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UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo. [ABSTRACT FROM AUTHOR]

Flowering and flower formation are defining features of angiosperms and the control of these developmental processes involves a common repertoire of genes which are shared among different species of flowering plants. These genes were first identified using various homeotic and flowering time mutants of Arabidopsis and snapdragon, and homologous genes have subsequently been isolated from a wide range of different plant species based on the conservation of protein sequence and function. Using degenerate reverse-transcriptase polymerase chain reaction, we have isolated one APETALA3-like (CitMADS8) and two SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1)-like ( CsSL1 and CsSL2) homologues from sweet orange (Citrus sinensis L.). Although the translated amino acid sequence of CitMADS8 shares many similarities with other higher plant APETALA3 proteins, CitMADS8 fails to complement the floral organ identity defects of the Arabidopsis ap3-3 mutant. By contrast, the two citrus SOC1-like genes, particularly CsSL1, are able to shorten the time taken to flower in the Arabidopsis wild-type ecotypes Columbia and C24, and functionally complement the late flowering phenotype of the soc1 mutant, essentially performing the endogenous function of Arabidopsis SOC1. Once flowering has commenced, interactions between specific flowering genes and a gene required for meristem maintenance, WUSCHEL, ensure that the Arabidopsis flower is a determinate structure with four whorls. We have isolated a citrus WUSCHEL homologue (CsWUS) that is capable of restoring most of the meristem function to the shoots and flowers of the Arabidopsis wus-1 mutant, implying that CsWUS is the functional equivalent of Arabidopsis WUSCHEL. [ABSTRACT FROM AUTHOR]

Flowering is an integral developmental process in angiosperms, crucial to reproductive success and continuity of the species through time. Some angiosperms complete their life cycle within a year (annual plants), and others have a longer reproductive life, which is characterized by the generation of new flowering and vegetative shoots every year (perennial plants). Despite the differences in their lifespan, the underlying genetics of flower induction and floral organ formation appears to be similar among these plants. Hence, the knowledge gained from the study of flowering mechanism in Arabidopsis thaliana can be used to better understand similar processes in other plant species, especially the perennials, which usually have a long generation time and are not amenable to genetic analysis. Using Arabidopsis as a model, we briefly discuss the current understanding of the transition from vegetative to reproductive growth and the subsequent formation of individual floral organs, and how this knowledge has been successfully applied to the identification of homologous genes from perennial crops. Although annuals appear to share many similarities with perennials in terms of gene function, they differ in their commitment to flowering. Once an annual reaches the reproductive phase, all meristems are typically converted into either floral or inflorescence meristems. In contrast, each year, each meristem of a mature perennial has the choice to produce either a vegetative or a reproductive shoot. The physiology and genetics of flowering in Citrus are used to highlight the complexity of reproductive development in perennials, and to discus possible future research directions. [ABSTRACT FROM AUTHOR]