The physiological and neuroendocrine correlates of hunger in the Red Junglefowl (Gallus gallus).

The ability to regulate food intake is critical to survival. The hypothalamus is central to this regulation, integrating peripheral signals of energy availability. Although our understanding of hunger in rodents is advanced, an equivalent understanding in birds is lacking. In particular, the relationship between peripheral energy indices and hypothalamic 'hunger' peptides, agouti-related protein (AgRP), pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) is poorly understood. Here, we compare AgRP, POMC and NPY RNA levels in the hypothalamus of Red Junglefowl chicks raised under ad libitum, chronic restriction and intermittent feeding regimens. Hypothalamic gene expression differed between chronically and intermittently restricted birds, confirming that different restriction regimens elicit different patterns of hunger. By assessing the relationship between hypothalamic gene expression and carcass traits, we show for the first time in birds that AgRP and POMC are responsive to fat-related measures and therefore represent long-term energy status. Chronically restricted birds, having lower indices of fat, show elevated hunger according to AgRP and POMC. NPY was elevated in intermittently fasted birds during fasting, suggesting a role as a short-term index of hunger. The different physiological and neuroendocrine responses to quantitative versus temporal feed restriction provide novel insights into the divergent roles of avian hunger neuropeptides.

Analysis of the petunia MADS-box transcription factor family.

Transcription factors are key regulators of plant development. One of the major groups of transcription factors is the MADS-box family, of which at least 80 members are encoded in the Arabidopsis genome. In this study, 23 members of the petunia MADS-box transcription factor family were investigated by Northern hybridisation, phylogenetic and yeast two-hybrid analyses. Many of the genes characterised appeared to have one or more close relatives that shared similar expression patterns. Comparison of the binding interactions of these proteins revealed that some show similar interaction patterns, and hence are likely to be functionally redundant. From an evolutionary point of view, their coding genes are probably derived from a recent duplication event. Furthermore, protein-protein interaction patterns, in combination with expression patterns and phylogenetic classification, appear to offer good criteria for the identification of functional homologues. Based on comparison of such data between petunia and Arabidopsis, functions can be predicted for several MADS-box transcription factors in both species.

NEC1, a novel gene, highly expressed in nectary tissue of Petunia hybrida.

To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RT-PCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating that the protein is incorporated into the cell membrane or the cytoplast membrane. Immunolocalization revealed that NEC1 protein is present in the nectaries. Northern blot analyses showed that NEC1 is highly expressed in nectary tissue and weakly in the stamen. GUS expression driven by the NEC1 promoter revealed GUS activity in the outer nectary parenchyma cells, the upper part of the filament and the anther stomium. The same expression pattern was observed in Brassica napus. GUS expression was observed as blue spots on the surface of very young nectaries that do not secrete nectar and do accumulate starch. GUS expression was highest in open flowers in which active secretion of nectar and starch hydrolysis had taken place. Ectopic expression of NEC1 resulted in transgenic plants that displayed a phenotype with leaves having 3-4 times more phloem bundles in mid-veins than the wild-type Petunia. The possible role of NEC1 gene in sugar metabolism and nectar secretion is discussed.

A petunia MADS box gene involved in the transition from vegetative to reproductive development.

We have identified a novel petunia MADS box gene, PETUNIA FLOWERING GENE (PFG), which is involved in the transition from vegetative to reproductive development. PFG is expressed in the entire plant except stamens, roots and seedlings. Highest expression levels of PFG are found in vegetative and inflorescence meristems. Inhibition of PFG expression in transgenic plants, using a cosuppression strategy, resulted in a unique nonflowering phenotype. Homozygous pfg cosuppression plants are blocked in the formation of inflorescences and maintain vegetative growth. In these mutants, the expression of both PFG and the MADS box gene FLORAL BINDING PROTEIN26 (FBP26), the putative petunia homolog of SQUAMOSA from Antirrhinum, are down-regulated. In hemizygous pfg cosuppression plants initially a few flowers are formed, after which the meristem reverts to the vegetative phase. This reverted phenotype suggests that PFG, besides being required for floral transition, is also required to maintain the reproductive identity after this transition. The position of PFG in the hierarchy of genes controlling floral meristem development was investigated using a double mutant of the floral meristem identity mutant aberrant leaf and flower (alf) and the pfg cosuppression mutant. This analysis revealed that the pfg cosuppression phenotype is epistatic to the alf mutant phenotype, indicating that PFG acts early in the transition to flowering. These results suggest that the petunia MADS box gene, PFG, functions as an inflorescence meristem identity gene required for the transition of the vegetative shoot apex to the reproductive phase and the maintenance of reproductive identity.

Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate.

The C function in Arabidopsis, which specifies stamen and carpel identity, is represented by a single gene called AGAMOUS (AG). From both petunia and cucumber, two MADS box genes have been isolated. Both share a high degree of amino acid sequence identity with the Arabidopsis AG protein. Their roles in specifying stamen and carpel identity have been studied by ectopic expression in petunia, resulting in plants with different floral phenotypes. Cucumber MADS box gene 1 (CUM1) induced severe homeotic transformations of sepals into carpelloid structures and petals into stamens, which is similar to ectopic AG expression in Arabidopsis plants. Overexpression of the other cucumber AG homolog, CUM10, resulted in plants with partial transformations of the petals into antheroid structures, indicating that CUM10 is also able to promote floral organ identity. From the two petunia AG homologs pMADS3 and Floral Binding Protein gene 6 (FBP6), only pMADS3 was able to induce homeotic transformations of sepals and petals. Ectopic expression of both pMADS3 and FBP6, as occurrs in the petunia homeotic mutant blind, phenocopies the pMADS3 single overexpresser plants, indicating that there is no additive effect of concerted expression. This study demonstrates that in petunia and cucumber, multiple AG homologs exist, although they differ in their ability to induce reproductive organ fate.

A novel class of MADS box genes is involved in ovule development in petunia.

We isolated and characterized two ovule-specific MADS box cDNAs from petunia, designated floral binding protein (fbp) genes 7 and 11. The putative protein products of these genes have approximately 90% of their overall amino acid sequence in common. In situ RNA hybridization experiments revealed that both genes are expressed in the center of the developing gynoecium before ovule primordia are visible. At later developmental stages, hybridization signals were observed only in the ovules, suggesting that these genes are involved in ovule formation. To test this hypothesis, we raised transgenic petunia plants in which both fbp7 and fbp11 expression was inhibited by cosuppression. In the ovary of these transformants, spaghetti-shaped structures developed in positions normally occupied by ovules. These abnormal structures morphologically and functionally resemble style and stigma tissues. Our results show that these MADS box genes belong to a new class of MADS box genes involved in proper ovule development in petunia.

Functional interaction between the homeotic genes fbp1 and pMADS1 during petunia floral organogenesis.

The petunia MADS box floral binding protein (fbp) gene 1 represents a class B homeotic gene determining the identity of second and third floral whorl organs. Suppression of fbp1, which is highly homologous to the Antirrhinum gene globosa and Arabidopsis gene pistillata, results in the conversion of petals to sepals and stamens to carpels. In contrast to fbp1, the petunia homeotic gene pMADS1, encoding a protein homologous to the Antirrhinum protein DEFICIENS, has been shown to be involved in the formation of petals only. We demonstrated that the induction of fbp1 is established independent of pMADS1, whereas at later developmental stages, fbp1 is up-regulated by pMADS1 in petals. On the other hand, the induction and maintenance of pMADS1 expression are not affected by fbp1. To obtain information about the functional interaction between fbp1 and pMADS1, an fbp1 cosuppression mutant with mild phenotypic alterations was crossed with a green petals mutant in which pMADS1 expression was abolished. Progeny plants, heterozygous for the pMADS1 gene, had flowers with a more pronounced reversion from petals into sepals than was observed for the parent fbp1 mutant. The morphology of the third whorl organs was not changed. These observations, together with expression levels of pMADS1 and fbp1 in mutant flowers, provide evidence for functional control of fbp1 by PMADS1 in vivo.

Co-suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem.

The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.

Petal and stamen formation in petunia is regulated by the homeotic gene fbp1.

For Arabidopsis and Antirrhinum, the so-called ABC model has been developed, which postulates that the determination of floral organ primordia is controlled by the action of three classes of homeotic genes. A number of these ABC genes encode putative transcription factors with the MADS box DNA binding motif. This paper reports on the functional analysis of the petunia MADS box gene fbp1. The temporal and spatial expression of fbp1 has been investigated in detail in transgenic plants containing the beta-glucuronidase (GUS) reporter gene fused to an fbp1 promoter fragment. fbp1-driven GUS activity was specifically detected in emerging petal and stamen primordia, suggesting a function of fbp1 in the control of second and third floral whorl identity. To test this hypothesis, transgenic petunia plants were generated in which fbp1 expression was inhibited by a co-suppression approach. The flowers of such plants exhibited homeotic conversions of petals towards sepals and stamens towards carpels. Occasionally, the third whorl carpels are fused forming a pentalocular gynoecium. This dominant fbp1 mutation acted as a single Mendelian trait in genetic crosses. These results strongly indicate that fbp1 is a petunia class B homeotic gene which is required for the correct initiation and determination of petals and stamens.

Differential expression of two MADS box genes in wild-type and mutant petunia flowers.

We isolated and characterized two flower-specific genes from petunia. The protein products of these genes, designated floral binding protein 1 (FBP1) and 2 (FBP2), are putative transcription factors with the MADS box DNA binding domain. RNA gel blot analysis showed that the fbp1 gene is exclusively expressed in petals and stamen of petunia flowers. In contrast, the FBP1 protein was only detectable in petals and not in stamens, suggesting post-transcriptional regulation of the fbp1 gene in these tissues. The fbp2 gene is expressed in petals, stamen, carpels, and at a very low level in sepals but not in vegetative tissues. We analyzed the spatial expression of these fbp genes in floral organs of two homeotic flower mutants. In the blind mutant, whose flower limbs are transformed into antheroid structures on top of normal tubes, identical expression levels of both genes were observed in the antheroid structures as in normal anthers. In the homeotic mutant green petals, the petals are replaced by sepaloid organs in which the expression of fbp1 is strongly reduced but not completely abolished. Our results suggest a regulation of the fbp1 gene expression by the green petals (gp) gene. Expression of the fbp2 gene was not affected in the green petals mutant. In contrast to the proposed models describing floral morphogenesis, our data indicated that homeotic genes can be functional in one whorl only.

Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene.

The expression in Aspergillus is described of genes, coding for intracellular and extracellular proteins controlled by the promoter region of the constitutively and efficiently expressed glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Aspergillus nidulans. Both the homologous gpdA and the heterologous Escherichia coli beta-galactosidase (lacZ) and beta-glucuronidase (uidA) genes could be expressed intracellularly at levels as high as 10-25% of total soluble protein. Efficient extracellular production of A. niger glucoamylase could be achieved with a fusion-gene containing the region of the glucoamylase gene coding for the mature protein preceded by a synthetic fungal signal sequence. Extracellular production of a heterologous protein, E. coli beta-glucuronidase, with such a fusion was much less efficient. Only very low levels of beta-glucuronidase were detected in the culture fluid, whereas considerable enzyme activity was detected in the mycelium.