SummaryLike their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and ⁄ or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face.

Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis. We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10 -18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination.genome maintenance ͉ microhomology ͉ recombination

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response.

DNA polymerases play a central role in the process of DNA replication. Yet, the proteins in charge of the replication of plant organelle DNA have not been unambiguously identified. There are however many indications that a family of proteins homologous to bacterial DNA polymerase I (PolI) is implicated in organelle DNA replication. Here, we have isolated mutant lines of the PolIA and PolIB genes of Arabidopsis (Arabidopsis thaliana) to test this hypothesis. We find that mutation of both genes is lethal, thus confirming an essential and redundant role for these two proteins. However, the mutation of a single gene is sufficient to cause a reduction in the levels of DNA in both mitochondria and plastids. We also demonstrate that polIb, but not polIa mutant lines, are hypersensitive to ciprofloxacin, a small molecule that specifically induces DNA double-strand breaks in plant organelles, suggesting a function for PolIB in DNA repair. In agreement with this result, a cross between polIb and a plastid Whirly mutant line yielded plants with high levels of DNA rearrangements and severe growth defects, indicating impairments in plastid DNA repair pathways. Taken together, this work provides further evidences for the involvement of the plant PolI-like genes in organelle DNA replication and suggests an additional role for PolIB in DNA repair.

The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the a-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases.Tryptophan decarboxylase (TDC; EC 4.1.1.28, formerly EC 4.1.1.27) catalyzes the conversion of L-tryptophan to tryptamine. This enzyme has been detected in numerous plant systems, and it has been suggested that its primary role is to supply possible precursors for auxin biosynthesis (1-3). In the Gramineae, TDC catalyzes the synthesis ofprecursors for the protoalkaloids, which have considerable physiological activity in higher animals (4). Tryptophan-derived tryptamines are also precursors of the tricyclic 8-carboline alkaloids formed by condensation with a one-or two-carbon moiety (5). In periwinkle (Catharanthus roseus), TDC produces tryptamine for biosynthesis of two commercially important antineoplastic monoterpenoid indole alkaloids, vinblastine and vincristine (6).The TDC from C. roseus has been purified to homogeneity (ref. 7; J. Alvarez, T. Owen, W. Kurz, and V.D.L., unpublished work). It occurs as a dimer consisting of two identical subunits of Mr 54,000, and it requires pyridoxal phosphate for activity (7). The enzyme is characteristic of plant aromatic decarboxylases, which usually exhibit high substrate specificity. For example, TDC will decarboxylate L-tryptophan and 5-hydroxy-L-tryptophan but is inactive toward Lphenylalanine and L-tyrosine (7), whereas the tyrosine decarboxylases from Syringa vulgaris (8), Thalictrum rugosum (9), and Escholtzia californica (9) accept L-tyrosine and L-dopa (3,4-dihydroxy-L-phenylalanine) as substrates but not L-tryptophan or 5-hydroxy-L-tryptophan. The aromatic Lamino acid decarboxylases [dopa decarboxylase (DDC), EC 4.1.1.28] of Drosophila melanogaster (10-12) and mammals (13) have a broader substrate specificity, with L-dopa, tyrosine, phenylalanine, and possibly histidine also serving as substrates (14). In animals, the role of aromatic L-amino acid decarboxylase is to produce the major neurotransmitters dopamine a...

Elicitor-induced activation of the potato pathogenesis-related gene PR-10a requires a 30-bp promoter sequence termed the ERE (elicitor response element) that is bound by the nuclear factor PBF-2 ( PR-10a binding factor 2). In this study, PBF-2 has been purified to near homogeneity from elicited tubers through a combination of anion-exchange and DNA affinity chromatography. Evidence demonstrates that inactive PBF-2 is stored in the nuclei of fresh tubers and becomes available for binding to the ERE upon elicitation. A protein with an apparent molecular mass of 24 kD (p24) is a DNA binding component of PBF-2. A cDNA encoding p24 has been cloned and encodes a novel protein with a potential transcriptional activation domain that could also act as a single-stranded DNA binding domain. Both PBF-2 and the cDNA-encoded protein bind with high affinity to the single-stranded form of the ERE in a sequence-specific manner. The inverted repeat sequence of the ERE, TGACAnnnnTGTCA, is critical for binding of this factor in vitro and for PR-10a expression in vivo, supporting the role of PBF-2 as a transcriptional regulator. INTRODUCTIONPlants defend themselves against fungal pathogens by a variety of mechanisms, including preexisting physical barriers and inducible defenses (Lamb et al., 1989). Attack by an avirulent strain of pathogen results in a rapid localized necrosis at the site of infection (termed the hypersensitive response), which contributes to pathogen limitation (Keen, 1992). With a few exceptions, deployment of the inducible defenses requires massive gene induction (Lamb et al., 1989). Despite the importance of transcriptional activation during the plant defense response, very little is known about the players involved and the exact mechanisms that lead to defense gene induction.PR (pathogenesis-related) genes are among the best characterized genes induced by pathogens. Heterogeneous in structure and function, PR genes are subdivided into 11 groups (Van Loon et al., 1994). Although the function of certain PR proteins is unknown, some display in vitro antifungal properties (Schlumbaum et al., 1986;Vigers et al., 1991;Ponstein et al., 1994;Niderman et al., 1995). Genes of the PR-10 group are present in numerous dicots (Somssich et al., 1988;Breiteneder et al., 1989;Matton and Brisson, 1989;Walter et al., 1990) and monocots (Warner et al., 1992;Moons et al., 1997). Evidence is accumulating that some PR-10 proteins might possess ribonuclease activity (Moiseyev et al., 1994;Bufe et al., 1996;Swoboda et al., 1996). More recently, structural and sequential homology between the PR-10 proteins and a group of latex proteins has been described (Osmark et al., 1998). Genes of the PR-10 group encode small, primarily acidic intracellular proteins with molecular masses ranging from 15 to 18 kD and have been shown to be transcriptionally regulated (Linthorst, 1991).In only two cases have cis elements and their trans -acting factors been characterized in the promoters of PR-10 genes. These studies revealed that the processes of tr...

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