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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.

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.

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.

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.

The highly conserved protein ubiquitin is encoded by five polyubiquitin genes in Arabidopsis thaliana ecotype Columbia that have been divided into two subtypes, the UBQ3/UBQ4 subtype and the UBQ10/UBQ11/UBQ14 subtype. Northern analysis using gene-specific oligonucleotides as hybridization probes and enzyme activity measurements from transgenic plants expressing beta-glucuronidase (GUS) under the control of individual polyubiquitin 5' flanking regions were used to determine the development and environmental regulation of polyubiquitin transcription and mRNA accumulation. Polyubiquitin mRNA levels within and between subtypes were independently modulated. UBQ3 mRNA levels were three-fold higher than UBQ4 mRNA levels in vegetative organs, but only two-thirds of the UBQ4 mRNA levels in flowers. UBQ3 mRNA was modulated by dark/light treatments, while mRNAs from UBQ and all members of the other subtype were unaffected. Similarly, within the UBQ10/UBQ11/UBQ14 subtype, UBQ11/UBQ14 mRNAs were modulated differently in seedlings after a two-hour heat-shock treatment. Among all the polyubiquitin genes, UBQ10 mRNA level was the most constant in all organs and environmental conditions examined. Transgenic plants transformed with a UBQ10 5' flanking region::GUS gene contained higher levels of GUS activity than transgenic plants expressing GUS under the control of UBQ3 5' flanking regions. In conclusion, the relative abundance of different Arabidopsis polyubiquitin mRNAs, even those produced from highly similar genes within a subtype, appears to be modulated independently in response to developmental and environmental cues.

The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, Ks value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.

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.