AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis.

EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A two-hybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene (Zhang et al., 1992, Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle.

A molecular link between stem cell regulation and floral patterning in Arabidopsis.

The homeotic gene AGAMOUS (AG) has dual roles in specifying organ fate and limiting stem cell proliferation in Arabidopsis flowers. We show that the floral identity protein LEAFY (LFY), a transcription factor expressed throughout the flower, cooperates with the homeodomain protein WUSCHEL (WUS) to activate AG in the center of flowers. WUS was previously identified because of its role in maintaining stem cell populations in both shoot and floral meristems. The unsuspected additional role of WUS in regulating floral homeotic gene expression supports the hypothesis that floral patterning uses a general meristem patterning system that was present before flowers evolved. We also show that AG represses WUS at later stages of floral development, thus creating a negative feedback loop that is required for the determinate growth of floral meristems.

A genetic framework for floral patterning.

The initial steps of flower development involve two classes of consecutively acting regulatory genes. Meristem-identity genes, which act early to control the initiation of flowers, are expressed throughout the incipient floral primordium. Homeotic genes, which act later to specify the identity of individual floral organs, are expressed in distinct domains within the flower. The link between the two classes of genes has remained unknown so far. Here we show that the meristem-identity gene LEAFY has a role in controlling homeotic genes that is separable from its role in specifying floral fate. On the basis of our observation that LEAFY activates different homeotic genes through distinct mechanisms, we propose a genetic framework for the control of floral patterning.

The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development.

Previous studies have shown that recessive mutations at the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON1 (LEC1) loci lead to various abnormalities during mid-embryogenesis and late embryogenesis. In this study, we investigated whether these loci act in independent regulatory pathways or interact in controlling certain facets of seed development. Several developmental responses were quantified in abi3, fus3, and lec1 single mutants as well as in double mutants combining either the weak abi3-1 or the severe abi3-4 mutations with either fus3 or lec1 mutations. Our data indicate that ABI3 interacts genetically with both FUS3 and LEC1 in controlling each of the elementary processes analyzed, namely, accumulation of chlorophyll and anthocyanins, sensitivity to abscisic acid, and expression of individual members of the 12S storage protein gene family. In addition, both FUS3 and LEC1 regulate positively the abundance of the ABI3 protein in the seed. These results suggest that in contrast to previous models, the ABI3, FUS3, and LEC1 genes act synergistically to control multiple elementary processes during seed development.

Interactions between the ABI1 and the ectopically expressed ABI3 genes in controlling abscisic acid responses in Arabidopsis vegetative tissues.

In Arabidopsis, the abscisic acid (ABA)-Insensitive ABI1 and ABI3 genes have been proposed to act in separate ABA signalling cascades. Recessive mutations in ABI3 alter various physiological processes during seed development, whereas the dominant abi1 mutation inhibits ABA responses largely in vegetative tissues. The seed-specific ABI3 gene was ectopically expressed in the vegetative tissues of transgenic Arabidopsis plants carrying a transcriptional fusion between the CaMV 35S promoter and the ABI3 cDNA. Genetic interactions between the ectopically expressed ABI3 and endogenous ABI1 genes were investigated by monitoring diverse ABA responses in vegetative tissues. Ectopic expression of ABI3 conferred to plantlets the ability to accumulate the seed-specific At2S33 and AtEm1 mRNAs in response to ABA, and the abi1 mutation inhibited this ABI3-dependent induction of AtEm1 by ABA. Furthermore, ectopic expression of ABI3 also influenced ABI1-dependent responses that occur in wild-type vegetative tissues. Expression of ABI3 increased ABA induction of the Rab18 mRNA and ABA inhibition of root growth, and both responses were sensitive to the abi1 mutation in the presence as in the absence of ABI3. Finally, although ABI3 is thought to be a transcription activator and stomatal regulation is not known to involve transcriptional events, the ectopically expressed ABI3 gene suppressed the effect of the abi1 mutation on stomatal regulation. The present data demonstrate that ABI1 and ABI3 genetically interact in controlling diverse ABA responses in transgenic vegetative tissues. The possibility that the endogenous ABI1 and ABI3 genes may similarly act in a common ABA signalling pathway in seed is discussed with previous phenotypic studies of the abi1 and abi3 mutants.

Differential regulation of two ABA-inducible genes from Craterostigma plantagineum in transgenic Arabidopsis plants.

In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the beta-glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter.

Current advances in abscisic acid action and signalling.

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3 locus and of endogenous abscisic acid.

The accumulation kinetics of 18 mRNAs were characterized during Arabidopsis silique development. These marker mRNAs could be grouped in distinct classes according to their coordinate temporal expression in the wild type and provided a basis for further characterization of the corresponding regulatory pathways. The abscisic acid (ABA)-insensitive abi3-4 mutation modified the expression pattern of several but not all members of each of these wild-type temporal mRNA classes. This indicates that the ABI3 protein directly participates in the regulation of several developmental programs and that multiple regulatory pathways can lead to the simultaneous expression of distinct mRNA markers. The ABI3 gene is specifically expressed in seed, but ectopic expression of ABI3 conferred the ability to accumulate several seed-specific mRNA markers in response to ABA in transgenic plantlets. This suggested that expression of these marker mRNAs might be controlled by an ABI3-dependent and ABA-dependent pathway(s) in seed. However, characterization of the ABA-biosynthetic aba mutant revealed that the accumulation of these mRNAs is not correlated to the ABA content of seed. A possible means of regulating gene expression by developmental variations in ABA sensitivity is apparently not attributable to variations in ABI3 cellular abundance. The total content of ABI3 protein per seed markedly increased at certain developmental stages, but this augmentation appears to result primarily from the simultaneous multiplication of embryonic cells. Our current findings are discussed in relation to their general implications for the mechanisms controlling gene expression programs in seed.

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.