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Post-transcriptional regulation in Arabidopsis flower development We are interested in how cells in a floral meristem make the decision to undergo terminal differentiation and how cells acquire distinct identities during flower development in Arabidopsis thaliana. The floral homeotic C function gene AGAMOUS (AG) is a master regulator of cell identities and stem cell fate in the floral meristem. In severe loss-of-function ag mutants, stamens and carpels, the two reproductive organs, are transformed into petals and sepals, respectively. In addition, the floral meristem fails to terminate properly, resulting in a Ònested flowersÓ phenotype. Since genetic screens in the wild-type background resulted in the isolation of only ag alleles, little is known about the molecular mechanisms underlying the specification of reproductive organ identities and floral determinacy. Our goal is to identify genes that act in or modulate the AG pathway and determine their molecular functions in relation to their developmental functions. Through a sensitized screen, we identified two genes, HUA1 and HUA2, which act in the specification of stamen identity, carpel identity and floral determinacy. The weak floral phenotype of the hua1-1 hua2-1 double mutant allowed us to perform another sensitized screen aimed at the isolation of more genes acting similarly in flower development. Mutations in six HUA ENHANCER (HEN) loci have been isolated that result in ag-like flowers in the hua1-1 hua2-1 background. The studies on the hua1-1 hua2-1 hen mutants and the molecular cloning of the HEN genes shed light on the functions of not only the HEN genes, but also those of HUA1 and HUA2, in the regulation of the AG pathway. Furthermore, our studies strongly suggest that AG or other components in the pathway are under post-transcriptional control and implicate the existence of an RNAi-like mechanism in the modulation of the AG pathway. HEN4Two recessive mutations at the HEN4 locus cause stamen-to-petal and carpel-to-sepal transformation and loss of floral determinacy in the hua1-1 hua2-1 background such that the hua1-1 hua2-1 hen4 flowers resemble ag flowers. Consistent with the perianth identities in the inner two whorls of hua1-1 hua2-1 hen4 flowers, APETALA1 (AP1), an A function gene that specifies perianth identities, was found to be ectopically expressed in the inner two whorls. Introduction of ap1-1, a severe loss-of-function mutation in AP1, into hua1-1 hua2-1 hen4-1 resulted in the recovery of stamens in the third whorl, suggesting that the stamen-to-petal transformation in hua1-1 hua2-1 hen4 is largely due to ectopic AP1 expression. Consistent with the observed loss of floral determinacy in hua1-1 hua2-1 hen4 flowers, the expression of WUSCHEL, a stem cell fate promoting gene, was not terminated properly during flower development. We cloned HEN4 with a map-based strategy and found that HEN4 codes for a protein with five K homology (KH) domains, which are well-characterized RNA-binding motifs. Furthermore, we found that HEN4 is associated with HUA1, an RNA-binding protein with six CCCH zinc fingers, in a two-hybrid assay in yeast and in a fluorescence resonance energy transfer (FRET) assay in plants. In hua1-1 hua2-1 hen4 but not in any combinations of double mutants, AG mRNA abundance is greatly reduced and concomitantly two large aberrant AG RNAs that contain part of the second intron accumulate. The reduced amount of AG RNA in the triple mutant is likely responsible for the floral phenotype, because in previous studies similar phenotypes were found in plants in which AG RNA abundance was reduced to a similar level by RNAi. Over-expression of AG cDNA in the hua1-1 hua2-1 hen4-1 triple mutant rescued the homeotic phenotype. Therefore, HUA1, HUA2 and HEN4 promote AG pre-mRNA processing. HEN2
hua1-1 hua2-1 hen2-1 flowers show stamen-to-sepal and carpel-to-sepal transformation. The stamen-to-sepal transformation indicates that both floral homeotic B and C functions are compromised. Indeed, in situ hybridization showed that the expression of the class B genes APETALA3 and PISTILLATA and the class C gene AG is initiated correctly but not maintained during mid stages of flower development. Cloning of HEN2 with a map-based strategy showed that HEN2 is a putative RNA helicase. This implies the existence of post-transcriptional regulation in the maintenance of homeotic gene expression. HEN1
Two recessive mutations in HEN1 cause defects in reproductive organ identities and in the proper termination of the floral meristem in the hua1-1 hua2-1 background such that hua1-1 hua2-1 hen1 flowers resemble ag flowers. Detailed studies demonstrated that HEN1 behaves genetically as a C function gene in flower development, although its exact function in this pathway is unclear at the moment. The cloning of HEN1 by chromosome walking showed that HEN1 is a novel protein. hen1 single mutants exhibit vegetative phenotypes similar to those in plants with a mutation in CARPEL FACTORY, an ortholog of dicer from Drosophila and humans. Since dicer is involved in generating small double-stranded RNA in RNAi and small single-stranded microRNAs from their precursors during normal development, we tested whether HEN1 also participates in an RNAi-like mechanism. In collaboration with Dr. VaucheretÕs lab at INRA, we demonstrated that HEN1 is required for RNAi in plants. We hypothesize that the phenotypes associated with hen1 mutations result from defects in the proper regulation of endogenous targets by an RNA-based mechanism. Furthermore, in collaboration with Dr. MessingÕs lab, we isolated and confirmed the expression of 11 microRNAs from Arabidopsis and found that the accumulation of these microRNAs is absent or much reduced in hen1-1. Therefore, HEN1 acts in microRNA metabolism in Arabidopsis. The floral homeotic phenotypes caused by hen1 mutations indicate that microRNAs act in floral organ identity specification. Wonkeun Park, Junjie Li, Rentao Song, Joachim Messing, and Xuemei Chen. (2002) CARPEL FACTORY, the Dicer homologue, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Current Biology 12, 1484-1495. Tamara Western, Yulan Cheng, Jun Liu, and Xuemei Chen. (2002) HUA ENANCER2, a putative DExH-box RNA helicase, maintains homeotic B and C gene expression in Arabidopsis. Development 129, 1569-1581. Xuemei Chen, Jun Liu, Yulan Cheng, and Dongxuan Jia. (2002) HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development 129, 1085-1094. Junjie Li, and Dongxuan Jia, and Xuemei Chen. (2001) HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear zinc finger protein. Plant Cell 13, 2269-2281. Xuemei Chen, JosŽ Luis Riechmann, Dongxuan Jia and Elliot Meyerowitz (2000). The minimal regulatory region of the floral homeotic gene PISTILLATA does not contain a CArG box. Sexual Plant Reprod. 13(2), 85-94. Xuemei Chen, and Elliot M. Meyerowitz (1999). HUA1 and HUA2 are two members of the floral homeotic AGAMOUS pathway. Molecular Cell 3, 349-360. Lab Support Dr. Wonkeun Park, Research Assistant |
Research Summary