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Contenido archivado el 2022-12-23

Molecular Analysis of the Gibberellin-regulated Gene Expression in Petunia Flowers

Objetivo

- The growth regulator gibberellic acid (GA) affects several processes in plants, most notably seed germination, floral induction, and elongation of various tissues and organs. Mutants that are unable to synthesise GAs or unable to respond to GA, have a dwarf phenotype and usually do not flower. In normal plants, GA is also required for the elongation of corollas and the synthesis of anthocyanin pigments, which give the flower its characteristic colour. The long-term objective of this project is to understand the molecular mechanism by which gibberellins control these processes.
GA-activation of anthocyanin regulatory genes
- GA induces expression of pigmentation genes in the corolla at the transcriptional level that can be considered an end point of the GA signalling pathway. We demonstrated this by using an in vitro culture system of excised floral buds. In this system, expression of pigmentation genes can be repressed by an incubation in sucrose medium. Expression can be reactivated by adding GA. The re-induction kinetics of all the structural pigmentation genes tested is about the same; mRNA accumulation is detectable among 4 and 6 hours after the application of GA. These genes are however not the primary GA-response genes but are activated by regulatory proteins whose genes are more directly controlled by GA-generated signals. Several of these regulatory genes have been cloned from petunia, among which An1, An2 and An11. The proteins of these three genes are all required for the transcriptional activation of the pigmentation genes of the second half of the anthocyanin pathway, beginning with dihydroflavonol 4-reductase. As expected, in the in vitro flower bud assay, activation of these An regulators by GA occurs earlier than that of the structural genes. Thus, the promoters of these genes are excellent tools to move one step up in the GA-signalling pathway.


GA-induced MYB-type transcription factor genes
- Apart from An2, which encodes a MYB transcription factor, a number of other Myb genes have been cloned from corollas, by means of homology between the DNA-binding domains. The expression of the Myb27, Myb92, and Myb.Ph1 genes in petals requires the presence of GA3. However, examining the expression of Myb27 yielded a surprise. Myb27 is expressed in leaves and in almost all organs of the flower but mostly in corollas. By examining mutants and transgenic plants carrying a Myb27 promoter driven reporter gene we found that Myb27 expression is controlled by the same An1, An2, and An11 regulatory genes that control the pigmentation genes. Thus, the GA-activated expression of the regulator Myb27 may turn out to be very similar to that of the structural genes. The function and the target genes of the MYB27 protein are not yet known, despite the identification of more than 10 different transposon insertion alleles. All transposon insertions were found in one of the two introns or in the 3'UTR and which had no effect on mRNA synthesis. Although Myb27 is regulated by the An1, An2, and An1 gene products, which already act in young corollas, the level of Myb27 mRNA is the highest in relatively old corollas. How the differential effect on the An-target genes, myb27 and the pigmentation genes, is achieved by the same regulators is unknown. MYB27 seems not to be involved in transcriptional activation as it lacks a clear transcription activation domain in the C-terminal part of the protein. The C-terminal region is actually very short, so it might very well act as a repressor protein.
- MYB92, which contains a putative transcription activation domain, is expressed in almost all organs of the flower. However, its function remains to be determined as a plant carrying a Myb92 knock-out allele did not show an altered visible phenotype. As petunia contains several Myb92 homologues, this might be due to functional redundancy.
- The strongly GA-inducible Myb.Ph1 gene encodes for a protein that is required for the formation of the conical shape of epidermal cells of the corolla. The cells of a mutant in which the Myb.Ph1 gene was disrupted by a transposon were flat. This phenotype has been described before in Snapdragon and shown to be caused by a mutation in a MYB protein called mixta. Myb.Ph1 is therefore the petunia mixta homologue. The proteins are very homologous especially in the DNA binding domain. Myb.Ph1 expression in petals begins very early and is already at its maximum level in small yet unpigmented buds. At later stages the mRNA level declines. This indicates that GA activation of this gene is much earlier than that of the flavonoid genes. The promoter of Myb.Ph1 is therefore very interesting with respect to its mode of GA activation, because the response of this promoter to GA seems the highest of all GA-inducible Myb genes tested.

GIP1: a GA-induced cysteine-rich protein from elongating tissues
- A GAST homologue from petunia, Gip1, was isolated. This gene is strongly induced by GA in petals, in stem and in leaves. Induction of this gene depends much less on the presence of sucrose than other GA-responsive genes GIP1is a cysteine-rich protein of which the function is yet unknown. By examining a petunia seed library for transposon insertions in the Gip1 gene, one mutant allele was found to have a dTph1 insertion into the first intron. As it is located 21 bp from the 3' splice site it seems to affect pre-mRNA splicing because the mRNA level in this Gip1 mutant is severely reduced as compared to wild-type. Preliminary results suggest that this reduction leads to male sterility. No other phenotypes are visible that might be due to the partial inactivation of the gene.

GA-induced 'housekeeping' genes
- The effect of GA on plant tissues is very dramatic. There is usually an extensive elongation and it is conceivable that many cellular processes are up-regulated. It was therefore of interest to look at the response of various, so called 'housekeeping' genes, to GA. By different approaches several of these genes from petunia were isolated: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triose phosphate isomerase (TPI), which are involved in glycolysis, 5 enolpyruvyl shikimate-3-phosphate synthase (EPSPS) of the shikimate pathway, and S-adenosylmethionine synthase (SAM-S), which is required for the synthesis of SAM, a general methyl donor. In corollas, all these genes, except GAPDH, are up-regulated by GA. The pattern of this induction is very similar to that of the pigmentation genes. These first experiments, just by following mRNA levels, indeed suggest that several cellular processes are geared up by GA. However, the finding that several distinct genes are up-regulated in a comparable manner raises the question about the specificity of gene activation by GA. Since GA induces gene expression only in the presence of sucrose it was therefore of interest to examine the role sucrose as a possible, more general, signal molecule. The idea being that GA simply facilitates the uptake of sucrose. Sucrose uptake studies were done using 14C-sucrose as a tracer. This revealed that although GA stimulated sucrose uptake by 20-30%, GA was still needed as a specific stimulus. By inhibiting sucrose uptake by 40%, using the inhibitors PCMBS or vanadate, GA was still able to activate gene expression despite the much lower sucrose uptake. In the samples that had taken up much more sucrose, the examined genes remained unexpressed. These results indicate that genes are not simply activated by a high intracellular sucrose concentration. It clearly requires a specific GA generated signal.

Follow-up

- Up to now, the results of this project show that actually many genes in plants are up-regulated by GA. Expression of several housekeeping genes is enhanced above a certain basal level, whereas the expression of several of the transcription factor genes analysed and of gip1 appears to depend entirely on GA. The picture that is beginning to emerge is that GA may regulate genes in different ways, meaning that there is not a single linear pathway from GA-perception leading to promoter activation and gene expression. To get more insight into these different modes of activation, it will be necessary to examine in depth the steps that occur at the promoter of a primary GA-response gene in combination with the identification of second messenger signals that trigger these events in GA-stimulated cells.
- Little is known about the perception of GA, and GA-receptors have not been cloned yet. Since a number of genes that are specifically expressed in corollas have been cloned and characterised, we have taken the approach to study if and how these genes are regulated by GA. In this bottom-up approach we examine the last step in the GA signalling cascade, which is the transcriptional activation of specific genes. By analysing the promoters of these genes and the corresponding transcription factors, we intend to move up into the GA signalling pathway thereby being able to characterise the protein factors and signalling molecules involved. GA is only active in the presence of metabolic sugars. Therefore, at the physiological level, the GA-signalling is examined with respect to the role of these sugars. As GA may not be the only stimulus activating gene expression, other stimuli have also been examined, such as wounding and jasmonic acid. This may provide insight in how different signalling pathways are connected and share similar components.

Tema(s)

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Convocatoria de propuestas

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Régimen de financiación

CSC - Cost-sharing contracts

Coordinador

Vrije Universiteit Amsterdam
Aportación de la UE
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Dirección
1087,De Boelelaan
1081 HV Amsterdam
Países Bajos

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Coste total
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