Caiyun He

Next-generation sequencing-based mRNA and microRNA expression profiling analysis revealed pathways involved in the rapid growth of developing culms in Moso bamboo

BackgroundAs one of the fastest-growing lignocellulose-abundant plants on Earth, bamboos can reach their final height quickly due to the expansion of individual internodes already present in the buds; however, the molecular processes underlying this phenomenon remain unclear. Moso bamboo (Phyllostachys heterocycla cv. Pubescens) internodes from four different developmental stages and three different internodes within the same stage were used in our study to investigate the molecular processes at the transcriptome and post-transcriptome level.ResultsOur anatomical observations indicated the development of culms was dominated by cell division in the initial stages and by cell elongation in the middle and late stages. The four major endogenous hormones appeared to actively promote culm development. Using next-generation sequencing-based RNA-Seq, mRNA and microRNA expression profiling technology, we produced a transcriptome and post-transcriptome in possession of a large fraction of annotated Moso bamboo genes, and provided a molecular basis underlying the phenomenon of sequentially elongated internodes from the base to the top. Several key pathways such as environmental adaptation, signal transduction, translation, transport and many metabolisms were identified as involved in the rapid elongation of bamboo culms.ConclusionsThis is the first report on the temporal and spatial transcriptome and gene expression and microRNA profiling in a developing bamboo culms. In addition to gaining more insight into the unique growth characteristics of bamboo, we provide a good case study to analyze gene, microRNA expression and profiling of non-model plant species using high-throughput short-read sequencing. Also, we demonstrate that the integrated analysis of our multi-omics data, including transcriptome, post-transcriptome, proteome, yield more complete representations and additional biological insights, especially the complex dynamic processes occurring in Moso bamboo culms.

Temporal and spatial profiling of internode elongation-associated protein expression in rapidly growing culms of bamboo.

In natural conditions, culms of developing Moso bamboo, Phyllostachys heterocycla var. pubescens, reach their final height of more than ten meters within a short period of two to four months. To study this phenomenon, bamboo culm material collected from different developmental stages and internodes was analyzed. Histological observations indicated that the development of culm was dominated by cell division in the initial stages and by cell elongation in the middle and late stages. Development, maturation, and aging in different regions of the culm were studied systematically from the basal to the top internode. The four major endogenous hormones, indole acetic acid, gibberellic acid, zeatin riboside, and abscisic acid appeared to strongly influence the cell elongation phase. A total of 258 spots were differentially expressed in culm development. Of these, 213 spots were identified by MALDI-TOF/TOF MS and were involved in many physiological and metabolic processes including carbohydrate metabolism, cell division, cell expansion, protein synthesis, amino acid metabolism and redox homeostasis. These proteins with different expression patterns constructed an ingenious network to regulate the culm development. Developmental stage-specific and internode-specific protein expression patterns were identified. Protein abundance was regulated temporally and to some extent spatially, and the sequential development from base to apex of bamboo culm was implemented by temporal and spatial expression of enzymes. Results indicate that during development energy was mainly derived from sucrose degradation, as photosynthetic capacity was poor. The regulation of anaerobic and aerobic modes of respiration appeared to play an important role in energy generation. This is the first report on proteomic profiling in bamboo and helps in understanding the regulatory processes in developing culms.

Proteins responding to drought and high-temperature stress in Populus × euramericana cv. ‘74/76’

Proteomic analysis provides a powerful method of studying plant responses to stress at the protein level. In order to study stress-responsive molecular mechanisms for Populus × euramericana cv. ‘74/76’, one of the most important forest plantation tree species in subtropical and temperate regions, we analyzed the response of 2-year-old cuttings of P. × euramericana cv. ‘74/76’ to drought and high temperature using two-dimensional gel electrophoresis. More than 1,000 reproducible leaf proteins were detected in the controls and treatments, and 26 proteins were found to change notably in abundance. We identified 13 proteins affected by drought stress and 11 proteins affected by high temperature. These proteins are mainly involved in photosynthesis such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and putative photosystem I reaction center subunit II precursor, and detoxification (manganese superoxide dismutase and methionine sulfoxide reductase A). Furthermore, the level of the photosynthesis proteins affected greatly by the imposed stress conditions was consistent with the observed noticeable decrease in net photosynthesis rate. These studies provides a fundamental data for future research on responses to drought and high temperature, two major factors limiting the growth of forest trees during summer under recent climatic warming.

Genome-wide and molecular evolution analysis of the Poplar KT/HAK/KUP potassium transporter gene family

As the largest K+ transport gene family, KT/HAK/KUP family plays an important role in plant growth, development, and stress adaptation. However, there is limited information about this family in woody plant species. In this study, with genome-wide in-depth investigation, 31 Poplar KT/HAK/KUP transporter genes including six pairs of tandem duplicated and eight pairs of segmental duplicated paralogs have been identified, suggesting segmental and tandem duplication events contributed to the expansion of this family in Poplar. The combination of phylogenetic, exon structure and splice site, and paragon analysis revealed 11 pairs of Poplar KT/HAK/KUP duplicates. For these 11 pairs, all pairs are subject to purify selection, and asymmetric evolutionary rates have been found to occur in three pairs. This study might provide more insights into the underlying evolution mechanisms of trees acclimating to their natural habitat.

Genome-wide analysis of the AP2/ERF gene family in Salix arbutifolia.

AP2/ERF genes encode transcriptional regulators with a variety of functions in plant growth and development and in response to biotic and abiotic stresses. To date, there are no detailed classification and expression profiles for AP2/ERF genes inSalix. In this study, a comprehensive computational analysis identified 173 AP2/ERF superfamily genes in willow (Salix arbutifolia), by usingin silico cloning methods with the use of the AP2/ERF conserved domain amino acid sequence ofArabidopsis thaliana as a probe. Based on the results of phylogenetic analyses and the number of AP2/ERF domains, the AP2/ERF genes were classified into four groups: AP2, RAV, ERF and Soloist. The expression profile was analyzed using transcriptome data from different tissues. A comparative analysis ofAP2/ERF superfamily genes amongSalix,Populus andArabidopsis was performed. TheSalix DREB, AP2 and RAV groups had a similar number to those in Arabidopsis, and the size of the ERF subfamily inSalix was about 1.4‐fold that of Arabidopsis. TheSalix DREB subfamily was smaller compared toPopulus, while the other families were similar in size to those inPopulus. These results will be useful for future functional analyses of the ERF family genes.

De Novo Transcriptome and Small RNA Analysis of Two Chinese Willow Cultivars Reveals Stress Response Genes in Salix matsudana

Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar ‘Tortuosa’). De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs) and 36 different expressed miRNAs (DEMs). Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix.

Genes Responsive to Elevated CO2 Concentrations in Triploid White Poplar and Integrated Gene Network Analysis

Background The atmospheric CO2 concentration increases every year. While the effects of elevated CO2 on plant growth, physiology and metabolism have been studied, there is now a pressing need to understand the molecular mechanisms of how plants will respond to future increases in CO2 concentration using genomic techniques. Principal Findings Gene expression in triploid white poplar ((Populus tomentosa ×P. bolleana) ×P. tomentosa) leaves was investigated using the Affymetrix poplar genome gene chip, after three months of growth in controlled environment chambers under three CO2 concentrations. Our physiological findings showed the growth, assessed as stem diameter, was significantly increased, and the net photosynthetic rate was decreased in elevated CO2 concentrations. The concentrations of four major endogenous hormones appeared to actively promote plant development. Leaf tissues under elevated CO2 concentrations had 5,127 genes with different expression patterns in comparison to leaves under the ambient CO2 concentration. Among these, 8 genes were finally selected for further investigation by using randomized variance model corrective ANOVA analysis, dynamic gene expression profiling, gene network construction, and quantitative real-time PCR validation. Among the 8 genes in the network, aldehyde dehydrogenase and pyruvate kinase were situated in the core and had interconnections with other genes. Conclusions Under elevated CO2 concentrations, 8 significantly changed key genes involved in metabolism and responding to stimulus of external environment were identified. These genes play crucial roles in the signal transduction network and show strong correlations with elevated CO2 exposure. This study provides several target genes, further investigation of which could provide an initial step for better understanding the molecular mechanisms of plant acclimation and evolution in future rising CO2 concentrations.

Stand Diameter Distribution Modelling and Prediction Based on Richards Function

The objective of this study was to introduce application of the Richards equation on modelling and prediction of stand diameter distribution. The long-term repeated measurement data sets, consisted of 309 diameter frequency distributions from Chinese fir (Cunninghamia lanceolata) plantations in the southern China, were used. Also, 150 stands were used as fitting data, the other 159 stands were used for testing. Nonlinear regression method (NRM) or maximum likelihood estimates method (MLEM) were applied to estimate the parameters of models, and the parameter prediction method (PPM) and parameter recovery method (PRM) were used to predict the diameter distributions of unknown stands. Four main conclusions were obtained: (1) R distribution presented a more accurate simulation than three-parametric Weibull function; (2) the parameters p, q and r of R distribution proved to be its scale, location and shape parameters, and have a deep relationship with stand characteristics, which means the parameters of R distribution have good theoretical interpretation; (3) the ordinate of inflection point of R distribution has significant relativity with its skewness and kurtosis, and the fitted main distribution range for the cumulative diameter distribution of Chinese fir plantations was 0.4∼0.6; (4) the goodness-of-fit test showed diameter distributions of unknown stands can be well estimated by applying R distribution based on PRM or the combination of PPM and PRM under the condition that only quadratic mean DBH or plus stand age are known, and the non-rejection rates were near 80%, which are higher than the 72.33% non-rejection rate of three-parametric Weibull function based on the combination of PPM and PRM.

Genome-wide analysis of long non-coding RNAs at the mature stage of sea buckthorn (Hippophae rhamnoides Linn) fruit.

Long non-coding RNAs (lncRNAs), which are >200nt longer transcripts, potentially play important roles in almost all biological processes in plants and mammals. However, the functions and profiles of lncRNAs in fruit is less understood. Therefore, it is urgent and necessary to identify and analyze the functions of lncRNAs in sea buckthorns. Using RNA-sequencing, we synthetically identified lncRNAs in mature fruit from the red and yellow sea buckthorn. We obtained 567,778,938 clean reads from six samples and identified 3428 lncRNAs in mature fruit, including 2498 intergenic lncRNAs, 593 anti-sense lncRNAs, and 337 intronic lncRNAs. We also identified 3819 and 2295 circular RNAs in red and yellow sea buckthorn Fruit. In the aspects of gene architecture and expression, our results showed significant differences among the three lncRNA subtypes. We also investigated the effect of lncRNAs on its cis and trans target genes. Based on target genes analysis, we obtained 61 different expression lncRNAs (DE-lncRNAs) between these two sea buckthorns, including 23 special expression lncRNAs in red fruit and 22 special expression lncRNAs in yellow fruit. Importantly, we found a few DE-lncRNAs play cis and trans roles for genes in the Carotenoid biosynthesis, ascorbate and aldarate metabolism and fatty acid metabolism pathways. Our study provides a resource for lncRNA studies in mature fruit. It probably encourages researchers to deeply study fruit-coloring. It expands our knowledge about lncRNA biology and the annotation of the sea buckthorn genome.

Integrated analysis of multiomic data reveals the role of the antioxidant network in the quality of sea buckthorn berry

Berries of sea buckthorn, known as the “king of vitamin C,” are abundant in antioxidants, have attractive colors, and are an excellent material with which to study the relationships between berry color, antioxidants, and berry quality. No study has yet determined the molecular basis of the relationship between sea buckhorn berries and their color and antioxidant levels. By using RNA‐seq, LC–MS/MS, and LC/GC‐MS technology and selecting red (darkest colored) and yellow (lightest colored) sea buckthorn berries at different development stages, this study showed that the red and yellow berry resulted from a higher ratio of lycopene to β‐carotene and of β‐carotene to lycopene content, respectively. The uronic acid pathway—a known animal pathway—in ascorbic acid synthesis was found in sea buckthorn berries, and the higher expression of UDP‐glucuronosyltransferase in red berries was consistent with the higher content of ascorbic acid. In summary, multiomic data showed that the color of sea buckthorn berries is mainly determined by β‐carotene and lycopene; red sea buckthorn berries were richer than yellow berries in antioxidants, such as carotenoids, flavonoids, and ascorbic acid; and the animal pathway might be operating in sea buckthorn.—He, C., Zhang, G., Zhang, J., Zeng, Y., Liu, J. Integrated analysis of multiomic data reveals the role of the antioxidant network in the quality of sea buckthorn berry. FASEB J. 31, 1929–1938 (2017). www.fasebj.org