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Carpels are essential for sexual plant reproduction because they house the ovules and subsequently develop into fruits that protect, nourish and ultimately disperse the seeds. The AGAMOUS ( AG ) gene is necessary for plant sexual reproduction because stamens and carpels are absent from ag mutant flowers 1 , 2 . However, the fact that sepals are converted into carpelloid organs in certain mutant backgrounds even in the absence of AG activity indicates that an AG -independent carpel-development pathway exists 2 . AG is a member of a monophyletic clade of MADS-box genes that includes SHATTERPROOF1 ( SHP1 ), SHP2 and SEEDSTICK ( STK ) 3 , indicating that these four genes might share partly redundant activities. Here we show that the SHP genes are responsible for AG -independent carpel development. We also show that the STK gene is required for normal development of the funiculus, an umbilical-cord-like structure that connects the developing seed to the fruit, and for dispersal of the seeds when the fruit matures. We further show that all four members of the AG clade are required for specifying the identity of ovules, the landmark invention during the course of vascular plant evolution that enabled seed plants to become the most successful group of land plants 4 .

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes, slr1736 and HPT1, that encode HPT from Synechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystis sp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803 slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1 in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.

THE first step in flower development is the transition of an inflorescence meristem into a floral meristem. Each floral meristem differentiates into a flower consisting of four organ types that occupy precisely defined positions within four concentric whorls. Genetic studies in Arabidopsis thaliana and Antirrhinum majus have identified early-acting genes that determine the identity of the floral meristem, and late-acting genes that determine floral organ identity 1–5 . In Arabidopsis, at least two genes, APETALA1 and LEAFY, are required for the transition of an inflorescence meristem into a floral meristem 1 . We have cloned the APETALA1 gene and here we show that it encodes a putative transcription factor that contains a MADS-domain 2 . APETALA1 RNA is uniformly expressed in young flower primordia, and later becomes localized to sepals and petals. Our results suggest that APETALA1 acts locally to specify the identity of the floral meristem, and to determine sepal and petal development.

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G[rightwards arrow]A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.

The fruit, which mediates the maturation and dispersal of seeds, is a complex structure unique to flowering plants. Seed dispersal in plants such as Arabidopsis occurs by a process called fruit dehiscence, or pod shatter. Few studies 1 , 2 , 3 have focused on identifying genes that regulate this process, in spite of the agronomic value of controlling seed dispersal in crop plants such as canola 4 , 5 . Here we show that the closely related SHATTERPROOF ( SHP1 ) and SHATTERPROOF2 ( SHP2 ) MADS-box genes are required for fruit dehiscence in Arabidopsis . Moreover, SHP1 and SHP2 are functionally redundant, as neither single mutant displays a novel phenotype. Our studies of shp1 shp2 fruit, and of plants constitutively expressing SHP1 and SHP2 , show that these two genes control dehiscence zone differentiation and promote the lignification of adjacent cells. Our results indicate that further analysis of the molecular events underlying fruit dehiscence may allow genetic manipulation of pod shatter in crop plants.

MADS box genes play important roles in specifying floral meristem and floral organ identity. We characterized the temporal and spatial expression patterns of two members of this gene family AGL4 and AGL5 (for AGAMOUS [AG]-like). AGL4 RNA initially accumulates after the onset of expression of the floral meristem identity genes but before the onset of expression of the floral organ identity genes. AGL4 is therefore a putative target of the floral meristem identity genes and/or a potential regulator of the floral organ identity genes. AGL5 is initially expressed early in carpel development shortly after the onset of AG expression. The 1088 of AGL5 expression in flowers of ag mutants the activation of AGL5 by ectopic expression of AG and the specific binding of AG to an element in the AGL5 promoter identify AGL5 as a putative direct target of AG. Our study provides possible links between the establishment of floral meristem and floral organ identity as well as subsequent steps in flower development

Genetic studies demonstrate that two Arabidopsis genes, CAULIFLOWER and APETALA1, encode partially redundant activities involved in the formation of floral meristems, the first step in the development of flowers. Isolation of the CAULIFLOWER gene from Arabidopsis reveals that it is closely related in sequence to APETALA1. Like APETALA1, CAULIFLOWER is expressed in young flower primordia and encodes a MADS-domain, indicating that it may function as a transcription factor. Analysis of the cultivated garden variety of cauliflower (Brassica oleracea var. botrytis) reveals that its CAULIFLOWER gene homolog is not functional, suggesting a molecular basis for one of the oldest recognized flower abnormalities

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes, slr1736 and HPT1, that encode HPT from Synechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystis sp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803 slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1 in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.

Upon appropriate environmental and internal cues, plants generate flowers in a tightly programmed manner, such that the flowers nearly always arise in the correct position at the correct time and have a fixed pattern of floral organs. The plant's decision of how, where and when to generate complex flowers has intrigued scientists for centuries. The recent use of molecular genetic tools has allowed for great progress in the field of flower development, and many of these questions are beginning to be addressed. The work described in my thesis provides insight into both early and later events of Arabidopsis flower development. Genetic studies have shown that CAULIFLOWER (CAL) and APETALA1(AP1) are functionally redundant for specifying floral meristems. In this thesis I describe the isolation and characterization of CAL, providing further insights into its role in determining floral meristem identity as well as its relationship to AP1. Sequence analysis of CAL revealed that it is a MADS-box gene and is very closely related to AP1. RNA in situ hybridizations show that CAL and AP1 are coordinately regulated, as both are expressed in the earliest arising floral meristems. CAL and AP1 are also functionally redundant for activating the floral organ identity gene, AGAMOUS, which in turn negatively regulates CAL and AP1 in the third and fourth whorls. Although the isolation of a loss of function allele of cal demonstrates it is not required for flower development, ectopic expression of CAL is sufficient to convert apical and lateral shoots into flowers. Characterization of another MADS-box gene, AGL5, through RNA in situ hybridizations and the study of trangenic plants, provides insight into its role in carpel development. RNA in situ hybridiztions revealed that AGL5 is carpel-specific and is positively regulated by AG. AG binds specifically to an AG consensus binding site within the AGL5 promoter, and ectopic AG can activate the AGL5 promoter in leaves, identifying AGL5 as a putative direct target of AG. Furthermore, constitutive expression of AGL5 can rescue carpel development in ag mutants, providing the first example of a gene that is involved in early floral organ differentiation.