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Biogenesis of plastid ribosomes is facilitated by auxiliary factors that process and modify ribosomal RNAs (rRNAs) or are involved in ribosome assembly. In comparison with their bacterial and mitochondrial counterparts, the biogenesis of plastid ribosomes is less well understood, and few auxiliary factors have been described so far. In this study, we report the functional characterization of CONSERVED ONLY IN THE GREEN LINEAGE20 (CGL20) in Arabidopsis (Arabidopsis thaliana; AtCGL20), which is a Pro-rich, ∼10-kD protein that is targeted to mitochondria and chloroplasts. In Arabidopsis, CGL20 is encoded by segmentally duplicated genes of high sequence similarity (AtCGL20A and AtCGL20B). Inactivation of these genes in the atcgl20ab mutant led to a visible virescent phenotype and growth arrest at low temperature. The chloroplast proteome, pigment composition, and photosynthetic performance were significantly affected in atcgl20ab mutants. Loss of AtCGL20 impaired plastid translation, perturbing the formation of a hidden break in the 23S rRNA and causing abnormal accumulation of 50S ribosomal subunits in the high-molecular-mass fraction of chloroplast stromal extracts. Moreover, AtCGL20A-eGFP fusion proteins comigrated with 50S ribosomal subunits in Suc density gradients, even after RNase treatment of stromal extracts. Therefore, we propose that AtCGL20 participates in the late stages of the biogenesis of 50S ribosomal subunits in plastids, a role that presumably evolved in the green lineage as a consequence of structural divergence of plastid ribosomes.

Photoreceptors, especially the far-red light-absorbing phytochrome A, play a crucial role in early seedling development, triggering the transition from etiolated to photomorphogenic growth. Here, we describe the biological functions of two GRAS proteins from Arabidopsis (Arabidopsis thaliana), SCARECROW-LIKE21 (SCL21) and PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1), which are specifically involved in phytochrome A signal transduction. Loss-of-function mutants show an elongated hypocotyl under far-red light and are impaired in other far-red high-irradiance responses. The SCL21 transcript itself is down-regulated by far-red light in a phytochrome A- and PAT1-dependent manner. Our results demonstrate that both SCL21 and PAT1 are positive regulators of phytochrome A signal transduction for several high-irradiance responses. Genetic and biochemical evidence suggest a direct interaction of the two proteins.

Since the sequencing of the nuclear genome of Arabidopsis thaliana ten years ago, various large-scale analyses of gene function have been performed in this model species. In particular, the availability of collections of lines harbouring random T-DNA or transposon insertions, which include mutants for almost all of the ~27,000 A. thaliana genes, has been crucial for the success of forward and reverse genetic approaches. In the foreseeable future, genome-wide phenotypic data from mutant analyses will become available for Arabidopsis, and will stimulate a flood of novel in-depth gene-function analyses. In this review, we consider the present status of resources and concepts for systematic studies of gene function in A. thaliana. Current perspectives on the utility of loss-of-function and gain-of-function mutants will be discussed in light of the genetic and functional redundancy of many A. thaliana genes.

The GRAS protein family is present not only in all higher plant species, but also in lower plants and at least one alga. Recently, it has been shown that bacterial proteins, belonging to the Rossmann-fold SAM-dependent methyltransferases, cluster with GRAS proteins. GRAS proteins are involved in many diverse signaling and developmental processes and play an important role in transcriptional control. The GRAS domain itself is highly conserved with several nearly invariant amino acids and a strict spatial distribution, suggesting the importance of these amino acids for a conserved function. In higher plants about 15 clades of GRAS proteins can be discriminated, but not all of them are present in all plant species, suggesting that adaptive processes probably played a role in the establishment and maintenance of specific clades and the numbers of members within each.

The GRAS protein family has been analyzed in several plant species and our knowledge of their biological role has increased steadily since they were first identified in 1996. GRAS proteins play important roles in many developmental processes. GRAS proteins involved in root development, meristem maintenance, and colonization of roots with fungi or bacteria have been especially well studied over the years. Furthermore, GRAS proteins play a role in several signaling pathways such as hormones, light, and stress. Changes in transcriptional activity can often be attributed to GRAS proteins and although their direct role as transcription factors is not really clear, they associate with several classes of transcription factors and chromatin remodeling factors suggesting that they are important regulators of transcription.

Phytochrome photoreceptors enable plants to perceive divergent light signals leading to adaptive changes in response to differing environmental conditions. However, the mechanism of light signal transduction is not fully understood. Here we report the identification of a new signaling intermediate from Arabidopsis thaliana, Scarecrow-like (SCL)13, which serves as a positive regulator of continuous red light signals downstream of phytochrome B (phyB). SCL13 antisense lines exhibit reduced sensitivity towards red light, but only a distinct subset of phyB-mediated responses is affected, indicating that SCL13 executes its major role in hypocotyl elongation during de-etiolation. Genetic evidence suggests that SCL13 is also needed to modulate phytochrome A (phyA) signal transduction in a phyB-independent way. The SCL13 protein is localized in the cytoplasm, but can also be detected in the nucleus. Overexpression of both a nuclear and cytoplasmic localized SCL13 protein leads to a hypersensitive phenotype under red light indicating that SCL13 is biologically active in both compartments. SCL13 is a member of the plant-specific GRAS protein family, which is involved in various different developmental and signaling pathways. A previously identified phytochrome A signaling intermediate, PAT1, belongs to the same subbranch of GRAS proteins as SCL13. Although both proteins are involved in phytochrome signaling, each is specific for a different light condition and regulates a different subset of responses.

GRAS proteins are a recently discovered family of plant-specific proteins named after GAI, RGA and SCR, the first three of its members isolated. Although the Arabidopsis genome encodes at least 33 GRAS protein family members only a few GRAS proteins have been characterized so far. However, it is becoming clear that GRAS proteins exert important roles in very diverse processes such as signal transduction, meristem maintenance and development. Here we present a survey of the different GRAS proteins and review the current knowledge of the function of individual members of this protein family.

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings.

Expression of chimeric uidA gene fusions (for bacterial beta-glucuronidase) with 5'-flanking sequences of the spinach AtpC and PetE genes (encoding the subunit gamma of the chloroplast ATP synthase and plastocyanin, respectively) requires sequences for the 5'-untranslated leaders. The sequence for the PetE leader does not exhibit significant similarities to those of other leader sequences. Closer inspection of PetE uncovered that the crucial region is located in the vicinity of the transcription start site (+5/+15, TTGTCATTTCT). In contrast, 3' deletions of sequences for the AtpC leader revealed that the region in the vicinity of the translation initiation codon is essential for uidA gene expression (+103/+176). This segment contains a CT-rich sequence (TTCTCTCTCCT), which is found identically or in a slightly modified form in sequences for 85 plant leaders deposited in the EMBL data bank. Site-directed mutagenesis of the CT-rich sequence resulted in a three-fold reduction of the transcription of the transgene. It is concluded (1) that different elements in the sequences for the spinach PetE and AtpC leaders control the expression of the uidA gene, (2) that these elements operate transcriptionally rather than post-transcriptionally and (3) that a CT-rich sequence represents a crucial cis element for the transcription of the AtpC::uidA gene fusion.

Nuclear-encoded genes for proteins of the photosynthetic machinery represent a particular subset of genes. Their expression is cooperatively stimulated by discrete factors including the developmental stage of plastids and light. We have analyzed in transgenic tobacco the plastid- and light-dependent expression of a series of 5' promoter deletions of various nuclear genes from spinach, of fusions of defined promoter segments with the 90-bp 35S RNA CaMV minimal promoter, as well as with mutations in sequences with homologies to characterized cis-elements, to address the question of whether the plastid signal and light operate via the same or different cis-acting elements. In none of the 160 different transgenic lines (representing 32 promoter constructs from seven genes) analyzed, could significant differences be identified in the responses to the two regulatory pathways. The data are compatible with the idea that both signals control the expression of nuclear genes for plastid proteins via the same cis-acting elements.

Owing to duplication events in its progenitor, more than 90% of the genes in the Arabidopsis thaliana genome are members of multigene families. A set of 2108 gene families, each consisting of precisely two unlinked paralogous genes, was identified in the nuclear genome of A. thaliana on the basis of sequence similarity. A systematic method for the creation of double knock-out lines for such gene pairs, designated as DUPLO lines, was established and 200 lines are now publicly available. Their initial phenotypic characterisation led to the identification of seven lines with defects that emerge only in the adult stage. A further six lines display seedling lethality and 23 lines were lethal before germination. Another 14 lines are known to show phenotypes under non-standard conditions or at the molecular level. Knock-out of gene pairs with very similar coding sequences or expression profiles is more likely to produce a mutant phenotype than inactivation of gene pairs with dissimilar profiles or sequences. High coding sequence similarity and highly similar expression profiles are only weakly correlated, implying that promoter and coding regions of these gene pairs display different degrees of diversification.

The level of plastidic glutamine synthetase (GS; EC 6.3.1.2) in the cotyledonary whorl of the Scots pine (Pinus sylvestris L.) seedling was previously reported to be regulated by light. In the present paper we report on the control by light of the GS transcript level. A full-length GS cDNA clone of Scots pine was isolated (pGS1), sequenced and employed to measure GS transcript levels. Using dichromatic light treatments it was found that the transcript level is regulated by phytochrome. The strong specific effect of blue light is to be attributed to an increase of the responsiveness to phytochrome. Since no direct correlation between the transcript level and the rate of GS protein synthesis was observed, it was concluded that GS gene expression is only coarsely regulated at the level of transcript accumulation. Synthesis of GS protein is by itself light-dependent (light-mediated fine tuning of gene expression). This control at the translational level is also exerted via phytochrome with blue light determining the responsiveness of the process toward phytochrome. If the level of the far-red absorbing form of phytochrome (Pfr) is kept very low, blue light is not capable of bringing about synthesis of GS protein.

With the completion of the Arabidopsis thaliana genome sequencing project the next major challenge is the assignment of biological functions to the more than 25,000 genes. Reverse genetics is a powerful tool to elucidate gene function in Arabidopsis. Increasingly sophisticated genetic approaches are being developed for reverse genetics with the long-term goal of understanding how the coordinated activity of all proteins rises to a complex organism. Identification of a biological function for each gene is often doomed to fail as many loss- or gain-of-function lines exhibit no obvious phenotypes under normal propagation conditions . Here we provide an overview on how to phenotype plants during their development. This phenotypic streamlined analysis is based on a series of defined growth stages (germination, seedling, vegetative, and reproductive stages), which can be used for the profiling of mutants.

The disruption or modulation of signal transduction pathways does not always lead to drastic changes in plant growth and development. Therefore, many loss- or gain-of-function lines do not exhibit an obvious phenotype under normal greenhouse conditions. To be able to assign biological functions to these genes, the mutants need to be evaluated with a broad spectrum of assays to uncover conditional phenotypes.Here we provide an overview on how to evaluate plants in their development and their response to abiotic factors such as light, hormones, and different stressors. The assessment of the behavior of a plant under these conditions can be used to correlate a biological role with a genotype. This phenotypic analysis can be used for profiling of mutants.

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1(long after far-red light1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed thatlaf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1encodes a nuclear protein with strong homology with the R2R3–MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO).

Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsismutant, pat (phytochrome Asignal transduction)1-1, which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncatedPAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.

The promoters of the spinach ferredoxin-NADP + -oxidoreductas gene and one member of the gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase contain ACGT sequences relevant for gene expression. Site-directed mutagenesis revealed that these sequences operate quantitatively and are not involved in the light response. We have isolated a cDNA for a basic leucine zipper protein (bZIP) from spinach. After transcription and translation in cell-free systems, the protein binds in vitro to double-stranded oligonucleotides designed according to both sequences, although with different efficiencies. The genomic DNA segment for this bZIP contains 10 introns, The bZIP gene promoter harbors also an ACGT sequence; however, promoter uidA gene fusions revealed that these nucleotides are not cssential for expression. At least three other genes with high similarities are present in the spinach genome; however, they appear to be either pseudogenes, because they contain in-frame stop codons in highly conserved epitopes, or must be posttranscriptionally modified in order to code for functional proteins. The high sequence similarities suggest that all four sequences derive from gene duplications.

Plastid- and light-regulated expression of the spinach PsaD gene in transgenic tobacco requires sequences downstream of the transcription start site, sand promoter sequences alone are not sufficient to respond to these stimuli. The spinach PsaD mRNA level in transgenic tobacco is still plastid- and light-responsive when the expression of the intron-containing transcription unit is driven by the 35S RNA CaMV promoter indicating that PsaD contains (a) gene-internal control element(s). If the genomic PsaD sequence in the latter construct was replaced by the cDNA, a constitutive expression of the PsaD transcript level was observed. It is concluded that the intron sequence contributes to the plastid- and light-dependent expression of the spinach PsaD gene.

Run-on assays with isolated nuclei demonstrate that the transcription rates of AtpC and AtpD (gene products: the CF1 subunits gamma and delta of the chloroplast ATP synthase) are comparable in spinach seedlings. However, chimeric GUS gene fusions with 5'-flanking regions of the AtpC gene direct an approximately 10-fold lower GUS level in transgenic tobacco compared with equivalent fragments from the AtpD gene. Both promoters contain sequences in the vicinity of the respective TATA boxes, which are sufficient to direct light-regulated, plastid-dependent and organ-specific expression of the GUS gene. In contrast, the upstream regions of both promoters differ the higher GUS level directed by the AtpD promoter is caused by enhancer-like elements located upstream of the region involved in the regulated expression, while nucleotides upstream of -73 in the AtpC promoter contribute relatively little to the promoter activity. 5'-Deletion analyses and site-directed mutagenesis studies indicated that the -73/-48 bp AtpC region contains cis-elements crucial for this regulated expression. If five nucleotides within this region (-59/-55) are exchanged, the GUS gene is constitutively expressed and the activity in etiolated seedlings, in seedlings with photobleached plastids and in roots increases to the level detectable in green cotyledons. It is concluded that signal transduction pathways from different regulators converge prior to gene regulation and that these five nucleotides are part of a cis-element which functions as a repressor in darkness, in tissues with impaired plastids and in roots.

We have analyzed plastid and nuclear gene expression in tobacco seedlings using the carotenoid biosynthesis inhibitor nor-flurazon. mRNA levels for three nuclear-encoded chlorophyll-binding proteins of photosystem I and photosystem II (CAB I and II and the CP 24 apoprotein) are no longer detectable in photobleached seedlings, whereas those for other components of the thylakoid membrane (the 33- and 23-kD polypeptides and Rieske Fe/S polypeptide) accumulate to some extent. Transgenic tobacco seedlings with promoter fusions from genes for thylakoid membrane proteins exhibit a similar expression behavior: a CAB-[beta]-glucuronidase (GUS) gene fusion is not expressed in herbicide-treated seedlings, whereas PC-, FNR-, PSAF-, and ATPC-promoter fusions are expressed, although at reduced levels. All identified segments in nuclear promoters analyzed that have been shown to respond to light also respond to photodamage to the plastids. Thus, the regulatory signal pathways either merge prior to gene regulation or interact with closely neighboring cis elements. These results indicate that plastids control nuclear gene expression via different and gene-specific cis-regulatory elements and that CAB gene expression is different from the expression of the other genes tested. Finally, a plastid-directing import sequence from the maize Waxy gene is capable of directing the GUS protein into the photodamaged organelle. Therefore, plastid import seems to be functional in photobleached organelles.

The promoter region -118/-29 of the spinach PetH gene encoding the ferredoxin-NADP(+)-oxidoreductase contains crucial cis-elements for the regulated expression, while sequences for the 5'-untranslated leader determine the quantitative expression of chimeric GUS gene fusions in transgenic tobacco. Deletion of leader sequences in chimeric GUS gene fusions of the spinach PetE and PsaF genes (for plastocyanin and the subunit III of photosystem I, respectively) results also in a decline in the GUS activity. Appropriate gene constructs and run-on transcription assays demonstrate unambiguously that the leaders of all three genes are involved in transcription rather than in post-transcriptional processes. They appear to contain gene-specific control elements rather than cis-determinants for general initiation factors. Expression-relevant segments in the PsaF and PetH leaders contain two CT-rich sequences, designated CT-LB and CT-B, of which at least the former binds to a protein factor in gel mobility shift assays. These motifs are not found in the PetE leader. The findings imply that leader sequences may contain cis-elements that are essential for the transcription, that they influence GUS gene expression quantitatively rather than qualitatively, and that these elements, as those of promoters, can be quite variable in sequence.

The light-regulated expression of eight nuclear-encoded genes for plastid proteins from spinach (Spinacia oleracea) (RBCS-1 and CAB-1; ATPC and ATPD, encoding the subunits [gamma] and [delta] of the ATP synthase; PC and FNR; PSAD and PSAF, encoding the subunits II and III of photosystem I reaction center) was analyzed with promoter/[beta]-glucuronidase (GUS) gene fusions in transgenic tobacco (Nicotiana tabacum and Nicotiana plumbaginifolia) seedlings and mature plants under standardized light and growth conditions. Unique response patterns were found for each of these promoters. GUS activities differed more than 30-fold. Strong promoters were found for the PC and PSAD genes. On the other hand, the ATPC promoter was relatively weak. Expression of the CAB/GUS gene fusion in etiolated material was at the detection limit; all other chimeric genes were expressed in the dark as well. Light stimulation of GUS activities ranged from 3- (FNR promoter) to more than 100-fold (CAB-1 promoter). The FNR promoter responded only to red light (RL) and not significantly to blue light (BL), whereas the PC promoter contained regions with different sensitivities toward RL and BL. Furthermore, different RNA accumulation kinetics were observed for the PSAF, CAB, FNR, and PC promoter/GUS gene fusions during de-etiolation, which, at least in the case of the PSAF gene, differed from the regulation of the corresponding endogenous genes in spinach and tobacco. The results suggest either that not all cis elements determining light-regulated and quantitative expression are present on the spinach promoter fragments used or that the spinach cis-regulatory elements respond differently to the host (tobacco) regulatory pathway(s). Furthermore, as in tobacco, but not in spinach, the trans-gene hardly responds to single light pulses that operate through phytochrome. Taken together, the results suggest that the genes have been independently translocated from the organelle to the nucleus during phylogeny. Furthermore, each gene seems to have acquired a unique set of regulatory elements.

We describe a genomic DNA segment from spinach that bears part of the single-copy gene for ferredoxin-NADP(+)-oxidoreductase (FNR) including a 3.4 kb promoter sequence. Dissection of this DNA segment and its analysis in GUS (beta-glucuronidase) gene fusions in transgenic tobacco demonstrated that the promoter differs in structure from all other promoters for thylakoid protein genes studied to date. Two regions with light-responsive elements were identified. One is located within the first 118 bp upstream of the transcription initiation site. A second fragment covering nucleotide positions -220 to -119 is capable of conferring light-dependent GUS gene expression on two different minimal promoters. The latter fragment binds a transacting factor in gel-shift assays. None of the fragments carries cis elements known from other genes to be involved in light-controlled expression. Comparison of the light responsiveness of GUS gene fusions controlled by the -753/+231 and -118/+231 regions indicates that they respond differentially to phytochrome-dependent signals and that their expression in tobacco is not restricted to tissue with functional chloroplasts.