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Jurisdiction - Indonesia
Indonesia – New Rules, And Worries, For E-Commerce Companies.
Legal News & Analysis – Asia Pacific – Indonesia – Corporate/M&A
E-commerce in Indonesia is still in its early stages compared to more mature markets like Europe and North America. Nevertheless, the online shopping sector has grown rapidly in Indonesia in recent years and this is expected to continue as it attracts more entrepreneurs eager to tap into the power that e-commerce gives them to reach customers not only in Indonesia but globally.
According to data from the Ministry of Communication and Informatics, in 2012 the value of e-commerce transactions in Indonesia was around Rp 120 trillion, or about US$12.3 billion at the current exchange rate. That is almost double the value of e-commerce transactions in 2011, which stood at Rp 63 trillion.
Indonesia is touted by e-commerce players as a fast-growing market with huge potential, given the country's population of more than 240 million and economic growth that has been above 6 percent in recent years. Lazada, one of the fastest growing e-commerce companies in Indonesia, saw its revenue grow by at least 20 percent month-on-month in 2012, and Maximilian Bittner, the CEO of Lazada Southeast Asia, told The Jakarta Post that he expected to sustain that pace through this year. He also told the newspaper that Indonesia had become Lazada's number one source of revenue in the region in just a year.
To accommodate online business players and this fast-growing sector, the Indonesian Government late last year issued Government Regulation No. 82 of 2012 regarding the Implementation of Electronic Transactions and Systems ("GR 82″). This is an implementing regulation for Law No. 11 of 2008 regarding Electronic Information and Electronic Transactions, which was issued on April 21, 2008. GR 82 basically sets out the obligations for electronic system providers, including individuals, government institutions and companies, e-commerce companies included.
It is likely that GR 82 will be followed by other regulations that will provide more detail on the implementation of the e-commerce business in the country. The Government has said it plans to issue new regulations on e-commerce that will provide a clearer roadmap for the sector. Azhar Hasyim, director of e-business at the Ministry of Communication and Informatics, confirmed to The Jakarta Globe that they were considering new regulations for the registration of electronic systems that would affect e-commerce.
The major requirement in GR 82, and the one that all e-commerce business players should take note of, is that every electronic system provider operating in Indonesia must be registered in the country and have a data center and disaster recovery center here. The new regulations, which according to Azhar are likely to be implemented later this year, would re-emphasize this requirement that electronic system providers register in Indonesia.
This requirement is aimed at helping the government control or access the relevant data in the event a consumer inside or outside Indonesia encounters trouble while engaging in online business or shopping. The Ministry of Communication and Informatics says that in the past few years it has received a large number of complaints from e-commerce consumers in Indonesia and abroad about websites with "dot-com" domain names (1). It says there is little it can do because the websites are not registered with the Indonesian Government and the Government does not possess data on e-commerce websites with foreign domain names. If the data is located outside the territory of Indonesia it is difficult for the Government to obtain such data.
However, for a number of reasons many e-commerce players remain reluctant to register in Indonesia and locate data centers and disaster recovery centers in the country. One concern is the availability of a reliable power supply in Indonesia (2) with the sole provider being state-owned electricity company PLN. It seems likely that major online business players such as Google, Facebook and BlackBerry will be hesitant to rely on PLN as the lone power supplier for their data centers. There will also be concerns about protecting data centers from natural disasters such as earthquakes and floods, which are not uncommon in Indonesia.
The discussed new regulations would also require e-commerce players to operate their platforms using an Indonesian "dot-id" domain name. Azhar explained that the purpose of this was also to help the government control and track e-commerce websites, and ultimately ensure they are not operating outside the legal framework of the country (3).
Although the new regulations have not been issued, online businesses are already concerned about the "dot-id" requirement. The CEO of GoIndonesia, Yusuf Ijsseldijk, told Metro News he worried the requirement could curb the growth of the e-commerce industry in Indonesia. While there are few details about the proposed "dot-id" requirement, it is probably safe to assume that the process for e-commerce players to obtain a "dot-id" domain name would take additional time. The "dot-id" requirement could also prevent Indonesian e-commerce sites from competing on a global level.
E-commerce companies that existed prior to the issuance of these new regulations would be exempt from the requirement that they operate under a "dot-id" domain name. They would be allowed to continue operating under "dot-com" or any other domain name. However, they would still have to register in Indonesia and have a data center in the country.
The purpose behind GR 82 and the discussed new regulations seems to be to protect the interests of consumers and to provide legal certainty to e-commerce business activities in Indonesia. Azhar told The Jakarta Globe that the regulations would help prevent fraud and improve consumer trust in online shopping services, which would benefit the industry.
In practice, it seems likely that e-commerce businesses would have trouble implementing the requirements in GR 82 and the discussed new regulations, considering the lack of facilities for e-commerce business players to create proper data centers in Indonesia. And the obligation to use a "dot-id" domain name could cause hesitation among investors thinking about starting an e-commerce business in Indonesia.
It is also apparent that the existing and expected regulations on e-commerce in Indonesia are more focused on practical matters, while the actual operations of e-commerce businesses themselves have not been specifically regulated. Any stipulations on e-commerce transactions, however, will likely be scattered across a number of regulations rather than gathered in a single piece of legislation.
The confusion and general consternation that the issuance of GR 82 and the discussed new regulations have caused among e-commerce business players and the Government itself should not be a great surprise, considering that e-commerce in Indonesia is still very young. And with e-commerce growing so rapidly in Indonesia it is unlikely that this is the last time these kinds of issues will emerge as the Government puts in place a regulatory framework for the sector
1- http://finance.detik.com/read/2013/03/22/200449/2201623/4/situs-dagang-online-domain-dotcom-banyak-penipuan
2 – http://property.okezone.com/read/2013/03/26/55/781708/index_news.html
3 – http://www.thejakartaglobe.com/business/indonesian-government-to-regulate-online-shopping/581592
Saprita Tahir, Soewito Suhardiman Eddymurthy Kardono
Soewito Suhardiman Eddymurthy Kardono Corporate/M&A Practice Profile in Indonesia
Corporate/M&A Law Firms in Indonesia


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You are here: Home Organisations and movements »NGO's and self-help groups
Al-Arish Needlework Project
The Al-Arish Needlework Project was founded in 1973 in Al-Arish, northern Sinai, Egypt, by the NGO, the Mennonite Central Committee of North America. It was set up in order to help Bedouin and village women to make a living by creating and selling needlework, notably embroidery and beadwork.
Freedom Quilting Bee
The Freedom Quilting Bee was a quilting co-operative of rural African-American women, founded in Rehobeth, Alabama (USA), in 1966. The co-operative had two major goals: to raise money for the civil rights movement and to improve living standards for the quilters' families.
Golden Needle Ladies Sewing Classes (Herat, Afghanistan)
See The Sewing Circles of Herat: a Memoir of Afghanistan.
Hansiba
Hansiba is the name of an Indian fashion brand created by the Self-Employed Women's Association Trade Facilitation Centre (SEWATFC). The brand is named after the first and oldest Self-Employed Women's Association (SEWA) member, a traditional embroiderer called Hansiba.
Kala Raksha Trust
The Kala Raksha Trust is a social development organisation based in Sumrasar Sheikh, in the Kutch region of Gujarat (western India). The Trust's main aim is to preserve traditional arts, in particular embroidery, by helping artisans to become economically self-sustaining.
Kaross is an embroidery initiative founded in 1989 in the Letsitele and Giyani areas of the Limpopo Province, South Africa. Kaross was established by five Shangaan embroiderers and Irma van Rooyen, a visual artist, who was also the creative director and founder. Sitting on local blankets (kaross; hence their name), the ladies started to create works of art reflecting their lives and cultural background.
Parzor Foundation
The Parzor Foundation for the Preservation of Vulnerable Human Heritage, founded in 2002, developed out of the UNESCO sponsored Parzor project for the preservation and promotion of Parsi Zoroastrian culture and heritage, that was started in 1999. The Foundation supports social activities and academic research in the field of Parsi cultural heritage, including Parsi embroidery.
Self-Employed Women's Association (SEWA)
The Self-Employed Women's Association (SEWA) is a trade union in India of approximately 1.2 million members. SEWA is working with the United Nations Women's Department to organize home-based women workers throughout South Asia.
See Self-Employed Women's Association.
SEWA Trade Facilitation Centre (SEWATFC)
The SEWA Trade Facilitation Centre (SEWATFC) is a non-profit company in India that was founded in 2003. It is the commercial arm of the Self-Employed Women's Association (SEWA). The women are the company shareholders and suppliers. SEWATFC helps women to develop and market products throughout India and internationally from two centres, in Ahmadabad and New Delhi (India).
Shrujan (India)
Shrujan is a non-profit organization (NGO) founded in 1969 by Ms. Chandaben Shroff. Shroff had just visited the Kutch area in Gujarat, India, in order to help with famine relief after a severe drought. Realizing that many of local women were excellent embroiderers, she began an income-generating project with women producing up-market embroidered saris. This decision resulted in Shrujan.
Siwa Creations
Siwa Creations is an Egyptian company, founded by Laila Neamatallah, working with hand embroiderers in the Siwa oasis, Egypt. Siwa Creations was set up in 2001 with the aim of helping Siwan women and girls develop and sell their embroideries.
Swat Valley Guild (Pakistan)
The Swat Valley Guild is an organisation working in the Swat Valley north of Peshawar, in northern Pakistan. It supports artisans to retain their craft and help develop their access to the global market.
Tambani
Tambani is a South African self-help group that is making embroideries and appliqués based on local, Venda folklore. The Venda live in northeastern South Africa, in an arid area with little employment. The Tambani group grew out of the work of a South African academic, Ina le Rouz, who in 1989 carried out a research project about Venda folk tales at the University of Venda.

redpajama_set_name: RedPajamaCommonCrawl


The technological components of a "smart city," including everything from smart grids and driverless cars to automated buildings and advanced sensors, can be complicated. But the core question behind the purpose of a smart city is quite simple: does it make human lives better? That's the key theme explored in the webcast "The Connected City: Trends and Developments Driving Smart City Innovation," produced by MIT Technology Review and IEEE Collabratec. Three influential subject matter experts with different backgrounds in developing smart cities delve into how these cities influence their human populations.


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A comparative gene co-expression analysis using self-organizing maps on two congener filmy ferns identifies specific desiccation tolerance mechanisms associated to their microhabitat preference
Enrique Ostria-Gallardo ORCID: orcid.org/0000-0003-0544-66881,2,
Giovanni Larama3,
Graciela Berríos2,
Ana Fallard2,
Ana Gutiérrez-Moraga ORCID: orcid.org/0000-0002-4540-34904,
Ingo Ensminger5 &
León A. Bravo ORCID: orcid.org/0000-0003-4705-48422,6
Filmy-ferns (Hymenophyllaceae) are poikilohydric, homoiochlorophyllous desiccation-tolerant (DT) epiphytes. They can colonize lower and upper canopy environments of humid forest. Filmy-ferns desiccate rapidly (hours), contrasting with DT angiosperms (days/weeks). It has been proposed that desiccation tolerance in filmy-ferns would be associated mainly with constitutive features rather than induced responses during dehydration. However, we hypothesize that the inter-specific differences in vertical distribution would be associated with different dynamics of gene expression within the dehydration or rehydration phases. A comparative transcriptomic analysis with an artificial neural network was done on Hymenophyllum caudiculatum (restricted to lower canopy) and Hymenophyllum dentatum (reach upper canopy) during a desiccation/rehydration cycle.
Raw reads were assembled into 69,599 transcripts for H. dentatum and 34,726 transcripts for H. caudiculatum. Few transcripts showed significant changes in differential expression (DE). H. caudiculatum had ca. twice DE genes than H. dentatum and higher proportion of increased-and-decreased abundance of genes occurs during dehydration. In contrast, the abundance of genes in H. dentatum decreased significantly when transitioning from dehydration to rehydration. According to the artificial neural network results, H. caudiculatum enhanced osmotic responses and phenylpropanoid related pathways, whilst H. dentatum enhanced its defense system responses and protection against high light stress.
Our findings provide a deeper understanding of the mechanisms underlying the desiccation tolerance responses of two filmy ferns and the relationship between the species-specific response and the microhabitats these ferns occupy in nature.
Plant evolution has shaped several acclimation mechanisms and adaptations to withstand short and long-term periods of water deficit. However, most plants cannot survive desiccation, except for a small group of so-called resurrection plants [1, 2]. Desiccation tolerance is a complex trait that involves the coordination of a cascade of molecular events divided into constitutive and inducible mechanisms to protect/repair cells and tissues against oxidative damage, and disruptions of metabolism and cell ultrastructure [2, 3]. In general, the physiological and metabolic components of desiccation tolerance in resurrection plants resemble a combination of processes underlying drought stress responses and seed maturation [4, 5]; however, the molecular "switches" and regulatory pathways behind desiccation tolerance are largely unknown [6]. Resurrection plants are classified into two groups according to their sensitivity, mechanisms of responses, and the velocity of water loss [7]. Those that can survive rapid water loss, such as mosses, possess constitutive (pre-existing) morpho-physiological desiccation tolerance mechanisms employing cellular repair mechanisms induced after rehydration, in contrast to those that survive only if water loss is gradual, which rely upon cellular protection mechanisms induced during dehydration.
Surprisingly, resurrection plants are also found in humid environments such as tropical and temperate rain forests [8]. One example is Lindernia brevidens, an angiosperm species from tropical rain forests [9]. The epiphytic ferns of the Hymenophyllaceae family (Pteridophyta) are another group of resurrection plants living in humid ecosystems [8]. The members of Hymenophyllaceae are called filmy ferns because they possess membranous fronds of a single layer of cells, normally lack cuticles, with undifferentiated epidermis, and lack of stomata [10, 11]. Because the features of their leaves and the recurrent exposure to rapid desiccation-rehydration events, these ferns evolved a poikilohydric and homoiochlorophyllous strategy, most typical of bryophytes, showing a high desiccation tolerance and the capacity to rapidly rehydrate [10, 12, 13].
The Hymenophyllaceae family is represented by 16 endemic species to the Chilean temperate rain forest. They are an important component of the epiphytic species diversity. Filmy ferns can colonize mostly all vertical strata of their host tree, although intraspecific habitat requirements have been reported [11, 14, 15]. For example, Hymenophyllum caudiculatum and Hymenophyllum pectinatum are restricted to the lower portion of the host trunk (from the ground to 1 m of vertical height), where light availability is very low (10–100 μmol photons m− 2 s− 1), and humidity is high. Other species (e.g., H. dentatum, H. plicatum) extend their vertical distribution to heights above 10 m (were PDF eventually reach ≥1000 μmol photons m− 2 s− 1) [11, 13, 15]. Both light intensity (PFD) and vapor pressure deficit (VPD) increase with the height of the host plant, whereas relative humidity decreases. Therefore, a species that reaches the top of trunks is prone to suffer frequent desiccation and photoinhibition. The wide spectrum of habitats, from very sheltered, high steady humidity environments to higher light, lower humidity along the vertical distribution over host trees offers a unique opportunity to address questions about how molecular and physiological mechanisms are shaped in congeneric populations differing in microhabitat preferences and ability to tolerate desiccation. Questions such as: are the molecular mechanisms responsible for protection/repair of these filmy ferns against tissue desiccation inducible during dehydration? or constitutive (pre-existing), such as those exhibited by rapidly desiccating bryophytes? Is the dynamic of gene expression similar between filmy fern species from lower light, higher humidity environments with species from higher light, lower humidity environments? Do these filmy ferns invoke similar gene functions during a cycle of desiccation-rehydration? As filmy ferns are frequently and rapidly dehydrated, it has been proposed that desiccation tolerance in these ferns would be associated mainly with constitutive features rather than induced responses during dehydration. However, we hypothesize that the inter-specific differences in vertical distribution would be associated with different dynamics of gene expression within the dehydration or rehydration phases. Published data partially support this idea [16, 17]. Advances in Next Generation Sequencing (NGS) tools have brought important advantages to uncover underlying mechanisms that control plant responses associated with natural constraints explored in situ or under experimental conditions. Here we used RNA-seq on the Illumina Hi-seq platform to study and compare the transcriptional responses of H. caudiculatum and H. dentatum, two Hymenophyllaceae species with contrasting vertical microhabitat preferences along host trees and different rates of water loss (Fig. 1). Specifically, we examined at the dynamics of gene expression and a Weighted Gene Co-expression Network (WGCNA) [18] coupled with neural artificial networks [19,20,21] of fronds subjected to experimental desiccation-rehydration cycles to identify commonalities and differences on gene expression dynamics associated to its water status underlying their resurrection strategy.
Filmy ferns in their natural environment. At the left of the image a reference of a host tree covered with a carpet of epiphytic filmy ferns. At the right, the two species of resurrection filmy ferns studied, attached to the trunk of their host tree
Changes in ferns' relative water content and maximum quantum efficiency during desiccation-rehydration cycle
The two filmy fern species showed a similar rate of dehydration during the first three hours after cessation of irrigation (beginning of dehydration), both reaching ca. 60% of relative water content (RWC) (Fig. 2a). From 3 to 25 h without irrigation, H. dentatum losses water faster than H. caudiculatum, reaching 18 and 30% RWC respectively. During this period of dehydration, the maximum quantum efficiency (Fv/Fm) drastically decayed from about 0.7 to 0.2 in H. dentatum but remained nearly 0.78 in H. caudiculatum (Fig. 2a insert). After a week without irrigation, both reached a RWC between 11 to 17% and a Fv/Fm near to 0.2 (Fig. 2a). When irrigation was reestablished, H. caudiculatum had a faster rehydration and recovery of Fv/Fm compared to H. dentatum. Nevertheless, both species reached similar maximum photochemical efficiency (Fv/Fm) by the end of the experiment. Recovery of photochemical efficiency at the whole frond level was confirmed by imaging fluorescence (Fig. 2b).
Characterization of desiccation-rehydration cycle in H. caudiculatum and H. dentatum. a Changes in relative water content (RWC) and photochemical efficiency parameter (Fv/Fm, inserted panel) of H. caudiculatum and H. dentatum during a dehydration and rehydration process. Red arrows indicate sampling of fronds during the dehydration-rehydration process used for the RNAseq library construction. b Average Y(II) images measured on dark adapted detached frond of H. caudiculatum and H. dentatum at full hydration, dehydrated, and rehydrated states
Transcriptional profile and transcripts annotation
A total of 111,495,169 and 110,988,488 paired-end reads (101 bp) were obtained after sequencing libraries of H. caudiculatum and H. dentatum, on the Illumina HiSeq2000 platform (Additional file 1: Table S1). Following the removal of low-quality reads and duplicated reads, we performed a de novo transcriptome assemblies with Trinity software by using a set of ~ 85 million reads for H. caudiculatum and ~ 87 million reads for H. dentatum (Additional file 1: Table S1). The initial assemblies resulted in 161,689 contigs for H. caudiculatum and 332,003 contigs for H. dentatum, which were refined to remove low supported transcripts. Transcripts with an estimated abundance lower than 1 FPKM and highly similar or redundant transcripts with a sequence similarity higher than 95% were removed. The resulting transcriptomes are represented by 34,726 contigs for H. caudiculatum and 69,599 contigs for H. dentatum (Additional file 1: Table S2). Although the number of transcripts decreased significantly during the refinement, ca. 80 and 70% of high-quality reads were mapped to the H. caudiculatum and H. dentatum transcriptomes, respectively.
The final transcriptome assemblies were aligned to the SwissProt database for annotation, with an alignment rate of ca. 50% for the transcripts of each transcriptome. In spite of the low identification rate, most of the unknown transcripts (~ 80% in H. dentatum, and ~ 65% H. caudiculatum; Additional file 1: Table S3) belonged to small size transcripts (< 1000 bp; see Fig. 3a). An insight into the taxonomic distribution of top blast hits of transcripts revealed that both H. caudiculatum and H. dentatum had among their top hits a high amount of sequences belonging to the model moss Physcomitrella patens and the lycophyte Sellaginella moellendorffii (Fig. 3b), which is consistent with their poikilohydry strategy and the regressive evolution hypothesis [22].
a Transcript size distribution showing the proportion those sequences with blast hits (blue) and without blast hits (red) in the final transcriptome assemblies. b Top-hit species distribution of the final transcriptome of H. caudiculatum and H. dentatum showing abundance of top hits to sequences of Bryophyta, Lycophyta, and Pinophyta
Differential expression analysis and functional annotation
After quality filtering and refinement, we examined the expression dynamics of annotated genes during the desiccation-rehydration cycle by pairwise comparisons by using a fold change ≥2 and a FDR < 0.05 as cut-off (Additional file 2: Dataset S1 and S2). In both species, few transcripts showed significant changes in differential expression (DE). For H. caudiculatum, the highest number of DE genes occurred during the dehydration process with a total of 265 DE genes, where most of them [139] showed an increase in their abundance (Fig. 4). In H. dentatum, the number of DE genes that increase and decrease their abundance were similar. However, among the different hydration states, the abundance of genes decreases significantly when transitioning from dehydration to rehydration. When comparing both species, H. caudiculatum presents ca. twice DE genes of H. dentatum and a higher proportion of both, increase and decrease abundance of genes under the dehydration process (Fig. 4).
Venn diagrams showing up-and-down regulated genes in H. caudiculatum (a) and H. dentatum (b). The size of each circle is proportional to the number of unique, annotated, and non-duplicated up-or-down regulated genes for each of the species. Numbers inside the circles indicate the number of genes differentially expressed by each pair-wise comparison among the hydration states of the fronds (FH, full hydrated; DH, dehydrated; RH, rehydrated). In H. caudiculatum, the comparison between FH X DH contributes the largest number of differentially expressed genes (up-and-down regulated). For H. dentatum, the comparison between FH X DH contributes the highest number of up-regulated genes, whereas DH X RH contributes the highest number of down-regulated genes. Venn diagrams were generated by using BioVenn (Hulsen et al., 2008)
From the differentially expressed transcripts we explored the function of the gene products by conducting a Gene Ontology analysis (GO) (see Material and Method for details). Both species showed similar enrichment pattern of sequences for each GO category (Fig. 5). For example, at the biological process category (BP), there was a high number of sequences in the metabolic process (> 4000 sequences) and in cellular process (> 2000 sequences). In the cellular component category (CC), organelle and cell part components showed the highest enrichment of sequences (e.g., ca. 1000 and 1500 sequences for organelle, for H. dentatum and H. caudiculatum, respectively). Finally, in the molecular function category (MF), the highest accumulation of sequences was found to be in the antioxidant activity, followed by binding process (Fig. 5).
GO-category distribution of annotated genes for H. caudiculatum and H. dentatum among level 2 GO categories: biological process (BP), cellular component (CC), and molecular function (MF)
In parallel to the differential expression analysis, we also observed all those highly abundant transcripts based on the FPKM value but with a fold change vale < 2, i.e., constitutive highly abundant genes without significant change along the dehydration-rehydration cycle. A total of 102 transcripts for H. caudiculatum, and 128 for H. dentatum show that there are broadly three main processes involved in the desiccation tolerance of these two filmy ferns, namely translation, photosynthesis and antioxidant activity (See Additional file 2: Dataset S3 for the detailed list of the highly abundant constitutive transcripts in each species).
Transcripts clustering patterns and gene co-expression network of H. caudiculatum and H. dentatum across dehydration-rehydration cycle
From the annotated DE genes, we studied the dynamics of gene expression in these two resurrection filmy ferns to identify, firstly, transcripts with similar accumulation patterns in response to a given hydration status, and secondly, the complexity of the interaction and co-expression of genes related with their desiccation and rehydration responses. The self-organizing maps (SOM) partitioned the DE genes into six clusters (hereafter nodes) arranged as a map (Fig. 6 a-c). The underlying topology of the SOM shows distinct accumulation patterns of genes membership across nodes (Fig. 6 a-c), and prominent densities of transcripts for a given hydration state within a node (Fig. 6 b-d), which in topological terms, reflects similar accumulation patterns. Thus, SOM Node 6 in H. caudiculatum and SOM Node 1 in H. dentatum have a prominent density of transcripts associated with the full hydrated state (FH). For the dehydrated and re-hydrated conditions, respectively, the enrichment of transcripts was observed in Nodes 3 and 2 in H. caudiculatum (Fig. 6b), and Nodes 2 and 6 in H. dentatum (Fig. 6d). In order to ascertain if SOM-based clustering yields biologically relevant information, we determined GO enrichment in those nodes. Specifically, we found that for H. caudiculatum, transcripts of the dehydrated state (Node 3) were enriched in functional categories related to stress signaling and response, photosynthesis and photosystem II stabilization and repair, unsaturation of fatty acid, and lignin biosynthetic process. Next, transcripts in the rehydration state (Node 2) were enriched in responses to oxidative stress, lignin biosynthetic process, photosynthesis, protein-chromophore linkage, cellular redox homeostasis, and translation. On the other hand, in H. dentatum, the dehydrated state (Node 2) was enriched in functional categories related mainly with antioxidant responses such as glutathione metabolic process and ROS detoxification systems, drought response, transcription, translation regulators, photosystems II stabilization, ATP synthesis and proton transport, photoprotection, and ABA non-regulated stress responses. For the rehydrated state (Node 6), transcripts were enriched in functional categories corresponding mainly to ethylene and abscisic acid related signaling, photosynthesis, proton transport, plasmodesmata-mediated intercellular transport, response to stress and to toxic substances.
Mapping quality, clustering results for self-organizing maps (SOM) and GO enrichment of transcripts with similar and significant accumulation patterns associated to a given frond hydration state in H. caudiculatum (a-c) and H. dentatum (d-f). The heatmaps (a and d) shows the number of genes (counts) assigned to each SOM Node (numbered from 1 to 6) and the mean Euclidean distance of genes (distance) mapped to the particular Nodes. The constructed maps with a 2 × 3 hexagonal topology shows a reasonable spread out and small distances over the maps, indicating a good mapping process. Nodes with similar codebook vectors (i.e., the patterns of gene neighboring at each hydration state) lie closer to each other. Red color indicates low count and distance, whereas light yellow indicates high count and distance. From the total six Nodes defined after SOM, boxplots were used to visualize and select those Nodes describing the highest pattern of differentially expressed genes for a given hydration state in H. caudiculatum (b) and H. dentatum (e) (see details in the Result section). For each boxplot, horizontal line represents the median, and bars represent the maximum and minimum values of the scaled gene abundance. The pie charts indicate the GO enrichment terms for the annotated transcripts with the highest pattern of differential expression for each hydration state in H. caudiculatum (c) and H. dentatum (f). X axis labels in boxplots read as follow. FH = Full Hydration; DH = Dehydration; and RH = Rehydration
From these SOM nodes (2, 3 and 6 in H.caudiculatum, and 1, 2 and 6 in H. dentatum), we carefully reviewed the annotated genes with high scale expression to construct weighted gene coexpression networks for each node (Fig. 7; see Materials and Methods for details). Based on the Fast Greedy modularity optimization algorithm for finding community structure, all gene-coexpression networks had two modules. The networks obtained from the full hydrated state of H. caudiculatum (Node 6, Fig. 7a), and from the dehydrated state of H. dentatum (Node 2, Fig. 7b) showed the highest gene connectivity, i.e., interactions between genes (6216 and 866 connections, respectively). An overview of the resultant gene-coexpression network for each hydration state of H. caudiculatum (Fig. 7a) showed that at full hydration, twelve hub genes (> 200 connections) composed a core network connecting both modules, which were involved in: protective system against oxidative stress (GPX7, CAT3); light harvesting complexes and reaction centers of photosystems I and II (e.g., CB23, CP24, LHCA 4, LHCB 2, PSAA, PSBA); lipid metabolism and transport (ACLA3, NLTP5). Under desiccation, the resultant network showed six hub genes (> 100 connections), and they were part of one of the modules. Hub genes were involved in: cell wall reinforcement (WUN1); glutathione metabolism (e.g., GSTUJ, GSTX4); mitochondrial uncoupling protein (PUMP4); glycosylation (U85A3); nitrogen mobilization (NRTs). At the rehydration state, the resultant network contained only eighteen genes, all with the same number of connections among them. Two main process were represented by these genes, namely cell wall structure and architecture (e.g., PME53, PRP1, CSE), and stress response and signaling (e.g., GRP, ASR1, DSP22, TET8).
Gene co-expression networks analysis for the full hydrated (FH), dehydrated (DH) and rehydrated (RH) states of (a) H. caudiculatum and (b) H. dentatum fronds. The genes used for the network construction were obtained from Nodes 2, 3, and 6 for H. caudiculatum, and Nodes 1, 2 and 6 in H. dentatum (See Fig. 6b and d). Each network included two modules indicated by the colors of the circles (green and light blue for H. caudiculatum, yellow and blue for H. dentatum). Hub genes (> 100 connections) are in red. Each of the modules contain transcripts with denser connections representing predicted interactions. The names of those genes showing higher connections within a given hydration state are indicated and their specific functions are discussed on the text
Regarding H. dentatum, there were no hub genes in none of the resultant gene-coexpression networks (Fig. 7b). At the full hydration state, the network contained twelve coexpressed genes, equally connected among them. They were grouped into different functional categories, such as plant defense and abiotic stress resistance (e.g., DIR5, ERF17, BURP16), chloroplast development (e.g., PBP1), and cell wall structure and architecture (e.g., PME53). Under desiccation, the resultant network contained a total of forty-two coexpressed genes. Among them, we found genes involved in flavonoid biosynthesis (CHSY), structure of photosystems I and II (PSAA, PSBS), stress response (GL82), ubiquitination (UBIQP), aldo-keto reductases (ALKR4), immune system and Salicylic acid homeostasis (DLO2), non-symbiotic hemoglobins (HBL) which function in signal transduction pathways of several hormones, Jasmonic acid precursor (OPR7). Lastly, at the rehydration state the resultant network contained only 6 coexpressed genes. They were mainly involved in membrane metabolism (e.g., GDPD), stress response and signaling (e.g., DIR5, GRG1, DRE2A), and plant immunity (e.g., PUB25, TGA2.1), and sugar metabolism (GRG1).
Previous studies have indicated at different scales, from cellular to ecophysiological approaches, how Hymenophyllaceae species from temperate rain forest responds to desiccation [12, 13, 15,16,17, 23]. However, the link among desiccation response, habitat preferences and gene expression have never been addressed before in Hymenophyllaceae until our present study. Both de novo assembled transcriptomes (the first for the Hymenophyllaceae family) exhibited good quality parameters, comparable to other non-model species transcriptomes sequenced with the same Illumina platform and using the same criteria for subsequent downstream assembly pipelines [24,25,26]. Thus, the robustness of our transcriptomes allowed us to explore differential gene expression and to contrast the dynamical patterns of high-and-low abundance of genes during the transition from dehydration to rehydration versus the constitutive gene expression, thereby contributing to a comprehensive view of the response mechanisms involved in a fast dehydration-rehydration process. Finding patterns in massive gene expression data sets consistent with a biological function is always a challenge. Despite the undeniable advantages of exploratory statistical models such hierarchical clustering [27] and k-means clustering [28], these methods are subjective due to human bias based on arbitrary statistical significance threshold and does not consider the topology between clusters [29]. The characteristics of the artificial neural networks, considering topology in clusters neighboring [19, 20], provides an excellent tool to depict clustering patterns of gene expression across multiple factors [19, 30, 31]. With the differential gene expression analysis, we found similar patterns of molecular responses between both filmy ferns during the desiccation-rehydration cycle. Then, by combining the Self-Organizing Maps with weighted gene co-expression network analysis, we showed clear patterns of transcript accumulation and found a clear relationship between core genes that are co-expressed in a given hydration state. Thus, we were able to elucidate both shared and species-specific molecular components such as mechanisms of signaling and responses to desiccation [6, 32], disturbance of chloroplast homeostasis [33, 34], and photoinhibition-related stresses [35,36,37], associated with frond hydration state and the spectrum of microhabitat that H. caudiculatum and H. dentatum occupy along the vertical distribution over host trees.
At a fully hydrated state, the patterns of transcripts accumulation in both species reflect normal functioning of primary metabolic process such as photosynthesis and respiration. After dehydration, the clustering patterns and abundance of differentially expressed identified transcripts, shared mechanisms associated with the maintenance of redox homeostasis, stabilization and maintenance of the photosynthetic apparatus, and chloroplast operational signaling. In contrast, the species-specific responses were particularly associated to phenolic compounds biosynthetic pathways (e.g., phenylpropanoid metabolism) and photoinhibition related processes. Both general and species-specific responses agree with physiological, ultrastructural, and chemical changes, as with the homoiochlorophyllous strategy of these filmy ferns [11, 13, 15, 23].
Shared patterns of transcriptional responses during the dehydration-rehydration cycle
It must be noted that because the relatively few transcripts showing differential expression, the desiccation tolerance response of these two filmy ferns would rely mainly on constitutive mechanisms, as it has been suggested by proteomic analyses [16]. Based on our results, both filmy-ferns have high abundance of transcripts involved in translation, photosynthesis and antioxidant capacity regardless the hydration state of the frond. An interesting result from the highly abundant constitutive genes is the expression of the LATE EMBRYOGENESIS ABUNDANT PROTEIN 14 gene (LEA14) which has been reported as an atypical LEA protein localized in the cytoplasm and nucleus, with important roles against abiotic stress, particularly osmotic and drought stress [38, 39].
The patterns of differential gene expression shared by the two species (as shown in Fig. 4) suggest conserved mechanisms of responses regardless their habitat preferences along the vertical gradient of host trees. Thus, during dehydration both species increase the abundance of genes encoding for Glutathione-S-Transferases (GST), while there is a decrease of GST genes following rehydration. GST enzymes scavenge a wide variety of toxic compounds (e.g., hydrophobic, electrophilic and cytotoxic substrates; see [40]). When the fronds of both filmy ferns turn desiccated, they must deal with the cytotoxic effects of reactive oxygen species associated with multiple and simultaneous stress to which they are subjected, such as water deficit and high light stress [41]. Efficient detoxification and antioxidant defense systems are key components for desiccation tolerance [3, 6, 36]., Given that GST enzymes inactivates a wide variety of toxic compounds (e.g., hydrophobic, electrophilic and cytotoxic substrates; see [38]), these two ferns appear to have evolved a mechanism to finely tune the GST responses against oxidative damage during the desiccated state. We found the same dynamic in genes encoding for ferritins which could be a more specific mechanism coupled with the aforementioned for GST. Ferritin is a putative iron storage protein that has been linked with ROS metabolism in plants. Specifically, ferritin synthesis is activated at the transcriptional level by cellular iron and H2O2 as well as by high light intensity, enhancing ROS detoxifying enzymes [42]. Evidence that links ferritin and ROS metabolism points out that the control of ferritin synthesis would be required for a proper maintenance of the redox status of fronds cells. Moreover, it is known that ferritin domains are part of the structure of desiccation related proteins (DRP, [43]), therefore besides the antioxidant role of ferritin, the two filmy ferns may use this protein for protection of membrane structures, such as photosynthetic structures [44].
Both species also shared patterns of gene expression involved in the control of structure and function of photosystems. During desiccation both species accumulate transcripts encoding for components of reaction centers of Photosystem I and II while there is a decrease in transcripts abundance encoding for light-harvesting antenna and oxygen-evolving complexes. This response differs with what has been reported for the model resurrection plant Craterostigma pumilum, in which the inverse pattern occurs [36]. A plausible explanation is that because of the homoichlorophyllous nature of Hymenophyllaceae species, i.e. the retention of their chloroplast when desiccated, this genes dynamics would help during the time-lapse of desiccation-rehydration against the combined effects of severe dehydration and intermittent occurrences of high irradiances inputs by sunflecks to deal with reactive oxygen species, oxidative damage [45], and the need to minimize the potential for light capture and the oxidation of water in order to decrease redox reactions but to maintain a stock for reaction centers.
The coordination of the complex mechanisms such as those mentioned above require intertwined signaling pathways for systemic responses against the internal and external constraints associated with the resurrection strategy. The increases of transcript abundance of AAA-ATPase genes which are known to be involved in ROS signaling and the presence of components of signal transduction such as Auxin and calcium signaling, oxylipin metabolism, and ethylene and ABA responsive transcription factors points to the chloroplast as the signaling orchestrator [34]. Because the fronds of these ferns ranges from one to a few cells layers, and given that plastid-to-nucleus signaling is essential for the coordination and adjustment of cellular responses to external and internal cues [33, 46, 47], environmental perturbations imposing restrictions for the chloroplast homeostasis would have a great impact in whole frond form and function. Fluctuations in frond water status will initiate an operational signaling to change the expression of thousands of genes to adjust chloroplast and the whole cell functioning [34]. According to this, both species showed an increase of transcript abundance that encode for AP2/ERF transcription factors during desiccation (e.g., DREB, ERF, RAP), which are known to be involved in responses to environmental stimuli, especially for redox, osmotic and drought stress [33, 34]. Moreover, a coordination of chloroplastic and nuclear gene expression would be mediated by the redox status [48], activating a protein kinase phosphorylation cascades and AP2/ERF transcription factors. Our results are consistent with the above-mentioned mechanism, being a key mechanism contributing to the resurrection strategy used by the two studied filmy ferns.
Species-specific patterns of transcriptional responses during dehydration and rehydration
One of the most ambitious goals of our study was to associate the different niche preference of these two filmy fern species with species-specific transcriptional responses to have insights into particular physiological conditions that may prevail under different frond hydration states. In general, based on the DE analysis and the pairwise comparison among hydration states (Table 1), the specific responses in H. caudiculatum underpins enhanced osmotic responses and phenylpropanoid related pathways, whereas in H. dentatum the specific responses point to enhanced protection against oxidative damage and high light stress. By the other hand, the gene co-expression networks analysis provides a detailed interaction of central genes conferring each species-specific response. Examining the responses of H. caudiculatum during the dehydration-rehydration cycle, we observed an increase in the abundance of the DOG1 gene when full hydrated and rehydrated. This gene is involved in controlling the timing of seed dormancy in response to environmental signals such as temperature by altering gibberellin metabolism [49]. Given that during embryo development and maturation the acquisition of desiccation tolerance is a key process for the viability of the majority of seeds, it is logical that resurrection plants use part of the molecular components involved in the control of seed dormancy. However, the changes in the abundance of DOG1 gene in H. caudiculatum suggest an induced mechanism linked to ABA and GA balance by a common intermediary such as DELLAs proteins [50]. Based on the gene-coexpression network results, we observed for the dehydration state that core genes are related to minimize ROS formation and oxidative stress through NAD metabolism and mitochondrial uncoupling proteins [51, 52], while for rehydration all co-expressed genes are involved mainly in cell wall and protein stability [53], membrane stability and signaling, and the reestablishment of glucose metabolism (Fig. 7a).
Table 1 Differential expression of transcripts of interest (FDR < 0.05) during experimental desiccation-rehydration cycle in H. caudiculatum and H. dentatum fronds. The table shows the pairwise comparison of the increase/decrease abundance of transcripts between the transition of fronds from one to another hydration state, namely full hydrated (FH), dehydrated (DH, and rehydrated (RH). Gene identities were obtained with GO-enrichment by Panther Classification System V11.0. The level of regulation of each gene was estimated by value of the logarithm of fold change (logFC, on brackets) above and below 1.0 and − 1.0, for up and down regulation, respectively
We observed an increase in the abundance of the ELI6 gene which encodes an early light inducible protein (Table 1) in H. dentatum when full hydrated and rehydrated. The same patter was observed for the dehydration response element transcription factor DRE2A. ELIPs proteins are induced by light, and act as sinks for excitation energy under high light. The increase in abundance ELI6 and DRE2A only in H. dentatum (Fig. 7b) would be a response associated with its distribution toward more illuminated and low humidity micro-sites along the vertical direction of host trees. Other specific responses are linked with the activation of defense responses driven by Ethylene and Salicylic Acid, and the scavenging of specific ROS such as hydrogen peroxide.
The present work provides a deeper understanding of the mechanisms underlying the desiccation tolerance responses of H. caudiculatum and H. dentatum. Here we have deciphered the transcriptional dynamics, gene co-expression networks and key genes involved in the signaling and response mechanisms during the process of dehydration and rehydration for both species. In addition, the identification of species-specific mechanisms contributes to explain their ecophysiological traits and microhabitat preferences they show in their natural environment. Thus, while the lower-canopy species H. caudiculatum seems to enhance osmotic responses and phenylpropanoid related pathways, the upper-canopy species H. dentatum enhanced its defense system responses and protection against high light stress.
Finally, much more work is needed to understand the roles and contribution of specific molecules (e.g., RNA-binding proteins) and posttranscriptional regulations that may have central roles in the desiccation tolerance strategy of these species when subjected to rapid loss-and-gain of water as occurs in the natural environment. Also, studies under an evolutionary developmental biology approach would help to decipher and understand the genetic basis of physiological, developmental and morphological variation associated with the microclimatic conditions in which these resurrection filmy ferns have evolved.
Plant material and growth conditions
Individuals of Hymenophyllum caudiculatum (Mart.) var. productum (K. Presl.) and Hymenophyllum dentatum (Cav.) (Fig. 1) were collected from Katalapi Park (41°31′07.5″ S, 72°45′2.2″ W) Cordillera de Quillaipe, Los Lagos Region, Chile. The collecting site correspond to a coastal evergreen temperate rainforest. Details on microclimatic conditions of the site have been previously published [11]. Small pieces of bark or fallen trees covered with epiphytic filmy ferns were collected in late Spring (Nov-Mid Dec, Southern hemisphere) and transported to a shaded experimental nursery garden with automated irrigation at the Universidad de La Frontera. Further description of nursery conditions and plant set up can be found in [17]. Briefly, plants were acclimated to nursery garden conditions under shade (25–30 μmol photons m − 2 s − 1), nebulized by an automated irrigation system (6 daily waterings, 2 min each) for 25 days prior to experimental procedures.
It is worth to note that the deposit of specimens to an herbarium was not necessary, since they already exist in the collection of the Herbarium of the Universidad de Concepción (http://www2.udec.cl/~herbconc/index.htm, Herbario CONC, Departamento de Botánica, Universidad de Concepción, Barrio Universitaro s/n, Casilla 160-C, Concepción, Chile.)
Experimental design and sample collection
The experimental design was a desiccation-rehydration experiment under nursery conditions during late spring (Nov-mid Dec, southern hemisphere) in which fronds of H. dentatum and H. caudiculatum were studied at three different hydration states; full hydrated (FH), dehydrated (DH), and rehydrated (RH) (detailed in Fig. 2a). At each hydration state, we used attached fronds for monitoring the changes in maximal quantum efficiency (Fv/Fm), and two set of detached fronds; one to determine their relative water content (RWC), and the other for RNA isolation. To reach a fully hydrated state, ferns were subjected to irrigation pulses of three minutes at intervals of twenty minutes each, during four hours; a single frond from each plant (3 plants per species, 6 plants total) were taken at this hydration state (FH) to obtain its relative water content (RWC). Then, ferns went through a desiccation-rehydration process by adjusting the irrigation settings and monitoring the changes in RWC and Fv/Fm of the fronds during the next seven days. AT the first day without irrigation, the first DH sampling (fronds from three different individuals per species) was taken when fronds reached about 60% of RWC. A second DH sample was obtained when plants reached a value equal or below the critical relative water content reported for these species [11], achieved at day seven (Fig. 2a). Both DH samples were pooled in order to have coverage for early and late desiccation-regulated transcripts. Finally, the rehydration was carried out resuming irrigation until at least a 90% of RWC was recovered. During rehydration (RH) two samplings were taken, one was obtained when plants recovered 60–70% of RWC, and the second sampling was taken at ca. 90% of RWC. Both samplings were pooled as RH in order to have coverage for early and late rehydration-regulated transcripts. Samples were stored at − 80 °C until processed for RNA isolation for comparisons of the transcriptional responses associated to each hydration state, either within or between species.
Fluorescence measurements
Maximal quantum efficiency (Fv/Fm) was calculated from chlorophyll a fluorescence signals obtained from attached fronds using a modulated fluorometer (FMS2, Hansatech, UK) as previously described in [17]. Briefly, attached fronds were carefully cover and dark acclimated for 30 min using FMS2 leaf clips. After dark acclimation the modulated light was turned on to obtain F0, then a saturating pulse (800 mS at ~ 3000 μmol m− 2 s− 1) was applied to obtain the maximum fluorescence (Fm). Variable fluorescence (Fv) and Fv/Fm ratio were calculated according to [54] at ambient temperature (17 C° approx.). Data was analyzed after check normality assumptions under an ANOVA test with P value ≤0.05. When data did not meet the normality assumptions, we used the non-parametric test of Kruskal-Wallis. Additionally, fluorescence images of Fv/Fm were obtained using detached fronds under each hydration condition FH, DH and RH using a Maxi-Imaging PAM (Walz, Effeltrich, Germany) to observe the rate of recovery of the quantum yield of fluorescence (YII) at the whole frond level of both species (Fig. 2b).
RNA-seq and De novo Transcriptome analysis
Total RNA was isolated using UltraClean™ Plant RNA Isolation Kit (Mo Bio, Carlsbad, CA, USA) and purified with Total RNA I kit (Omega Bio-Tek, Norcross, GA, USA) according to manufacturer's instructions. The yield and quality of the RNA isolation samples was determined by an Agilent 2100 Bioanalyzer. The RNA was precipitated with two volumes of acetate:ethanol solution (1:10 v/v) and sent for sequencing to Macrogen Inc., Korea. A total of six samples were sequenced in a single lane of an Illumina HiSeq 2000 platform (Illumina Inc. San Diego, CA, USA) obtaining 100 bp paired end reads. After quality filtering with the NGSQC Toolkit v2.3 ([55], http://www.nipgr.res.in/ngsqctoolkit.html) and Q-score composition (Additional file 3: Figure S1), we conducted a de novo assembly with the Trinity software package v2.1.1 [56, 57]. Transcriptome assembly was performed at Troquil Linux cluster at Centro de Modelación y Computación Científica (CMCC, Universidad de La Frontera) using 12 processors Intel Xeon E5–4640 and 192 GB of shared memory.
Reads from each sample were mapped to their corresponding final transcriptome using default RSEM parameters (RSEM = RNA-Seq by Expectation Maximization) written in the align_and_estimate_abundance.pl script. The resulting RSEM-estimated gene abundances for each fern were merged in a matrix and analyzed with run_DE_analysis.pl script from Trinity, which involves the Bioconductor package edgeR in R statistical environment [58, 59]. Transcripts with very low estimated counts (2 for combined groups), were not considered for edgeR pair-wise comparison of hydration states. To judge significance of gene expression, we used a False Discovery Rate value (FDR) lower than 0.05 and a minimum fold change (FC) of 2 as thresholds (Additional file 2: Dataset S1 and S2). To quantify transcript abundance, normalized RSEM-estimated counts were used for clustering assembled contigs based on expression patterns [29]. Finally, the resulting transcripts of each transcriptome were aligned into the SwissProt database using BLAST+ with an e-value filter of 1-e− 10 as threshold [60]. Functional annotation, classification, and over-or under-represented groups of genes were performed using PANTHER ([61], www.pantherdb.org).
Self-organizing maps (SOM) analysis
Normalized RSEM-estimated counts of both H. caudiculatum and H dentatum that met the expression values determined from the model described above were used for the SOM clustering method [29]. Specifically, only genes that vary significantly in expression across fronds hydration state of both filmy fern species were analyzed. To focus only on gene expression profile, and at the same time avoid biases with the differences in the magnitude of gene expression, expression values were mean centered, selected from the upper 50% quartile of coefficient of variation, and variance scaled using the scale function (R base package [58];) separately for H. caudiculatum and H. dentatum. Scaled expression values were used to cluster genes in both species across fronds hydration states into a multidimensional 2 × 3 hexagonal SOM using the Kohonen package on R [19]. One hundred training iterations were used during clustering with a decrease in the alpha learning rate from ca. 0.0018 to 0.0010 (Additional file 3: Figure S2). After the iteration process, the final assignment of genes to the winning units shaped the clusters of genes (termed nodes) associated to the hydration states. SOM outcome was visualized into pie charts for codebook vectors to obtain the counts number and mean distance of the genes assigned to each node ([19], Additional file 3: Figure S3). The box plot option from the ggplot2 package on R was used to visualize the gene accumulation patterns associated to the hydration states of the fronds in each Node. Finally, the genes of each Node were analyzed for GO enrichments terms at a 0.05 false discovery rate cutoff (Additional file 4: Dataset S6 and S7).
Gene co-expression network analysis
In order to go further the gene co-expression patterns obtained from the SOM analysis, a further Gene Regulatory Network-based (GRN) approach was used to study the interactions between gene expression and fronds hydration state of each fern species. From the SOM clustering method, a subset of 67 and 183 annotated genes from H. dentatum and H. caudiculatum were selected, respectively. For the selection of genes, those transcripts from SOM nodes with the highest scaled accumulation patterns for a given hydration state were chosen. Then, only those genes with GO annotation were selected. (Additional file 2: Dataset S4 and S5). These genes were used to construct a weighted gene co-expression network according to [31]. Briefly, features of two R packages, namely, Weighted Gene Coexpression Analysis (WGCNA [18];) and igraph [21], were combined to visualize the genes interaction. To calculate the adjacency matrix with the WGCNA package, a soft threshold (β) value of 9 was used to achieve the scale free topology criteria. Then, the network connectivity and modularity were visualized by using the algorithms of the graph generator and community structure functions of the igraph package, whilst custom graph functions were used for network visualization (available at https://github.com/eostria/Gavel).
Sequence submission
The quality-filtered, barcode-sorted, and trimmed short read data set used for transcriptome assembly and gene expression analysis, was deposited in the NCBI Sequence Read Archive (SRA) under accession SRR5195043, SRR5272488, SRR5272490, SRR6452149, SRR6452987, and SRR6453153. Additionally, the gene expression profiles of the assembled transcriptomes were deposited in the NCBI Gene Expression Omnibus database (GEO). For confidential access to the public GEO records use the following links: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140234 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140238.
The transcriptomes of the ferns species used in this study are available in the NCBI Sequence Read Archive (SRA) under accession SRR5195043, SRR5272488, SRR5272490, SRR6452149, SRR6452987, and SRR6453153. The gene expression datasets are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140234 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140238.
The custom script used for the analysis and construction of the gene co-expression networks is available at https://github.com/eostria/Gavel.
All data generated and analyzed in this study are available and included in this manuscript, as well as its supplementary information files.
Differential expression
DH:
FDR:
False Discovery Rate
FH:
Full Hydrated
FPKM:
Fragments per kilobase of exon model per million reads
Fv/Fm:
Maximum quantum efficiency
GO:
PFD:
Photon flux density
RH:
Rehydrated
ROS:
RSEM:
RNA-Seq by Expectation Maximization
RWC:
Relative Water Content
Self-Organizing Maps
WGCNA:
Weighted Gene Co-expression Network Analysis
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Magwanga RO, Lu P, Kirungu JN, Lu H, Wang X, Cai X, et al. Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton. BMC Genet. 2018;15:6.
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Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R. Reactive oxygen species homeostasis and signaling during drough and salinity. Plant Cell Environ. 2010;33:453–67.
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Carniel FC, Gerdol M, Montagner A, Banchi E, De Moro G, Manfrin C, et al. New features of desiccation tolerance in the lichen photobiont Trebouxi gelatinosa are revealed by a transcriptomic approach. Plant Mol Biol. 2016;91:319–39.
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Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protocol. 2013; 8: 1494–512.
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Rodríguez R, Alarcón D, Espejo J. Helechos Nativos del Centro y Sur de Chile. Guía de Campo. 1st ed. Chile: Corporación Chilena de la Madera, Concepción; 2009.
The authors thanks Katalapi Park for their hospitality and excellent research facilities for plant collection. Also, thanks Dr. Charles L. Guy for the critical revision and English editing.
This work was supported by the Projects FONDECYT 1120964, FONDECYT-CONICYT grant for the Postdoctoral Project No 3160446, and NEXER Project (NXR17–0002). The funding bodies had no role in the design, collection, analysis, and interpretation of data nor in the writing the manuscript.
Laboratorio de Fisiología Vegetal, Centro de Estudios Avanzados en Zonas Áridas CEAZA, La Serena, Chile
Enrique Ostria-Gallardo
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile
Enrique Ostria-Gallardo, Graciela Berríos, Ana Fallard & León A. Bravo
Centro de Excelencia de Modelación y Computación Científica, Facultad de Ingeniería y Ciencias, Universidad de La Frontera, Temuco, Chile
Giovanni Larama
Universidad Autónoma de Chile, Santiago, Chile
Ana Gutiérrez-Moraga
Department of Biology, University of Toronto, Toronto, ON, Canada
Ingo Ensminger
Laboratorio de Fisiología y Biología Molecular Vegetal, Departamento de Cs. Agronómicas y Recursos Naturales, Facultad de Cs. Agropecuarias y Forestales, Instituto de Agroindustria, Universidad de La Frontera, Temuco, Chile
León A. Bravo
Graciela Berríos
Ana Fallard
GL, GB, AG, and LB conceived the study. AG and LB provided materials. GB. AF and LB coordinated sampling and desiccation-rehydration experiment. GB and AF isolated the RNA. GL assembled de novo the transcriptomes. GL and EO-G analyzed the transcriptomic data sets. EO-G and IE worked on interpretation of transcriptomic data. EO-G generated and analyzed the cluster and artificial networks.; EO-G, LB and G.L. wrote the article with contribution of all other authors; all authors read, edited, and approved the article.
Correspondence to Enrique Ostria-Gallardo or León A. Bravo.
Plant collection was made on the Katalapi Park Foundation, a non-profit institution for research and environmental education. Dr. Luis J. Corcuera, President of the Foundation provided the permission to access and collect the plant material from the Park. There were no need for special permissions from any other government or private agent for collecting the plant material used in our study. The fern species were identified by EO and LB based on the description of the species published on [62]. The experimental design was made following the institutional normative of the Ethics Committee of the University of La Frontera.
The authors declare that they have no competing interest.
Additional file 1:
Table S1. Statistics for RNA-seq libraries sequencing of H. caudiculatum (Hca) and H. dentatum (Hdent) for each of their hydrated states. Statistics shows total number of reads (No. of reads), high quality reads that passed quality control (No. of HQ reads), reads that were mapped to transcriptomes (No. of mapped reads) and their percentage of mapping (Mapping rate) Table S2. Assembly statistics for H. caudiculatum and H. dentatum transcriptomes before (raw) and after refinement (filtered). The total number of transcripts (No. transcripts), number of trinity components (Trinity components), putative transcriptome in mega base pairs (Transcriptome Size), basic statistics on transcript lengths (Average, median, minimum and maximum) and assembly quality as N50 (N50) Table S3. Blast results for H. caudiculatum and H. dentatum against SwissProt database. Table shows the counts distribution per sequence size (Size range) of blast hit (Blast Hit) and missing hits (Unknow) and the cumulative percentages (Cums %)
Additional file 2: Dataset S1.
Matrix of the DE genes among pairwise comparissons between frond's hydration states of H. caudiculatum. Columns indicates de sequnce ID (id), fold change (log FC), counts per million (log CPM), the significance (PValue), the false discovery rate value (FDR), and the normalized counts for each hydration state (HcaFH.matrix, HcaD.matrix, and HcaRH.matrix), Dataset S2. Matrix of the DE genes among pairwise comparissons between frond's hydration states of H. dentatum. Columns indicates de sequnce ID (id), fold change (log FC), counts per million (log CPM), the significance (PValue), the false discovery rate value (FDR), and the normalized counts for each hydration state (HdFH, HdD, and HdRH), Dataset S3. Matrix showing those highly abundant genes without significant changes between frond's hydration states of H. caudiculatum and H. dentatum. Columns shows the gene id, transcript id, the counts abundance for each hydration state, the UniProt ID, the percentage of similitud of the Blast query, and the Protein Name, Dataset S4. Matrix of the Self-Organizing Maps output for the Nodes 2, 3 and 6 for H. caudiculatum, Dataset S5. Matrix of the Self-Organizing Maps output for the Nodes 1, 2 and 4 to 6 for H. dentatum.
Additional file 3
: Figure S1. Quality scores and accuracy of Illumina Hiseq 100-bp paired end reads for H. caudiculatum and H. dentatum after quality filters. Bad quality reads that showed values 20 on the histogram were discarded. All reads above the value of 30 indicates 99.9% of accuracy, regarding to bases correctly read by the sequencer, Figure S2. Training progress of the average distances of genes of H. caudiculatum and H. dentatum using Self-Organizing Maps showing the effect of neighborhood shrinking to include the winning unit, i.e., when the vectors in the dataset reach the closest similarity. Figure S3. Codebbook vectors for H. caudiculatum and H. dentatum showing the clusters of differentially expresed genes with maximum neighbouring after training process. The codebook vectors represent the expression profile of genes associated to a given state after the constructionof the map
Compiled GO enrichment data for H. caudiculatum, Dataset S7. Compiled GO enrichment data for H. dentatum.
Ostria-Gallardo, E., Larama, G., Berríos, G. et al. A comparative gene co-expression analysis using self-organizing maps on two congener filmy ferns identifies specific desiccation tolerance mechanisms associated to their microhabitat preference. BMC Plant Biol 20, 56 (2020). https://doi.org/10.1186/s12870-019-2182-3
Hymenophyllaceae
Poikilohydric
Homoiochlorophyllous
Temperate rainforest
RNA-seq transcriptome
Gene co-expression
Plant-abiotic interactions


Vancouver---By 7:20 a.m. on a Thursday morning, one hundred and seventy-five people had formed a line around the corner of Cambie and Sixteenth.
At the front of the line, the truly determined, sat on folding chairs and sipped coffee from Starbucks cups. They had risen before six a.m. to get their places. Lured by the promise of a "free bag of groceries" for the first two hundred, bargain-hunting folk continued to line up way past the free bag mark to participate in the grand opening of the new Capers store. Among the crowd also were nine-year-olds and four-year-olds who just liked the idea of winning a prize.
Fairview MLA Gregor Robertson, dressed to the nines in an elegant silvery green suit, surveyed the crowd and said, "I've never seen anything like this."
Then he went to the front of the line to cut the ribbon, and welcome his constituents to the newest community market, at 8 a.m.
A savvy marketer from the Hollyhock Educational Centre which is located on Cortes Island handed out brochures to the centre.
"It helps the time pass," said a woman with a newborn in a carriage, leafing through pages about meditation, leadership training and personal growth.
At seven-thirty a.m., Capers employees bracleted the first lucky two hundred and marked them as winners.
The doors opened at 8 a.m. Once the first two hundred people had entered the stores, more kept coming. It became impossible to move, much less to shop. The back-up to get the free and previously prepared bags became huge. Shopping carts couldn't fit through the ailes.
Nevertheless, with patience, people walked away with bounty.
"We bagged it," said Elsie Jones, who lived in the Cambie in 1924, but moved away to Marpole, and then settled on Granville and thirtieth, straightening her pink rimmed glasses and beaming at her free red cloth bag stuffed with...well, stuff.
Samples, to be exact. A lot of tea, including a bag of Spicey Chai Green Tea, a jar of jelly, a box of soy milk, one piece of fruit gummy bear candy, a box of organic raisin bran cereal, a sample of iron supplement, a small, lovely smelling bar of soap, two organic oreo cookies, various small pacakges of pasta, one Cherry Spritzer, a small container of coconut milk drink, a small water in a plastic container, a tiny shampoo, and equally small body lotion container, a sampler of ginsing supplement and a variety of other samples the ordinary shopper might or might not find useful.
But a lot of marketing happened in a congenial setting on a sunny morning. For Capers, it was a grand and successful marketing caper. The people got stuff and the store got stuffed with people.
There was the lady passing out brochures for the educational centre on Cortes Island, and the politician cutting the ribbon and greeting his constiutents.
And at 9 a.m., the party wasn't over. That night, an employee told shoppers, Capers would be hosting a party. Come one, come all. For free.


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text:
April 30, 2015 07:00:48
Exclusive: Check Out Cupcakes and Cashmere's Birchbox Collaboration
Cupcakes and Cashmere founder Emily Schuman is the newest name on Bi...
by Carly Ostroff
Cupcakes and Cashmere founder Emily Schuman is the newest name on Birchbox's roster. Schuman is the first lifestyle blogger to partner with the beauty e-commerce site on a limited-edition collection of curated sample boxes, available later this month in Canada.
The one-off boxes include a selection of Schuman's favourite products, from brands like W3LL PEOPLE, 12 Benefits, Not Soap Radio, Benefit Cosmetics and LAQA & Co.
"I'm a total beauty junkie and I think Birchbox and Cupcakes and Cashmere have similar visions," Schuman said exclusively to The Kit. "At the end of the day, both of us are trying to focus on the little things to make every day extraordinary."
The draw was equally strong for Birchbox. "As a long-time fan of Cupcakes and Cashmere, it's been inspiring to watch Emily grow the blog into an incredibly successful business," said Katia Beauchamp, Birchbox co-founder and co-CEO. "We're proud to welcome her and her readers into the Birchbox world, and we know Emily's 'elevate everyday' approach to beauty will truly resonate with our subscribers."
Fans of the blogger can expect products which reflect Schuman's newest role, as a new mom to three-month-old Sloan. "After becoming a mom, the time I spend on my beauty routine has dramatically decreased. If there is a multi-tasking product, I am on it. A two-in-one cheek and lip tint is a lifesaver," says Schuman. In addition to product selection, Schuman has developed tips and tutorials, which will be live on Birchbox's social media channels.
Sign up for the Cupcakes and Cashmere box by May 17 at birchbox.com/cupcakesandcashmere, available to new subscribers for $10, plus $5 shipping. Schuman's new book Cupcakes and Cashmere at Home rolls out on birchbox.ca May 19.
For the latest in fashion and beauty, pick up a copy of The Kit's print edition found inside The Toronto Star on Thursdays, Edmonton Journal, Calgary Herald and Ottawa Citizen on Fridays and The Vancouver Sun and Montreal Gazette on Saturdays.


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text:
Елчо () — переписна місцевість (CDP) в США, в окрузі Ланґлейд штату Вісконсин. Населення — 339 осіб (2010).

Географія
Елчо розташоване за координатами  (45.436765, -89.188825).  За даними Бюро перепису населення США в 2010 році переписна місцевість мала площу 3,11 км², з яких 2,75 км² — суходіл та 0,35 км² — водойми.

Демографія
Згідно з переписом 2010 року, у переписній місцевості мешкало 339 осіб у 161 домогосподарстві у складі 93 родин. Густота населення становила 109 осіб/км².  Було 199 помешкань (64/км²).

Расовий склад населення:

До двох чи більше рас належало 3,5 %. Частка іспаномовних становила 2,7 % від усіх жителів.

За віковим діапазоном населення розподілялося таким чином: 23,0 % — особи молодші 18 років, 54,0 % — особи у віці 18—64 років, 23,0 % — особи у віці 65 років та старші. Медіана віку мешканця становила 45,6 року. На 100 осіб жіночої статі у переписній місцевості припадало 89,4 чоловіків;  на 100 жінок у віці від 18 років та старших — 94,8 чоловіків також старших 18 років.

Середній дохід на одне домашнє господарство  становив  доларів США (медіана — ), а середній дохід на одну сім'ю —  доларів (медіана — ). Медіана доходів становила  доларів для чоловіків та  долар для жінок. За межею бідності перебувало 14,4 % осіб, у тому числі 0,0 % дітей у віці до 18 років та 0,0 % осіб у віці 65 років та старших.

Цивільне працевлаштоване населення становило 163 особи. Основні галузі зайнятості: освіта, охорона здоров'я та соціальна допомога — 36,8 %, виробництво — 14,7 %, роздрібна торгівля — 12,3 %, мистецтво, розваги та відпочинок — 11,7 %.

Примітки

Джерела

Переписні місцевості Вісконсину
Населені пункти округу Ланглейд (Вісконсин)

redpajama_set_name: RedPajamaWikipedia


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text:
EBONYLIFE GROUP
EBONYLIFE MEDIA
The Crunch
HNC spokesperson comments on latest round of peace talks in Geneva
Mar 28, 2016 Syrian opposition High Negotiations Committee spokesperson Salim Muslet recently spoke about the committee's positions compared with those of the Syrian government during the latest round of peace talks in Geneva.
Speaking during an interview, Muslet said:
"We diverge a lot with the government. We came to settle the Syrian crisis peacefully, but the government is still using air raids to strike the people. We represent the people, but the Syrian government only cares about the fate of one person (Bashar al-Assad), not that of the 23 million Syrians,"
Although the committee says it represents the interests of Syrians, however the fact that many of its members reside outside the war-torn country has elicited doubt on the legitimacy of the claim.
Muslet moved to defend this arrangement saying:
"Those who have emigrated overseas used to be in the government prison for many years. They were afraid of being purged after leaving prison, so they fled to other countries. Most of us, however, are still from within Syria. Although they (those who have emigrated) live overseas, they stay in touch with those within the border, and their families are in the country. So it is not the case that those residing overseas do not care about the people living within the country,"
The latest round of negotiations between the Syrian government and opposition forces is expected to continue in April.
Nigerians call for transparency in spending funds recovered from graft →← Day wins match but heads for treatment after hurting back
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As a member of the Maurice A. Deane School of Law's alumni community, you receive many benefits and services. For more information on these benefits and services, please contact the Office of Alumni Relations at 516-463-2586 or lawalum@hofstra.edu.
For more information on these benefits and to receive your free Alumni ID card, please contact Allyson, director of alumni relations, at allyson.t.lehrer@hofstra.edu.


text:
Eduard Boas, född 18 januari 1815 och död 12 juni 1853, var en tysk författare.

Boas stod i sina lyriska dikter den romantiska skolan nära, företog resor i södra och norra Europa och skildrade sina intryck i bland annat Des Kriegskommissars Pipitz Reise nach Italien (1841) och In Skandinavien. Nordlichter (1844).

Källor
 Svensk uppslagsbok, Malmö 1939.

Tyska författare
Födda 1815
Avlidna 1853
Män
Svensk uppslagsbok


Youngstown Home Learners is an educational association brought together for the purpose of educational excellence. We accept all races and all religions. Religious practices are studied for their historical attributes only. We are a secular group that provides supplimentary classes for our school age children. Classes are taught by parent professionals and by outside speakers brought in by parents. Music and Art Classes are also offered. Field trips and fun days are also part of our organization.
Youngstown Home Learners meet every 1st Friday of the month, September through May for Parent's organizational meetings. Meetings are now taking place at St. Dominic's Sienna Hall on the corner of Avondale and Southern Boulevard at 6:30 pm. We are in the process of finding another meeting place as the hall will soon be torn down. Anyone with information concerning a free meeting place for our meetings and for our children's classes, please e-mail us at musik1@earthlink.net . You may also email this address for more information.
Currently, membership is $15.00 which includes our Parent's Forum, part of which is presented on the following pages. This newsletter is presented and sent by snail mail monthly. It covers many community and organization events as well as providing homeschool information, mini lessons for our children, exciting web sites and other educational material. Please join us in the Youngstown Ohio area and anyone may subscribe to the Parent Forum.