Timothy W. McNellis

Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.

The Arabidopsis protein COP1, encoded by the constitutive photomorphogenic locus 1, is an essential regulatory molecule that plays a role in the repression of photomorphogenic development in darkness and in the ability of light-grown plants to respond to photoperiod, end-of-day far-red treatment, and ratio of red/far-red light. The COP1 protein contains three recognizable structural domains: starting from the N terminus, they are the zinc binding motif, the putative coiled-coil region, and the domain with multiple WD-40 repeats homologous to the beta subunit of trimeric G-proteins (G beta). To understand the functional implications of these structural motifs, 17 recessive mutations of the COP1 gene have been isolated based on their constitutive photomorphogenic seedling development in darkness. These mutations define three phenotypic classes: weak, strong, and lethal. The mutations that fall into the lethal class are possible null mutations of COP1. Molecular analysis of the nine mutant alleles that accumulated mutated forms of COP1 protein revealed that disruption of the G beta-protein homology domain or removal of the very C-terminal 56 amino acids are both deleterious to COP1 function. In-frame deletions or insertions of short amino acid stretches between the putative coiled-coil and G beta-protein homology domains strongly compromised COP1 function. However, a mutation resulting in a COP1 protein with only the N-terminal 282 amino acids, including both the zinc binding and the coiled-coil domains, produced a weak phenotypic defect. These results indicated that the N-terminal half of COP1 alone retains some activity and a disrupted C-terminal domain masks this remaining activity.

Arabidopsis COP8, COP10, and COP11 genes are involved in repression of photomorphogenic development in darkness.

Wild-type Arabidopsis seedlings are capable of following two developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. Screening of Arabidopsis mutants for constitutive photomorphogenic development in darkness resulted in the identification of three new loci designated COP8, COP10, and COP11. Detailed examination of the temporal morphological and cellular differentiation patterns of wild-type and mutant seedlings revealed that in darkness, seedlings homozygous for recessive mutations in COP8, COP10, and COP11 failed to suppress the photomorphogenic developmental pathway and were unable to initiate skotomorphogenesis. As a consequence, the mutant seedlings grown in the dark had short hypocotyls and open and expanded cotyledons, with characteristic photomorphogenic cellular differentiation patterns and elevated levels of light-inducible gene expression. In addition, plastids of dark-grown mutants were defective in etioplast differentiation. Similar to cop1 and cop9, and in contrast to det1 (deetiolated), these new mutants lacked dark-adaptive change of light-regulated gene expression and retained normal phytochrome control of seed germination. Epistatic analyses with the long hypocotyl hy1, hy2, hy3, hy4, and hy5 mutations suggested that these three loci, similar to COP1 and COP9, act downstream of both phytochromes and a blue light receptor, and probably HY5 as well. Further, cop8-1, cop10-1, and cop11-1 mutants accumulated higher levels of COP1, a feature similar to the cop9-1 mutant. These results suggested that COP8, COP10, and COP11, together with COP1, COP9, and DET1, function to suppress the photomorphogenic developmental program and to promote skotomorphogenesis in darkness. The identical phenotypes resulting from mutations in COP8, COP9, COP10, and COP11 imply that their encoded products function in close proximity, possibly with some of them as a complex, in the same signal transduction pathway.

Light Control of Seedling Morphogenetic Pattern

Plant development is characterized by a high degree of plasticity in response to environmental signals. As s e d e organisms, plants cannot actively move away from sources of stress, nor can they seek out a location with optimal nutrient and light resources. Instead, they must tailor their developmental pattern in a way that maximizes their chances of survival and reproduction. A plant’s “choice” of developmental pattern is based largely on environmental cues, one of the most important of these being light. Given the importance of photosynthesis to plant survival, it comes as no surprise that higher plants respond to light signals by assuming a growth pattern that enhances their access and exposure to light. This control of plant form by ambient light conditions is generally termed photomorphogenesis (Kendrick and Kronenberg, 1994). The light environment in nature is complex. Unobstructed sunlight consists of a wide continuum of photon wavelengths that is conveniently divided into three large spectral domains: UV ( 700 nm) light (Figure 1A). The spectral quality, or relative photon distribution, at different wavelengths can vary greatly, depending on the location and the time of day. For example, within the canopy, the light available is essentially depleted in the visible and UV regions, and far-red light is highly represented (Figure 1A). Furthermore, twilight normally has a higher farred to red ratio than daylight (Smith, 1994). Although higher plants effectively utilize only visible light for photosynthesis, they have the capability to sense and respond to a much wider range of the spectrum, including UV and far-red light. For example, the effectiveness of different wavelengths of continuous light at inhibiting hypocotyl elongation of dark-grown Sinapis alba seedlings (Beggs et al., 1980) is shown in Figure 1B. It is evident that multiple spectral regions of light, including blue, red, and far-red, all are very effective at inhibiting hypocotyl elongation, suggesting that S. alba seedlings are capable of perceiving all of these light signals and utilizing them to control seedling morphogenesis. Plant responses to light are especially evident in the young seedling, although they occur throughout the life of the plant.

Functional dissection of Arabidopsis COP1 reveals specific roles of its three structural modules in light control of seedling development.

Arabidopsis COP1 acts as a repressor of photomorphogenesis in darkness, and light stimuli abrogate the repressive ability and nuclear abundance of COP1. COP1 has three known structural modules: an N‐terminal RING‐finger, followed by a predicted coiled‐coil and C‐terminal WD‐40 repeats. A systematic study was undertaken to dissect the functional roles of these three COP1 domains in light control of Arabidopsis seedling development. Our data suggest that COP1 acts primarily as a homodimer, and probably dimerizes through the coiled‐coil domain. The RING‐finger and the coiled‐coil domains can function independently as light‐responsive modules mediating the light‐controlled nucleocytoplasmic partitioning of COP1. The C‐terminal WD‐40 domain functions as an autonomous repressor module since the overexpression of COP1 mutant proteins with intact WD‐40 repeats are able to suppress photomorphogenic development. This WD‐40 domain‐mediated repression can be at least in part accounted for by COP1s direct interaction with and negative regulation of HY5, a bZIP transcription factor that positively regulates photomorphogenesis. However, COP1 self‐association is a prerequisite for the observed interaction of the COP1 WD‐40 repeats with HY5. This work thus provides a structural basis of COP1 as a molecular switch.

Rootstock effects on gene expression patterns in apple tree scions.

Like many fruit trees, apple trees (Malus pumila) do not reproduce true-to-type from seed. Desirable cultivars are clonally propagated by grafting onto rootstocks that can alter the characteristics of the scion. For example, the M.7 EMLA rootstock is semi-dwarfing and reduces the susceptibility of the scion to Erwiniaamylovora, the causal agent of fire blight disease. In contrast, the M.9 T337 rootstock is dwarfing and does not alter fire blight susceptibility of the scion. This study represents a comprehensive comparison of gene expression patterns in scions of the ‘Gala’ apple cultivar grafted to either M.7 EMLA or M.9 T337. Expression was determined by cDNA-AFLP coupled with silver staining of the gels. Scions grafted to the M.9 T337 rootstock showed higher expression of a number of photosynthesis-related, transcription/translation-related, and cell division-related genes, while scions grafted to the M.7 EMLA rootstock showed increased stress-related gene expression. The observed differences in gene expression showed a remarkable correlation with physiological differences between the two graft combinations. The roles that the differentially expressed genes might play in tree stature, stress tolerance, photosynthetic activity, fire blight resistance, and other differences conferred by the two rootstocks are discussed.

EXPRESSION OF AN N-TERMINAL FRAGMENT OF COP1 CONFERS A DOMINANT-NEGATIVE EFFECT ON LIGHT-REGULATED SEEDLING DEVELOPMENT IN ARABIDOPSIS

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.

Maintenance of chloroplast components during chromoplast differentiation in the tomato mutant green flesh

During ripening of tomato (Lycopersicon esculentum) fruit, chloroplasts develop into chromoplasts. The chloroplast-chromoplast transition is marked by the accumulation of carotenoids and the disappearance of chlorophyll, the degradation of the highly structured thylakoid membrane system, and a reduction in the levels of proteins and mRNAs associated with photosynthesis. In the tomato mutant green flesh (gf), detectable amounts of chlorophyll remain in the ripe, mutant fruit, giving rise to a rusty red fruit color and suggesting that at least chlorophyll degradation is defective in the mutant. We show here that the ultrastructure of the plastids in the ripe gf fruit maintained significant amouonts of the chloroplast thylakoid grana along with structures characteristic of tomato chromoplasts. The maintenance of chloroplast structure in the gf ripe fruit was paralleled on the molecular level by the retention of plastid photosynthetic components that normally decline significantly in ripening tomato fruits. These included the light-harvesting chlorophyll a/b-binding proteins of photosystem II, the second electron accepting plastoquinone of photosystem II binding protein, the large and small subunits of ribulose bisphosphate carboxylase/oxygenase, the 33-kD oxygen evolution protein, and cytochrome b559. Similarly, photosynthetic transcripts, cab, psbA, rbcL, rbcS, and psbE mRNAs, also accumulated to higher levels in ripening gf fruit than wild type. It is interesting that the levels of some of these transcripts, especially cab mRNA, were noticeably higher in the mature gf green fruit than in the corresponding wild-type fruit. This suggests that the onset of the effect from the gf mutation might be earlier than fruit ripening. We also observed that when chloroplast formation was blocked during the development and ripening of gf fruit, these mutant fruits were bright red and their chromoplasts were indistinguishable from those found in wild-type ripe fruits grown and ripened either in the dark or in the light. These results suggest that the lesion in gf may alleviate conditions associated with chloroplast deterioration during the chloroplast-chromoplast transition in tomato ripening but has no direct effect on chromoplast differentiation per se. The ultrastructure of gf provides unequivocal evidence that, in ripening tomato, chromoplasts indeed differentiate from preexisting chloroplasts; on the other hand, chromoplast differentiation in the dark-matured and -ripened tomato fruits indicates that chromoplast development can be a process entirely independent of the chloroplasts.

Fibrillin protein function: the tip of the iceberg?

Fibrillins are nuclear-encoded, plastid proteins associated with chromoplast fibrils and chloroplast plastoglobules, thylakoids, photosynthetic antenna complexes, and stroma. There are 12 sub-families of fibrillins. However, only three of these sub-families have been characterized genetically or functionally. We review evidence indicating that fibrillins are involved in plastoglobule structural development, chromoplast pigment accumulation, hormonal responses, protection of the photosynthetic apparatus from photodamage, and plant resistance to a range of biotic and abiotic stresses. The area of fibrillin research has substantial growth potential and will contribute to better understanding of mechanisms of plant stress tolerance and plastid structure and function.

Mutational Analysis of the Arabidopsis RPS2 Disease Resistance Gene and the Corresponding Pseudomonas syringae avrRpt2 Avirulence Gene

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.

Regulation of Arabidopsis COPINE 1 Gene Expression in Response to Pathogens and Abiotic Stimuli

The copines are a widely distributed class of calcium-dependent, phospholipid-binding proteins of undetermined biological function. Mutation of the Arabidopsis CPN1 (COPINE 1) gene causes a humidity-sensitive lesion mimic phenotype with increased resistance to a bacterial and an oomyceteous pathogen, constitutive pathogenesis-related gene expression, and an accelerated hypersensitive cell death defense response. Here, we show that the disease resistance phenotype of the cpn1-1 mutant was also temperature sensitive, demonstrate increased CPN1 gene transcript accumulation in wild-type plants under low-humidity conditions, and present a detailed analysis of CPN1 gene transcript accumulation in response to bacterial pathogens. In wild-type plants, CPN1 transcript accumulation was rapidly, locally, and transiently induced by both avirulent and virulent strains of Pseudomonas syringae pv tomato bacteria. However, induction of CPN1 transcript accumulation by avirulent bacteria was much faster and stronger than that induced by virulent bacteria. Bacterial induction of CPN1 transcript accumulation was dependent on a functional type III bacterial protein secretion system. In planta expression of the avrRpt2 avirulence gene was sufficient to trigger rapid CPN1 transcript accumulation. CPN1 transcript accumulation was induced by salicylic acid treatment but was not observed during lesion formation in the lesion mimic mutants lsd1 and lsd5. These results are consistent with CPN1 playing a role in plant disease resistance responses, possibly as a suppressor of defense responses including the hypersensitive cell death defense response. The results also suggest that CPN1 may represent a link between plant disease resistance and plant acclimation to low-humidity and low-temperature conditions.