Pi-starvation induced transcriptional changes in barley revealed by a comprehensive RNA-Seq and degradome analyses

Background Small RNAs (sRNAs) are 20–30 nt regulatory elements which are responsible for plant development regulation and participate in many plant stress responses. Insufficient inorganic phosphate (Pi) concentration triggers plant responses to balance the internal Pi level. Results In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) RNA-Seq data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. We find that differentially and significantly expressed miRNAs (DEMs, Bonferroni adjusted p-value < 0.05) are represented by 15 molecules in shoot and 13 in root; mainly various miR399 and miR827 isomiRs. The remaining small RNAs (i.e., those without perfect match to reference sequences deposited in miRBase) are considered as differentially expressed other sRNAs (DESs, p-value Bonferroni correction < 0.05). In roots, a more abundant and diverse set of other sRNAs (DESs, 1796 unique sequences, 0.13% from the average of the unique small RNA expressed under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (DESs, 199 unique sequences, 0.01%). More than 80% of differentially expressed other sRNAs are up-regulated in both organs. Additionally, in barley shoots, up-regulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3′-5′ exonuclease). This suggests that most small RNAs may be generated upon nucleolytic cleavage to increase the internal Pi pool. Transcriptomic profiling of Pi-starved barley shoots identifies 98 differentially expressed genes (DEGs). A majority of the DEGs possess characteristic Pi-responsive cis-regulatory elements (P1BS and/or PHO element), located mostly in the proximal promoter regions. GO analysis shows that the discovered DEGs primarily alter plant defense, plant stress response, nutrient mobilization, or pathways involved in the gathering and recycling of phosphorus from organic pools. Conclusions Our results provide comprehensive data to demonstrate complex responses at the RNA level in barley to maintain Pi homeostasis and indicate that barley adapts to Pi-starvation through elicitation of RNA degradation. Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.

Pi-starvation induced transcriptional changes in barley revealed by a comprehensive RNA-Seq and degradome analyses

Background: Small RNAs (sRNAs) are 20–30 nt regulatory elements which are responsible for plant development regulation and participate in many plant stress responses. Insufficient inorganic phosphate (Pi) concentration triggers plant responses to balance the internal Pi level. Results: In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) RNA-Seq data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. We find that differentially and significantly expressed miRNAs (DEMs, p-value < 0.05) are represented by 162 (44.88 % of total differentially expressed small RNAs) molecules in shoot and 138 (7.14 %) in root; mainly various miR399 and miR827 isomiRs. The remaining small RNAs (i.e., those without perfect match to reference sequences deposited in miRBase) are considered as differentially expressed other sRNAs (DESs, p-value Bonferroni correction < 0.05). In roots, a more abundant and diverse set of other sRNAs (DESs, 1796 unique sequences, 0.13 % from total unique reads obtained under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (DESs, 199 unique sequences, 0.01 %). More than 80 % of differentially expressed other sRNAs are up-regulated in both organs. Additionally, in barley shoots, up-regulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3’-5’ exonuclease). This suggests that most small RNAs may be generated upon endonucleolytic cleavage to increase the internal Pi pool. Transcriptomic profiling of Pi-starved barley shoots identifies 98 differentially expressed genes (DEGs). A majority of the DEGs possess characteristic Pi-responsive cis-regulatory elements (P1BS and/or PHO element), located mostly in the proximal promoter regions. GO analysis shows that the discovered DEGs primarily alter plant defense, plant stress response, nutrient mobilization, or pathways involved in the gathering and recycling of phosphorus from organic pools.Conclusions: Our results provide comprehensive data to demonstrate complex responses at the RNA level in barley to maintain Pi homeostasis and indicate that barley adapts to Pi-starvation through elicitation of RNA degradation. Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.

Differential Gene Expression Responding to Low Phosphate Stress in Leaves and Roots of Maize by cDNA-SRAP

Phosphate (Pi) deficiency in soil can have severe impacts on the growth, development, and production of maize worldwide. In this study, a cDNA-sequence-related amplified polymorphism (cDNA-SRAP) transcript profiling technique was used to evaluate the gene expression in leaves and roots of maize under Pi stress for seven days. A total of 2494 differentially expressed fragments (DEFs) were identified in response to Pi starvation with 1202 and 1292 DEFs in leaves and roots, respectively, using a total of 60 primer pairs in the cDNA-SRAP analysis. These DEFs were categorized into 13 differential gene expression patterns. Results of sequencing and functional analysis showed that 63 DEFs (33 in leaves and 30 in roots) were annotated to a total of 54 genes involved in diverse groups of biological pathways, including metabolism, photosynthesis, signal transduction, transcription, transport, cellular processes, genetic information, and organismal system. This study demonstrated that (1) the cDNA-SRAP transcriptomic profiling technique is a powerful method to analyze differential gene expression in maize showing advantageous features among several transcriptomic methods; (2) maize undergoes a complex adaptive process in response to low Pi stress; and (3) a total of seven differentially expressed genes were identified in response to low Pi stress in leaves or roots of maize and could be used in the genetic modification of maize.

Integrated mRNA and miRNA expression profiling analyses of Pinus massoniana roots and shoots in response to phosphate deficiency

Background Masson pine ( Pinus massoniana ) is primarily present in subtropical and tropical areas of China, which are severely deficient in inorganic phosphate (Pi). Although some studies identified transcriptomic and proteomic responses to Pi deficiency in Masson pine seedlings, different tissues, especially the roots, that exhibit primary responses to low-Pi stress, have not been well studied. To shed further light on the complex responses of Masson pine to Pi deficiency, a spatiotemporal experiment was performed to identify differentially expressed mRNAs and miRNAs under Pi-deficient conditions. Results Spatiotemporal analyses of 72 RNA sequencing libraries provided a comprehensive overview of the dynamic responses of Masson pine to low-Pi stress. Differentially expressed gene analysis revealed several high-affinity phosphate transporters and nitrate transporters, reflecting the crosstalk between nitrate and Pi homeostasis in plants. The MYB family was the most abundant transcription factor family identified. miRNA differential expression analysis identified several families that were associated with Pi deficiency, such as miR399. In addition, some other families, including novel miRNA families in Masson pine, were dramatically changed in response to Pi starvation. GO and KEGG analyses of these mRNAs and targets of miRNAs indicated that metabolic processes were most enriched under Pi deficiency. Conclusions This study provided abundant spatiotemporal transcriptomic information to functionally dissect the response of Masson pine seedlings to Pi deficiency, which will aid in further elucidation of the biological regulatory mechanisms of pines in response to low-Pi stress.

Pi-starvation induced transcriptional changes in barley revealed by a comprehensive RNA-Seq and degradome analyses

Background: Small RNAs (sRNAs) are 18–24 nt regulatory elements which are responsible for plant development regulation and participate in many plant stress responses. Insufficient inorganic phosphate (Pi) concentration triggers plant responses to balance the internal Pi level. Results: In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. We find that differentially and significantly expressed miRNAs (DEMs, p-value < 0.05) are represented by 162 (44.88 % of total differentially expressed small RNAs) molecules in shoot and 138 (7.14 %) in root; mainly various miR399 and miR827 isomiRs. The remaining small RNAs (i.e., those without perfect match to reference sequences deposited in miRBase) are considered as differentially expressed other sRNAs (DESs, Bonferroni correction). In roots, a more abundant and diverse set of other sRNAs (1796 unique sequences, 0.13 % from total unique reads obtained under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (199 unique sequences, 0.01 %). More than 80 % of differentially expressed other sRNAs are upregulated in both organs. Additionally, in barley shoots, upregulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3’-5’ exonuclease). This suggests that most small RNAs may be generated upon endonucleolytic cleavage to increase the internal Pi pool. Transcriptomic profiling of Pi-starved barley shoots identify 98 differentially expressed genes (DEGs). A majority of the DEGs possess characteristic Pi-responsive cis-regulatory elements (P1BS and/or PHO element), located mostly in the proximal promoter regions. GO analysis shows that the discovered DEGs primarily alter plant defense, plant stress response, nutrient mobilization, or pathways involved in the gathering and recycling of phosphorus from organic pools.Conclusions: Our results provide comprehensive data to demonstrate complex responses at the RNA level in barley to maintain Pi homeostasis and indicate that barley adapts to Pi scarcity through elicitation of RNA degradation. Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.

Cloning, Characterization and Expression Analysis of the Phosphate Starvation Response Gene, ClPHR1, from Chinese Fir

The study on the function and sequence of PHR1 (Phosphate Starvation Response gene 1) gene, which plays a central role in plant phosphorus (Pi) signal regulatory network, is of great significance to further study response mechanisms to Pi deficiency. In this work, the previously selected Pi-efficient Chinese fir clone M32 was used as research material to obtain the full-length sequence of ClPHR1 transcription factors in Chinese fir by RACE (Rapid Amplification of cDNA Ends) full-length cloning technique, and the structure, function and subcellular localization of ClPHR1 gene encoding protein were analyzed. The temporal and spatial expression characteristics of ClPHR1 transcription factors in Chinese fir under low Pi stress were also analyzed, and the overexpression of ClPHR1 gene in transgenic Arabidopsis thaliana was obtained to verify the function of ClPHR1 gene under low Pi stress. The results showed that the length of the ClPHR1 gene obtained by rapid amplification of cDNA ends technique was 1954 bp, of which 1512 bp was an open reading frame. ClPHR1 was predicted to be an unstable hydrophilic protein with only one possible transmembrane domain. The ClPHR1 gene had a highly conserved MYB-CC domain, which is similar to the PHR1 gene of other plants. Phylogenetic tree analysis showed that the sequence had high homology with PHR1 genes in the Prunus species. The ClPHR1 was expressed in all organs of Chinese fir, with the highest expression in the roots, followed by the leaves with the lowest expression in stems. ClPHR1 expression in roots was reduced dramatically at the beginning of Pi stress treatment and followed by an increase at 7days; in leaves, it increased dramatically at the beginning of Pi starvation treatment and showed a decreasing trend after 3 days; in stems, the expression level of ClPHR1 increased after 7 days of Pi stress treatment. The transient expression vector was introduced into plant cells, and it was found that ClPHR1 was located in the nucleus and was a MYB-CC transcription factor expressed in the cell nucleus. The ClPHR1 overexpression vector was constructed, and then introduced into Arabidopsis thaliana by agrobacterium infection inflorescence method. The expressions of Pi transporter genes, AtPHT1;1, AtPHT1;2, AtPHT1;8 and AtPHT1;9, was significantly higher in the overexpressing strain than that in the wild type strain. The results suggest that the ClPHR1 transcription factor could regulate the regulation of downstream Pi transporter gene and increase Pi utilization efficiency of the Chinese fir under Pi stress.

Transcriptome analysis of low phosphate stress response in the roots of masson pine (Pinus massoniana) seedlings

Background: Masson pine (Pinus massoniana) is primarily present within subtropical and tropical areas in China, and a number of these regions have a severe deficiency in inorganic phosphate (Pi). As a macronutrient, phosphorus plays a crucial role in plant development. Although several studies have studied the responses of masson pine to Pi starvation at the global level using RNA-Seq and comparative proteomic analyses, the detailed features in the roots that primarily respond to low Pi stress have not yet been studied. Results: This study examined the masson pine of the response of masson pine roots to a deficiency in Pi. Approximately 1,117 unigenes were shown to respond to Pi-deficiency by differential expression when analyzed using RNA-Seq. A total of 819 and 298 of these transcripts were up- and down-regulated, respectively. The results identified several phosphate transporters (PHT1, PHO88), ABC transporters and metal transporters. In particular, the ethylene response factor (ERF) was the most abundant transcription factor. Analyses of these genes, including gene ontology enrichment and the KEGG pathway analysis, indicated that the metabolic processes are the most enriched under abiotic stresses, including Pi-deficiency. Conclusions: This study provided abundant transcriptomic information to functionally dissect the response of masson pine roots to a deficiency of Pi, which will provide additional aid to elucidate the biological regulatory mechanisms that the pines use to respond to low Pi stress.

Early Transcriptomic Response to Phosphate Deprivation in Soybean Leaves as Revealed by RNA-Sequencing

Low phosphate (Pi) availability is an important limiting factor affecting soybean production. However, the underlying molecular mechanisms responsible for low Pi stress response and tolerance remain largely unknown, especially for the early signaling events under low Pi stress. Here, a genome-wide transcriptomic analysis in soybean leaves treated with a short-term Pi-deprivation (24 h) was performed through high-throughput RNA sequencing (RNA-seq) technology. A total of 533 loci were found to be differentially expressed in response to Pi deprivation, including 36 mis-annotated loci and 32 novel loci. Among the differentially expressed genes (DEGs), 303 were induced and 230 were repressed by Pi deprivation. To validate the reliability of the RNA-seq data, 18 DEGs were randomly selected and analyzed by quantitative RT-PCR (reverse transcription polymerase chain reaction), which exhibited similar fold changes with RNA-seq. Enrichment analyses showed that 29 GO (Gene Ontology) terms and 8 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were significantly enriched in the up-regulated DEGs and 25 GO terms and 16 KEGG pathways were significantly enriched in the down-regulated DEGs. Some DEGs potentially involved in Pi sensing and signaling were up-regulated by short-term Pi deprivation, including five SPX-containing genes. Some DEGs possibly associated with water and nutrient uptake, hormonal and calcium signaling, protein phosphorylation and dephosphorylation and cell wall modification were affected at the early stage of Pi deprivation. The cis-elements of PHO (phosphatase) element, PHO-like element and P responsive element were present more frequently in promoter regions of up-regulated DEGs compared to that of randomly-selected genes in the soybean genome. Our transcriptomic data showed an intricate network containing transporters, transcription factors, kinases and phosphatases, hormone and calcium signaling components is involved in plant responses to early Pi deprivation.