Cristina Ferrándiz

Conservation of Arabidopsis Flowering Genes in Model Legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.

The Role of the REPLUMLESS Homeodomain Protein in Patterning the Arabidopsis Fruit

The outside of the Arabidopsis thaliana fruit consists of three principal tissues: the valves or seedpod walls, the replum or central ridge between the valves, and the valve margins where the valves separate from the replum to disperse the seeds. Previous studies have shown that valve margin formation is specified by the SHATTERPROOF MADS-box transcription factors and that valve development is controlled by the FRUITFULL MADS-box transcription factor. FRUITFULL negatively regulates SHATTERPROOF to prevent the valves from adopting a valve margin cell fate. Here we identify a gene called REPLUMLESS that is required for replum development. REPLUMLESS encodes a homeodomain protein that prevents replum cells from adopting a valve margin cell fate by negatively regulating expression of the SHATTERPROOF genes. Both REPLUMLESS and FRUITFULL are required to limit SHATTERPROOF expression to a narrow stripe of cells so that the valve margin differentiates precisely at the valve/replum boundary.

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.

The NGATHA Genes Direct Style Development in the Arabidopsis Gynoecium

The gynoecium is the most complex floral organ, designed to protect the ovules and ensure their fertilization. Correct patterning and tissue specification in the developing gynoecium involves the concerted action of a host of genetic factors. In addition, apical-basal patterning into different domains, stigma and style, ovary and gynophore, appears to depend on the establishment and maintenance of asymmetric auxin distribution, with an auxin maximum at the apex. Here, we show that a small subfamily of the B3 transcription factor superfamily, the NGATHA (NGA) genes, act redundantly to specify style development in a dosage-dependent manner. Characterization of the NGA gene family is based on an analysis of the activation-tagged mutant named tower-of-pisa1 (top1), which was found to overexpress NGA3. Quadruple nga mutants completely lack style and stigma development. This mutant phenotype is likely caused by a failure to activate two auxin biosynthetic enzymes, YUCCA2 and YUCCA4, in the apical gynoecium domain. The NGA mutant phenotypes are similar to those caused by multiple combinations of mutations in STYLISH1 (STY1) and additional members of its family. NGA3/TOP1 and STY1 share almost identical patterns of expression, but they do not appear to regulate each other at the transcriptional level. Strong synergistic phenotypes are observed when nga3/top1 and sty1 mutants are combined. Furthermore, constitutive expression of both NGA3/TOP1 and STY1 induces the conversion of the ovary into style tissue. Taken together, these data suggest that the NGA and STY factors act cooperatively to promote style specification, in part by directing YUCCA-mediated auxin synthesis in the apical gynoecium domain.

INDEHISCENT and SPATULA Interact to Specify Carpel and Valve Margin Tissue and Thus Promote Seed Dispersal in Arabidopsis

Mobile signals provided by hormones and morphogens are essential to organize multicellular structures. This article demonstrates that the joint activity of two bHLH transcription factors is required at two separate stages during Arabidopsis gynoecium and fruit development. In both instances, these factors mediate their function by ensuring appropriate distribution of the plant hormone auxin. Structural organization of organs in multicellular organisms occurs through intricate patterning mechanisms that often involve complex interactions between transcription factors in regulatory networks. For example, INDEHISCENT (IND), a basic helix-loop-helix (bHLH) transcription factor, specifies formation of the narrow stripes of valve margin tissue, where Arabidopsis thaliana fruits open on maturity. Another bHLH transcription factor, SPATULA (SPT), is required for reproductive tissue development from carpel margins in the Arabidopsis gynoecium before fertilization. Previous studies have therefore assigned the function of SPT to early gynoecium stages and IND to later fruit stages of reproductive development. Here we report that these two transcription factors interact genetically and via protein–protein contact to mediate both gynoecium development and fruit opening. We show that IND directly and positively regulates the expression of SPT, and that spt mutants have partial defects in valve margin formation. Careful analysis of ind mutant gynoecia revealed slight defects in apical tissue formation, and combining mutations in IND and SPT dramatically enhanced both single-mutant phenotypes. Our data show that SPT and IND at least partially mediate their joint functions in gynoecium and fruit development by controlling auxin distribution and suggest that this occurs through cooperative binding to regulatory sequences in downstream target genes.

Common regulatory networks in leaf and fruit patterning revealed by mutations in the Arabidopsis ASYMMETRIC LEAVES1 gene

Carpels and leaves are evolutionarily related organs, as the former are thought to be modified leaves. Therefore, developmental pathways that play crucial roles in patterning both organs are presumably conserved. In leaf primordia of Arabidopsis thaliana, the ASYMMETRIC LEAVES1 (AS1) gene interacts with AS2 to repress the class I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP), KNAT2 and KNAT6, restricting the expression of these genes to the meristem. In this report, we describe how AS1, presumably in collaboration with AS2, patterns the Arabidopsis gynoecium by repressing BP, which is expressed in the replum and valve margin, interacts in the replum with REPLUMLESS (RPL), an essential gene for replum development, and positively regulates the expression of this gene. Misexpression of BP in the gynoecium causes an increase in replum size, while the valve width is slightly reduced, and enhances the effect of mutations in FRUITFULL (FUL), a gene with an important function in valve development. Altogether, these findings strongly suggest that BP plays a crucial role in replum development. We propose a model for pattern formation along the mediolateral axis of the ovary, whereby three domains (replum, valve margin and valve) are specified by the opposing gradients of two antagonistic factors, valve factors and replum factors, the class I KNOX genes working as the latter.

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.

Flower development in pisum sativum: from the war of the whorls to the battle of the common primordia

The ontogeny of pea (Pisum sativum L.) flowers, as in many legume and nonlegume plant species, proceeds through a very different sequence of events from the same process in Antirrhinum majus and Arabidopsis thaliana. Using scanning electron microscopic analysis, we have characterized the early development of wild-type pea flowers and selected morphological characters or markers to break it down into different developmental stages. We used these markers as tools to characterize early alterations in flower development of several pea floral homeotic mutants. These mutants display phenotypes resembling those of: (1) floral meristem identity mutations, frondosus (brac); (2) class A mutations, calix carpellaris (cc); (3) class B mutations, stamina pistilloida (stp-1 and stp-2); and (4) class C mutations, petalosus (pe). According to the homeotic transformations observed in the pea floral mutants, it would appear feasible that the identity and developmental pattern of the four organ types in pea flowers are governed by at least the same three developmental functions, A, B, and C, proposed for the two model systems. However, our results suggest that, in pea, although these functions do have a similar role in the specification of organ identity shown by their counterparts in Arabidopsis or Antirrhinum, they may differ in the control of other processes, such as floral determinacy, organ number, or leaf development. The more remarkable features of pea flower ontogeny were the existence of four common primordia to petals and stamens, the early carpel primordium initiation, and the abaxial-adaxial unidirectional initiation of organ primordia within each different floral whorl, in contrast to the centripetal and sequential floral ontogeny in other plants. Organ differentiation within each of these common primordia appears to be a complex process that plays a central role in the ontogeny of pea flowers. Analysis of flower developmental pea homeotic mutants suggests that A, B, and C functions are necessary for the correct differentiation of organs from common primordia and that, in addition to its role in the specification of petals and stamens, B function, would be involved in conferring common primordia identity. Copyright 1999 Wiley-Liss, Inc.

Dynamic, auxin-responsive plasma membrane-to-nucleus movement of Arabidopsis BRX

In Arabidopsis, interplay between nuclear auxin perception and trans-cellular polar auxin transport determines the transcriptional auxin response. In brevis radix (brx) mutants, this response is impaired, probably indirectly because of disturbed crosstalk between the auxin and brassinosteroid pathways. Here we provide evidence that BRX protein is plasma membrane-associated, but translocates to the nucleus upon auxin treatment to modulate cellular growth, possibly in conjunction with NGATHA class B3 domain-type transcription factors. Application of the polar auxin transport inhibitor naphthalene phthalamic acid (NPA) resulted in increased BRX abundance at the plasma membrane. Thus, nuclear translocation of BRX could depend on cellular auxin concentration or on auxin flux. Supporting this idea, NPA treatment of wild-type roots phenocopied the brx root meristem phenotype. Moreover, BRX is constitutively turned over by the proteasome pathway in the nucleus. However, a stabilized C-terminal BRX fragment significantly rescued the brx root growth phenotype and triggered a hypocotyl gain-of-function phenotype, similar to strong overexpressors of full length BRX. Therefore, although BRX activity is required in the nucleus, excess activity interferes with normal development. Finally, similar to the PIN-FORMED 1 (PIN1) auxin efflux carrier, BRX is polarly localized in vascular cells and subject to endocytic recycling. Expression of BRX under control of the PIN1 promoter fully rescued the brx short root phenotype, suggesting that the two genes act in the same tissues. Collectively, our results suggest that BRX might provide a contextual readout to synchronize cellular growth with the auxin concentration gradient across the root tip.

Instructive roles for hormones in plant development

Plants, like animals, construct their body following modular sets of instructions that determine cell fate, morphogenesis and patterning, among other building requirements. Hormones regulate plant growth in different ways, and there is increasing evidence for a decisive function of certain hormones in the establishment of developmental programs, equivalent to the role of peptidic molecules and signals of another nature in animal embryo development. Here, we review this role of hormones as instructive agents, and illustrate it with examples such as the generation of morphogenetic gradients by auxin (which determine organ patterning and phyllotaxis), the specification of cell fate at the shoot meristem by gibberellins and cytokinins, the switch between alternative developmental programs (photo- and skotomorphogenesis) by gibberellins and brassinosteroids, and the decision between pistil senescence or fruit growth after anthesis.