The duplicated B-class MADS-box genes display dualistic characters in orchid floral organ identity and growth

Orchidaceae are an excellent model to examine perianth development because of their sophisticated floral architecture. In this study, we identified 24 APETALA3 (AP3)-like and 13 PISTILLA (PI)-like genes from 11 species of orchids and characterized them into four AP3- and two PI-duplicated homologs. The first duplication event in AP3 homologs occurring in the early evolutionary history of the Orchidaceae gave rise to AP3A and AP3B clades. Further duplication events resulted in four subclades, namely AP3A1, AP3A2, AP3B1 and AP3B2, during the evolution of Orchidaceae. The AP3 paralogous genes were expressed throughout inflorescence and floral bud development. From the in situ hybridization results, we noticed that the transition timings from ubiquitous to constrained expression in floral organs for both clades are different. The transition point of expression of the AP3A clade (clades 3 and 4) was at the late floral organ primordia stage. In contrast, that for the AP3B clade (clades 1 and 2) was not observed until the late inflorescence and floral bud stages. In addition, the AP3 orthologous genes revealed diverse expression patterns in various species of orchids, whereas the PI homologs were uniformly expressed in all floral whorls. AP3A2 orthologs play a noticeable role in lip formation because of their exclusive expression in the lip. Further evidence comes from the ectopic expression of AP3A2 detected in the lip-like petals extending from the lip in four sets of peloric mutants. Finally, a Homeotic Orchid Tepal (HOT) model is proposed, in which dualistic characters of duplicated B-class MADS-box genes are involved in orchid perianth development and growth.     

Are Cape floral clades the same age? Contemporaneous origins of two lineages in the genistoids s.l. (Fabaceae)

The hypothesis that the elements of the modern species-rich flora of the Cape Floristic Region (CFR), South Africa, originated more or less simultaneously at the Miocene/Pliocene boundary, in response to the development of a mediterranean climate, has been challenged by numerous molecular dating estimates of Cape floral clades. These studies reveal a more gradual emergence, with the oldest clades originating in the Eocene, but others appearing later, some as recently as the Pliocene. That there are factors which might affect the dates recovered, such as choice of calibration point, analysis method, sampling density and the delimitation of Cape floral clades, suggests a need for further critical evaluation of the age estimates presented to date. In this study, the dates of origin of two Cape floral clades (the legume Crotalarieae p.p. and Podalyrieae) are estimated, constrained by a shared calibration point in a single analysis using an rDNA ITS phylogeny in which 633 taxa are sampled. The results indicate that these two clades arose contemporaneously 44-46 mya, not at the Miocene/Pliocene boundary as had been previously supposed. The contemporaneous origin of these Cape floral clades suggests that additional more inclusive analyses are needed before rejecting the hypothesis that a single environmental trigger explains the establishment of Cape floral clades.     

Expression Divergence and Functional Redundancy of Polygalacturonases in Floral Organ Abscission

The importance of cell separation in plant development cannot be overemphasized. The polygalacturonases (PGs) are the one of cell wall hydrolytic enzyme families that has been associated with various cell separation processes in plant development including seed germination, dehiscence, organ abscission, and fruit ripening. Both Arabidopsis and rice PG gene family have expanded in a lineage-specific fashion after the split more than 150 million years ago. Tandem duplications and large-scale duplications are the major contributors to the current PG family size in Arabidopsis. The spatial expression analysis of the 66 Arabidopsis PG family members have led us to conclude that different duplication mechanisms affect the expression divergence differently. This becomes more apparent when temporal examination of expression is conducted in five developmental stages of floral organ abscission in Arabidopsis. Nine distinct patterns of PGs are identified during floral organ abscission in Arabidopsis. Four PGs are specifically upregulated during abscission associated with cell separation process. Careful understanding of relationships among Arabidopsis PGs in a context of evolution together with expression analysis of these PGs will facilitate the functional study of PGs specifically in floral organ abscission in Arabidopsis.Key Words: cell separation, polygalacturonases, gene duplication, abscissionCell separation processes have been recognized for their involvement in plant development and more importantly, agricultural traits such as pollen dehiscence and the abscission of organs including leaves, floral organs, and fruits.^1–6 Among many cell wall hydrolytic enzymes, the PGs (polygalaturonases) have been shown to be associated with a wide range of these plant developmental programs such as seed germination, organ abscission, pod and anther dehiscence, pollen grain maturation, fruit softening and decay, xylem cell formation, and pollen tube growth.^4,7–9 Although the roles of the PG members in the various developmental programs have been hypothesized, many scientists still face challenges in developing a comprehensive understanding of the biological functions of the PG family, due to both size and possible redundancy.^10,11It has been previously reported that PGs from both tomato and Arabidopsis located in tandem clusters were derived from tandem duplications.^10,12 In addition, it has been shown by several groups that the Arabidopsis genome contains large blocks of related regions derived from whole genome duplication events.^13–15We have recently conducted a comparative analysis of PGs from Arabidopsis and rice in attempts to address following questions: (1) the patterns and extent of expansion of PG gene family in Arabidopsis and rice, (2) the mechanisms that contribute to the expansion of this gene family, (3) the degree of both spatial and temporal expression divergence amongst Arabidopsis PGs, and (4) possible mechanisms of duplicates retention and the biological roles in floral organ abscission.¹⁶In this study, we have shown that with the identification of the nodes that lead to each Arabidopsis-specific and rice-specific subfamily, there are at least 21 immediate ancestors before the split between these two organisms.¹⁶ Subsequent expansion events have been followed in a lineage specific mode in both organisms. We found that in Arabidopsis the PG family is the product of both tandem duplications and large-scale duplications, similar to other gene families such as the NBS-LRR¹⁷ and the RLK/Pelle gene family.¹⁸ As a matter of fact, more than one third of the Arabidopsis PGs (24 out of 66) were found in tandem clusters. In addition, using both AGI (Arabidopsis Genome initiative) and BHW (Blanc, Hokamp, Wolfe) blocks almost 90% (59 out of 66) of the PGs were included within large-scale duplication events.^13,15,16Interestingly, the correlation between the synonymous substitution rate (Ks) and the expression profile for the related PGs in the tandem clusters was not significant. However, the related PGs in the duplicated blocks from large-scale duplication events tend to have a similar expression pattern within the five major tissues (flowers, siliques, stems, rosette and cauline leaves, and roots). Thus, indicating tandem-duplicated PGs have higher levels of expression divergence compared with PGs found in the large-scale duplication blocks.Further examination of PG expression during specific aspects of plant growth such as the five developmental stages of floral organ abscission has led us to conclude that expression divergence between all of the PGs that showed no difference at the tissue level most likely have differences. Thus, by looking more closely at specific tissue types and time of development, the divergence in expression patterns can be revealed. This is also supported by the hypothesis that duplication mechanisms may contribute to divergence of expression differently. In addition, by identifying nine distinct patterns of PG expression during the five developmental stages of floral organ abscission, we provided candidate PGs important for studies of abscission. Considering the proposed roles of PGs in pectin modification and/or breakdown, four of the PGs (At2g41850, At2g43880, At2g43890, and At3g07970) that showed upregulated expression during floral organ abscission associated with cell separation were identified as “best candidates” for understanding floral organ abscission (Fig. 1A).Open in a separate windowFigure 1Floral organ abscission specific PGs and their T-DNA insertion lines. (A) Expression of four PGs that showed specific upregulation during floral organ abscission is demonstrated. X-axis represents the developmental stages of floral organ abscission. Pos. 1/2 and Pos. 4/5 represent pre-abscission, Pos. 7/8 represents during abscission, and Pos. 10/11 and Pos. 13/14 represent post-abscission. Y-axis represents the relative level of expression of these four PGs and the names of each gene are shown in the z-axis. Four arrows indicate the positions that are associated with cell separation and upregulation of the PGs during abscission. (B) Gene structures of abscission specific PGs and the positions of the T-DNA insertions in each mutant. Each arrow indicates the position of the T-DNA insertion for the mutant. At2g41850 (SALK_035098, red arrow head) is further examined for petal abscission zones using SEM as shown in (C). (C) Schematic diagram of flower positions in wild type in Arabidopsis and SEM of petal abscission zones from SALK_035098 and wild type. Flower position 1 denotes the flower position where white petals protrude right after anthesis and subsequent positions correspond to older stages of flowers. The days after anthesis are demonstrated with flower positions in the right. Note that at flower position 7/8 floral organs are abscised. When comparing during-and post-abscission (Pos. 7/8, Pos. 10/11 and Pos. 13/14) between the mutant and wild type, difference of a delay in floral organ abscission is not observed.In order to better understand the functions of these PGs in floral organ abscission, we also isolated homozygous T-DNA insertion lines (http://signal.salk.edu/cgi-bin/tdnaexpress) for three of these PGs (At2g41850, At2g43890, and At2g07970) associated with cell separation during abscission. We examined these mutants for a delay in floral organ abscission (Fig. 1B and C); however, nearly all of the single insertion lines examined did not display any notable phenotype regarding floral organ abscission even though in the T-DNA disruption line of At2g41850 showed slight delay positions around 7/8 (Fig. 1 and ​and1C).1C). One possible explanation is that even though each of these PGs is specifically upregulated right before or during the cell separation process of abscission, there is still redundancy in their functions. One of the approaches to resolve this problem may be the generation of multiple mutants. For example, At2g41850 and At3g07970 are derived from the same ancestor. Thus, examination of double mutants between these two diverged PGs may provide a clue as to the abscission function. In addition, At2g43880, At2g43890 and At2g41850 are also derived from the large-scale duplication events while they share different immediate ancestors. Making double mutants between the two PGs At2g43890 and At2g41850 may provide a clue as to PG function during development. Ultimately, generating triple mutants among At2g41850, At2g43890, and At3g07970 may also provide insights into the possible functions of these PGs in floral organ abscission. Studying functions of a large gene family has been always a challenge due to the size and the redundancy of the family, but these recent findings will facilitate this task.From our study, we addressed the questions as to how PG gene family has expanded and/or duplicated in the evolution, how the duplicates were retained with the possible biological functions, and how functional studies of PGs in floral organ abscission can be conducted in the future.     

A comparative analysis of the evolution,expression, and <Emphasis Type="Italic">cis</Emphasis>-regulatory element of polygalacturonase genes in grasses and dicots

Cell walls are a distinguishing characteristic of plants essential to their survival. The pectin content of primary cell walls in grasses and dicots is distinctly different. Polygalacturonases (PGs) can degrade pectins and participate in multiple developmental processes of plants. This study comprehensively compared the evolution, expression, and cis-regulatory element of PGs in grasses and dicots. A total of 577 PGs identified from five grasses and five dicots fell into seven clades. Evolutionary analysis demonstrated the distinct differences between grasses and dicots in patterns of gene duplication and loss, and evolutionary rates. Grasses generally contained much fewer clade C and F members than dicots. We found that this disparity was the result of less duplication and more gene losses in grasses. More duplications occurred in clades D and E, and expression analysis showed that most of clade E members were expressed ubiquitously at a high overall level and clade D members were closely related to male reproduction in both grasses and dicots, suggesting their biological functions were highly conserved across species. In addition to the general role in reproductive development, PGs of clades C and F specifically played roles in root development in dicots, shedding light on organ differentiation between the two groups of plants. A regulatory element analysis of clade C and F members implied that possible functions of PGs in specific biological responses contributed to their expansion and preservation. This work can improve the knowledge of PGs in plants generally and in grasses specifically and is beneficial to functional studies.     

Proteoglycan control of cell movement during wound healing and cancer spreading.

By virtue of their multifunctional nature, proteoglycans (PGs) are thought to govern the process of cell movement in numerous physiological and pathological contexts, spanning from early embryonic development to tumour invasion and metastasis. The precise mode by which they influence this process is still fragmentary, but evidence is accruing that they may affect it in a multifaceted manner. PGs bound to the plasma membrane mediate the polyvalent interaction of the cell with matrix constituents and with molecules of the neighbouring cells' surfaces; they modulate the activity of receptors implicated in the recognition of these components; and they participate in the perception and convergence of growth- and motility-promoting cues contributed by soluble factors. Through some of these interactions several PGs transduce to pro-motile cells crucial intracellular signals that are likely to be essential for their mobility. A regulated shedding of certain membrane-intercalated PGs seems to provide an additional level of control of cell movement. Coincidentally, matrix-associated PGs may govern cell migration by structuring permissive and non-permissive migratory paths and, when directly secreted by the moving cells, may alternatively create favourable or hostile microenvironments. To exert this latter, indirect effect on cell movement, matrix PGs strongly rely upon their primary molecular partners, such as hyaluronan, link proteins, tenascins, collagens and low-affinity cell surface receptors, whereas a further finer control is provided by a highly regulated proteolytic processing of the PGs accounted by both the migrating cells themselves and cells of their surrounding tissues. Overall, PGs seem to play an important role in determining the migratory phenotype of a cell by initiating, directing and terminating cell movement in a spatio-temporally controlled fashion. This implies that the "anti-adhesive and/or "anti-migratory" properties that have previously been assigned to certain PGs may be re-interpreted as being a means by which these macromolecules elaborate haptotaxis-like mechanisms imposing directionality upon the moving cells. Since these conditions would allow cells to be led to given tissue locations and become immobilized at these sites, a primary function may be ascribed to PGs in the dictation of a "stop or go" choice of the migrating cells.     

Similar genetic mechanisms underlie the parallel evolution of floral phenotypes

The repeated origin of similar phenotypes is invaluable for studying the underlying genetics of adaptive traits; molecular evidence, however, is lacking for most examples of such similarity. The floral morphology of neotropical Malpighiaceae is distinctive and highly conserved, especially with regard to symmetry, and is thought to result from specialization on oil-bee pollinators. We recently demonstrated that CYCLOIDEA2-like genes (CYC2A and CYC2B) are associated with the development of the stereotypical floral zygomorphy that is critical to this plant-pollinator mutualism. Here, we build on this developmental framework to characterize floral symmetry in three clades of Malpighiaceae that have independently lost their oil bee association and experienced parallel shifts in their floral morphology, especially in regard to symmetry. We show that in each case these species exhibit a loss of CYC2B function, and a strikingly similar shift in the expression of CYC2A that is coincident with their shift in floral symmetry. These results indicate that similar floral phenotypes in this large angiosperm clade have evolved via parallel genetic changes from an otherwise highly conserved developmental program.     

Regulation of heme oxygenase expression by cyclopentenone prostaglandins

Prostaglandins (PGs) originate from the degradation of membranar arachidonic acid by cyclooxygenases (COX-1 and COX-2). The prostaglandin actions in the nervous system are multiple and have been suggested to play a significant role in neurodegenerative disorders. Some PGs have been reported to be toxic and, interestingly, the cyclopentenone PGs have been reported to be cytoprotective at low concentration and could play a significant role in neuronal plasticity. They have been shown to be protective against oxidative stress injury; however, the cellular mechanisms of protection afforded by these PGs are still unclear. It is postulated that the cascade leading to neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer's disease, would be mediated by free radical damage. We tested the hypothesis that the neuroprotective action of cyclopentanone could be caused partially by an induction of heme oxygenase 1 (HO-1). We and others have previously reported that modulation of HO total activity may well have direct physiological implications in stroke and in Alzheimer's disease. HO acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin that is rapidly converted into bilirubin. Using mouse primary neuronal cultures, we demonstrated that PGs of the J series induce HO-1 in a dose-dependent manner (0, 0.5, 5, 10, 20, and 50 micro g/ml) and that PGJ(2) and dPGJ(2) were more potent than PGA(2), dPGA(2), PGD(2), and PGE(2). No significant effects were observed for HO-2 and actin expression. In regard to HO-3 expression found in rat, with its protein deducted sequence highly homologous to HO-2, no detection was observed in HO-2(-/-) mice, suggesting that HO-3 protein would not be present in mouse brain. We are proposing that several of the protective effects of PGJ(2) could be mediated through beneficial actions of heme degradation and its metabolites. The design of new mimetics based on the cyclopentenone structure could be very useful as neuroprotective agents and be tested in animal models of stroke and Alzheimer's disease.     

Gene structure dynamics and divergence of the polygalacturonase gene family of plants and fungus.

Whole copies of the polygalacturonase (PG) genes from rice (Oryza sativa subsp. japonica) and a filamentous fungus (Aspergillus oryzae) were isolated. The orthologs of the rice PGs were also retrieved from other plant species. The 106 plant PGs analyzed were divided into 5 clades, A, B, C, D, and E. The fungus PGs were classified into 3 clades, of which one formed a loose cluster with clade E of the plant PGs. Four domain motifs (I, II, III, IV) were identified in all PGs. Motifs II and III were split by introns such as G/DDC and CGPGHGIS/IGSLG, respectively. In plant PGs there were 446 introns in total and 3.98 introns per gene. Intron phase distribution was 65.5% for phase 0, 19.7% for phase 1, and 14.8% for phase 2 in plant PGs. In the PGs of A. oryzae there were 37 introns of phase 0 (59.5%), phase 1 (24.3%), and phase 2 (16.2%), with 2.47 introns per gene. The 5 clades of plant PGs were divided into 3 basic gene structure lineages. Intron positions and phases were conserved among the PGs in the first 2 lineages. The third lineage consisted of PGs of clade E, which also carried highly conserved introns at different positions from other PGs. Intron positions were not as highly conserved in fungus PGs as in plant PGs. The introns in the current PGs have been present since before the divergence of monocots from dicots. The results obtained show that differential losses of introns created gene diversity, which was followed by segmental and tandem duplication in plant PGs.     

Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a human macrophage cell line

Prostaglandins (PGs) play regulatory roles in a variety of physiological and pathological processes, including the immune response, cytoprotection and inflammation. Desferrioxamine (DFX), an iron chelator, is known to reduce free radical-mediated cell injury and to upregulate certain inflammatory mediators. We investigated the effects of DFX on the production of PGs and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGs, using a human macrophage cell line, U937. Our results showed that COX-2 expression and PGE(2) production are upregulated by DFX treatment and that this upregulation is dependent on both COX-2 promoter activity and alteration of mRNA stability. COX-2 promoter activity may be, at least in part, mediated by activation of the extracellular signal-regulated kinase pathway. These findings suggest that iron metabolism may regulate inflammatory processes by modulating PGs as well as other inflammatory mediators.     

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F.?graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F.?graminearum. Our data suggest that PGs likely play a role in F.?graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.     

THE INFLUENCE OF POLLINATOR PHYLOGEOGRAPHY AND MATE PREFERENCE ON FLORAL DIVERGENCE IN A SEXUALLY DECEPTIVE DAISY

Divergent mate preferences and subsequent genetic differentiation between populations has been demonstrated, but its effects on interspecific interactions are unknown. Associated species exploiting these mate preferences, for example, may diverge to match local preferences. We explore this idea in the sexually deceptive, fly‐mimicking daisy, Gorteria diffusa, by testing for association between genetic structure in the fly pollinator (a proxy for mate preference divergence) and geographic divergence in floral form. If genetic structure in flies influences interactions with G. diffusa, we expect phylogeographically distinct flies to be associated with different floral forms. Flies associated with forms exploiting only feeding behavior often belonged to several phylogeographic clades, whereas flies associated with forms exploiting male‐mating behavior always belonged to distinct clades, indicating the possibility of pollinator‐mediated floral divergence through phylogeographic variation in mating preferences of male flies. We tested this hypothesis with reciprocal presentations using male flies from distinct clades associated with separate floral forms. Results show that males from all clades exhibit similar preferences, making pollinator driven divergence through geographic variation in mate preference unlikely. Males, however, showed evidence of learned resistance to deceptive traits, suggesting antagonistic interactions between plants and pollinators may drive deceptive floral trait evolution in G. diffusa.     

Conservation and divergence in the AGAMOUS subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization events

The MADS-box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule-specific expression might have evolved more than twice. Although sub- and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.     

Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.     

Expression of HtKNOT1, a class I KNOX gene, overlaps cell layers and development compartments of differentiating cells in stems and flowers of Helianthus tuberosus

In plant, post-embryonic development relies on the activities of indeterminate cell populations termed meristems, spatially clustered cell lineages, wherein a subset divides indeterminately. For correct growth, the plant must maintain a constant flow of cells through the meristem, where the input of dividing pluripotent cells offsets the output of differentiating cells. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the plant meristems and play important roles in maintaining meristematic cell identity. We have analyzed the expression pattern of HtKNOT1, a class I KNOX gene of Helianthus tuberosus, in stems, inflorescence meristems, floral meristems and floral organs. HtKNOT1 is expressed in cambial cells, phloem cells and xylematic parenchyma within apical stem internodes, while in basal internodes HtKNOT1 expression was restricted to the presumptive initials and recently derived phloem cells. In the reproductive phase, HtKNOT1 mRNAs were detected in both the inflorescence and floral meristems as well within lateral organ primordia (i.e. floral bracts, petals, stamens and carpels). In more differentiated flowers, the expression of HtKNOT1 was restricted to developing ovules and pollen mother cells. HtKNOT1 may play a dual role being required to maintain the meristem initials as well as initiating differentiation and/or conferring new cell identity. In particular, it is possible that HtKNOT1 cooperates at floral level with additional factors that more specifically control floral organs and pollen development in H. tuberosus.     

Evolution of reproductive traits in the mahagony family (Meliaceae)

Meliaceae are a mostly pantropical family in the Sapindales, bearing flowers typically provided with a staminal tube, formed by filaments that are fused partially or totally. Nevertheless, several genera of subfamily Cedreloideae have free stamens, which may be adnate to an androgynophore in some taxa. The fact that the family exhibits a wide diversity of floral and fruit features, as well as of sexual systems and pollination syndromes, presents interesting questions on the evolutionary processes that might have taken place during its history. In this study, we analyzed the distribution of 20 reproductive morphological traits of Meliaceae, upon an available molecular phylogenetic framework, using 31 terminals from the family's two main clades (Cedreloideae and Melioideae), plus six Simaroubaceae taxa as outgroup. We aimed to identify and/or confirm synapomorphies for clades within the family and to develop hypotheses on floral evolution and sexual systems in the group. Our reconstruction suggests that the ancestor of Meliaceae was possibly provided with united stamens and unisexual flowers in dioecious individuals, with a subsequent change to free stamens and monoecy in the ancestor of Cedreloideae. Most characters studied show some degree of homoplasy, but some are unique synapomorphies of clades, such as the haplostemonous androecium. An androgynophore defines the Cedrela‐Toona clade. The comparative approach of our study and the evolutionary hypotheses generated herein reveal several aspects demanding further structural investigation, and possible evolutionary pathways of the reproductive structures along with the lineages' diversification, mostly related to the specialization of sexual systems, floral biology, and dispersal strategies.     

Activation of Basal Cells of the Apical Meristem during Sepal Formation in Tomato

Although great advances have been made in research on the regulation of primordium fate in the floral meristem, our understanding of the molecular events occurring during the floral transition remains incomplete. Via a careful analysis of the expression patterns of five genes encoding housekeeping functions during the floral transition in tomato (using both in situ hybridization and enzyme histochemistry), we identified a particular phase of floral development (sepal initiation) at which cells located toward the base of the meristem show a high level of cellular metabolism, whereas cells at the tip of the meristem dome show little activity. At other stages of floral development, the probes used showed genespecific patterns of expression generally consistent with our previous investigation of the vegetative apical meristem. Our data, in conjunction with other reports in the literature, enabled us to postulate that relative activation of basal cells of the meristem may be of general occurrence during the transition to flowering. Such a hypothesis could account for recent observations using periclinal chimeras on the effect of L3 genotypes on flower development and have a bearing on the expected mechanism by which the number of primordia generated by a floral meristem is regulated.     

Prostaglandins act as neurotoxin for differentiated neuroblastoma cells in culture and increase levels of ubiquitin and β-amyloid

Summary Although chronic inflammatory reactions have been proposed to cause neuronal degeneration associated with Alzheimer’s disease (AD), the role of prostaglandins (PGs), one of the secretory products of inflammatory reactions, in degeneration of nerve cells has not been studied. Our initial observation that PGE₁-induced differentiated neuroblastoma (NB) cells degenerate in vitro more rapidly than those induced by RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, has led us to postulate that PGs act as a neurotoxin. This study has further investigated the effects of PGs on differentiated NB cells in culture. Results showed that PGA₁ was more effective than PGE₁ in causing degeneration of differentiated NB cells as shown by the cytoplasmic vacuolation and fragmentation of soma, nuclei, and neurites. Because increased levels of ubiquitin and β-amyloid have been implicated in causing neuronal degeneration, we studied the effects of PGs on the levels of these proteins during degeneration of NB cells in vitro by an immunostaining technique, using primary antibodies to ubiquitin and β-amyloid. Results showed that PGs increased the intracellular levels of ubiquitin and β-amyloid prior to degeneration, whereas the degenerated NB cells had negligible levels of these proteins. These data suggest that PGs act as external neurotoxic signals which increase levels of ubiquitin and β-amyloid that represent one of the intracellular signals for initiating degeneration of nerve cells.