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To investigate the molecular response of ancient plants to heavy metal stress and to explore the feature of DNA methylation in endangered plants after exposure to heavy metals, the Isoetes sinensis, an endangered plant, was stressed with three different concentrations of two heavy metals lead (Pb) and cadmium (Cd), respectively. Then the degrees and the patterns of DNA methylation in the leaves were measured on the 14th day using Methylation Sensitive Amplified Polymorphism (MSAP) technique. The results showed that the DNA methylated profile of I. sinensis was affected by Pb and Cd stress. There was no significant difference in the amount of DNA methylation among control check (CK), Pb stress group, and Cd stress group (CK 46.96%, Pb 48.23%, and Cd 48.1%). However, full-methylation level of Pb stress group (28.34%) and Cd stress group (20.25%) was lower than control (33.91%), in contrast, hemi-methylation level Pb stress group (19.89%) and Cd stress group (27.85%) were higher than control (13.04%). The change of patterns from no methylation or hemi-methylation of internal and external cytosines into full-methylation of internal and external cytosines accounted for a large proportion in enhanced methylation aspects. The full-methylation into no methylation or hemi- or full-methylation of internal and external cytosines occupied most of demethylation. The proportion of DNA methylation (including hypermethylation) by both Pb and Cd stresses is nearly equal (39.04% and 39.71%), but the proportion of DNA demethylation by Cd is higher than that by Pb (46.86% than 33.92%).

Isoetes sinensis Palmer is a critically endangered, first-class protected plant in China. Until now, researchers have primarily focused on the ultrastructure, phylogeny, and transcriptomes of the plant. However, flavonoid profiles and bioactivity of I. sinensis have not been extensively investigated. To develop the endangered I. sinensis for edible and medicinal purposes, flavonoid content, chemical constitution, and antioxidant activities were investigated in this study. Results revealed the following. 1) The total flavonoid content was determined as 10.74 ± 0.25 mg/g., 2) Antioxidant activities were stronger than most ferns, especially ABTS free radical scavenging activities. 3) Four flavones, containing apigenin, apigenin-7-glucuronide, acacetin-7-O-glcopyranoside, and homoplantageninisoetin; four flavonols, namely, isoetin, kaempferol-3-O-glucoside, quercetin-3-O-[6”-O-(3-hydroxy-3-methylglutaryl)-β-D-glucopyranoside], and limocitrin-Neo; one prodelphinidin (procyanidins;) and one nothofagin (dihydrochalcone) were tentatively identified in the mass spectrometry-DAD (254nm) chromatograms. This study was the first to report on flavonoid content and antioxidant activities of I. sinensis. Stronger antioxidant activity and flavonoid content suggests that the endangered I. sinensis is an important and potentially edible and medicinal plant.

Relatively high frequencies of polyploidy are generally found in high-altitude regions. However, more diploid than polyploid species of Isoetes are distributed in plateau environments in China. The low-altitude species Isoetes sinensis originates from the allopolyploidization between a plateau parent and another low altitude species. To study potential ecological adaptation to plateau environments in the allopolyploid I. sinensis, we transplanted individuals of I. sinensis to different altitudes. RNA sequencing was performed to elucidate the molecular response of this species’ ecological adaptation to plateaus. More unique differentially expressed genes (DEGs) were detected in individuals transplanted to the Qinghai–Tibet Plateau (QTP) than elsewhere, such as genes for ribosomal proteins (RPs) and succinate dehydrogenase (SDH), which helped them adapt to higher altitudes under both terrestrial and submerged conditions. In both terrestrial and submerged conditions, the expression of a large number of genes changed to help plants adapt to exposure to the high levels of ultraviolet (UV) radiation on plateaus, such as the genes associated with the accumulation of flavonoids, decreasing chlorophyll content, and reducing light harvesting activity. The expression of vacuolar-type H+-transporting ATPase (V-ATPase) and phosphoenolpyruvate carboxylase (PEPC) increased with altitude, revealing that the intensity of the crassulacean acid metabolism (CAM) of I. sinensis might be positively associated with altitude. In general, our results reveal that although I. sinensis only lives at low altitudes, it can still adapt to plateau stress by regulating the expression of certain key genes. The potential ecological adaptation to plateau environments in the allopolyploid I. sinensis may have been inherited from the plateau parent.

Light-dark cycle exercises great influences on plant growth and metabolism. Isoetes L. is one of basal vascular plants with unique phylogenetic position. Isoetes sinensis is an amphibious plant, alternating frequently between terrestrial and aquatic environments. Here, we performed RNA-sequencing to investigate different expression profiles of I. sinensis in response to light-dark shift under terrestrial (TC) and submerged conditions (SC), respectively. Four different samples were sequenced, including terrestrial leaves in the light (LTC) and dark (DTC), as well as submerged materials in the light (LSC) and dark (DSC). Totally, there were 1154 differentially expressed genes (DEGs) were enriched in LTC vs. DTC, and 1212 DEGs in LSC vs. DSC. Moreover, 102 and 103 transcription factors (TFs) were significantly enriched in LTC vs. DTC and LSC vs. DSC, respectively. Co-expression analysis revealed that the differently expressed TFs were distinctly categorized into two subsets for the two pairwise comparisons. The network analysis demonstrated that NAC played key roles in the light-dark shift under TC. The dataset might provide new insights in understanding I. sinensis in response to light-dark shift under different conditions and serve as an important platform for further researches in Isoetes.

Isoetes L. is an ancient genus of heterosporous lycopsids with a unique phylogenetic position. Repeated adaptations to environmental changes over time have contributed to occupying a variety of niches in Isoetes. However, we know little about how they adapt to the environmental changes, and the sequence resources are very limited in public databases. Isoetes sinensis is an amphibious plant in this genus, alternating frequently between terrestrial and aquatic environments. In this study, I. sinensis was applied to investigate the adaptations under terrestrial (TC) and submerged (ST) conditions using Illumina RNA-sequencing technology. Approximately 87 million high-quality reads were yielded and assembled into 31,619 unigenes with an average length of 1618 bp. Overall, 28,208 unigenes were annotated against the National Center of Biotechnology Information (NCBI), Non-redundant (Nr), Cluster of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, we identified 1740 differentially expressed genes with 1146 up-regulated and 594 down-regulated genes under TC. GO annotation revealed that stress-relevant categories were remarkably enriched, and KEGG enrichment analysis showed that the phytohormone signalings and carbohydrate metabolism were significantly influenced. Furthermore, a total of 1646 transcription factors (TF) were identified and classified into 54 TF families; among them, 180 TFs were dynamic between terrestrial and submerged conditions. This study is the first report for Isoetes to generate numerous sequences and establish general understandings about the adaptations in the changing environments. The dataset provides a foundation for novel gene discoveries, comparative genomics, functional genomics, and phylogenetics in Isoetes. Electronic supplementary material The online version of this article (doi:10.1007/s11105-015-0906-6) contains supplementary material, which is available to authorized users.

Photosynthetic inorganic carbon (C-i) acquisition mechanisms were surveyed in 30 freshwater macrophytes grown under uniform, nutrient-sufficient, low C-i conditions where C-i is a potentially limiting factor on plant photosynthesis. In pH-drift experiments there was evidence for HCO3- use in 21 of the 30 species that were able to raise the solution pH above 10.0. Titratable acidity had distinct diel fluctuations in the leaves of Nechamandra alternifolia, Egeria densa, Vallisneria spinulosa, Deinostema violaceum, and Isoetes sinensis (aerial and submersed), which suggests some degree of Crassulacean Acid Metabolism (CAM) in these species. Incubation of D. violaceum under low C-i conditions substantially increased the amount of acidity and the difference between night and day. The phosphoenolpyruvate carboxylase (PEPC) activity in Hydrilla verticillata, V. spinulosa, E. densa, D. violaceum, Ottelia acuminate, and O. alismoides ranged from 32.4 +/- 6.6 to 156.8 +/- 5.3 mu mol mg(-1) protein h(-1) and the ratio of PEPC to ribulose bisphosphate carboxylase-oxygenase (Rubisco) activity ranged from 1.0 to 2.9. When O. alismoides and D. violaceum were incubated under conditions that greatly reduced daytime C-i the PEPC activity was around 1.5 times higher than in untreated leaves. A high PEPC to Rubisco activity ratio, and the increased ratio under low C-i conditions, is consistent with a facultative C-4 or CAM system. These results indicated that most of the freshwater macrophytes tested can use HCO3- as a carbon source, while O. alismoides, and D. violaceum may possess C-4 or CAM characteristics like H. verticillata and I. howellii, and thus are worthy of further study. (C) 2016 Published by Elsevier B.V.

Although the palynology and sporophyte stage of Isoetes sinensis have been well studied, the biology of its gametophyte and embryo is less well understood. To date, the functions of several genes of I. sinensis and the molecular mechanisms of enzymes encoded by them remain to be studied. In the present study, the Fe-SOD gene of I. sinensis was successfully cloned using RT-PCR and rapid amplification of cDNA ends (RACE), and termed IsFeSOD. IsFeSOD has certain reference value in the classification of system evolution. The study also accumulated data for further research on the SOD gene. Bioinformatic analysis was employed to compare IsFeSOD with gene sequences obtained from other plants present in the GenBank. Furthermore, the recombinant pET32-FeSOD plasmids were transformed into Escherichia coli BL21 for expression. IsFeSOD was observed to have 1469 nucleotides that were predicted to encode 247 amino acids. The bioinformatic analysis revealed that IsFeSOD contained conserved TGGGA sequences, similar to eight other species, in addition to five other conserved sequences. The recombinant protein was about 43 kDa. Recombinant FeSOD was expressed, purified, and confirmed by western blotting. Alignment of complete Fe-SOD mRNA sequences from 9 species revealed several conserved sequences. A phylogenetic tree was constructed using MEGA4.1 and ClustalX multiple-sequence alignment programs. This study could be helpful in further characterization of SOD genes and for classification of system evolution status.

Isoetes sinensis is a critically endangered quillwort. To facilitate studies on the conservation genetics of this species, we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 50,063 unigenes were predicted by transcriptome sequencing, 5294 (10.6%) of which significantly matched 3011 Gene Ontology annotations and 2363 were assigned to Kyoto Encyclopedia of Genes and Genomes metabolic pathways. Most of these (2297) were involved in metabolism. A total of 1982 SSR motifs were identified, with trinucleotides being the dominant repeat motif, and 1438 (72.6%) SSR primers were designed. Eighteen randomly selected primer pairs were used to genotype 24 I. sinensis accessions, which confirmed the suitability of these novel markers for molecular studies of I. sinensis. The heterozygosity index value ranged between 0.0799 and 0.9106, while the Shannon-Wiener diversity index value ranged between 0.1732 and 2.5589. The EST-SSRs reported in this study are linked to genic sequences, and are therefore ideal for investigating the evolutionary history of I. sinensis. These markers, together with the large EST dataset generated in this study, will greatly facilitate conservation genetic studies of I. sinensis.

Isoetes sinensis is a critically endangered aquatic quillwort, occurring in two fragmented sites of China (Xiuning county of Anhui; Jiande county of Zhejiang). During a five-year period (2004–2009), the areas and sizes of the two populations diminished dramatically due to intensive human activities. Genetic structure of the species was investigated using simple sequence repeat makers (SSRs). For seven nuclear microsatellites, high levels of genetic diversity were found within populations ( H E = 0.324–0.447). Strong genetic differentiation was detected between populations ( G ST = 0.376), while weak genetic differentiation was found within populations ( G ST = 0.026–0.080). Notably, in contrast to the source–sink model suggested by previous study ( Chen et al., 2009 ), the migration pattern of I. sinensis along the Xin’an River is best explained by the linear symmetrical non-adjacent flow model (LSNF), which indicates that intensive human activities of recent years have greatly affected the gene exchange pattern among I. sinensis subpopulations.

Isoetes sinensis (Isoetaceae), an aquatic quillwort which occurs only in two fragmented sites of China as an allotetraploid, is critically endangered. Genetic variation among eight subpopulations of I. sinensis was examined in the Xin’an River (119°14′–15′E, 29°28′N) by using allozyme polymorphism. Eighteen loci of 10 enzyme systems were examined and used for the analysis of population genetic parameters. As expected for allotetraploids, fixed heterozygosity was found at four loci. A high level of genetic diversity was observed in the population, with mean number of alleles per loci of 1.8, and mean percentage of polymorphic loci of 55.6%, which were much higher than the average values in fern species. The genetic variation within each subpopulation was not positively correlated with its size, which may be explained by high gene flow (Nm = 2.57), clonal reproduction and fixed heterozygosity of allopolyploid. The I. sinensis population contained high clonal diversity (PD = 0.39, D = 0.95), indicating the successful seedling recruitment of the population. Significant positive relationship was detected between clonal diversity and the size of subpopulation. Partitioning the genetic diversity indicated that 91.1% of the genetic variation was within subpopulations and only 8.9% existed among subpopulations. The migration pattern of I. sinensis along the Xin’an River is best explained by a source–sink model, but with unidirectional gene flow among subpopulations underlined by hydrochoric force. The results were then discussed in relation to both in situ and ex situ conservation efforts of the population.

Chromosomal abnormalities including cytomixis occurred in meiotic process of pollen mother cells of many angiosperm species at high frequency, but similar phenomenon was rarely reported in low vascular plants. This study we investigated the microsporogenesis of Isoetes sinensis Palmer, a quillwort of ancient heterosporous lycopsids Isoetaceae plants. Irregular chromosomal behavior especially cytomixis was emphasized. It appeared in each stage of meiotic division from prophase I to telophase II. The cytoplasmic channels between microspore mother cells were narrow and stretched. The genetic material connection was maintained mainly by visible chroma- tin fiber. The cytomixis was just occurred between two adjacent microspore mother cells of same stages. A variety of other abnormal chromosomal behaviors including chromosomal stickiness, fragmentation, bridge, laggards, micronucleus, were observed, albeit with low frequency. All sorts of chromosomal abnormalities were counted at two stages, anaphase I and anaphase II. They were approximately 24.4% and 26.9% respectively. This number was slightly higher than that of microspore abortion which was about 14.8%. I. sinensis was listed as a critical endangered species due to habitat fragmentalization, water pollution and wetland degradation. Human being activities are without doubt the immediate forces resulting in the population decreasing of I. sinensis. Whether the individual numbers dropping or habitat withdraw has consequently caused the producing of the overall high abnormalities of chromosomal behaviours is not determined yet. However, cytomixis and other irregular chromo- somal phenomenon seemed not to be a natural process.

Crassulacean acid metabolism (CAM) is widespread in terrestrial and aquatic species, plastic in response to environmental changes. Isoetes L. is one of the earliest basal vascular plants and CAM is popular in this genus. Isoetes sinensis Palmer is an amphibious species, alternating frequently between terrestrial and aquatic environments. Given this, we investigated and compared photosynthetic characteristics over a diurnal cycle under submerged condition (SC) and terrestrial condition (TC). The results suggest that I. sinensis possesses a stronger CAM capacity under SC. Compared with under TC, titratable acidity levels and organic acid concentrations were more enriched under SC, whereas soluble sugar or starch and protein levels were lower under SC. Transcript analyses for nine photosynthetic genes revealed that CAM-associated genes possessed high transcripts under SC, but C3-related transcripts were highly expressed under TC. In addition, the enzyme activity measurements demonstrated that PEPC activity over a diurnal cycle was slightly higher under SC, whereas Rubisco activity during the daytime was greater under TC. This comprehensive study probably facilitates general understandings about the CAM photosynthetic characteristics of Isoetes in response to the environmental changes.

Isoetes sinensis is a rare and endangered plant in wetlands of southeastern China. Previous studies have reported the chromosome number, geographic distribution, and ecology of this Asian endemic, but there has not been an analysis of diagnostic characters associated with the sporophylls of I. sinensis in China. Therefore, morphological and anatomical variation of sporophylls in the three known Chinese populations of I. sinensis were evaluated and compared. The variation found is discussed in relation to the present taxonomy of I. sinensis.

Isoetes sinensis Palmer is an East Asia endemic fern that faces impending extirpation from the expansive area of Mainland China. Presently, only two natural populations are known to exist in this vast region. Both populations have decreased by about 50% within the last 4 years. This reduction is most probably related to the decline in the size of the wetlands since reproductive activity, spore production, and recruitment were noticeably lower at the microsites occurring in smaller ponds. The decline in population size seems to be associated with succession from marsh to dry land at the habitats of both populations. The study revealed that individuals in both populations produced copious numbers of spores suggesting adequate fecundity, furthermore high levels of spore germination were obtained in laboratory experiments. However, inefficient spore dispersal appears to have contributed to the limited spread of the species. The sex ratio in both populations was male‐biased. Several factors are identified here...

Chromosome behavior during meiosis of the tetraploid Isoetes sinensis was observed to be similar to that of basic diploid Isoetes species. This observation was consistent with the previous report that polyploid species of Isoetes are allopolyploids. Meiosis was generally similar in both megaspore and microspore mother cells, but differences were observed. Due to the smaller size of the microspore mother cells, during prophase I, chromosomes are not as dispersed as those in megaspore mother cells. Cytomixis was observed in all stages of meiosis in microspore mother cells, but it was not seen at any stage of meiosis in megaspore mother cells. Cytomixis, lagging chromosomes, chromosome bridges, chromosome fragments, and micronuclei, observed during meiosis in I. sinensis, can affect sexual reproduction, but it does not appear to be a major cause of population decline in this species. The major cause of decline is habitat degradation from human disturbance. The few remaining populations of the Chines...

The progressive decline in populations of Isoetes sinensis Palmer in China has been attributed to deteriorating water quality; however, no data have been adduced to support this assumption. In this study we identified locations in China that presently have populations of I. sinensis and locations where the species has been extirpated. We measured various water chemistry parameters at these sites, as well as at similar sites within the distribution range of I. sinensis but from where the species has never been recorded. The results indicate that distribution and occurrence of I. sinensis are closely correlated with water chemistry. Water samples drawn from habitats from where I. sinensis has been extirpated had higher concentrations of heavy metal ions, particularly chronium VI (Cr6+. In addition, these samples consistently yielded higher readings for dissolved carbon dioxide, water hardness, and concentration of nitrates.

Isoetes sinensis Palmer (Isoetaceae) is an aquatic or amphibious plant that is critically endangered in China. Previous studies have revealed the crassulacean acid metabolism (CAM)-like photosynthetic pathway occurs commonly in submerged leaves in genusIsoetes. Water chemistry parameters and the titratable acidity content of the plant extract were measured from samples obtained in the early morning (7∶00) and late afternoon (15∶00) from twoI. sinensis populations in China. One population occurs in the eulittoral zone of a freshwater tidal river at low elevation (134 m) and another occurs in a densely vegetated, high elevation (1 100 m) alpine shallow pool. Significant differences in pH and titratable acidity of the plant extract were detected between the morning and afternoon samples. These changes are associated with diurnal changes in water chemistry. Our results provide the first evidence for the existence of the CAM pathway in the East Asian endemicIsoetes sinensis Palmer. The magnitude of fluctuations in the titratable acidity of the plant extract may be correlated with the severe carbon limitation imposed on the plants by its aquatic habitat.

PREMISE OF THE STUDY The first microsatellite primers were developed for Isoetes hypsophila, an endangered quillwort species endemic to the Qinghai-Tibetan Plateau in China, to further describe its genetic variability and population structure. We also examined their cross-amplification in a congeneric species, I. sinensis. METHODS AND RESULTS Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, nine microsatellite loci were isolated and characterized in 32 samples from four natural populations of I. hypsophila. The primers amplified di- and hexanucleotide repeats with three to 11 alleles per locus. Seven of nine primers were cross-amplified in I. sinensis with two to seven alleles per locus. CONCLUSION The microsatellite loci primers will be useful for studies of genetic diversity and gene flow in natural populations of Isoetes species.

Isoetes sinensis Palmer (Isoetaceae) is a critically endangered fern that is a marsh plant (that is an aquatic or amphibious plant) in China. To evaluate damage or influence of lead (Pb) on cell ultrastructure in I. sinensis, we used 2000mg·L-1 Pb(NO3)2 solution to treat I. sinensis for 35d, and used transmission electron microscope (TEM) to observe the cell ultrastructure of leaf blades and roots of the plant. Our results indicated that Pb induced distinct changes of the organelles including chloroplast, mitochondria, nucleolus and vacuole. The level of damage organ was lower leaf > upper leaf > root The typical performance of the damages caused by lead shown that part of the nucleolus cracked; the cristae dilated, matrix vacuolized and membrane structure blurred in mitochondria; the vacuole cracked; grana lamella decreased, stroma lamella loosed, starch grains decreased, and membrane structure was disrupted in chloroplasts; Pb deposits were present on cell wall. The damages to chloroplasts and mitochondria were relatively severe, while damage to the nucleus was relatively lighter. The damage to the cell ultrastructure of leaf blades with direct contact with Pb was more severe than that without direct contact with Pb.