Morphological and anatomical traits during development: Highlighting extrafloral nectaries in Passiflora organensis.

Passiflora organensis is a small herbaceous vine with characteristic morphological variations throughout its development. The plant bears button-shaped extrafloral nectaries exclusively in adult leaves. Extrafloral nectaries are structures that secrete nectar and play an important role in plant-animal interactions as a strategy for protecting plants against herbivory. In this work, we performed anatomical and ultrastructural studies to characterize P. organensis extrafloral nectaries during their secretory phase. We showed extrafloral nectaries in Passiflora organensis are composed of three distinct regions: nectary epidermis, nectariferous parenchyma, and subnectariferous parenchyma. Our data suggests that all nectary regions constitute a functional unit involved in nectar production and release. The high metabolic activity in the nectary cells is characterized by the juxtaposition of organelles such as mitochondria and plastids together plasmalemma. In addition, calcium oxalate crystals are frequently associated to the nectaries. An increasing concentration of calcium during leaf development and nectary differentiation was observed, corresponding to the calcium deposition as calcium oxalate crystals. This is the first description of extrafloral nectaries in Passiflora organensis that is a promising tropical model species for several studies. RESEARCH HIGHLIGHTS: The anatomical and ultrastructural characteristics and the presence of calcium oxalate crystals in the nectary tissue suggest novel strategies against herbivory in the genus Passiflora.

Passiflora organensis FT/TFL1 gene family and their putative roles in phase transition and floral initiation.

KEY MESSAGE: Comprehensive analysis of the FT/TFL1 gene family in Passiflora organensis results in understanding how these genes might be involved in the regulation of the typical plant architecture presented by Passiflora species. Passion fruit (Passiflora spp) is an economic tropical fruit crop, but there is hardly any knowledge available about the molecular control of phase transition and flower initiation in this species. The florigen agent FLOWERING LOCUS T (FT) interacts with the bZIP protein FLOWERING LOCUS D (FD) to induce flowering in the model species Arabidopsis thaliana. Current models based on research in rice suggest that this interaction is bridged by 14-3-3 proteins. We identified eight FT/TFL1 family members in Passiflora organensis and characterized them by analyzing their phylogeny, gene structure, expression patterns, protein interactions and putative biological roles by heterologous expression in Arabidopsis. PoFT was highest expressed during the adult vegetative phase and it is supposed to have an important role in flowering induction. In contrast, its paralogs PoTSFs were highest expressed in the reproductive phase. While ectopic expression of PoFT in transgenic Arabidopsis plants induced early flowering and inflorescence determinacy, the ectopic expression of PoTSFa caused a delay in flowering. PoTFL1-like genes were highest expressed during the juvenile phase and their ectopic expression caused delayed flowering in Arabidopsis. Our protein-protein interaction studies indicate that the flowering activation complexes in Passiflora might deviate from the hexameric complex found in the model system rice. Our results provide insights into the potential functions of FT/TFL1 gene family members during floral initiation and their implications in the special plant architecture of Passiflora species, contributing to more detailed studies on the regulation of passion fruit reproduction.

SCI1 Is a Direct Target of AGAMOUS and WUSCHEL and Is Specifically Expressed in the Floral Meristematic Cells.

The specified floral meristem will develop a pre-established number of floral organs and, thus, terminate the floral meristematic cells. The floral meristematic pool of cells is controlled, among some others, by WUSCHEL (WUS) and AGAMOUS (AG) transcription factors (TFs). Here, we demonstrate that the SCI1 (Stigma/style cell-cycle inhibitor 1) gene, a cell proliferation regulator, starts to be expressed since the floral meristem specification of Nicotiana tabacum and is expressed in all floral meristematic cells. Its expression is higher in the floral meristem and the organs being specified, and then it decreases from outside to inside whorls when the organs are differentiating. SCI1 is co-expressed with N. tabacum WUSCHEL (NtWUS) in the floral meristem and the whorl primordia at very early developmental stages. Later in development, SCI1 is co-expressed with NAG1 (N. tabacum AG) in the floral meristem and specialized tissues of the pistil. In silico analyses identified cis-regulatory elements for these TFs in the SCI1 genomic sequence. Yeast one-hybrid and electrophoresis mobility shift assay demonstrated that both TFs interact with the SCI1 promoter sequence. Additionally, the luciferase activity assay showed that NAG1 clearly activates SCI1 expression, while NtWUS could not do so. Taken together, our results suggest that during floral development, the spatiotemporal regulation of SCI1 by NtWUS and NAG1 may result in the maintenance or termination of proliferative cells in the floral meristem, respectively.

The MADS-box gene Agamous-like 11 is essential for seed morphogenesis in grapevine.

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype.

Perspectives for a Framework to Understand Aril Initiation and Development.

A differentiated structure called "aril" has been described in seeds of several plant species during the course of evolution and might be considered as a supernumerary integument. Besides its ecological function in seed dispersal, the structure also represents a relevant character for systematic classification and exhibits important properties that impart agronomic value in certain species. Little is known about the molecular pathways underlying this morphological innovation because it is absent in currently used model species. A remarkable feature of the seeds of Passiflora species is the presence of a conspicuous aril. This genus is known for the ornamental, medicinal, and food values of its species. In view of the molecular resources and tools available for some Passiflora species, we highlight the potential of these species as models for developmental studies of the aril.

MAEWEST expression in flower development of two petunia species.

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription-quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions.

Does variation in genome sizes reflect adaptive or neutral processes? New clues from Passiflora.

One of the long-standing paradoxes in genomic evolution is the observation that much of the genome is composed of repetitive DNA which has been typically regarded as superfluous to the function of the genome in generating phenotypes. In this work, we used comparative phylogenetic approaches to investigate if the variations in genome sizes (GS) should be considered as adaptive or neutral processes by the comparison between GS and flower diameters (FD) of 50 Passiflora species, more specifically, within its two most species-rich subgenera, Passiflora and Decaloba. For this, we have constructed a phylogenetic tree of these species, estimated GS and FD of them, inferred the tempo and mode of evolution of these traits and their correlations, using both current and phylogenetically independent contrasted values. We found significant correlations among the traits, when considering the complete set of data or only the subgenus Passiflora, whereas no correlations were observed within Decaloba. Herein, we present convincing evidence of adaptive evolution of GS, as well as clues that this pattern is limited by a minimum genome size, which could reduce both the possibilities of changes in GS and the possibility of phenotypic responses to environment changes.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development.

A cDNA encoding a small lysine-rich protein of unknown function was identified in a tobacco (Nicotiana tabacum) stigma/style suppression subtractive hybridization cDNA library. After its characterization, the corresponding gene was designated stigma/style cell cycle inhibitor 1 (SCI1). Fluorescence microscopy with an SCI1-GFP protein fusion demonstrated its nuclear localization, which was confined to the interchromatic region. Real-time RT-PCR and in situ hybridization experiments showed that SCI1 is stigma/style-specific and developmentally regulated. SCI1 RNAi knockdown and overexpression plants had stigmas/styles with remarkably enlarged and reduced areas, respectively, which was attributable to differences in cell numbers. These results indicate that SCI1 is a tissue-specific negative cell cycle regulator. The differences in cell division had an effect on the timing of the differentiation of the stigmatic papillar cells, suggesting that their differentiation is coupled to stigma cell divisions. This is consistent with a role for SCI1 in triggering differentiation through cell proliferation control. Our results revealed that SCI1 is a novel tissue-specific gene that controls cell proliferation/differentiation, probably as a component of a developmental signal transduction pathway.

MADS: the missing link between identity and growth?

Size and shape are intrinsic characteristics of any given plant organ and, therefore, are inherently connected with its identity. How the connection between identity and growth is established at the molecular level remains one of the key questions in developmental biology. The identity of floral organs is determined by a hierarchical combination of transcription factors, most of which belong to the MADS box family. Recent progress in finding the target genes of these master regulators reopened the debate about the missing link between identity and floral organ growth. Here, we review these novel findings and integrate them into a model, to show how MADS proteins, in concert with co-factors, could fulfill their role at later stages of floral organ development when size and shape are established.

Characterization of ScMat1, a putative TFIIH subunit from sugarcane.

The general transcription factor TFIIH is a multiprotein complex with different enzymatic activities such as helicase, protein kinase and DNA repair. MAT1 (ménage à trois 1) is one of the TFIIH subunits that has kinase activity and it is the third subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), CDK7- cyclin H. The main objective of this work was to characterize ScMAT1, a sugarcane gene encoding a MAT1 homolog. Northern blots and in situ hybridization results showed that ScMAT1 was expressed in sugarcane mature leaf, leaf roll and inflorescence, and it was not differentially expressed in any of the other tissues analyzed such us bud and roots. In addition, ScMAT1 was not differentially expressed during different stress conditions and treatment with hormones. In situ hybridization analyses also showed that ScMAT1 was expressed in different cell types during leaf development. In order to identify proteins that interact with ScMAT1, a yeast two hybrid assay with ScMAT1 as bait was used to screen a sugarcane leaf cDNA library. The screening of yeast two hybrids yielded 14 positive clones. One of them is a cytochrome p450 family protein involved in oxidative degradation of toxic compounds. Other clones isolated are also related to plant responses to stress. To determine the subcellular localization of ScMAT1, a ScMAT1-GFP fusion was assayed in onion epidermal cell and the fluorescence was localized to the nucleus, in agreement with the putative role of ScMAT1 as a basal transcription factor.

A genomic approach to elucidating grass flower development

Como na maior parte das gramíneas, num determinado momento do seu ciclo de vida, o meristema vegetativo da cana-de-açúcar é convertido em meristema reprodutivo. Em cana-de-açúcar há pelo menos duas conversões meristemáticas distintas entre a induçäo para o florescimento e a formaçäo do florete. Em espécies dicotiledôneas modelo, a conversäo sucessiva das identidades dos meristemas, bem como o arranjo concêntrico de órgäos florais säo controlados geneticamente. Todos os genes e/ou proteínas sabidamente envolvidos no desenvolvimento floral foram anotados e identificados no banco de dados do SUCEST (Sugarcane EST Project). Comparações de seqüências entre genes reconhecidamente envolvidos no controle do desenvolvimento floral revelaram a conservaçäo evolutiva entre os mecanismos de formaçäo do padräo de desenvolvimento floral entre mono- e dicotiledôneas, bem como entre as gramíneas. Nossos estudos se concentraram na análise das famílias multigênicas dos fatores de transcriçäo do tipo MADS-box e AP2, uma vez que estes têm um papel importante na regulaçäo do desenvolvimento reprodutivo vegetal. Também säo apresentadas considerações sobre a genética evolutiva do desenvolvimento das flores de gramíneas e sua relaçäo com o modelo ABC do desenvolvimento floral.