Ana Berbel

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.

Control of dissected leaf morphology by a Cys(2)His(2) zinc finger transcription factor in the model legume Medicago truncatula

Plant leaves are diverse in their morphology, reflecting to a large degree the plant diversity in the natural environment. How different leaf morphology is determined is not yet understood. The leguminous plant Medicago truncatula exhibits dissected leaves with three leaflets at the tip. We show that development of the trifoliate leaves is determined by the Cys(2)His(2) zinc finger transcription factor PALM1. Loss-of-function mutants of PALM1 develop dissected leaves with five leaflets clustered at the tip. We demonstrate that PALM1 binds a specific promoter sequence and down-regulates the expression of the M. truncatula LEAFY/UNIFOLIATA orthologue SINGLE LEAFLET1 (SGL1), encoding an indeterminacy factor necessary for leaflet initiation. Our data indicate that SGL1 is required for leaflet proliferation in the palm1 mutant. Interestingly, ectopic expression of PALM1 effectively suppresses the lobed leaf phenotype from overexpression of a class 1 KNOTTED1-like homeobox protein in Arabidopsis plants. Taken together, our results show that PALM1 acts as a determinacy factor, regulates the spatial-temporal expression of SGL1 during leaf morphogenesis and together with the LEAFY/UNIFOLIATA orthologue plays an important role in orchestrating the compound leaf morphology in M. truncatula.

VEGETATIVE1 is essential for development of the compound inflorescence in pea

Unravelling the basis of variation in inflorescence architecture is important to understanding how the huge diversity in plant form has been generated. Inflorescences are divided between simple, as in Arabidopsis, with flowers directly formed at the main primary inflorescence axis, and compound, as in legumes, where they are formed at secondary or even higher order axes. The formation of secondary inflorescences predicts a novel genetic function in the development of the compound inflorescences. Here we show that in pea this function is controlled by VEGETATIVE1 (VEG1), whose mutation replaces secondary inflorescences by vegetative branches. We identify VEG1 as an AGL79-like MADS-box gene that specifies secondary inflorescence meristem identity. VEG1 misexpression in meristem identity mutants causes ectopic secondary inflorescence formation, suggesting a model for compound inflorescence development based on antagonistic interactions between VEG1 and genes conferring primary inflorescence and floral identity. Our study defines a novel mechanism to generate inflorescence complexity.

Functional conservation of PISTILLATA activity in a pea homolog lacking the PI motif.

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.

Regulation of compound leaf development by PHANTASTICA in Medicago truncatula

A MYB domain protein maintains petiole identity by repressing expression of a motor organ identity gene and promoting petiole and stipule development in Medicago truncatula. Plant leaves, simple or compound, initiate as peg-like structures from the peripheral zone of the shoot apical meristem, which requires class I KNOTTED-LIKE HOMEOBOXI (KNOXI) transcription factors to maintain its activity. The MYB domain protein encoded by the ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) gene, together with other factors, excludes KNOXI gene expression from incipient leaf primordia to initiate leaves and specify leaf adaxial identity. However, the regulatory relationship between ARP and KNOXI is more complex in compound-leafed species. Here, we investigated the role of ARP and KNOXI genes in compound leaf development in Medicago truncatula. We show that the M. truncatula phantastica mutant exhibited severe compound leaf defects, including curling and deep serration of leaf margins, shortened petioles, increased rachises, petioles acquiring motor organ characteristics, and ectopic development of petiolules. On the other hand, the M. truncatula brevipedicellus mutant did not exhibit visible compound leaf defects. Our analyses show that the altered petiole development requires ectopic expression of ELONGATED PETIOLULE1, which encodes a lateral organ boundary domain protein, and that the distal margin serration requires the auxin efflux protein M. truncatula PIN-FORMED10 in the M. truncatula phantastica mutant.

Genetic control of inflorescence architecture in legumes.

The architecture of the inflorescence, the shoot system that bears the flowers, is a main component of the huge diversity of forms found in flowering plants. Inflorescence architecture has also a strong impact on the production of fruits and seeds, and on crop management, two highly relevant agronomical traits. Elucidating the genetic networks that control inflorescence development, and how they vary between different species, is essential to understanding the evolution of plant form and to being able to breed key architectural traits in crop species. Inflorescence architecture depends on the identity and activity of the meristems in the inflorescence apex, which determines when flowers are formed, how many are produced and their relative position in the inflorescence axis. Arabidopsis thaliana, where the genetic control of inflorescence development is best known, has a simple inflorescence, where the primary inflorescence meristem directly produces the flowers, which are thus borne in the main inflorescence axis. In contrast, legumes represent a more complex inflorescence type, the compound inflorescence, where flowers are not directly borne in the main inflorescence axis but, instead, they are formed by secondary or higher order inflorescence meristems. Studies in model legumes such as pea (Pisum sativum) or Medicago truncatula have led to a rather good knowledge of the genetic control of the development of the legume compound inflorescence. In addition, the increasing availability of genetic and genomic tools for legumes is allowing to rapidly extending this knowledge to other grain legume crops. This review aims to describe the current knowledge of the genetic network controlling inflorescence development in legumes. It also discusses how the combination of this knowledge with the use of emerging genomic tools and resources may allow rapid advances in the breeding of grain legume crops.

Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture

Highlight Plants carefully control where and when flowers are made through activators and repressors. We show that spatially the shoot meristem is key in responding to the repressors of flowering TFL1.

Pea VEGETATIVE2 Is an FD Homolog That Is Essential for Flowering and Compound Inflorescence Development

Mutant phenotypes reveal a central role for VEG2 in flowering and compound inflorescence formation and suggest that transcription of FT/TFL1 genes is regulated by VEG2-FT protein complexes. As knowledge of the gene networks regulating inflorescence development in Arabidopsis thaliana improves, the current challenge is to characterize this system in different groups of crop species with different inflorescence architecture. Pea (Pisum sativum) has served as a model for development of the compound raceme, characteristic of many legume species, and in this study, we characterize the pea VEGETATIVE2 (VEG2) locus, showing that it is critical for regulation of flowering and inflorescence development and identifying it as a homolog of the bZIP transcription factor FD. Through detailed phenotypic characterizations of veg2 mutants, expression analyses, and the use of protein-protein interaction assays, we find that VEG2 has important roles during each stage of development of the pea compound inflorescence. Our results suggest that VEG2 acts in conjunction with multiple FLOWERING LOCUS T (FT) proteins to regulate expression of downstream target genes, including TERMINAL FLOWER1, LEAFY, and MADS box homologs, and to facilitate cross-regulation within the FT gene family. These findings further extend our understanding of the mechanisms underlying compound inflorescence development in pea and may have wider implications for future manipulation of inflorescence architecture in related legume crop species.

AUXIN RESPONSE FACTOR3 Regulates Compound Leaf Patterning by Directly Repressing PALMATE-LIKE PENTAFOLIATA1 Expression in Medicago truncatula

Diverse leaf forms can be seen in nature. In Medicago truncatula, PALM1 encoding a Cys(2)His(2) transcription factor is a key regulator of compound leaf patterning. PALM1 negatively regulates expression of SGL1, a key regulator of lateral leaflet initiation. However, how PALM1 itself is regulated is not yet known. To answer this question, we used promoter sequence analysis, yeast one-hybrid tests, quantitative transcription activity assays, ChIP-PCR analysis, and phenotypic analyses of overexpression lines and mutant plants. The results show that M. truncatula AUXIN RESPONSE FACTOR3 (MtARF3) functions as a direct transcriptional repressor of PALM1. MtARF3 physically binds to the PALM1 promoter sequence in yeast cells. MtARF3 selectively interacts with specific auxin response elements (AuxREs) in the PALM1 promoter to repress reporter gene expression in tobacco leaves and binds to specific sequences in the PALM1 promoter in vivo. Upregulation of MtARF3 or removal of both PHANTASTICA (PHAN) and ARGONAUTE7 (AGO7) pathways resulted in compound leaves with five narrow leaflets arranged in a palmate-like configuration. These results support that MtARF3, in addition as an adaxial-abaxial polarity regulator, functions to restrict spatiotemporal expression of PALM1, linking auxin signaling to compound leaf patterning in the legume plant M. truncatula.

Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones

Key messageExpression analysis of theAG-subfamily members fromG. hirsutumduring flower and fruit development.Abstract Reproductive development in cotton, including the fruit and fiber formation, is a complex process; it involves the coordinated action of gene expression regulators, and it is highly influenced by plant hormones. Several studies have reported the identification and expression of the transcription factor family MADS-box members in cotton ovules and fibers; however, their roles are still elusive during the reproductive development in cotton. In this study, we evaluated the expression profiles of five MADS-box genes (GhMADS3, GhMADS4, GhMADS5, GhMADS6 and GhMADS7) belonging to the AGAMOUS-subfamily in Gossypium hirsutum. Phylogenetic and protein sequence analyses were performed using diploid (G. arboreum, G. raimondii) and tetraploid (G. barbadense, G. hirsutum) cotton genomes, as well as the AG-subfamily members from Arabidopsis thaliana, Petunia hybrida and Antirrhinum majus. qPCR analysis showed that the AG-subfamily genes had high expression during flower and fruit development in G. hirsutum. In situ hybridization analysis also substantiates the involvement of AG-subfamily members on reproductive tissues of G. hirsutum, including ovule and ovary. The effect of plant hormones on AG-subfamily genes expression was verified in cotton fruits treated with gibberellin, auxin and brassinosteroid. All the genes were significantly regulated in response to auxin, whereas only GhMADS3, GhMADS4 and GhMADS7 genes were also regulated by brassinosteroid treatment. In addition, we have investigated the GhMADS3 and GhMADS4 overexpression effects in Arabidopsis plants. Interestingly, the transgenic plants from both cotton AG-like genes in Arabidopsis significantly altered the fruit size compared to the control plants. This alteration suggests that cotton AG-like genes might act regulating fruit formation. Our results demonstrate that members of the AG-subfamily in G. hirsutum present a conserved expression profile during flower development, but also demonstrate their expression during fruit development and in response to phytohormones.