the albino bamboo mutants with aberrant chloroplast genome. The combined use of this SSH library with microarray analysis will provide a powerful analytical tool for future studies of the bamboo genome.

Postglacial range expansion and the role of ecological factors in driving adaptive evolution of Musa basjoo var. formosana

Genetic variation evolves during postglacial range expansion of a species and is important for adapting to varied environmental conditions. It is crucial for the future survival of a species. We investigate the nuclear DNA sequence variation to provide evidence of postglacial range expansion of Musa basjoo var. formosana, a wild banana species, and test for adaptive evolution of amplified fragment length polymorphic (AFLP) loci underlying local adaptation in association with environmental variables. Postglacial range expansion was suggested by phylogeographical analyses based on sequence variation of the second intron of copper zinc superoxide dismutase 2 gene. Two glacial refugia were inferred by the average FST parameter (mean FST of a population against the remaining populations). Using variation partitioning by redundancy analysis, we found a significant amount of explained AFLP variation attributed to environmental and spatially-structured environmental effects. By combining genome scan methods and multiple univariate logistic regression, four AFLP loci were found to be strongly associated with environmental variables, including temperature, precipitation, soil moisture, wet days, and surface coverage activity representing vegetation greenness. These environmental variables may have played various roles as ecological drivers for adaptive evolution of M. basjoo var. formosana during range expansion after the last glacial maximum.

Characterization of differential expression and leader intron function of Arabidopsis atTOC159 homologous genes by transgenic plants

BackgroundAccurate import of thousands of nuclear-encoded proteins is an important step in plastid biogenesis. However, the import machinery of cytosolic precursor proteins to plastids relies on the Toc and Tic (translocons on the outer envelope and inner envelope membrane of chloroplasts) complexes. Toc159 protein was identified in pea (Pisum sativum) as a major receptor for the precursor proteins. In Arabidopsis thaliana, four psToc159 homologs are identified, termed atToc159, atToc132, atToc120 and atToc90. The expression of these protein-encoding genes has to be properly regulated, because their gene products must be correctly integrated to appropriate apparatus to perform their functions.ResultsIn order to elucidate the regulatory mechanisms of atTOC159 homologous gene expression, transgenes containing various lengths of the upstream regulatory sequences of atTOC159/atTOC132/atTOC120/atTOC90 and GUS coding sequence were transferred to wild type Arabidopsis. In accordance with the analysis of GUS activity in these transgenic plants at various developmental stages, these homologous genes had distinct expression patterns. AtTOC159 and atTOC90 are preferentially expressed in above-ground tissues, such as cotyledons and leaves. In mature roots, atTOC159 and atTOC132 are expressed at higher levels, while atTOC120 and atTOC90 are expressed at the basal level. All four genes have increased expression level during flower and fruit development, particularly a remarkably high expression level of atTOC159 in later stage of fruit development. Furthermore, leader intron in the 5′ UTR induces the expression level of atTOC159 members in a tissue-specific manner. This is able to up-regulate the atTOC120 expression in roots/leaves/flowers, and the atTOC90 expression in cotyledons/leaves/anthers.ConclusionsThe differential expression of atTOC159 gene members is essential during plastid development, because proper atToc159 isoforms are required to import distinct proteins to the plastids of different tissues.