Francisco Madueño

Orchestration of Floral Initiation by APETALA1

Flower Power The transcription factor APETALA1 (AP1) controls the transition from vegetative growth to flower production in the plant Arabidopsis. A handful of factors that control AP1 have been identified, as well as some targets that AP1 controls. Kaufmann et al. (p. 85) now apply genome-wide microarray analysis to identify over a thousand genes whose transcription is regulated by AP1. By proximity to AP1 binding sites, over two thousand genes are implicated as putative AP1 targets. Analysis of this network of interactions indicates that AP1 functions first to repress vegetative identity, then to help establish floral primordia, and finally to shape the differentiation of floral parts. The master transcription factor APETALA1 dynamically regulates a complex genetic network to guide flower development. The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. Although AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning, and hormonal pathways.

Different roles of flowering-time genes in the activation of floral initiation genes in Arabidopsis.

We have analyzed double mutants that combine late-flowering mutations at four flowering-time loci (FVE, FPA, FWA, and FT) with mutations at the LEAFY (LFY), APETALA1 (AP1), and TERMINAL FLOWER1 (TFL1) loci involved in the floral initiation process (FLIP). Double mutants between ft-1 or fwa-1 and lfy-6 completely lack flowerlike structures, indicating that both FWA and FT act redundantly with LFY to control AP1. Moreover, the phenotypes of ft-1 ap1-1 and fwa-1 ap1-1 double mutants are reminiscent of the phenotype of ap1-1 cal-1 double mutants, suggesting that FWA and FT could also be involved in the control of other FLIP genes. Such extreme phenotypes were not observed in double mutants between fve-2 or fpa-1 and lfy-6 ap1-1. Each of these showed a phenotype similar to that of ap1-1 or lfy-6 mutants grown under noninductive photoperiods, suggesting a redundant interaction with FLIP genes. Finally, the phenotype of double mutants combining the late-flowering mutations with tfl1-2 were also consistent with the different roles of flowering-time genes.

How Floral Meristems are Built

The formation of flowers involves the activity of a genetic network that acts in meristems to specify floral identity. The main output of this network is the initiation of a developmental patterning program for the generation of floral organs. The first characteristic of meristem identity genes is their capacity to integrate the environmental and endogenous cues that regulate the onset of flowering. This mechanism synchronizes temporal and spatial information, ensuring that flowers arise in the correct location at the appropriate time. The second characteristic of this network is the mutual regulatory interactions established between meristem identity genes. These interactions provide flexibility and robustness against environmental noise and prevent reversion once the decision to flower has been made. Finally, the third feature is the overlap between the meristem identity and other developmental programs that operate simultaneously to regulate different aspects of the construction of flowers.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for “modified traffic to the vacuole”) mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative δ-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues.

Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

STENOFOLIA Regulates Blade Outgrowth and Leaf Vascular Patterning in Medicago truncatula and Nicotiana sylvestris

This study shows that a WUSCHEL-like gene, STENOFOLIA (STF), is required for blade outgrowth, and its deletion accounts for the classical bladeless lam1 phenotype of tobacco (Nicotiana sylvestris). STF confers morphogenetic competence to leaf primordial margins and coordinates auxin/cytokinin homeostasis and hormone crosstalk with sugar metabolism, integrating metabolic and developmental signals. Dicot leaf primordia initiate at the flanks of the shoot apical meristem and extend laterally by cell division and cell expansion to form the flat lamina, but the molecular mechanism of lamina outgrowth remains unclear. Here, we report the identification of STENOFOLIA (STF), a WUSCHEL-like homeobox transcriptional regulator, in Medicago truncatula, which is required for blade outgrowth and leaf vascular patterning. STF belongs to the MAEWEST clade and its inactivation by the transposable element of Nicotiana tabacum cell type1 (Tnt1) retrotransposon insertion leads to abortion of blade expansion in the mediolateral axis and disruption of vein patterning. We also show that the classical lam1 mutant of Nicotiana sylvestris, which is blocked in lamina formation and stem elongation, is caused by deletion of the STF ortholog. STF is expressed at the adaxial–abaxial boundary layer of leaf primordia and governs organization and outgrowth of lamina, conferring morphogenetic competence. STF does not affect formation of lateral leaflets but is critical to their ability to generate a leaf blade. Our data suggest that STF functions by modulating phytohormone homeostasis and crosstalk directly linked to sugar metabolism, highlighting the importance of coordinating metabolic and developmental signals for leaf elaboration.

Clustering of genes involved in nitrate assimilation in the cyanobacterium Synechococcus

SummaryA region of the genome of the cyanobacterium Synechococcus R2, that bears a cluster of genes involved in nitrate assimilation, has been cloned and the relative positions of some of the genes in the region have been determined. Mutations generated by insertion of an antibiotic-resistance gene cassette into the gene encoding nitrite reductase are associated with reduced expression of nitrate reductase; cotranscription of nitrate assimilation genes in the cluster is inferred from this finding.

The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.

A cytoplasmic-membrane protein repressible by ammonium in Synechococcus R2: altered expression in nitrate-assimilation mutants

A protein of ∼48 kDa was present in the cytoplasmic membrane of cells of Synechococcus R2 grown on nitrate or incubated in the absence of combined nitrogen, but not in ammonium‐grown cells. The level of this polypeptide was diminished in mutant strains that also exhibit reduced levels of other components of the nitrate‐reductive system. On the other hand, a mutant constitutive for nitrate uptake also synthesized constitutively the 48 kDa protein.