Impact of High Light Intensity and Low Temperature on the Growth and Phenylpropanoid Profile of Azolla filiculoides.

Exposure to high light intensity (HL) and cold treatment (CT) induces reddish pigmentation in Azolla filiculoides, an aquatic fern. Nevertheless, how these conditions, alone or in combination, influence Azolla growth and pigment synthesis remains to be fully elucidated. Likewise, the regulatory network underpinning the accumulation of flavonoids in ferns is still unclear. Here, we grew A. filiculoides under HL and/or CT conditions for 20 days and evaluated the biomass doubling time, relative growth rate, photosynthetic and non-photosynthetic pigment contents, and photosynthetic efficiency by chlorophyll fluorescence measurements. Furthermore, from the A. filiculoides genome, we mined the homologs of MYB, bHLH, and WDR genes, which form the MBW flavonoid regulatory complex in higher plants, to investigate their expression by qRT-PCR. We report that A. filiculoides optimizes photosynthesis at lower light intensities, regardless of the temperature. In addition, we show that CT does not severely hamper Azolla growth, although it causes the onset of photoinhibition. Coupling CT with HL stimulates the accumulation of flavonoids, which likely prevents irreversible photoinhibition-induced damage. Although our data do not support the formation of MBW complexes, we identified candidate MYB and bHLH regulators of flavonoids. Overall, the present findings are of fundamental and pragmatic relevance to Azolla's biology.

Light and Temperature Shape the Phenylpropanoid Profile of Azolla filiculoides Fronds.

Azolla is a genus of floating freshwater ferns. By their high growth and N2 fixation rates, Azolla species have been exploited for centuries by populations of South-east Asia as biofertilizers in rice paddies. The use of Azolla species as a sustainable plant material for diverse applications, such as feeding, biofuel production, and bioremediation, has encountered a growing interest over the last few years. However, high levels of feed deterrent flavonoids in their fronds have discouraged the use of these ferns as a sustainable protein source for animal consumption. Additionally, information on how and to what extent environmental determinants affect the accumulation of secondary metabolites in these organisms remains poorly understood. Moving from these considerations, here, we investigated by an untargeted metabolomics approach the profiles of phenylpropanoid compounds in the fronds of Azolla filiculoides sampled under control and pigment-inducing stress conditions. In parallel, we assayed the expression of essential structural genes of the phenylpropanoid pathway by quantitative RT-PCR. This study provides novel information concerning A. filiculoides phenylpropanoid compounds and their temporal profiling in response to environmental stimuli. In particular, we show that besides the already known 3-deoxyanthocyanidins, anthocyanidins, and proanthocyanidins, this fern can accumulate additional secondary metabolites of outstanding importance, such as chemoattractants, defense compounds, and reactive oxygen species (ROS) scavengers, and crucial as dietary components for humans, such as dihydrochalcones, stilbenes, isoflavones, and phlobaphenes. The findings of this study open an opportunity for future research studies to unveil the interplay between genetic and environmental determinants underlying the elicitation of the secondary metabolites in ferns and exploit these organisms as sustainable sources of beneficial metabolites for human health.

An ancient RAB5 governs the formation of additional vacuoles and cell shape in petunia petals.

Homologous ("canonical") RAB5 proteins regulate endosomal trafficking to lysosomes in animals and to the central vacuole in plants. Epidermal petal cells contain small vacuoles (vacuolinos) that serve as intermediate stations for proteins on their way to the central vacuole. Here, we show that transcription factors required for vacuolino formation in petunia induce expression of RAB5a. RAB5a defines a previously unrecognized clade of canonical RAB5s that is evolutionarily and functionally distinct from ARA7-type RAB5s, which act in trafficking to the vacuole. Loss of RAB5a reduces cell height and abolishes vacuolino formation, which cannot be rescued by the ARA7 homologs, whereas constitutive RAB5a (over)expression alters the conical cell shape and promotes homotypic vacuolino fusion, resulting in oversized vacuolinos. These findings provide a rare example of how gene duplication and neofunctionalization increased the complexity of membrane trafficking during evolution and suggest a mechanism by which cells may form multiple vacuoles with distinct content and function.

Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal-maternal relationship. To this end, we used reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus-mother relationship can be hypothesized.

The Role of APOSTART in Switching between Sexuality and Apomixis in Poa pratensis.

The production of seeds without sex is considered the holy grail of plant biology. The transfer of apomixis to various crop species has the potential to transform plant breeding, since it will allow new varieties to retain valuable traits thorough asexual reproduction. Therefore, a greater molecular understanding of apomixis is fundamental. In a previous work we identified a gene, namely APOSTART, that seemed to be involved in this asexual mode of reproduction, which is very common in Poa pratensis L., and here we present a detailed work aimed at clarifying its role in apomixis. In situ hybridization showed that PpAPOSTART is expressed in reproductive tissues from pre-meiosis to embryo development. Interestingly, it is expressed early in few nucellar cells of apomictic individuals possibly switching from a somatic to a reproductive cell as in aposporic apomixis. Moreover, out of 13 APOSTART members, we identified one, APOSTART_6, as specifically expressed in flower tissue. APOSTART_6 also exhibited delayed expression in apomictic genotypes when compared with sexual types. Most importantly, the SCAR (Sequence Characterized Amplified Region) derived from the APOSTART_6 sequence completely co-segregated with apomixis.

The Influence of Light on Olive (Olea europaea L.) Fruit Development Is Cultivar Dependent.

In olive, the response to environmental conditions, such as light availability, is under genetic control and requires a combination of biochemical and physiological events. We investigated the effect of irradiance in fruit development in two Italian cultivars, Leccino and Frantoio. Morphological and cyto-histological analyses, as well as water and oil content determination, were carried out in fruits exposed to a different light regime (named as light and shade fruits). Results demonstrated that the influence of light availability on fruit development depends on the cultivar. In Leccino, the fresh and the dry weight, the percentage of dry matter, the kernel and fruit diameter, the mesocarp thickness and the mesocarp cell size were higher in the light exposed fruits than in the ones grown in the shade. In Frantoio, differences between light and shade fruits were observed only at 140 DAF (Days After Flowering) and only in the kernel and fruit diameter and in the dry and fresh weight, which were higher in the light exposed fruits. Leccino, therefore, showed a greater sensitivity to the light availability. This may be related to the observed delay in the endocarp lignification as compared to the Frantoio cultivar. In each cultivar, moreover, shade and light fruits did not show differences in the timing of cell differentiation. Finally, the investigation of oil storage carried out in cyto-histological studies demonstrated that differences in oil content between fruit subjected to different light regimes correlated with the number of oil containing cells, rather than the oil content per cell. A different behaviour was observed in the two cultivars: in Leccino, the mesocarp cell size was almost twice of Frantoio, while oil drops were only 30% larger; therefore, the percentage of cell volume occupied by the oil drops was lower in Leccino than in Frantoio. The chemical analysis confirmed this observation.

Metabolite Storage in Theobroma cacao L. Seed: Cyto-Histological and Phytochemical Analyses.

Cocoa (Theobroma cacao L.), an economically important tropical-fruit crop as source of chocolate, has recently gained a considerable attention; its seeds contain a large amount of different bioactive compounds that have attracted interest because may be beneficial to humans by improving cardiovascular health, by cancer chemo-preventive effects and also through neuroprotective activities. The morphological and anatomical characteristics of cocoa seeds are closely related to the aroma and to the nutritional properties. This study aimed to provide more information about the storage of some metabolites in the various components of cocoa seed by microscopical and phytochemical analyses. Polyphenols, sterols, tocopherols and fatty acids were detected in different portions of the seeds (teguments, cotyledons, embryo axis and pulp). Quali and quantitative differences were observed and a characteristic polyphenol pattern was detected in the different portions of the seed; cytological analysis demonstrated the presence of these compounds in big vacuolated polyphenolic cells. Among the analyzed fatty acids, the stearic and oleic acids were the most abundant in all the seed components (teguments, cotyledons and embryo axis). Fatty acids, usually found in the form of esters, thioesters and amides, represent one of the storage substances of cocoa seed probably localized in lipid globules, which in our observations occupied almost the entire volume of small isodiametric cells of cotyledon mesophyll. In the cocoa seeds we observed also a different distribution of sterols: ß-sitosterol and Δ5-avenasterol were the most abundant, above all in the embryo axis; stigmasterol and campesterol were less present in embryo axis and more abundant in teguments; campestanol level was again higher in teguments but lower in cotyledons. The specific localization of different kind of sterols was probably related to a peculiar function. Our experiments demonstrated that all seed components contribute to the metabolites storage, but with interesting differences in the localization and amount of each metabolite.

Applying predictive models to decipher rhizobacterial modifications in common reed die-back affected populations.

The microbiota inhabiting the soil, as well as the rhizosphere, represents a key determinant of several plant functions. Like for humans, dysbiosis of the plant-associated microbiota may be a co-causal agent in disease with still obscure eziology. In the last decades, the common reed Phragmites australis has been deeply studied for its disappearance from natural stands, but no clear causative agents have been identified and no laboratory models of such "reed die-back syndrome" (RDBS) have been developed. In this study, we try to shed light on the RDBS, by comparing the rhizosphere microbiota of five Italian P. australis populations with different degrees of decline. Results obtained showed a biogeographical meaningful pattern of rhizosphere microbiota, coupled with an impact of RDBS. Obtained data allowed to construct a two-steps predictive model which enabled the prediction of the plant health status from the microbiota taxonomic composition, independently from their geographic location. In conclusion, this study represents one of the first overviews that statistically links RDBS to alteration of rhizosphere microbiota and suggests a model for the analysis of plant-bacteria relationships in nature.

AFLP Approach Reveals Variability in Phragmites australis: Implications for Its Die-Back and Evidence for Genotoxic Effects.

Phragmites australis is a subcosmopolitan species typical of wetlands being studied in Europe for its disappearance from natural stands, a phenomenon called reed die-back syndrome (RDBS). Although it is conjectured that low genetic variability contributes to RDBS, this aspect remains neglected to this day. Using a molecular fingerprinting approach and a sequence analysis of the trnT-trnL/rbcL-psaI regions of cpDNA, this study aimed to compare the genetic structure of stable vs. RDBS-affected P. australis stands from five wetlands of central Italy. Beforehand, in order to characterize the health condition of reed populations, the occurrence of the main macromorphological descriptors for RDBS was considered on 40 reed stands. Soil samples were also collected to examine the total content of heavy metals. The current study analyzed cpDNA in 19 samples and AFLP profiles in 381 samples to investigate the genetic structure of Phragmites populations. Based on the multinomial-Dirichlet model, an analysis of candidate loci under selective pressure was also performed. The relationships among AFLP data, RDBS descriptors and chemicals were evaluated with the use of Linear Mixed Models. The analysis of the cpDNA shows the occurrence of the haplotypes M (the most widespread), and K here recorded for the first time in Italy. Three new haplotypes were also described. The DNA fingerprinting analysis has produced a total of 322 loci (98% polymorphic) and shows the medium-to-high amount of genetic diversity. The significant genetic differentiation among wetlands (Fst = 0.337) suggests either low gene flow or small effective population size. Moreover, the low amount of outlier loci (only 5; l.5% of the total), seems to indicate the scarce occurrence of selective pressure upon the reed's genome. Genetic diversity increased in relationship to the decrease in diameter and of flowering buds of the reed, two of the trends associated with the die-back. The current study rejects the hypothesis that genetic diversity massively contributed to RDBS. Moreover, significant relationships between genetic diversity and the total concentration of some heavy metals (Cr, Cu, and Zn) were highlighted, indicating possible genotoxic effects on P. australis. The current study represents a fact-finding background useful for the conservation of common reed.

Oomycete Communities Associated with Reed Die-Back Syndrome.

Phragmites australis (Cav.) Trin. ex Steud. die-back is a widely-studied phenomenon that was first discovered in northern Europe and that, until recently, was almost unknown in the Mediterranean basin. It has been described as a complex syndrome affecting reed populations leading to their retreat and decline, with significant impacts on valuable ecosystem services. Among the factors that cause the decline, soil-living microorganisms can be crucial. The aims of this study were to analyze the diversity of oomycetes communities associated with reed stands, and to understand whether they could play a key role in the decline. Variations in the structure of oomycetes communities were studied by metabarcoding of the internal transcribed spacer (ITS) 1 region of ribosomal DNA, from the sediments of five Italian freshwater ecosystems. They were chosen to cover a large variability in terms of surface area, water depth, microclimate, and presence of documented reed retreat. From 96 samples collected from reed roots, rhizosphere, and bulk soil, we assembled 207661 ITS1 reads into 523 OTUs. We demonstrated that oomycete communities were structured by several factors, among which the most important was die-back occurrence. Our study also indicates that Pythiogeton spp. could be potentially involved in the development of die-back. The role of heavy metals in the soil was also explored, and cadmium concentration was shown to affect oomycetes distribution. This study represents a significant step forward for the characterization of microbial communities associated with reed die-back syndrome and helps to gain knowledge of the complexity of these important wet ecosystems.

A Tonoplast P3B-ATPase Mediates Fusion of Two Types of Vacuoles in Petal Cells.

It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV) of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation.

Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32) hybrids, the latter being the result of bilateral sexual polyploidization (BSP). These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture.

The hidden geometries of the Arabidopsis thaliana epidermis.

The quest for the discovery of mathematical principles that underlie biological phenomena is ancient and ongoing. We present a geometric analysis of the complex interdigitated pavement cells in the Arabidopsis thaliana (Col.) adaxial epidermis with a view to discovering some geometric characteristics that may govern the formation of this tissue. More than 2,400 pavement cells from 10, 17 and 24 day old leaves were analyzed. These interdigitated cells revealed a number of geometric properties that remained constant across the three age groups. In particular, the number of digits per cell rarely exceeded 15, irrespective of cell area. Digit numbers per 100 µm(2) cell area reduce with age and as cell area increases, suggesting early developmental programming of digits. Cell shape proportions as defined by length:width ratios were highly conserved over time independent of the size and, interestingly, both the mean and the medians were close to the golden ratio 1.618034. With maturity, the cell area:perimeter ratios increased from a mean of 2.0 to 2.4. Shape properties as defined by the medial axis transform (MAT) were calculated and revealed that branch points along the MAT typically comprise one large and two small angles. These showed consistency across the developmental stages considered here at 140° (± 5°) for the largest angles and 110° (± 5°) for the smaller angles. Voronoi diagram analyses of stomatal center coordinates revealed that giant pavement cells (≥ 500 µm(2)) tend to be arranged along Voronoi boundaries suggesting that they could function as a scaffold of the epidermis. In addition, we propose that pavement cells have a role in spacing and positioning of the stomata in the growing leaf and that they do so by growing within the limits of a set of 'geometrical rules'.

Morphological and cytological development and starch accumulation in hermaphrodite and staminate flowers of olive (Olea europaea L.).

In olive (Olea europaea L.), the formation of functionally staminate flowers rather than fully functional hermaphrodites is one of the major factors limiting fruit set, as flowers with aborted pistils are incapable of producing fruit. Studies conducted on various angiosperm species have shown a correlation between flower abortion and starch content. Thus, it is important to know if starch content plays a role in regulating pistil development in olive and if so, what mechanism regulates starch distribution. Cyto-histological observations of staminate and hermaphrodite olive flowers show that pistil development in staminate flowers is interrupted after the differentiation of the megaspore mother cell. At that stage, starch grains were only detected in the ovary, style and stigma of the hermaphrodite flowers. No starch was observed in the pistils of the staminate flowers. This finding suggests a tight correlation between starch content and pistil development. The secondary origin of starch within the flower is indicated by low chlorophyll content in the gynoecium, undetectable Rubisco activity in the pistils of these two kinds of flowers and by the ultrastructure of the plastids observed by transmission electron microscope analysis. The plastids have few thylakoid membranes and grana and in the staminate flowers appeared very similar to proplastids. Considering differences in starch content between staminate and hermaphrodite flowers and the secondary origin of the starch, differences in pistil development in the staminate and hermaphrodite flowers could be related to differences in the sink strength of these two types of flowers.

Isolation of genes from female sterile flowers in Medicago sativa.

A better knowledge of female sporogenesis and gametogenesis could have several practical applications, from commercial hybrid seed production to gene containment in GM crops. With the purpose of isolating genes involved in the megasporogenesis process, the cDNA-AFLP technique was employed to isolate transcript-derived fragments (TDF) differentially expressed between female-fertile and female-sterile full-sib alfalfa plants. This female sterility trait involves female-specific arrest of sporogenesis at early prophase associated with ectopic, massive callose deposition within the nucellus. Ninety-six TDFs were generated and BLAST analyses revealed similarities with genes involved in different Gene Ontology categories. Three TDFs were selected based on their putative functions: showing high similarity to a soybean flower-expressed beta 1,3-glucanase, to an Arabidopsis thaliana MAPKKK, and to an A. thaliana eukaryotic initiation translation factor eIF4G III, respectively. The full length mRNA sequences were obtained. RT-PCR and in situ hybridizations were performed to confirm differential expression during flower development. The genomic organization of the three genes was assessed through sequencing and Southern experiments. Sequence polymorphisms were found between sterile and fertile plants. Our approach based on differential display and bulked segregant analysis was successful in isolating genes that were differentially expressed between fertile and sterile alfalfa plants.

NO release by nitric oxide donors in vitro and in planta.

Artificial nitric oxide (NO) donors are widely used as tools to study the role of NO in plants. However, reliable and reproducible characterisation of metabolic responses induced by different NO donors is complicated by the variability of their NO release characteristics. The latter are affected by different physical and biological factors including temperature and light. Here we critically evaluate NO release characteristics of the donors sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO) and nitric oxide synthase (NOS), both in vitro and in planta (Nicotiana tabacum L. cv. BelW3) and assess their effects on NO dependent processes such as the transcriptional regulation of the mitochondrial alternative oxidase gene (AOX1a), accumulation of H(2)O(2) and induction of cell death. We demonstrate that, contrary to NOS and SNP, GSNO is not an efficient NO generator in leaf tissue. Furthermore, spectrophotometric measurement of NO with a haemoglobin assay, rather than diaminofluorescein (DAF-FM) based detection, is best suited for the quantification of tissue NO. In spite of the different NO release signatures by SNP and NOS in tissue, the NO dependent responses examined were similar, suggesting that there is a critical threshold for the NO response.

An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour.

The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H(+)-ATPase (V-ATPase), which, in plants, act together with H(+)-pyrophosphatases (PPase), whereas distinct P-type H(+)-ATPases in the cell membrane control the pH in the cytoplasm and energize the plasma membrane. Flower colour mutants have proved useful in identifying genes controlling the pH of vacuoles where anthocyanin pigments accumulate. Here we show that PH5 of petunia encodes a P(3A)-ATPase proton pump that, unlike other P-type H(+)-ATPases, resides in the vacuolar membrane. Mutation of PH5 reduces vacuolar acidification in petals, resulting in a blue flower colour and abolishes the accumulation of proanthocyanidins (condensed tannins) in seeds. Expression of PH5 is directly activated by transcription regulators of the anthocyanin pathway, in conjunction with PH3 and PH4. Thus, flower coloration, a key-factor in plant reproduction, involves the coordinated activation of pigment synthesis and a specific pathway for vacuolar acidification.

cGMP in ozone and NO dependent responses.

We have recently reported that ozone (O(3)) can inhibit mitochondrial respiration and induce activation of the alternative oxidase (AOX) pathway and in particular AOX1a in tobacco. While O(3) causes mitochondrial H(2)O(2), early leaf nitric oxide (NO) as well as transient ethylene (ET) accumulation, the levels of jasmonic acid and 12-oxo-phytodienoic acid remained unchanged. It was shown that both, NO and ET dependent pathways can induce AOX1a transcription by O(3). AOX plays a role in reducing reactive oxygen species (ROS) which in turn are linked to biotic and abiotic plant stresses, much like the second messengers guanosine 3', 5'-cyclic monophosphate (cGMP). The goal is to unravel specific cGMP signatures and induction pathways downstream from O(3) and NO, including transcription of AOX1a. Here we propose that some late (>3 h) responses to NO, e.g., the accumulation of phenylalanine lyase (PAL) transcripts, are critically cGMP dependent, while the early (<2 h) responses, including AOX1a induction are not.

Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone-treated tobacco plants.

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O(3)). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O(3) response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O(3) fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O(3)-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.

SERK and APOSTART. Candidate genes for apomixis in Poa pratensis.

Seed production generally requires the mating of opposite sex gametes. Apomixis, an asexual mode of reproduction, avoids both meiotic reduction and egg fertilization. The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A more profound knowledge of the mechanisms that regulate reproductive events would contribute fundamentally to understanding the genetic control of the apomictic pathway. In Poa pratensis, we isolated and characterized two genes, PpSERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) and APOSTART. These full-length genes were recovered by rapid amplification of cDNA ends and their temporal and spatial expression patterns were assessed by reverse transcription-polymerase chain reaction and in situ hybridization, respectively. The expression of PpSERK and APOSTART differed in apomictic and sexual genotypes. Their putative role in cell-signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis, and embryogenesis in plants is reported and discussed. We propose that, in nucellar cells of apomictic genotypes, PpSERK is the switch that channels embryo sac development and that it may also redirect signaling gene products to compartments other than their typical ones. The involvement of APOSTART in meiosis and programmed cell death is also discussed.