Dual RNA-seq of maize and H. seropedicae ZAE94 association, in different doses of nitrate, reveals novel insights into Plant-PGPB-environment relationship.

The interactions between plants, beneficial bacteria and their environment are profoundly shaped by various environmental factors, including light, temperature, water availability, and soil quality. Despite efforts to elucidate the molecular mechanisms involved in the association between plants and beneficial bacteria, like Plant Growth-Promoting Bacteria (PGPB), with many studies focusing on the transcriptional reprogramming in the plant, there is no report on the modulation of genetic controls from both plant and associated bacteria standpoints, in response to environment. The main goal of this study was to investigate the relationship between plant-bacteria-environment signaling, using as a model maize plants inoculated with H. seropedicae ZAE94 and cultivated with different doses of N (0.3 and 3 mM). For this purpose, we performed rRNA-depleted RNA-seq to determine the global gene expression of both maize roots and associated H. seropedicae ZAE94. Our results revealed a differential modulation of maize nitrogen metabolism, phytohormone and cell wall responses when associated with H. seropedicae ZAE94 at different N concentrations. In parallel, a modulation of the bacterial metabolism could be observed, by regulating genes involved in transport, secretion system, cell mobility, oxidoreductases, and chemotaxis, when bacteria were associated with maize roots and cultivated at different doses of N. The molecular and phenotypic data of maize plantlets suggested that different doses of N fertilization differentially regulated the beneficial effects of bacterial inoculation, as higher doses (3 mM) favored shoot elongation and lower doses (0.3 mM) favored increase in plant biomass. Our results provide a valuable integrated overview of differentially expressed genes in both maize and associated H. seropedicae ZAE94 in response to different N availability, revealing new insights into pathways involved in grass-PGPB associations.

The regeneration conferring transcription factor complex ERF115-PAT1 coordinates a wound-induced response in root-knot nematode induced galls.

The establishment of root-knot nematode (RKN; Meloidogyne spp.) induced galls in the plant host roots likely involves a wound-induced regeneration response. Confocal imaging demonstrates physical stress or injury caused by RKN infection during parasitism in the model host Arabidopsis thaliana. The ERF115-PAT1 heterodimeric transcription factor complex plays a recognized role in wound-induced regeneration. ERF115 and PAT1 expression flanks injured gall cells likely driving mechanisms of wound healing, implying a local reactivation of cell division which is also hypothetically involved in gall genesis. Herein, functional investigation revealed that ectopic ERF115 expression resulted in premature induction of galls, and callus formation adjacent to the expanding female RKN was seen upon PAT1 upregulation. Smaller galls and less reproduction were observed in ERF115 and PAT1 knockouts. Investigation of components in the ERF115 network upon overexpression and knockdown by qRT-PCR suggests it contributes to steer gall wound-sensing and subsequent competence for tissue regeneration. High expression of CYCD6;1 was detected in galls, and WIND1 overexpression resulted in similar ERF115OE gall phenotypes, also showing faster gall induction. Along these lines, we show that the ERF115-PAT1 complex likely coordinates stress signalling with tissue healing, keeping the gall functional until maturation and nematode reproduction.

Cell wall formation pathways are differentially regulated in sugarcane contrasting genotypes associated with endophytic diazotrophic bacteria.

MAIN CONCLUSION: Differences in cell wall components between two BNF-contrasting sugarcane genotypes might result from genetic variations particular to the genotype and from the efficiency in diazotrophic bacteria association. Sugarcane is a plant of the grass family (Poaceae) that is highly cultivated in Brazil, as an important energy resource. Commercial sugarcane genotypes may be successfully associated with beneficial endophytic nitrogen-fixing bacteria, which can influence several plant metabolic pathways, such as cell division and growth, synthesis of hormones, and defense compounds. In this study, we investigated how diazotrophic bacteria associated with sugarcane plants could be involved in the regulation of cell wall formation pathways. A molecular and structural characterization of the cell wall was compared between two genotypes of sugarcane with contrasting rates of Biological Nitrogen Fixation (BNF): SP70-1143 (high BNF) and Chunee (low BNF). Differentially expressed transcripts were identified in transcriptomes generated from SP70-1143 and Chunee. Expression profiles of cellulose and lignin genes, which were more expressed in SP70-1134, and callose genes, which were more expressed in Chunee, were validated by RT-qPCR and microscopic analysis of cell wall components in tissue sections. A similar expression profile in both BNF-contrasting genotypes was observed in naturally colonized plants and in plants inoculated with G. diazotrophicus. Cell walls of the high BNF genotype have a greater cellulose content, which might contribute to increase biomass. In parallel, callose was concentrated in the vascular tissues of the low BNF genotype and could possibly represent a barrier for an efficient bacterial colonization and dissemination in sugarcane tissues. Our data show a correlation between the gene profiles identified in the BNF-contrasting genotypes and a successful association with endophytic diazotrophic bacteria.

The Armadillo BTB Protein ABAP1 Is a Crucial Player in DNA Replication and Transcription of Nematode-Induced Galls.

The biogenesis of root-knot nematode (Meloidogyne spp.)-induced galls requires the hyperactivation of the cell cycle with controlled balance of mitotic and endocycle programs to keep its homeostasis. To better understand gall functioning and to develop new control strategies for this pest, it is essential to find out how the plant host cell cycle programs are responding and integrated during the nematode-induced gall formation. This work investigated the spatial localization of a number of gene transcripts involved in the pre-replication complex during DNA replication in galls and report their akin colocation with the cell cycle S-phase regulator Armadillo BTB Arabidopsis Protein 1 (ABAP1). ABAP1 is a negative regulator of pre-replication complex controlling DNA replication of genes involved in control of cell division and proliferation; therefore, its function has been investigated during gall ontogenesis. Functional analysis was performed upon ABAP1 knockdown and overexpression in Arabidopsis thaliana. We detected ABAP1 promoter activity and localized ABAP1 protein in galls during development, and its overexpression displayed significantly reduced gall sizes containing atypical giant cells. Profuse ABAP1 expression also impaired gall induction and hindered nematode reproduction. Remarkably, ABAP1 knockdown likewise negatively affected gall and nematode development, suggesting its involvement in the feeding site homeostasis. Microscopy analysis of cleared and nuclei-stained whole galls revealed that ABAP1 accumulation resulted in aberrant giant cells displaying interconnected nuclei filled with enlarged heterochromatic regions. Also, imbalanced ABAP1 expression caused changes in expression patterns of genes involved in the cell division control as demonstrated by qRT-PCR. CDT1a, CDT1b, CDKA;1, and CYCB1;1 mRNA levels were significantly increased in galls upon ABAP1 overexpression, possibly contributing to the structural changes in galls during nematode infection. Overall, data obtained in galls reinforced the role of ABAP1 controlling DNA replication and mitosis and, consequently, cell proliferation. ABAP1 expression might likely take part of a highly ordered mechanism balancing of cell cycle control to prevent gall expansion. ABAP1 expression might prevent galls to further expand, limiting excessive mitotic activity. Our data strongly suggest that ABAP1 as a unique plant gene is an essential component for cell cycle regulation throughout gall development during nematode infection and is required for feeding site homeostasis.

ABAP1 Plays a Role in the Differentiation of Male and Female Gametes in Arabidopsis thaliana.

The correct development of a diploid sporophyte body and a haploid gametophyte relies on a strict coordination between cell divisions in space and time. During plant reproduction, these divisions have to be temporally and spatially coordinated with cell differentiation processes, to ensure a successful fertilization. Armadillo BTB Arabidopsis protein 1 (ABAP1) is a plant exclusive protein that has been previously reported to control proliferative cell divisions during leaf growth in Arabidopsis. Here, we show that ABAP1 binds to different transcription factors that regulate male and female gametophyte differentiation, repressing their target genes expression. During male gametogenesis, the ABAP1-TCP16 complex represses CDT1b transcription, and consequently regulates microspore first asymmetric mitosis. In the female gametogenesis, the ABAP1-ADAP complex represses EDA24-like transcription, regulating polar nuclei fusion to form the central cell. Therefore, besides its function during vegetative development, this work shows that ABAP1 is also involved in differentiation processes during plant reproduction, by having a dual role in regulating both the first asymmetric cell division of male gametophyte and the cell differentiation (or cell fusion) of female gametophyte.

Cell wall dynamics and gene expression on soybean embryonic axes during germination.

MAIN CONCLUSION: Identification of the structural changes and cell wall-related genes likely involved in cell wall extension, cellular water balance and cell wall biosynthesis on embryonic axes during germination of soybean seeds. Cell wall is a highly organized and dynamic structure that provides mechanical support for the cell. During seed germination, the cell wall is critical for cell growth and seedling establishment. Although seed germination has been widely studied in several species, key aspects regarding the regulation of cell wall dynamics in germinating embryonic axes remain obscure. Here, we characterize the gene expression patterns of cell wall pathways and investigate their impact on the cell wall dynamics of embryonic axes of germinating soybean seeds. We found 2143 genes involved in cell wall biosynthesis and assembly in the soybean genome. Key cell wall genes were highly expressed at specific germination stages, such as expansins, UDP-Glc epimerases, GT family, cellulose synthases, peroxidases, arabinogalactans, and xyloglucans-related genes. Further, we found that embryonic axes grow through modulation of these specific cell wall genes with no increment in biomass. Cell wall structural analysis revealed a defined pattern of cell expansion and an increase in cellulose content during germination. In addition, we found a clear correlation between these structural changes and expression patterns of cell wall genes during germination. Taken together, our results provide a better understanding of the complex transcriptional regulation of cell wall genes that drive embryonic axes growth and expansion during soybean germination.

Roles of Non-Coding RNA in Sugarcane-Microbe Interaction.

Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408-a copper-microRNA-was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5'RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly.

Ontogenia de los estróbilos, desarrollo de los esporangios y esporogénesis de Equisetum giganteum (Equisetaceae) en los Andes de Colombia

Ontogeny of strobili, sporangia development and sporogenesis in Equisetum giganteum (Equisetaceae) from the Colombian Andes. Studies on the ontogeny of the strobilus, sporangium and reproductive biology of this group of ferns are scarce. Here we describe the ontogeny of the strobilus and sporangia, and the process of sporogenesis using specimens of E. giganteum from Colombia collected along the Rio Frio, Distrito de Sevilla, Piedecuesta, Santander, at 2 200m altitude. The strobili in different stages of development were fixed, dehydrated, embedded in paraffin, sectioned using a rotatory microtome and stained with the safranin O and fast green technique. Observations were made using differential interference contrast microscopy (DIC) or Nomarski microscopy, an optical microscopy illumination technique that enhances the contrast in unstained, transparent. Strobili arise and begin to develop in the apical meristems of the main axis and lateral branches, with no significant differences in the ontogeny of strobili of one or other axis. Successive processes of cell division and differentiation lead to the growth of the strobilus and the formation of sporangiophores. These are formed by the scutellum, the manubrium or pedicel-like, basal part of the sporangiophore, and initial cells of sporangium, which differentiate to form the sporangium wall, the sporocytes and the tapetum. There is not formation of a characteristic arquesporium, as sporocytes quickly undergo meiosis originating tetrads of spores. The tapetum retains its histological integrity, but subsequently the cell walls break down and form a plasmodium that invades the sporangial cavity, partially surrounding the tetrads, and then the spores. Towards the end of the sporogenesis the tapetum disintegrates leaving spores with elaters free within the sporangial cavity. Two layers finally form the sporangium wall: the sporangium wall itself, with thickened, lignified cell walls and an underlying pyknotic layer. The mature spores are chlorofilous, morphologically similar and have exospore, a thin perispore and two elaters. This study of the ontogeny of the spore-producing structures and spores is the first contribution of this type for a tropical species of the genus. Fluorescence microscopy indicates that elaters and the wall of the sporangium are autofluorescent, while other structures induced fluorescence emitted by the fluorescent dye safranin O. The results were also discussed in relation to what is known so far for other species of Equisetum, suggesting that ontogenetic processes and structure of characters sporoderm are relatively constant in Equisetum, which implies important diagnostic value in the taxonomy of the group. Rev. Biol. Trop. 59 (4): 1845-1858. Epub 2011 December 01.

Estudios sobre la ontogenia del estróbilo, los esporangios y la biología reproductiva de Equisetum son escasos, por lo tanto, para la especie E. giganteum, se estudiaron estos aspectos en especímenes recolectados a orillas del Río Frío, Santander, Colombia (2 200m). Los estróbilos en diferentes etapas de maduración fueron fijados, deshidratados, embebidos en parafina, seccionados en micrótomo rotatorio y teñidos con safranina O-fast green. Las observaciones se efectuaron mediante un microscopio óptico de alta resolución con contraste diferencial de interferencia (DIC) y microscopio de fluorescencia. Los estróbilos se inician a partir del meristemo apical, tanto en el eje principal como en los laterales, sin diferencias en el proceso de ontogenia y esporogénesis entre estróbilos de diferentes ejes. Sucesivas mitosis y diferenciación celular conducen al crecimiento del estróbilo, y a la formación de los esporangióforos peltados, formados por el manubrio, o porción basal con aspecto de pedicelo, el escutelo, o porción apical aplanada y las iniciales del esporangio, los cuales se diferenciarán para formar la pared del esporangio, los esporocitos y el tapete. No se forma arquesporio y los esporocitos experimentan meiosis para formar tétradas de esporas. El tapete mantiene la integridad histológica hasta la formación de las tétradas y en esa etapa forma un plasmodio que invade la cavidad esporangial la cual rodea parcialmente las tétradas y luego las esporas, y aparecen las cámaras plasmodiales, un término propuesto aquí para las formaciones designadas en inglés "tapetal gaps". La pared del esporangio queda reducida a dos capas celulares: una externa con engrosamientos lignificados en todas las paredes celulares y una interna picnótica. Al finalizar la esporogénesis, el tapete degenera, y las esporas, con exosporio, perisporio delgado, casi membranáceo y eláteres quedan libres en la cavidad esporangial. El esporodermo, los núcleos y nucléolos presentan fluorescencia roja, inducida por coloración con safranina O, mientras que los eláteres y las células de la pared del esporangio presentan autofluorescencia amarillo-naranja.

[Ontogeny of strobili, sporangia development and sporogenesis in Equisetum giganteum (Equisetaceae) from the Colombian Andes]. / Ontogenia de los estróbilos, desarrollo de los esporangios y esporogénesis de Equisetum giganteum (Equisetaceae) en los Andes de Colombia.

Studies on the ontogeny of the strobilus, sporangium and reproductive biology of this group of ferns are scarce. Here we describe the ontogeny of the strobilus and sporangia, and the process of sporogenesis using specimens of E. giganteum from Colombia collected along the Rio Frio, Distrito de Sevilla, Piedecuesta, Santander, at 2200m altitude. The strobili in different stages of development were fixed, dehydrated, embedded in paraffin, sectioned using a rotatory microtome and stained with the safranin O and fast green technique. Observations were made using differential interference contrast microscopy (DIC) or Nomarski microscopy, an optical microscopy illumination technique that enhances the contrast in unstained, transparent. Strobili arise and begin to develop in the apical meristems of the main axis and lateral branches, with no significant differences in the ontogeny of strobili of one or other axis. Successive processes of cell division and differentiation lead to the growth of the strobilus and the formation of sporangiophores. These are formed by the scutellum, the manubrium or pedicel-like, basal part of the sporangiophore, and initial cells of sporangium, which differentiate to form the sporangium wall, the sporocytes and the tapetum. There is not formation of a characteristic arquesporium, as sporocytes quickly undergo meiosis originating tetrads of spores. The tapetum retains its histological integrity, but subsequently the cell walls break down and form a plasmodium that invades the sporangial cavity, partially surrounding the tetrads, and then the spores. Towards the end of the sporogenesis the tapetum disintegrates leaving spores with elaters free within the sporangial cavity. Two layers finally form the sporangium wall: the sporangium wall itself, with thickened, lignified cell walls and an underlying pyknotic layer. The mature spores are chlorofilous, morphologically similar and have exospore, a thin perispore and two elaters. This study of the ontogeny of the spore-producing structures and spores is the first contribution of this type for a tropical species of the genus. Fluorescence microscopy indicates that elaters and the wall of the sporangium are autofluorescent, while other structures induced fluorescence emitted by the fluorescent dye safranin O. The results were also discussed in relation to what is known so far for other species of Equisetum, suggesting that ontogenetic processes and structure of characters sporoderm are relatively constant in Equisetum, which implies important diagnostic value in the taxonomy of the group.

Ontogenia del esporangio y esporogénesis del licopodio Huperzia brevifolia (Lycopodiaceae) de las altas montañas de Colombia

Sporangia ontogeny and sporogenesis of the lycopodium Huperzia brevifolia (Lycopodiaceae) from the high mountains of Colombia. Huperzia brevifolia is one of the dominant species of the genus Huperzia living in paramos and superparamos from the Colombian Andes. A detailed study of the sporangium’s ontogeny and sporogenesis was carried out using specimens collected at 4200m above sea level, in Parque Natural Nacional El Cocuy, Colombia. Small pieces of caulinar axis bearing sporangia were fixed, dehydrated, paraffin embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made, stained with aqueous Toluidine Blue (TBO). The sporangia develops basipetally, a condition that allows observation of all the developmental stages taking place throughout the caulinar axis of adult plants. Each sporangium originates from a group of epidermal cells, axilar to the microphylls. These cells undergo active mitosis, and produce new external and internal cellular groups. The sporangium wall and the tapetum originate from the external group of cells, while the internal cellular group leads to the sporogenous tissue. Meiosis occur in the sporocytes and produce simultaneous types tetrads, each one giving rise four trilete spores, with foveolate ornamentation. During the sporangium ripening, the outermost layer of the wall develops anticlinally, and inner periclinal thickenings and the innermost one perform as a secretory tapetum, which persists until the spores are completely mature. All other cellular layers colapse. Rev. Biol. Trop. 57 (4): 1141-1152. Epub 2009 December 01.

Se describe la ontogenia y la esporogénesis en H. brevifolia, en material recolectado en el Parque Nacional Natural El Cocuy (Boyacá-Colombia) a 4200m de altitud. Los esporangios se desarrollan de forma basípeta sobre el eje caulinar: los iniciales y juveniles se localizan en el ápice y los adultos a maduros, en la base. El desarrollo se inicia a partir de un grupo de células epidérmicas localizadas en las axilas que forman los microfilos con el eje caulinar. Estas células se dividen activamente por mitosis formando una masa celular externa y otra interna. La primera da origen a la pared del esporangio, de varios estratos celulares; de éstos, el estrato externo desarrolla engrosamientos en las paredes anticlinales y en la periclinal interna. El estrato celular interno se diferencia para formar el tapete secretor. Los demás estratos celulares de la pared se degradan durante la maduración del esporangio. La masa celular interna da origen al tejido esporógeno que forma los esporocitos, que experimentan la meiosis I hasta la etapa de díada. La meiosis II concluye con la formación de tétradas, constituidas por esporas en disposición tetraédrica. Las esporas son foveoladas con abertura trilete y son liberadas del esporangio a través de la dehiscencia.

[Sporangia ontogeny and sporogenesis of the lycopodium Huperzia brevifolia (Lycopodiaceae) from the high mountains of Colombia]. / Ontogenia del esporangio y esporogénesis del licopodio Huperzia brevifolia (Lycopodiaceae) de las altas montañas de Colombia.

Huperzia brevifolia is one of the dominant species of the genus Huperzia living in paramos and superparamos from the Colombian Andes. A detailed study of the sporangium's ontogeny and sporogenesis was carried out using specimens collected at 4200m above sea level, in Parque Natural Nacional El Cocuy, Colombia. Small pieces of caulinar axis bearing sporangia were fixed, dehydrated, paraffin embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made, stained with aqueous Toluidine Blue (TBO). The sporangia develops basipetally, a condition that allows observation of all the developmental stages taking place throughout the caulinar axis of adult plants. Each sporangium originates from a group of epidermal cells, axilar to the microphylls. These cells undergo active mitosis, and produce new external and internal cellular groups. The sporangium wall and the tapetum originate from the external group of cells, while the internal cellular group leads to the sporogenous tissue. Meiosis occur in the sporocytes and produce simultaneous types tetrads, each one giving rise four trilete spores, with foveolate ornamentation. During the sporangium ripening, the outermost layer of the wall develops anticlinally, and inner periclinal thickenings and the innermost one perform as a secretory tapetum, which persists until the spores are completely mature. All other cellular layers colapse.