The Dynamics of Flower Development in Castanea sativa Mill.

The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein-protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.

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.

Unisexual flower initiation in the monoecious Quercus suber L.: a molecular approach.

Several plant species display a temporal separation of the male and female flower organ development to enhance outbreeding; however, little is known regarding the genetic mechanisms controlling this temporal separation. Quercus suber is a monoecious oak tree with accentuated protandry: in late winter, unisexual male flowers emerge adjacent to the swollen buds, whereas unisexual female flowers emerge in the axils of newly formed leaves formed during spring (4-8 weeks after male flowering). Here, a phylogenetic profiling has led to the identification of cork oak homologs of key floral regulatory genes. The role of these cork oak homologs during flower development was identified with functional studies in Arabidopsis thaliana. The expression profile throughout the year of flower regulators (inducers and repressors), in leaves and buds, suggests that the development of male and female flowers may be preceded by separated induction events. Female flowers are most likely induced during the vegetative flush occurring in spring, whereas male flowers may be induced in early summer. Male flowers stay enclosed within the pre-dormant buds, but complete their development before the vegetative flush of the following year, displaying a long period of anthesis that spans the dormant period. Our results portray a genetic mechanism that may explain similar reproductive habits in other monoecious tree species.

Genome-Wide Identification of Epigenetic Regulators in Quercus suber L.

Modifications of DNA and histones, including methylation and acetylation, are critical for the epigenetic regulation of gene expression during plant development, particularly during environmental adaptation processes. However, information on the enzymes catalyzing all these modifications in trees, such as Quercus suber L., is still not available. In this study, eight DNA methyltransferases (DNA Mtases) and three DNA demethylases (DDMEs) were identified in Q. suber. Histone modifiers involved in methylation (35), demethylation (26), acetylation (8), and deacetylation (22) were also identified in Q. suber. In silico analysis showed that some Q. suber DNA Mtases, DDMEs and histone modifiers have the typical domains found in the plant model Arabidopsis, which might suggest a conserved functional role. Additional phylogenetic analyses of the DNA and histone modifier proteins were performed using several plant species homologs, enabling the classification of the Q. suber proteins. A link between the expression levels of each gene in different Q. suber tissues (buds, flowers, acorns, embryos, cork, and roots) with the functions already known for their closest homologs in other species was also established. Therefore, the data generated here will be important for future studies exploring the role of epigenetic regulators in this economically important species.

Evolutionary Conservation of the Orchid MYB Transcription Factors DIV, RAD, and DRIF.

The MYB transcription factors DIVARICATA (DIV), DIV-and-RAD-Interacting-Factor (DRIF), and the small interfering peptide RADIALIS (RAD) can interact, forming a regulatory module that controls different plant developmental processes. In the snapdragon Antirrhinum majus, this module, together with the TCP transcription factor CYCLOIDEA (CYC), is responsible for the establishment of floral dorsoventral asymmetry. The spatial gene expression pattern of the OitDIV, OitDRIF, and OitRAD homologs of Orchis italica, an orchid with zygomorphic flowers, has suggested a possible conserved role of these genes in bilateral symmetry of the orchid flower. Here, we have identified four DRIF genes of orchids and have reconstructed their genomic organization and evolution. In addition, we found snapdragon transcriptional cis-regulatory elements of DIV and RAD loci generally conserved within the corresponding orchid orthologues. We have tested the biochemical interactions among OitDIV, OitDRIF1, and OitRAD of O. italica, showing that OitDRIF1 can interact both with OitDIV and OitRAD, whereas OitDIV and OitRAD do not directly interact, as in A. majus. The analysis of the quantitative expression profile of these MYB genes revealed that in zygomorphic orchid flowers, the DIV, DRIF1, and RAD transcripts are present at higher levels in the lip than in lateral inner tepals, whereas in peloric orchid flowers they show similar expression levels. These results indicate that MYB transcription factors could have a role in shaping zygomorphy of the orchid flower, potentially enriching the underlying orchid developmental code.

Successive Domain Rearrangements Underlie the Evolution of a Regulatory Module Controlled by a Small Interfering Peptide.

The establishment of new interactions between transcriptional regulators increases the regulatory diversity that drives phenotypic novelty. To understand how such interactions evolve, we have studied a regulatory module (DDR) composed by three MYB-like proteins: DIVARICATA (DIV), RADIALIS (RAD), and DIV-and-RAD-Interacting Factor (DRIF). The DIV and DRIF proteins form a transcriptional complex that is disrupted in the presence of RAD, a small interfering peptide, due to the formation of RAD-DRIF dimers. This dynamic interaction result in a molecular switch mechanism responsible for the control of distinct developmental processes in plants. Here, we have determined how the DDR regulatory module was established by analyzing the origin and evolution of the DIV, DRIF, and RAD protein families and the evolutionary history of their interactions. We show that duplications of a pre-existing MYB domain originated the DIV and DRIF protein families in the ancestral lineage of green algae, and, later, the RAD family in seed plants. Intraspecies interactions between the MYB domains of DIV and DRIF proteins are detected in green algae, whereas the earliest evidence of an interaction between DRIF and RAD proteins occurs in the gymnosperms, coincident with the establishment of the RAD family. Therefore, the DDR module evolved in a stepwise progression with the DIV-DRIF transcription complex evolving prior to the antagonistic RAD-DRIF interaction that established the molecular switch mechanism. Our results suggest that the successive rearrangement and divergence of a single protein domain can be an effective evolutionary mechanism driving new protein interactions and the establishment of novel regulatory modules.

Role of floral organ identity genes in the development of unisexual flowers of Quercus suber L.

Monoecious species provide an excellent system to study the specific determinants that underlie male and female flower development. Quercus suber is a monoecious species with unisexual flowers at inception. Despite the overall importance of this and other tree species with a similar reproductive habit, little is known regarding the mechanisms involved in the development of their male and female flowers. Here, we have characterised members of the ABCDE MADS-box gene family of Q. suber. The temporal expression of these genes was found to be sex-biased. The B-class genes, in particular, are predominantly, or exclusively (in the case of QsPISTILLATA), expressed in the male flowers. Functional analysis in Arabidopsis suggests that the B-class genes have their function conserved. The identification of sex-biased gene expression plus the identification of unusual protein-protein interactions suggest that the floral organ identity of Q. suber may be under control of specific changes in the dynamics of the ABCDE model. This study constitutes a major step towards the characterisation of the mechanisms involved in reproductive organ identity in a monoecious tree with a potential contribution towards the knowledge of conserved developmental mechanisms in other species with a similar sex habit.

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein.

Silk-elastin-like proteins (SELPs) are a family of genetically engineered recombinant protein polymers exhibiting mechanical and biological properties suited for a wide range of applications in the biomedicine and materials fields. They are being explored as the next generation of biomaterials but low productivities and use of antibiotics during production undermine their economic viability and safety. We have developed an industrially relevant, scalable, fed-batch process for the high level production of a novel SELP in E. coli in which the commonly used antibiotic selection marker of the expression vector is exchanged for a post segregational suicide system, the separate-component-stabilisation system (SCS). SCS significantly augments SELP productivity but also enhances the product safety profile and reduces process costs by eliminating the use of antibiotics. Plasmid content increased following induction but no significant differences in plasmid levels were discerned when using SCS or the antibiotic selection markers under the controlled fed-batch conditions employed. It is suggested that the absence of competing plasmid-free cells improves host cell viability and enables increased productivity with SCS. With the process developed, 12.8 g L-1 purified SELP was obtained, this is the highest SELP productivity reported to date and clearly demonstrates the commercial viability of these promising polymers.

Arabinogalactan proteins and pectin distribution during female gametogenesis in Quercus suber L.

BACKGROUND AND AIMS: Quercus suber L. (cork oak) is one of the most important monoecious tree species in semi-arid regions of Southern Europe, with a high ecological value and economic potential. However, as a result of its long reproductive cycle, complex reproductive biology and recalcitrant seeds, conventional breeding is demanding. In its complex reproductive biology, little is known about the most important changes that occur during female gametogenesis. Arabinogalactan proteins (AGPs) and pectins are the main components of plant cell walls and have been reported to perform common functions in cell differentiation and organogenesis of reproductive plant structures. AGPs have been shown to serve as important molecules in several steps of the reproductive process in plants, working as signalling molecules, associated with the sporophyte-gametophyte transition, and pectins have been implicated in pollen-pistil interactions before double fertilization. In this study, the distribution of AGP and pectin epitopes was assessed during female gametogenesis. METHODS: Immunofluorescence labelling of female flower cells was performed with a set of monoclonal antibodies (mAbs) directed to the carbohydrate moiety of AGPs (JIM8 and JIM13) and pectic homogalacturonans (HGs) (mAbs JIM5 and JIM7). KEY RESULTS: The selective labelling obtained with AGP and pectin mAbs JIM8, JIM13, JIM5 and JIM7 during Q. suber female gametogenesis shows that AGPs and pectic HG can work as markers for mapping gametophytic cell differentiation in this species. Pectic HG showed different distribution patterns, depending on their levels of methyl esterification. Methyl-esterified HGs showed a uniform distribution in the overall female flower cells before fertilization and a more specific pattern after fertilization. A low methyl-ester pectin distribution pattern during the different developmental stages appears to be related to the pathway that pollen tubes follow to reach the embryo sac. AGPs showed a more sparse distribution in early stages of development, but specific labelling is shown in the synergids and their filiform apparatus. CONCLUSIONS: The labelling obtained with anti-AGP and anti-pectin mAbs in Q. suber female flower cells showed a dynamic distribution of AGPs and pectic HGs, which may render these molecules useful molecular markers during female gametogenesis. Changes occurring during development will be determined in order to help describe cork oak ovule structural properties before and after fertilization, providing new insight to better understand Q. suber female gametogenesis.

The Quest for Molecular Regulation Underlying Unisexual Flower Development.

The understanding of the molecular mechanisms responsible for the making of a unisexual flower has been a long-standing quest in plant biology. Plants with male and female flowers can be divided mainly into two categories: dioecious and monoecious, and both sexual systems co-exist in nature in ca of 10% of the angiosperms. The establishment of male and female traits has been extensively described in a hermaphroditic flower and requires the interplay of networks, directly and indirectly related to the floral organ identity genes including hormonal regulators, transcription factors, microRNAs, and chromatin-modifying proteins. Recent transcriptomic studies have been uncovering the molecular processes underlying the establishment of unisexual flowers and there are many parallelisms between monoecious, dioecious, and hermaphroditic individuals. Here, we review the paper entitled "Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber" published in 2014 in the Frontiers of Plant Science (volume 5 |Article 599) and discussed it in the context of recent studies with other dioecious and monoecious plants that utilized high-throughput platforms to obtain transcriptomic profiles of male and female unisexual flowers. In some unisexual flowers, the developmental programs that control organ initiation fail and male or female organs do not form, whereas in other species, organ initiation and development occur but they abort or arrest during different species-specific stages of differentiation. Therefore, a direct comparison of the pathways responsible for the establishment of unisexual flowers in different species are likely to reveal conserved modules of gene regulatory hubs involved in stamen or carpel development, as well as differences that reflect the different stages of development in which male and/or female organ arrest or loss-of-function occurs.

Evaluation of the presence of arabinogalactan proteins and pectins during Quercus suber male gametogenesis.

BACKGROUND AND AIMS: Quercus suber (cork oak) is a dominant tree of the Fagaceae in forests of the south-west Iberian Peninsula. It is monoecious with a long progamic phase that provides a comprehensive system for comparative studies in development and sexual reproduction. In this study the distribution of arabinogalactan protein (AGPs) and pectin epitopes in anthers of Q. suber was assessed to map these hydroxyproline-rich glycoproteins and the galacturonate-rich acidic polysaccharides during pollen development. Methods Immunolocalization in male flowers was performed with a set of monoclonal antibodies directed against the carbohydrate moiety that recognizes AGPs and pectins. To identify AGP genes involved in cork oak male flower development, a search was conducted for annotated AGP genes in the available transcriptome data of the Cork Oak EST Consortium database (www.corkoakdb.org). KEY RESULTS: Ubiquitous labelling in all cell types was obtained with anti-homogalacturan antibodies for methyl-esterified pectins. In contrast, the antibody that labelled non-methyl-esterified homogalacturans had a preferential presence in microsporocyte cells walls at the beginning of pollen development. Intense labelling was obtained with anti-AGP antibodies both in the tapetum and in the intine wall near the pollen apertures and later in the generative cell wall and vegetative cell. Evaluation of the putative AGPs highly expressed in the male gametophyte was achieved by quantitative RT-PCR analysis in male and female cork oak flowers. CONCLUSIONS: Four putative AGP genes were identified that are preferentially expressed in the male flower compared with the female flower. The putative Arabidopsis thaliana orthologues of these genes are associated with preferential expression in pollen, suggesting that the AGPs probably play a significant role in cork oak reproduction.

Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber.

Monoecious species provide a comprehensive system to study the developmental programs underlying the establishment of female and male organs in unisexual flowers. However, molecular resources for most monoecious non-model species are limited, hampering our ability to study the molecular mechanisms involved in flower development of these species. The objective of this study was to identify differentially expressed genes during the development of male and female flowers of the monoecious species Quercus suber, an economically important Mediterranean tree. Total RNA was extracted from different developmental stages of Q. suber flowers. Non-normalized cDNA libraries of male and female flowers were generated using 454 pyrosequencing technology producing a total of 962,172 high-quality reads with an average length of 264 nucleotides. The assembly of the reads resulted in 14,488 contigs for female libraries and 10,438 contigs for male libraries. Comparative analysis of the transcriptomes revealed genes differentially expressed in early and late stages of development of female and male flowers, some of which have been shown to be involved in pollen development, in ovule formation and in flower development of other species with a monoecious, dioecious, or hermaphroditic sexual system. Moreover, we found differentially expressed genes that have not yet been characterized and others that have not been previously shown to be implicated in flower development. This transcriptomic analysis constitutes a major step toward the characterization of the molecular mechanisms involved in flower development in a monoecious tree with a potential contribution toward the knowledge of conserved developmental mechanisms in other species.

A subcellular tug of war involving three MYB-like proteins underlies a molecular antagonism in Antirrhinum flower asymmetry.

The establishment of meristematic domains with different transcriptional activity is essential for many developmental processes. The asymmetry of the Antirrhinum majus flower is established by transcription factors with an asymmetric pattern of activity. To understand how this asymmetrical pattern is established, we studied the molecular mechanism through which the dorsal MYB protein RADIALIS (RAD) restricts the activity of the MYB transcription factor DIVARICATA (DIV) to the ventral region of the flower meristem. We show that RAD competes with DIV for binding with other MYB-like proteins, termed DRIF1 and DRIF2 (DIV- and-RAD-interacting-factors). DRIF1 and DIV interact to form a protein complex that binds to the DIV-DNA consensus region, suggesting that the DRIFs act as co-regulators of DIV transcriptional activity. In the presence of RAD, the interaction between DRIF1 and DIV bound to DNA is disrupted. Moreover, the DRIFs are sequestered in the cytoplasm by RAD, thus, preventing or reducing the formation of DRIF-DIV heterodimers in the nuclei. Our results suggest that in the dorsal region of the Antirrhinum flower meristem the dorsal protein RAD antagonises the activity of the ventral identity protein DIV in a subcellular competition for a DRIF protein promoting the establishment of the asymmetric pattern of gene activity in the Antirrhinum flower.