Transcriptome analysis provides insights into the root response of Chinese fir to phosphorus deficiency

Background Phosphorus is one of the essential elements for plant growth and development, but available phosphorus (Pi) content in many soil types is low. As a fast-growing tree species for timber production, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir root responses to Pi deficiency are largely unexplored. In this study, seedlings of Yang 061 clone were grown under three Pi supply levels (0, 5 and 10 mg·L-1 P) and morphological attributes, organic acid content and enzyme activity were measured. The transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined. Results We annotated 50,808 Unigenes from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and 17 genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term Pi stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30 days of Pi deficiency. Conclusion Chinese fir roots showed increased expression of genes related with phosphorus starvation, citrate and malate synthesis genes, increased content of organic acids, and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of adaption to Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.

Physiological responses and transcriptomic changes reveal the mechanisms underlying adaptation of Stylosanthes guianensis to phosphorus deficiency

Background Phosphorus (P) is an essential macronutrient for plant growth that participates in a series of biological processes. Thus, P deficiency limits crop growth and yield. Although Stylosanthes guianensis (stylo) is an important tropical legume that displays adaptation to low phosphate (Pi) availability, its adaptive mechanisms remain largely unknown. Results In this study, differences in low-P stress tolerance were investigated using two stylo cultivars (‘RY2’ and ‘RY5’) that were grown in hydroponics. Results showed that cultivar RY2 was better adapted to Pi starvation than RY5, as reflected by lower values of relative decrease rates of growth parameters than RY5 at low-P stress, especially for the reduction of shoot and root dry weight. Furthermore, RY2 exhibited higher P acquisition efficiency than RY5 under the same P treatment, although P utilization efficiency was similar between the two cultivars. In addition, better root growth performance and higher leaf and root APase activities were observed with RY2 compared to RY5. Subsequent RNA-seq analysis revealed 8,348 genes that were differentially expressed under P deficient and sufficient conditions in RY2 roots, with many Pi starvation regulated genes associated with P metabolic process, protein modification process, transport and other metabolic processes. A group of differentially expressed genes (DEGs) involved in Pi uptake and Pi homeostasis were identified, such as genes encoding Pi transporter (PT), purple acid phosphatase (PAP), and multidrug and toxin extrusion (MATE). Furthermore, a variety of genes related to transcription factors and regulators involved in Pi signaling, including genes belonging to the PHOSPHATE STARVATION RESPONSE 1-like (PHR1), WRKY and the SYG1/PHO81/XPR1 (SPX) domain, were also regulated by P deficiency in stylo roots. Conclusions This study reveals the possible mechanisms underlying the adaptation of stylo to P deficiency. The low-P tolerance in stylo is probably manifested through regulation of root growth, Pi acquisition and cellular Pi homeostasis as well as Pi signaling pathway. The identified genes involved in low-P tolerance can be potentially used to design the breeding strategy for developing P-efficient stylo cultivars to grow on acid soils in the tropics.

Genome-Wide Analysis of Purple Acid Phosphatase Genes in Brassica rapa and Their Association with Pollen Development and Phosphorus Deprivation Stress

PAPs (purple acid phosphatases) belong to the metallo-phosphoesterase superfamily and play important roles in developmental processes, phosphorus foraging, and recycling. However, the specific functions of BrPAPs in Brassica rapa are poorly understood. In this study, 39 BrPAPs were identified and divided into three major clades and nine subgroups. In 8 of the 39 BrPAPs, some invariant amino acid residues were lost or shifted. Based on an expression profiling analysis, BrPAP11, 14, 20, 24, 29, and 34 were specifically expressed in fertile floral buds, indicating their critical roles during pollen development. A total of 21 BrPAPs responded to Pi deprivation in either shoots or roots. Of these, BrPAP4, 5, 19, and 21 were upregulated in roots under Pi depravation conditions, while BrPAP12 was upregulated in the roots in normal conditions. BrPAP28 was upregulated in shoots under Pi depravation conditions, indicating its function shifted compared with its Arabidopsis homolog, AtPAP26. The present work contributes to further investigation of BrPAPs as candidate genes for genetic improvement studies of low phosphorus tolerance as well as for creating male sterile lines based on gene editing methods in Brassica rapa.

Arabidopsis PAP17 is a dual-localized purple acid phosphatase up-regulated during phosphate deprivation, senescence, and oxidative stress

A 35 kDa monomeric purple acid phosphatase (APase) was purified from cell wall extracts of Pi starved (–Pi) Arabidopsis thaliana suspension cells and identified as AtPAP17 (At3g17790) by mass spectrometry and N-terminal microsequencing. AtPAP17 was de novo synthesized and dual-localized to the secretome and/or intracellular fraction of –Pi or salt-stressed plants, or senescing leaves. Transiently expressed AtPAP17–green fluorescent protein localized to lytic vacuoles of the Arabidopsis suspension cells. No significant biochemical or phenotypical changes associated with AtPAP17 loss of function were observed in an atpap17 mutant during Pi deprivation, leaf senescence, or salinity stress. Nevertheless, AtPAP17 is hypothesized to contribute to Pi metabolism owing to its marked up-regulation during Pi starvation and leaf senescence, broad APase substrate selectivity and pH activity profile, and rapid repression and turnover following Pi resupply to –Pi plants. While AtPAP17 also catalyzed the peroxidation of luminol, which was optimal at pH 9.2, it exhibited a low Vmax and affinity for hydrogen peroxide relative to horseradish peroxidase. These results, coupled with absence of a phenotype in the salt-stressed or –Pi atpap17 mutant, do not support proposals that the peroxidase activity of AtPAP17 contributes to the detoxification of reactive oxygen species during stresses that trigger AtPAP17 up-regulation.

Overlapping Functions of the Paralogous Proteins AtPAP2 and AtPAP9 in Arabidopsis thaliana

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), which is anchored to the outer membranes of chloroplasts and mitochondria, affects carbon metabolism by modulating the import of some preproteins into chloroplasts and mitochondria. AtPAP9 bears a 72% amino acid sequence identity with AtPAP2, and both proteins carry a hydrophobic motif at their C-termini. Here, we show that AtPAP9 is a tail-anchored protein targeted to the outer membrane of chloroplasts. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that both AtPAP9 and AtPAP2 bind to a small subunit of rubisco 1B (AtSSU1B) and a number of chloroplast proteins. Chloroplast import assays using [35S]-labeled AtSSU1B showed that like AtPAP2, AtPAP9 also plays a role in AtSSU1B import into chloroplasts. Based on these data, we propose that AtPAP9 and AtPAP2 perform overlapping roles in modulating the import of specific proteins into chloroplasts. Most plant genomes contain only one PAP-like sequence encoding a protein with a hydrophobic motif at the C-terminus. The presence of both AtPAP2 and AtPAP9 in the Arabidopsis genome may have arisen from genome duplication in Brassicaceae. Unlike AtPAP2 overexpression lines, the AtPAP9 overexpression lines did not exhibit early-bolting or high-seed-yield phenotypes. Their differential growth phenotypes could be due to the inability of AtPAP9 to be targeted to mitochondria, as the overexpression of AtPAP2 on mitochondria enhances the capacity of mitochondria to consume reducing equivalents.

A Balance between the Activities of Chloroplasts and Mitochondria Is Crucial for Optimal Plant Growth

Energy metabolism in plant cells requires a balance between the activities of chloroplasts and mitochondria, as they are the producers and consumers of carbohydrates and reducing equivalents, respectively. Recently, we showed that the overexpression of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), a phosphatase dually anchored on the outer membranes of chloroplasts and mitochondria, can boost the plant growth and seed yield of Arabidopsis thaliana by coordinating the activities of both organelles. However, when AtPAP2 is solely overexpressed in chloroplasts, the growth-promoting effects are less optimal, indicating that active mitochondria are required for dissipating excess reducing equivalents from chloroplasts to maintain the optimal growth of plants. It is even more detrimental to plant productivity when AtPAP2 is solely overexpressed in mitochondria. Although these lines contain high level of adenosine triphosphate (ATP), they exhibit low leaf sucrose, low seed yield, and early senescence. These transgenic lines can be useful tools for studying how hyperactive chloroplasts or mitochondria affect the physiology of their counterparts and how they modify cellular metabolism and plant physiology.

Transcriptome Analysis Provides Insights into the Root Response of Chinese Fir to Phosphorus Deficiency

Background: Phosphorus is one of the essential elements for plant growth and development, but the content of plant available phosphorus (Pi) in many soil types is low. As a fast-growing timber species, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir in response to Pi deficiency are largely unexplored. Results: In this study, seedlings of Yang 061 clone were grown under three Pi supply levels (0, 5 and 10 mg·L-1 P) and morphological attributes, organic acid content and enzyme activity were measured, the transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined. The results showed that there were 50,808 Unigenes annotated from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and seventeen genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30days of Pi deficiency. Conclusion: The Chinese fir roots showed increased expression of citrate and malate synthesis genes, increased content of organic acids and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.

Genome-Wide Characterization and Evolutionary Analyses of Purple Acid Phosphatase (PAP) Gene Family with Their Expression Profiles in Response to Low Phosphorus Stresses in Moso Bamboo (Phyllostachys edulis)

Low phosphorus increases acid phosphatase activity and transfers soluble phosphorus from the underground to the above-ground, but also inhibits the growth and development of the Moso bamboo root system. Purple acid phosphatase (PAP), a kind of acid phosphatase, plays an important role in phosphorus (P) uptake and metabolism. In our study of the Moso bamboo PAP gene family, we identified 17 Moso bamboo PAP genes (PePAP) in the entire genome and further analyzed their physical and chemical properties and functions PePAP. According to the analysis of the phylogenetic tree, special domains and conserved motifs, these 17 genes can be divided into four categories. The gene structure and conserved motifs are relatively conservative, but the 17 sequences of the PePAP domain are diverse. The prediction of the subcellular location indicated that PePAPs are mainly located in the secretory pathway. We have studied the expression levels of these PePAP in different organs, such as the roots, stems and leaves of Moso bamboo, and the results show that the expression of most PePAP genes in roots and stems seems to be higher than that in leaves. In addition to tissue-specific expression analysis, we also studied the expression of PePAPs under low phosphorus stress. Under such conditions, the PePAP genes show an increase in expression in the roots, stem and leaves, and the extent of this change varies between genes. In summary, our results reveal the evolution of the PePAP gene in the Moso bamboo genome and provide a basis for understanding the molecular mechanism of the PePAP-mediated response of Moso bamboo to low phosphorus.

Involvement of purple acid phosphatase gene into nitrogen uptake of oil palm (Elaeis guineensis)

Maryanto SD, Tanjung ZA, Roberdi, Sudania WM, Pujianto, Hairinsyah, Utomo C, Liwang T. 2021. Involvement of purple acid phosphatase gene into nitrogen uptake of oil palm (Elaeis guineensis). Biodiversitas 22: 1385-1390. Nitrogen is the most important nutrient element in terms of plant growth. Plant purple acid phosphatases (PAPs) are known to participate in the phosphate (Pi) acquisition and utilization. Moreover, PAP gene plays an important role in nitrogen fixation. A single nucleotide polymorphism (SNP) was previously detected in the exon 7 of EgPAP3, based on SNP mining analysis of oil palm (Elaeis guineensis Jacq.). genome database. This study was aimed to obtain a Cleaved Amplified Polymorphic Sequences (CAPS) marker based on SNP within EgPAP associated with efficient nitrogen uptake oil palm progenies. Primer pairs were designed and used for PCR amplification of 3 oil palm progenies that showed low N-content, 3 progenies with moderate N-content, and 3 progenies with high N-content. The amplicon was purified prior to single-pass DNA sequencing analysis. Based on Pearson’s chi-square and odds ratio statistical analysis, the SNP has strong positive correlation with the phenotype. The SNP is located at chromosome 13 with a distance of 17.7771 cM from start codon. The sequencing analysis revealed that three progenies with high N-content samples had GG allele motif, while moderate N-content progenies had GA allele and low N-content progenies had AA allele motifs respectively. In addition, a restriction site of NIaIV was found to be adjacent to the SNP location, thus the PCR products of all samples were digested with NIaIV restriction enzyme. NIaIV was able to distinguish between high, medium and low efficient DNA samples. Whole high N-content progenies with GG allele motifs were undigested indicating a single band size of 670 bp identical to the untreated PCR product (control). Moderate N-content progenies produced a 670 bp, 550 bp, and 120 bp bands because of digested by NIaIV. Low N-content progenies also resulted in double bands of 550 bp and 120 bp due to digested by NIaIV. Furthermore, NIaIV restriction enzyme was applied to digest other 54 oil palms DNA samples with unknown genotypes. Whole GG samples were consistently shown to have single band, GA and AA samples were also consistent in producing two bands with different lengths. Based on this result, CAPS marker based on SNP in EgPAP3 was successfully developed to screen between high and low efficient N-uptake of oil palm progenies.