Identification and characterization of pineapple leaf lncRNAs in crassulacean acid metabolism (CAM) photosynthesis pathway.
ABSTRACT: Long noncoding RNAs (lncRNAs) have been identified in many mammals and plants and are known to play crucial roles in multiple biological processes. Pineapple is an important tropical fruit and a good model for studying the plant evolutionary adaptation to the dry environment and the crassulacean acid metabolism (CAM) photosynthesis strategy; however, the lncRNAs involved in CAM pathway remain poorly characterized. Here, we analyzed the available RNA-seq data sets derived from 26 pineapple leaf samples at 13 time points and identified 2,888 leaf lncRNAs, including 2,046 long intergenic noncoding RNAs (lincRNAs) and 842 long noncoding natural antisense transcripts (lncNATs). Pineapple leaf lncRNAs are expressed in a highly tissue-specific manner. Co-expression analysis of leaf lncRNA and mRNA revealed that leaf lncRNAs are preferentially associated with photosynthesis genes. We further identified leaf lncRNAs that potentially function as competing endogenous RNAs (ceRNAs) of two CAM photosynthesis pathway genes, PPCK and PEPC, and revealed their diurnal expression pattern in leaves. Moreover, we found that 48% of lncRNAs exhibit diurnal expression patterns in leaves, suggesting their important roles in CAM. This study conducted a comprehensive genome-wide analysis of leaf lncRNAs and identified their role in gene expression regulation of the CAM photosynthesis pathway in pineapple.
Project description:BACKGROUND:Pineapple is the most important crop with CAM photosynthesis, but its molecular biology is underexplored. MADS-box genes are crucial transcription factors involving in plant development and several biological processes. However, there is no systematic analysis of MADS-box family genes in pineapple (Ananas comosus). RESULTS:Forty-eight MADS-box genes were identified in the pineapple genome. Based on the phylogenetic studies, pineapple MADS-box genes can be divided into type I and type II MADS-box genes. Thirty-four pineapple genes were classified as type II MADS-box genes including 32 MIKC-type and 2 M?-type, while 14 type I MADS-box genes were further divided into M?, M? and M? subgroups. A majority of pineapple MADS-box genes were randomly distributed across 19 chromosomes. RNA-seq expression patterns of MADS-box genes in four different tissues revealed that more genes were highly expressed in flowers, which was confirmed by our quantitative RT-PCR results. There is no FLC and CO orthologs in pineapple. The loss of FLC and CO orthologs in pineapple indicated that modified flowering genes network in this tropical plant compared with Arabidopsis. The expression patterns of MADS-box genes in photosynthetic and non-photosynthetic leaf tissues indicated the potential roles of some MADS-box genes in pineapple CAM photosynthesis. The 23% of pineapple MADS-box genes showed diurnal rhythm, indicating that these MADS-box genes are regulated by circadian clock. CONCLUSIONS:MADS-box genes identified in pineapple are closely related to flowering development. Some MADS-box genes are involved in CAM photosynthesis and regulated by the circadian clock. These findings will facilitate research on the development of unusual spiral inflorescences on pineapple fruit and CAM photosynthesis.
Project description:Circadian clock provides fitness advantage by coordinating internal metabolic and physiological processes to external cyclic environments. Core clock components exhibit daily rhythmic changes in gene expression, and the majority of them are transcription factors (TFs) and transcription coregulators (TCs). We annotated 1,398 TFs from 67 TF families and 80 TCs from 20?TC families in pineapple, and analyzed their tissue-specific and diurnal expression patterns. Approximately 42% of TFs and 45% of TCs displayed diel rhythmic expression, including 177?TF/TCs cycling only in the nonphotosynthetic leaf tissue, 247 cycling only in the photosynthetic leaf tissue, and 201 cycling in both. We identified 68?TF/TCs whose cycling expression was tightly coupled between the photosynthetic and nonphotosynthetic leaf tissues. These TF/TCs likely coordinate key biological processes in pineapple as we demonstrated that this group is enriched in homologous genes that form the core circadian clock in Arabidopsis and includes a STOP1 homolog. Two lines of evidence support the important role of the STOP1 homolog in regulating CAM photosynthesis in pineapple. First, STOP1 responds to acidic pH and regulates a malate channel in multiple plant species. Second, the cycling expression pattern of the pineapple STOP1 and the diurnal pattern of malate accumulation in pineapple leaf are correlated. We further examined duplicate-gene retention and loss in major known circadian genes and refined their evolutionary relationships between pineapple and other plants. Significant variations in duplicate-gene retention and loss were observed for most clock genes in both monocots and dicots.
Project description:Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ? duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.
Project description:Mesembryanthemum crystallinum, which switches the mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) upon high salt stress, was shown here to exhibit diurnal changes in not only the CO2 fixation pathway but also Chl fluorescence parameters under CAM-induced conditions. We conducted comprehensive time course measurements of M. crystallinum leaf Chl fluorescence using the same leaf throughout the CAM induction period. By doing so, we were able to distinguish the effect of CAM induction from that of photoinhibition and avoid the possible effects of differences in foliar age. We found that the diurnal change in the status of electron transfer could be ascribed to the formation of a proton gradient across thylakoid membranes presumably resulting from diurnal changes in the ATP/ADP ratio reported earlier. The electron transport by actinic illumination thus became limited at the step of plastoquinol oxidation by the Cyt b6/f complex in the 'night' period upon CAM induction, resulting in high levels of non-photochemical quenching. The actinically induced non-photochemical quenching in the 'night' period correlated well with the degree of CAM induction. Chl fluorescence parameters, such as NPQ or qN, could be used as a simple indexing system for the CAM induction.
Project description:BACKGROUND:Long noncoding RNAs (lncRNAs) play an important role in diverse biological processes and have been widely studied in recent years. However, the roles of lncRNAs in leaf pigment formation in ginkgo (Ginkgo biloba L.) remain poorly understood. RESULTS:In this study, lncRNA libraries for mutant yellow-leaf and normal green-leaf ginkgo trees were constructed via high-throughput sequencing. A total of 2044 lncRNAs were obtained with an average length of 702?nt and typically harbored 2 exons. We identified 238 differentially expressed lncRNAs (DELs), 32 DELs and 49 differentially expressed mRNAs (DEGs) that constituted coexpression networks. We also found that 48 cis-acting DELs regulated 72 target genes, and 31 trans-acting DELs regulated 31 different target genes, which provides a new perspective for the regulation of the leaf-color mutation. Due to the crucial regulatory roles of lncRNAs in a wide range of biological processes, we conducted in-depth studies on the DELs and their targets and found that the chloroplast thylakoid membrane subcategory and the photosynthesis pathways (ko00195) were most enriched, suggesting their potential roles in leaf coloration mechanisms. In addition, our correlation analysis indicates that eight DELs and 68 transcription factors (TFs) might be involved in interaction networks. CONCLUSIONS:This study has enriched the knowledge concerning lncRNAs and provides new insights into the function of lncRNAs in leaf-color mutations, which will benefit future selective breeding of ginkgo.
Project description:BACKGROUND:Long noncoding RNAs (lncRNAs) have emerged as playing crucial roles in abiotic stress responsive regulation, however, the mechanism of lncRNAs underlying drought-tolerance remains largely unknown in cassava, an important tropical and sub-tropical root crop of remarkable drought tolerance. RESULTS:In this study, a total of 833 high-confidence lncRNAs, including 652 intergenic and 181 anti-sense lncRNAs, were identified in cassava leaves and root using strand-specific RNA-seq technology, of which 124 were drought-responsive. Trans-regulatory co-expression network revealed that lncRNAs exhibited tissue-specific expression patterns and they preferred to function differently in distinct tissues: e.g., cell-related metabolism, cell wall, and RNA regulation of transcription in folded leaf (FL); degradation of major carbohydrate (CHO) metabolism, calvin cycle and light reaction, light signaling, and tetrapyrrole synthesis in full expanded leaf (FEL); synthesis of major CHO metabolism, nitrogen-metabolism, photosynthesis, and redox in bottom leaf (BL); and hormone metabolism, secondary metabolism, calcium signaling, and abiotic stress in root (RT). In addition, 27 lncRNA-mRNA pairs referred to cis-acting regulation were identified, and these lncRNAs regulated the expression of their neighboring genes mainly through hormone metabolism, RNA regulation of transcription, and signaling of receptor kinase. Besides, 11 lncRNAs were identified acting as putative target mimics of known miRNAs in cassava. Finally, five drought-responsive lncRNAs and 13 co-expressed genes involved in trans-acting, cis-acting, or target mimic regulation were selected and confirmed by qRT-PCR. CONCLUSIONS:These findings provide a comprehensive view of cassava lncRNAs in response to drought stress, which will enable in-depth functional analysis in the future.
Project description:Background and Aims:Leaf tissue CO2 partial pressure (pCO2) shows contrasting dynamics over a diurnal cycle in C3 and Crassulacean Acid Metabolism (CAM) plants. However, simultaneous and continuous monitoring of pCO2 and pO2 in C3 and CAM plants under the same conditions was lacking. Our aim was to use a new CO2 microsensor and an existing O2 microsensor for non-destructive measurements of leaf pCO2 and pO2 dynamics to compare a C3 and a CAM plant in an aquatic environment. Methods:A new amperometric CO2 microsensor and an O2 microsensor elucidated with high temporal resolution the dynamics in leaf pCO2 and pO2 during light-dark cycles for C3Lobelia dortmanna and CAM Littorella uniflora aquatic plants. Underwater photosynthesis, dark respiration, tissue malate concentrations and sediment CO2 and O2 were also measured. Key Results:During the dark period, for the C3 plant, pCO2 increased to approx. 3.5 kPa, whereas for the CAM plant CO2 was mostly below 0.05 kPa owing to CO2 sequestration into malate. Upon darkness, the CAM plant had an initial peak in pCO2 (approx. 0.16 kPa) which then declined to a quasi-steady state for several hours and then pCO2 increased towards the end of the dark period. The C3 plant became severely hypoxic late in the dark period, whereas the CAM plant with greater cuticle permeability did not. Upon illumination, leaf pCO2 declined and pO2 increased, although aspects of these dynamics also differed between the two plants. Conclusions:The continuous measurements of pCO2 and pO2 highlighted the contrasting tissue gas compositions in submerged C3 and CAM plants. The CAM leaf pCO2 dynamics indicate an initial lag in CO2 sequestration to malate, which after several hours of malate synthesis then slows. Like the use of O2 microsensors to resolve questions related to plant aeration, deployment of the new CO2 microsensor will benefit plant ecophysiology research.
Project description:Photosynthesis is a key reaction that ultimately generates the carbohydrates needed to form woody tissues in trees. However, the genetic regulatory network of protein-encoding genes (PEGs) and regulatory noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), underlying the photosynthetic pathway is unknown. Here, we integrated data from coexpression analysis, association studies (additive, dominance and epistasis), and expression quantitative trait nucleotide (eQTN) mapping to dissect the causal variants and genetic interaction network underlying photosynthesis in Populus. We initially used 30 PEGs, 6 miRNAs and 12 lncRNAs to construct a coexpression network based on the tissue-specific gene expression profiles of 15 Populus samples. Then, we performed association studies using a natural population of 435 unrelated Populus tomentosa individuals, and identified 72 significant associations (P ? 0.001, q ? 0.05) with diverse additive and dominance patterns underlying photosynthesis-related traits. Analysis of epistasis and eQTNs revealed that the complex genetic interactions in the coexpression network contribute to phenotypes at various levels. Finally, we demonstrated that heterologously expressing the most highly linked gene (PtoPsbX1) in this network significantly improved photosynthesis in Arabidopsis thaliana, pointing to the functional role of PtoPsbX1 in the photosynthetic pathway. This study provides an integrated strategy for dissecting a complex genetic interaction network, which should accelerate marker-assisted breeding efforts to genetically improve woody plants.
Project description:Salt stress impedes plant growth and development, and leads to yield loss. Recently, a halophyte species Mesembryanthemum crystallinum has become a model to study plant photosynthetic responses to salt stress. It has an adaptive mechanism of shifting from C3 photosynthesis to crassulacean acid metabolism (CAM) photosynthesis under stresses, which greatly enhances water usage efficiency and stress tolerance. In this study, we focused on investigating the morphological and physiological changes [e.g., leaf area, stomatal movement behavior, gas exchange, leaf succulence, and relative water content (RWC)] of M. crystallinum during the C3 to CAM photosynthetic transition under salt stress. Our results showed that in M. crystallinum seedlings, CAM photosynthesis was initiated after 6 days of salt treatment, the transition takes place within a 3-day period, and plants became mostly CAM in 2 weeks. This result defined the transition period of a facultative CAM plant, laid a foundation for future studies on identifying the molecular switches responsible for the transition from C3 to CAM, and contributed to the ultimate goal of engineering CAM characteristics into C3 crops.
Project description:Phalaenopsis is one of the most important potted plants in the ornamental market of the world. Previous reports implied that crassulacean acid metabolism (CAM) orchids at their young seedling stages might perform C3 or weak CAM photosynthetic pathways, but the detailed molecular evidence is still lacking. In this study, we used a key species in white Phalaenopsis breeding line, Phalaenopsis aphrodite subsp. formosana, to study the ontogenetical changes of CAM performance in Phalaenopsis. Based on the investigations of rhythms of day/night CO2 exchange, malate contents and phosphoenolpyruvate carboxylase (PEPC) activities, it is suggested that a progressive shift from C3 to CAM occurred as the protocorms differentiated the first leaf. To understand the role of phosphoenolpyruvate carboxylase kinase (PEPC kinase) in relation to its target PEPC in CAM performance in Phalaenopsis, the expression profiles of the genes encoding PEPC (PPC) and PEPC kinase (PPCK) were measured in different developmental stages. In Phalaenopsis, two PPC isogenes were constitutively expressed over a 24-h cycle similar to the housekeeping genes in all stages, whereas the significant day/night difference in PaPPCK expression corresponds to the day/night fluctuations in PEPC activity and malate level. These results suggest that the PaPPCK gene product is most likely involved in regulation of CAM performance in different developmental stages of Phalaenopsis seedlings.