Tielong Cheng

Selection of Reference Genes for Quantitative Gene Expression Studies in Platycladus orientalis (Cupressaceae) Using Real-Time PCR

Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

The complete chloroplast genome sequence of the relict woody plant Metasequoia glyptostroboides Hu et Cheng.

Metasequoia glyptostroboides Hu et Cheng is the only species in the genus Metasequoia Miki ex Hu et Cheng, which belongs to the Cupressaceae family. There were around 10 species in the Metasequoia genus, which were widely spread across the Northern Hemisphere during the Cretaceous of the Mesozoic and in the Cenozoic. M. glyptostroboides is the only remaining representative of this genus. Here, we report the complete chloroplast (cp) genome sequence and the cp genomic features of M. glyptostroboides. The M. glyptostroboides cp genome is 131,887 bp in length, with a total of 117 genes comprised of 82 protein-coding genes, 31 tRNA genes and four rRNA genes. In this genome, 11 forward repeats, nine palindromic repeats, and 15 tandem repeats were detected. A total of 188 perfect microsatellites were detected through simple sequence repeat (SSR) analysis and these were distributed unevenly within the cp genome. Comparison of the cp genome structure and gene order to those of several other land plants indicated that a copy of the inverted repeat (IR) region, which was found to be IR region A (IRA), was lost in the M. glyptostroboides cp genome. The five most divergent and five most conserved genes were determined and further phylogenetic analysis was performed among plant species, especially for related species in conifers. Finally, phylogenetic analysis demonstrated that M. glyptostroboides is a sister species to Cryptomeria japonica (L. F.) D. Don and to Taiwania cryptomerioides Hayata. The complete cp genome sequence information of M. glyptostroboides will be great helpful for further investigations of this endemic relict woody plant and for in-depth understanding of the evolutionary history of the coniferous cp genomes, especially for the position of M. glyptostroboides in plant systematics and evolution.

Quantitative proteomics analysis reveals that S-nitrosoglutathione reductase (GSNOR) and nitric oxide signaling enhance poplar defense against chilling stress

AbstractMain conclusionNO acts as the essential signal to enhance poplar tolerance to chilling stress via antioxidant enzyme activities and protein S-nitrosylation modification, NO signal is also strictly controlled by S-nitrosoglutathione reductase and nitrate reductase to avoid the over-accumulation of reactive nitrogen species. Poplar (Populus trichocarpa) are fast growing woody plants with both ecological and economic value; however, the mechanisms by which poplar adapts to environmental stress are poorly understood. In this study, we used isobaric tags for relative and absolute quantification proteomic approach to characterize the response of poplar exposed to cold stress. We identified 114 proteins that were differentially expressed in plants exposed to cold stress. In particular, some of the proteins are involved in reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism. Further physiological analysis showed that nitric oxide (NO) signaling activated a series of downstream defense responses. We further demonstrated that NO activated antioxidant enzyme activities and S-nitrosoglutathione reductase (GSNOR) activities, which would reduce ROS and RNS toxicity and thereby enhance poplar tolerance to cold stress. Suppressing NO accumulation or GSNOR activity aggravated cold damage to poplar leaves. Moreover, our results showed that RNS can suppress the activities of GSNOR and NO nitrate reductase (NR) by S-nitrosylation to fine-tune the NO signal and modulate ROS levels by modulating the S-nitrosylation of ascorbate peroxidase protein. Hence, our data demonstrate that NO signaling activates multiple pathways that enhance poplar tolerances to cold stress, and that NO signaling is strictly controlled through protein post-translational modification by S-nitrosylation.

Salinity-induced changes in protein expression in the halophytic plant Nitraria sphaerocarpa

Salinity is a major abiotic stress that inhibits plant growth and development. Plants have evolved complex adaptive mechanisms that respond to salinity stress. However, an understanding of how plants respond to salinity stress is far from being complete. In particular, how plants survive salinity stress via alterations to their intercellular metabolic networks and defense systems is largely unknown. To delineate the responses of Nitraria sphaerocarpa cell suspensions to salinity, changes in their protein expression patterns were characterized by a comparative proteomic approach. Cells that had been treated with 150 mM NaCl for 1, 3, 5, 7, or 9 days developed several stress-related phenotypes, including those affecting morphology and biochemical activities. Of ~1100 proteins detected in 2-DE gel patterns, 130 proteins showed differences in abundance with more than 1.5-fold when cells were stressed by salinity. All but one of these proteins was identified by MS and database searching. The 129 spots contained 111 different proteins, including those involved in signal transduction, cell rescue/defense, cytoskeleton and cell cycle, protein folding and assembly, which were the most significantly affected. Taken together, our results provide a foundation to understand the mechanism of salinity response.

Physiological and proteomic analyses of leaves from the halophyte Tangut Nitraria reveals diverse response pathways critical for high salinity tolerance

Soil salinization poses a serious threat to the environment and agricultural productivity worldwide. Studies on the physiological and molecular mechanisms of salinity tolerance in halophytic plants provide valuable information to enhance their salt tolerance. Tangut Nitraria is a widely distributed halophyte in saline-alkali soil in the northern areas of China. In this study, we used a proteomic approach to investigate the molecular pathways of the high salt tolerance of T. Nitraria. We analyzed the changes in biomass, photosynthesis, and redox-related enzyme activities in T. Nitraria leaves from plant seedlings treated with high salt concentration. Comparative proteomic analysis of the leaves revealed that the expression of 71 proteins was significantly altered after salinity treatments of T. Nitraria. These salinity-responsive proteins were mainly involved in photosynthesis, redox homeostasis, stress/defense, carbohydrate and energy metabolism, protein metabolism, signal transduction, and membrane transport. Results showed that the reduction of photosynthesis under salt stress was attributed to the down-regulation of the enzymes and proteins involved in the light reaction and Calvin cycle. Protein-protein interaction analysis revealed that the proteins involved in redox homeostasis, photosynthesis, and energy metabolism constructed two types of response networks to high salt stress. T. Nitraria plants developed diverse mechanisms for scavenging reactive oxygen species (ROS) in their leaves to cope with stress induced by high salinity. This study provides important information regarding the salt tolerance of the halophyte T. Nitraria.

Transcriptomic analysis reveals importance of ROS and phytohormones in response to short-term salinity stress in Populus tomentosa

Populus tomentosa (Chinese white poplar) is well adapted to various extreme environments, and is considered an important species to study the effects of salinity stress on poplar trees. To decipher the mechanism of poplars rapid response to short-term salinity stress, we firstly detected the changes in H2O2 and hormone, and then profiled the gene expression pattern of 10-week-old seedling roots treated with 200 mM NaCl for 0, 6, 12, and 24 h (h) by RNA-seq on the Illumina-Solexa platform. Physiological determination showed that the significant increase in H2O2 began at 6 h, while that in hormone ABA was at 24 h, under salt stress. Compared with controls (0 h), 3991, 4603, and 4903 genes were up regulated, and 1408, 2206, and 3461 genes were down regulated (adjusted P ≤ 0.05 and |log2Ratio|≥1) at 6, 12, and 24 h time points, respectively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation revealed that the differentially expressed genes (DEGs) were highly enriched in hormone- and reactive oxygen species-related biological processes, including “response to oxidative stress or abiotic stimulus,” “peroxidase activity,” “regulation of transcription,” “hormone synthetic and metabolic process,” “hormone signal transduction,” “antioxidant activity,” and “transcription factor activity.” Moreover, K-means clustering demonstrated that DEGs (total RPKM value>12 from four time points) could be categorized into four kinds of expression trends: quick up/down over 6 or 12 h, and slow up/down over 24 h. Of these, DEGs involved in H2O2- and hormone- producing and signal-related genes were further enriched in this analysis, which indicated that the two kinds of small molecules, hormones and H2O2, play pivotal roles in the short-term salt stress response in poplar. This study provides a basis for future studies of the molecular adaptation of poplar and other tree species to salinity stress.

The Complete Chloroplast Genome Sequence of a Relict Conifer Glyptostrobus pensilis: Comparative Analysis and Insights into Dynamics of Chloroplast Genome Rearrangement in Cupressophytes and Pinaceae.

Glyptostrobus pensilis, belonging to the monotypic genus Glyptostrobus (Family: Cupressaceae), is an ancient conifer that is naturally distributed in low-lying wet areas. Here, we report the complete chloroplast (cp) genome sequence (132,239 bp) of G. pensilis. The G. pensilis cp genome is similar in gene content, organization and genome structure to the sequenced cp genomes from other cupressophytes, especially with respect to the loss of the inverted repeat region A (IRA). Through phylogenetic analysis, we demonstrated that the genus Glyptostrobus is closely related to the genus Cryptomeria, supporting previous findings based on physiological characteristics. Since IRs play an important role in stabilize cp genome and conifer cp genomes lost different IR regions after splitting in two clades (cupressophytes and Pinaceae), we performed cp genome rearrangement analysis and found more extensive cp genome rearrangements among the species of cupressophytes relative to Pinaceae. Additional repeat analysis indicated that cupressophytes cp genomes contained less potential functional repeats, especially in Cupressaceae, compared with Pinaceae. These results suggested that dynamics of cp genome rearrangement in conifers differed since the two clades, Pinaceae and cupressophytes, lost IR copies independently and developed different repeats to complement the residual IRs. In addition, we identified 170 perfect simple sequence repeats that will be useful in future research focusing on the evolution of genetic diversity and conservation of genetic variation for this endangered species in the wild.

Phylogenetic studies and comparative chloroplast genome analyses elucidate the basal position of halophyte Nitraria sibirica (Nitrariaceae) in the Sapindales.

Abstract Nitraria sibirica is a halophyte and belongs to the family Nitrariaceae. The chloroplast genome of Nitraria sibirica (159,466 bp) has a quadripartite structure, which consists of a large single-copy (87,914 bp) region, a small single-copy (18,316 bp) region, and a pair of inverted repeats (26,618 bp). Sequencing analyses indicate that the chloroplast genome contains 113 distinct genes, including 79 peptide-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. We also identified 105 perfect simple sequence repeats, 12 most divergent non-coding regions, and 6 most divergent coding regions when compared to the chloroplast genomes of the Sapindales plants. Phylogenetic analyses using the concatenated amino acid sequences of 58 protein-coding genes from 48 species suggest that the ‘basal’ position of Nitraria sibirica belongs to the Sapindales clade. We also found that the inverted repeat expansion resulted in a duplication of rps19 in Nitraria sibirica when comparing its chloroplast genome structure with Theobroma cacao, Vitis vinifera, Eucalyptus erythrocorys and Arabidopsis thaliana. The duplication of rps19 gene was consistent with that in Zanthoxylum piperitum, Azadirachta indica, Sapindus mukorossi and Citrus sinensis, all of which belong to the order Sapindales, but different from most Rosids plants. In summary, the analyses of Nitraria sibirica chloroplast genome not only provide insights into comparative genome analysis, but also pave the way for a better understanding of the phylogenetic relationships within the Sapindales.

Phylogeny and molecular evolution analysis of PIN-FORMED 1 in angiosperm.

PIN-FORMED 1 (PIN1) is an important secondary transporter and determines the direction of intercellular auxin flow. As PIN1 performs the conserved function of auxin transport, it is expected that the sequence and structure of PIN1 is conserved. Therefore, we hypothesized that PIN1 evolve under pervasive purifying selection in the protein-coding sequences in angiosperm. To test this hypothesis, we performed detailed evolutionary analyses of 67 PIN1 sequences from 35 angiosperm species. We found that the PIN1 sequences are highly conserved within their transmembrane regions, part of their hydrophilic regions. We also found that there are two or more PIN1 copies in some of these angiosperm species. PIN1 sequences from Poaceae and Brassicaceae are representative of the modern clade. We identified 12 highly conserved motifs and a significant number of family-specific sites within these motifs. One family-specific site within Motif 11 shows a different residue between monocots and dicots, and is functionally critical for the polarity of PIN1. Likewise, the function of PIN1 appears to be different between monocots and dicots since the phenotype associated with PIN1 overexpression is opposite between Arabidopsis and rice. The evolution of angiosperm PIN1 protein-coding sequences appears to have been primarily driven by purifying selection, but traces of positive selection associated with sequences from certain families also seem to be present. We verified this observation by calculating the numbers of non-synonymous and synonymous changes on each branch of a phylogenetic tree. Our results indicate that the evolution of angiosperm PIN1 sequences involve strong purifying selection. In addition, our results suggest that the conserved sequences of PIN1 derive from a combination of the family-specific site variations and conserved motifs during their unique evolutionary processes, which is critical for the functional integrity and stability of these auxin transporters, especially in new species. Finally, functional difference of PIN1 is likely to be present in angiosperm because the positive selection is occurred in one branch of Poaceae.

Expansion and Functional Divergence of AP2 Group Genes in Spermatophytes Determined by Molecular Evolution and Arabidopsis Mutant Analysis

The APETALA2 (AP2) genes represent the AP2 group within a large group of DNA-binding proteins called AP2/EREBP. The AP2 gene is functional and necessary for flower development, stem cell maintenance, and seed development, whereas the other members of AP2 group redundantly affect flowering time. Here we study the phylogeny of AP2 group genes in spermatophytes. Spermatophyte AP2 group genes can be classified into AP2 and TOE types, six clades, and we found that the AP2 group homologs in gymnosperms belong to the AP2 type, whereas TOE types are absent, which indicates the AP2 type gene are more ancient and TOE type was split out of AP2 type and losing the major function. In Brassicaceae, the expansion of AP2 and TOE type lead to the gene number of AP2 group were up to six. Purifying selection appears to have been the primary driving force of spermatophyte AP2 group evolution, although positive selection occurred in the AP2 clade. The transition from exon to intron of AtAP2 in Arabidopsis mutant leads to the loss of gene function and the same situation was found in AtTOE2. Combining this evolutionary analysis and published research, the results suggest that typical AP2 group genes may first appear in gymnosperms and diverged in angiosperms, following expansion of group members and functional differentiation. In angiosperms, AP2 genes (AP2 clade) inherited key functions from ancestors and other genes of AP2 group lost most function but just remained flowering time controlling in gene formation. In this study, the phylogenies of AP2 group genes in spermatophytes was analyzed, which supported the evidence for the research of gene functional evolution of AP2 group.