Genome-wide analysis of bZIP transcription factors and their expression patterns in response to methyl jasmonate and low-temperature stresses in Platycodon grandiflorus.

Background: Platycodon grandiflorus belongs to the genus Platycodon and has many pharmacological effects, such as expectorant, antitussive, and anti-tumor properties. Among transcription factor families peculiar to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important, which exists widely in plants and participates in many biological processes, such as plant growth, development, and stress responses. However, genomic analysis of the bZIP gene family and related stress response genes has not yet been reported in P. grandiflorus. Methods: P. grandiflorus bZIP (PgbZIP) genes were first identified here, and the phylogenetic relationships and conserved motifs in the PgbZIPs were also performed. Meanwhile, gene structures, conserved domains, and the possible protein subcellular localizations of these PgbZIPs were characterized. Most importantly, the cis-regulatory elements and expression patterns of selected genes exposed to two different stresses were analyzed to provide further information on PgbZIPs potential biological roles in P. grandiflorus upon exposure to environmental stresses. Conclusions: Forty-six PgbZIPs were identified in P. grandiflorus and divided into nine groups, as displayed in the phylogenetic tree. The results of the chromosomal location and the collinearity analysis showed that forty-six PgbZIP genes were distributed on eight chromosomes, with one tandem duplication event and eleven segmental duplication events identified. Most PgbZIPs in the same phylogenetic group have similar conserved motifs, domains, and gene structures. There are cis-regulatory elements related to the methyl jasmonate (MeJA) response, low-temperature response, abscisic acid response, auxin response, and gibberellin response. Ten PgbZIP genes were selected to study their expression patterns upon exposure to low-temperature and MeJA treatments, and all ten genes responded to these stresses. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that the expression levels of most PgbZIPs decreased significantly within 6 h and then gradually increased to normal or above normal levels over the 90 h following MeJA treatment. The expression levels of all PgbZIPs were significantly reduced after 3 h of the low-temperature treatment. These results reveal the characteristics of the PgbZIP family genes and provide valuable information for improving P. grandiflorus's ability to cope with environmental stresses during growth and development.

Genome-Wide Identification of R2R3-MYB Family Genes and Their Response to Stress in Dendrobium nobile.

BACKGROUND: R2R3-MYB genes comprise one of the largest and most important gene families in plants, and are involved in the regulation of plant growth and development as well as responses to abiotic stresses. However, the functions of R2R3-MYB genes in Dendrobium nobile remains largely unknown. METHODS: Here, a comprehensive genome-wide analysis of D. nobile R2R3-MYB genes was performed, in which phylogenic relationships, gene structures, motif composition, chromosomal locations, collinearity analysis, and cis-acting elements were investigated. Moreover, the expression patterns of selected DnMYB genes were analyzed in various tissues and under different abiotic stresses. RESULTS: In total, 125 DnMYB genes were identified in the D. nobile genome, and were subdivided into 26 groups based on phylogenetic analysis. Most genes in the same subgroup showed similar exon/intron structure and motif composition. All the DnMYB genes were mapped to 19 chromosomes with the co-linearity relationship. Reverse transcription-quantitative real-time PCR (RT-qPCR) results showed that 8 DnMYBs exhibited different expression patterns in different plant tissues, and were differentially expressed in response to abscisic acid, methyl jasmonate, low-temperature stress. CONCLUSIONS: This work contributes to a comprehensive understanding of the R2R3-MYB gene family in D. nobile, and provides candidate genes for future research on abiotic stress in this plant.

Comprehensive analysis of complete chloroplast genome sequence of Plantago asiatica L. (Plantaginaceae).

Plantago asiatica L. is a representative individual species of Plantaginaceae, whose high reputation is owed to its edible and medicinal values. However, the phylogeny and genes of the P. asiatica chloroplast have not yet been well described. Here we report the findings of a comprehensive analysis of the P. asiatica chloroplast genome. The P. asiatica chloroplast genome is 164,992 bp, circular, and has a GC content of 37.98%. The circular genome contains 141 genes, including 8 rRNAs, 38 tRNAs, and 95 protein-coding genes. Seventy-two simple sequence repeats are detected. Comparative chloroplast genome analysis of six related species suggests that a higher similarity exists in the coding region than the non-coding region, and differences in the degree of preservation is smaller between P. asiatica and Plantago depressa than among others. Our phylogenetic analysis illustrates P. asiatica has a relatively close relationship with P. depressa, which was also divided into different clades with Plantago ovata and Plantago lagopus in the genus Plantago. This analysis of the P. asiatica chloroplast genome contributes to an improved deeply understanding of the evolutionary relationships among Plantaginaceae.

Transcriptomic responses to cold stress in Dendrobium huoshanense C.Z. Tang et S.J. Cheng.

Dendrobium huoshanense C.Z. Tang et S.J. Cheng is a perennial epiphytic herb of the family Orchidaceae. The main metabolites of D. huoshanense include polysaccharides and flavonoids. Low temperature is the main environmental factor that limits the growth and development of plants. However, changes that occur at the molecular level in response to low temperatures in D. huoshanense are poorly understood. We performed a transcriptome analysis at two time points of 0 d (control group) and 7 d (cold stress group) under culture of D. huoshanense at 4 °C. A total of 37.63 Gb transcriptomic data were generated using the MGI 2000 platform. These reads were assembled into 170,754 transcripts and 23,724 differentially expressed genes (DEGs) were obtained. Pathway analysis indicated that "flavonoid biosynthesis," "anthocyanin biosynthesis," "flavone and flavonol biosynthesis," and "plant hormone signal transduction" might play a vital role in the response of D. huoshanense to cold stress. Several important pathway genes were identified to be altered under cold stress, such as genes encoding polysaccharides, flavonoids, and plant hormone-signaling transduction kinase. In addition, the content of mannose and total flavonoids increased under cold stress. Twelve DEGs related to polysaccharides, flavonoid, and hormone pathways were selected from the transcriptome data for validation with real-time quantitative PCR (RT-qPCR). Our results provide a transcriptome database and candidate genes for further study of the response of D. huoshanense to cold stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01385-7.

ICP-MS based metallomics and GC-MS based metabolomics reveals the physiological and metabolic responses of Dendrobium huoshanense plants exposed to Fe3O4 nanoparticles.

It is found that the growth of Dendrobium huoshanense was dependent on Fe3O4, while the bioavailability of plants to ordinary Fe3O4 was low on the earth. In order to improve the growth, quality and yield of D. huoshanense, we used Fe3O4 NPs (100 or 200 mg/L) that was easily absorbed by plants as nano-fertilizer to hydroponically treat seedlings of D. huoshanense for 3 weeks. Fe3O4 NPs induced not only earlier flowering and increased sugar content and photosynthesis, but also stressed to plants, increased MDA content and related antioxidant enzymes activities. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) revealed that Fe3O4 NPs caused a significant accumulation of Fe and some other nutrient elements (Mn, Co, B, Mo) in stems of D. huoshanense. Metabolomics revealed that the metabolites were reprogrammed in D. huoshanense when under Fe3O4 NPs exposure. Fe3O4 NPs inhibited antioxidant defense-related pathways, demonstrating that Fe3O4 NPs have antioxidant capacity to protect D. huoshanense from damage. As the first study associating Fe3O4 NPs with the quality of D. huoshanense, it provided vital insights into the molecular mechanisms of how D. huoshanense responds to Fe3O4 NPs, ensuring the reasonable use of Fe3O4 NPs as nano-fertilizer.

Genome-wide identification of R2R3-MYB family genes and gene response to stress in ginger.

Ginger (Zingiber officinale Roscoe) is an important plant used worldwide for medicine and food. The R2R3-MYB transcription factor (TF) family has essential roles in plant growth, development, and stresses resistance, and the number of genes in the family varies greatly among different types of plants. However, genome-wide discovery of ZoMYBs and gene responses to stresses have not been reported in ginger. Therefore, genome-wide analysis of R2R3-MYB genes in ginger was conducted in this study. Protein phylogenetic relations and conserved motifs and chromosome localization and duplication, structure, and cis-regulatory elements were analyzed. In addition, the expression patterns of selected genes were analyzed under two different stresses. A total of 299 candidate ZoMYB genes were discovered in ginger. Based on groupings of R2R3-MYB genes in the model plant Arabidopsis thaliana (L.) Heynh., ZoMYBs were divided into eight groups. Genes were distributed across 22 chromosomes at uneven densities. In gene duplication analysis, 120 segmental duplications were identified in the ginger genome. Gene expression patterns of 10 ZoMYBs in leaves of ginger under abscisic acid (ABA) and low-temperature stress treatments were different. The results will help to determine the exact roles of ZoMYBs in anti-stress responses in ginger.

Comparative Proteome and Phosphoproteome Analyses Reveal Different Molecular Mechanism Between Stone Planting Under the Forest and Greenhouse Planting of Dendrobium huoshanense.

The highly esteemed Chinese herb, Dendrobium huoshanense, whose major metabolites are polysaccharides and alkaloids, is on the verge of extinction. The stone planting under the forest (SPUF) and greenhouse planting (GP) of D. huoshanense are two different cultivation methods of pharmaceutical Dendrobium with significantly differences in morphology, metabolites content and composition, and medication efficacy. Here, we conducted proteomics and phosphoproteomics analyses to reveal differences in molecular mechanisms between SPUF and GP. We identified 237 differentially expressed proteins (DEPs) between the two proteomes, and 291 modification sites belonging to 215 phosphoproteins with a phosphorylation level significantly changed (PLSC) were observed. GO, KEGG pathway, protein domain, and cluster analyses revealed that these DEPs were mainly localized in the chloroplast; involved in processes such as posttranslational modification, carbohydrate transport and metabolism, and secondary metabolite biosynthesis; and enriched in pathways mainly including linoleic acid metabolism, plant-pathogen interactions, and phenylpropanoid, cutin, suberin, and wax biosynthesis. PLSC phosphoproteins were mainly located in the chloroplast, and highly enriched in responses to different stresses and signal transduction mechanisms through protein kinase and phosphotransferase activities. Significant differences between SPUF and GP were observed by mapping the DEPs and phosphorylated proteins to photosynthesis and polysaccharide and alkaloid biosynthesis pathways. Phosphorylation characteristics and kinase categories in D. huoshanense were also clarified in this study. We analyzed different molecular mechanisms between SPUF and GP at proteomic and phosphoproteomic levels, providing valuable information for the development and utilization of D. huoshanense.

The complete chloroplast genome sequence of Lespedeza buergeri Miq. (Fabaceae).

The complete chloroplast genome sequence of Lespedeza buergeri is presented in this report. It is 149,065 bp in length and divided into four distinct regions: a small single copy (SSC) region of 18,934 bp, a large single copy (LSC) region of 82,476 bp, and a pair of inverted repeat regions of 23,826 bp. The annotation of the L. buergeri complete chloroplast genome predicted a total of 123 genes (77 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes). Phylogenetic analysis with the reported chloroplast genomes revealed that L. buergeri is nested in the genus Lespedeza of Fabaceae family. Furthermore, L. buergeri exhibited a close relationship with Lespedeza bicolor and Lespedeza cuneata. This results in this study might contribute to further investigating the evolutionary relationship of family Fabaceae.

Molecular Cloning and Functional Characterization of a ß-Glucosidase Gene to Produce Platycodin D in Platycodon grandiflorus.

Platycodin D (PD) is a deglycosylated triterpene saponin with much higher pharmacological activity than glycosylated platycoside E (PE). Extensive studies in vitro showed that the transformation of platycoside E to platycodin D can be achieved using ß-glucosidase extracted from several bacteria. However, whether similar enzymes in Platycodon grandiflorus could convert platycoside E to platycodin D, as well as the molecular mechanism underlying the deglycosylation process of platycodon E, remain unclear. Here, we identified a ß-glucosidase in P. grandiflorus from our previous RNA-seq analysis, with a full-length cDNA of 1,488 bp encoding 495 amino acids. Bioinformatics and phylogenetic analyses showed that ß-glucosidases in P. grandiflorus have high homology with other plant ß-glucosidases. Subcellular localization showed that there is no subcellular preference for its encoding gene. ß-glucosidase was successfully expressed as 6 × His-tagged fusion protein in Escherichia coli BL21 (DE3). Western blot analysis yielded a recombinant protein of approximately 68 kDa. In vitro enzymatic reactions determined that ß-glucosidase was functional and could convert PE to PD. RT-qPCR analysis showed that the expression level of ß-glucosidase was higher at night than during the day, with the highest expression level between 9:00 and 12:00 at night. Analysis of the promoter sequence showed many light-responsive cis-acting elements, suggesting that the light might regulate the gene. The results will contribute to the further study of the biosynthesis and metabolism regulation of triterpenoid saponins in P. grandiflorus.

Transcriptome-wide identification of WRKY transcription factors and their expression profiles in response to methyl jasmonate in Platycodon grandiflorus.

Platycodon grandiflorus, a perennial flowering plant widely distributed in China and South Korea, is an excellent resource for both food and medicine. The main active compounds of P. grandiflorus are triterpenoid saponins. WRKY transcription factors (TFs) are among the largest gene families in plants and play an important role in regulating plant terpenoid accumulation, physiological metabolism, and stress response. Numerous studies have been reported on other medicinal plants; however, little is known about WRKY genes in P. grandiflorus. In this study, 27 PgWRKYs were identified in the P. grandiflorus transcriptome. Phylogenetic analysis showed that PgWRKY genes were clustered into three main groups and five subgroups. Transcriptome analysis showed that the PgWRKY gene expression patterns in different tissues differed between those in Tongcheng City (Southern Anhui) and Taihe County (Northern Anhui). Gene expression analysis based on RNA sequencing and qRT-PCR analysis showed that most PgWRKY genes were expressed after induction with methyl jasmonate (MeJA). Co-expressing PgWRKY genes with triterpenoid biosynthesis pathway genes revealed four PgWRKY genes that may have functions in triterpenoid biosynthesis. Additionally, functional annotation and protein-protein interaction analysis of PgWRKY proteins were performed to predict their roles in potential regulatory networks. Thus, we systematically analyzed the structure, evolution, and expression patterns of PgWRKY genes to provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in triterpenoid biosynthesis.

Comparative proteomics reveals biochemical changes in Salvia miltiorrhiza Bunge during sweating processing.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge is a bulk medicinal material used in traditional Chinese medicine, that can cure cardiovascular diseases, neurasthenia, and other conditions. Sweating is a frequently used method of processing S. miltiorrhiza for medical applications. We previously demonstrated changes to the metabolic profile of linoleic acid, glyoxylate, and dicarboxylate after Sweating. However, this alteration has not been explained at the molecular level. MATERIALS AND METHODS: Fresh roots of Salvia miltiorrhiza Bunge were treated by the Sweating processing, and then the tandem mass tag technique was used to compare the proteome difference between Sweating S. miltiorrhiza and non-Sweating S. miltiorrhiza. RESULTS: We identified a total of 850 differentially expressed proteins after Sweating treatment in S. miltiorrhiza, including 529 upregulated proteins and 321 downregulated proteins. GO enrichment analysis indicated that these differentially expressed proteins are involved in external encapsulating structure, cell wall, oxidoreductase activity, ligase activity, and others. Further analysis showed that CYP450, the pathogenesis-related protein Bet v 1 family, and the peroxidase domain were the major protein domains. KEGG enrichment identified 18 pathways, of which phenylpropanoid biosynthesis is the most important one related to the metabolite profile and is the principal chemical component of S. miltiorrhiza. CONCLUSION: This study addressed potential molecular mechanisms in S. miltiorrhiza after Sweating, and the findings provide reasons for the changes in biochemical properties and metabolites changes which might cause pharmacological variation at the proteome level.

Qualitative Proteome-Wide Analysis Reveals the Diverse Functions of Lysine Crotonylation in Dendrobium huoshanense.

The lysine crotonylation of histone proteins is a newly identified posttranslational modification with diversified cellular functions. However, there are few reports on lysine crotonylation of non-histone proteins in medicinal plant cells. By using high-resolution liquid chromatography-mass spectrometry (LC-MS) coupled with highly sensitive-specific immune-affinity antibody analysis, a whole crotonylation proteome analysis of Dendrobium huoshanense was performed. In total, 1,591 proteins with 4,726 lysine crotonylation sites were identified; among them, 11 conserved motifs were identified. Bioinformatic analyses linked crotonylated proteins to the drought stress response and multiple metabolic pathways, including secondary metabolite biosynthesis, transport and catabolism, energy production and conversion, carbohydrate transport and metabolism, translation, and ribosomal structure and biogenesis. This study contributes toward understanding the regulatory mechanism of polysaccharide biosynthesis at the crotonylation level even under abiotic stress.

Comprehensive analysis of chloroplast genome of Albizia julibrissin Durazz. (Leguminosae sp.).

MAIN CONCLUSION: The Albizia julibrissin chloroplasts have a classical chloroplast genome structure, containing 93 coding genes and 34 non-coding genes. Our research provides basic data for plant phylogenetic evolutionary studies. There is limited genomic information available for the important Chinese herb Albizia julibrissin Durazz. In this study, we constructed the chloroplast (Cp) genome of A. julibrissin. The length of the assembled Cp genome was 175,922 bp consisting of four conserved regions: a 5145 bp small single-copy (SSC) region, a 91,323 bp large single-copy (LSC) region, and two identical length-inverted repeat (IR) regions (39,725 bp). This Cp genome included 34 non-coding RNAs and 93 unique genes, the former contains 30 transfer and 4 ribosomal RNA genes. Gene annotation indicated some of the coding genes (82) in the A. julibrissin Cp genome classified in the Leguminosae family, with some to other related families (11). The results show that low GC content (36.9%) and codon bias towards A- or T-terminal codons may affect the frequency of gene codon usage. The sequence analysis identified 30 forward, 18 palindrome, and 1 reverse repeat > 30 bp length, and 149 simple sequence repeats (SSR). Fifty-five RNA editing sites in the Cp of A. julibrissin were predicted, most of which are C-to-U conversions. Analysis of the reverse repeat expansion or contraction and divergence area between several species, including A. julibrissin, was performed. The phylogenetic tree revealed that A. julibrissin was most closely related to Albizia odoratissima and Albizia bracteata, followed by Samanea saman, forming an evolutionary branch with Mimosa pudica and Leucaena trichandra. The research results are helpful for breeding and genetic improvement of A. julibrissin, and also provide valuable information for understanding the evolution of this plant.

The complete chloroplast genome sequence of Buxus megistophylla Levl. (Buxaceae Dumort.).

Buxus megistophylla Levl. is one of the most common green horticultural plants in the city presently. Here, we assembled and annotated the complete chloroplast (cp) genome of B. megistophylla. The whole length of the genome is 157,611 bp and encodes a total of 124 genes, which contains 89 protein-coding genes, 31 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. Phylogenetic tree analysis showed that B. megistophylla is separated from two other species of the same family, but these three species, clustered in one clade, are relatively closer to each other compared to the species in other families. This cp genome sequencing and phylogenetic analysis offer genetic background for conservation and may contribute to further evolutionary studies of this species.

The complete chloroplast genome of Epilobium hirsutum L. (Onagraceae).

The complete chloroplast genome sequence of Epilobium hirsutum L. is presented here. It is 161,111 bp in length and divides into four distinct regions: a small single-copy region (SSC) of 17,310 bp, a large single-copy region (LSC) of 89,117 bp, and a pair of inverted repeat (IR) regions of 27,342 bp. The chloroplast genome of E. hirsutum includes a total of 125 genes, consisting of 31 tRNA genes, 8 rRNA genes, and 86 protein-coding genes. A phylogenetic tree was generated to evaluate the evolutionary relationship between E. hirsutum and relevant species. The chloroplast genome sequencing and phylogenetic analysis offer genetic background for conservation and phylogenetic studied of this species.

Putative genes in alkaloid biosynthesis identified in Dendrobium officinale by correlating the contents of major bioactive metabolites with genes expression between Protocorm-like bodies and leaves.

BACKGROUND: Dendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids. RESULTS: We used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + α-NAA 0.5 mg·L- 1 + 6-BA 1.0 mg·L- 1 + 2, 4-D 1.5-2.0 mg·L- 1 + potato juice 100 g·L- 1. Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine ß-D-Glucosidase, geissoschizine synthase and vinorine synthase in D. officinale. CONCLUSIONS: The identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

A candidate gene identified in converting platycoside E to platycodin D from Platycodon grandiflorus by transcriptome and main metabolites analysis.

Platycodin D and platycoside E are two triterpenoid saponins in Platycodon grandiflorus, differing only by two glycosyl groups structurally. Studies have shown ß-Glucosidase from bacteria can convert platycoside E to platycodin D, indicating the potential existence of similar enzymes in P. grandiflorus. An L9(34) orthogonal experiment was performed to establish a protocol for calli induction as follows: the optimal explant is stems with nodes and the optimum medium formula is MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L to obtain callus for experimental use. The platycodin D, platycoside E and total polysaccharides content between callus and plant organs varied wildly. Platycodin D and total polysaccharide content of calli was found higher than that of leaves. While, platycoside E and total polysaccharide content of calli was found lower than that of leaves. Associating platycodin D and platycoside E content with the expression level of genes involved in triterpenoid saponin biosynthesis between calli and leaves, three contigs were screened as putative sequences of ß-Glucosidase gene converting platycoside E to platycodin D. Besides, we inferred that some transcription factors can regulate the expression of key enzymes involved in triterpernoid saponins and polysaccharides biosynthesis pathway of P. grandiflorus. Totally, a candidate gene encoding enzyme involved in converting platycoside E to platycodin D, and putative genes involved in polysaccharide synthesis in P. grandiflorus had been identified. This study will help uncover the molecular mechanism of triterpenoid saponins biosynthesis in P. grandiflorus.

UPLC/MS-based untargeted metabolomics reveals the changes of metabolites profile of Salvia miltiorrhiza bunge during Sweating processing.

Salvia miltiorrhiza has numerous compounds with extensive clinical application. "Sweating", a processing method of Traditional Chinese Medicine (TCM), results in great changes in pharmacology and pharmacodynamics. Previously, chromatogram of 10 characteristic metabolites in S. miltiorrhiza showed a significant difference after "Sweating". Due to the complexity of TCM, changes in metabolites should be investigated metabolome-wide after "Sweating". An untargeted UPLC/MS-based metabolomics was performed to discover metabolites profile variation of S. miltiorrhiza after "Sweating". Multivariate analysis was conducted to screen differential metabolites. Analysis indicated distinct differences between sweated and non-sweated samples. 10,108 substance peaks had been detected altogether, and 4759 metabolites had been identified from negative and positive ion model. 287 differential metabolites were screened including 112 up-regulated and 175 down-regulated and they belong to lipids and lipid-like molecules, and phenylpropanoid and polyketides. KEGG analysis showed the pathway of linoleic acid metabolism, and glyoxylate and dicarboxylate metabolism were mainly enriched. 31 and 49 identified metabolites were exclusively detected in SSM and NSSM, respectively, which mainly belong to carboxylic acids and derivatives, polyketides and fatty acyls. By mapping tanshinones and salvianolic acids to 4759 identified metabolites library, 23 characteristic metabolites had been identified, among which 11 metabolites changed most. We conclude that "Sweating'' has significant effect on metabolites content and composition of S. miltiorrhiza.

Malonylome analysis in developing rice (Oryza sativa) seeds suggesting that protein lysine malonylation is well-conserved and overlaps with acetylation and succinylation substantially.

In recent years, lysine malonylation has garnered wide spread interest due to its potential regulatory roles. While studies have been performed in bacteria, mouse, and human, the involvement and the biological function of this modification in plant are still largely unknown. We examined the global proteome profile of lysine malonylation in developing rice seeds using affinity enrichment followed by LC-MS/MS analysis. We identified 421 malonylated lysine sites across 247 proteins. Functional analyses showed predominant presence of malonylated proteins in metabolic processes, including carbon metabolism, glycolysis/gluconeogenesis, TCA cycle, as well as photosynthesis. Malonylation was also detected on enzymes in starch biosynthesis pathway in developing rice seeds. In addition, we found a remarkable overlap among the malonylated, succinylated and acetylated sites identified in rice. Furthermore, malonylation at conserved sites of homologous proteins was observed across organisms of different kingdoms, including mouse, human, and bacteria. Finally, distinct motifs were identified when the rice malonylation sites were analyzed and conserved motifs were observed from bacterium to human and rice. Our results provide an initial understanding of the lysine malonylome in plants. The study has critical reference value for future understanding of the biological function of protein lysine malonylation in plants. BIOLOGICAL SIGNIFICANCE: Lysine malonylation is a newly discovered acylation with functional potential in regulating cellular metabolisms and activities. However, the malonylation status has not been reported in plants. Grain yield and quality, mainly determined during cereal seed development, are closely related to food security, human health and economic value. To evaluate malonylation level in plants and the possible regulatory functions of malonylation in seed development, we conducted comprehensive analyses of malonylome in developing rice seeds. A total of 421 malonylated lysine sites from 247 proteins were identified, which involved in multiple critical metabolic processes, including central carbon metabolism, lipid metabolism, photosynthesis, and starch biosynthesis. We found that charged amino acids, lysine and arginine, were the preferred residues in positions flanking the modified lysines. Highly conserved modification sites on both histone and non-histone proteins were observed among different organisms through sequence alignment analysis. More interestingly, a large number of modification sites shared by malonylation, acetylation and succinylation were identified in rice. The study presents a comprehensive understanding of malonylome in plants, which will serve as an initial platform for further investigation of the functions of lysine malonylation, especially in cereal seeds development.

Proteome-wide Analysis of Lysine 2-hydroxyisobutyrylation in Developing Rice (Oryza sativa) Seeds.

Lysine 2-hydroxyisobutyrylation is a recently identified protein post-translational modification that is known to affect the association between histone and DNA. However, non-histone protein lysine 2-hydroxyisobutyrylation remains largely unexplored. Utilizing antibody-based affinity enrichment and nano-HPLC/MS/MS analyses of 2-hydroxyisobutyrylation peptides, we efficaciously identified 9,916 2-hydroxyisobutyryl lysine sites on 2,512 proteins in developing rice seeds, representing the first lysine 2-hydroxyisobutyrylome dataset in plants. Functional annotation analyses indicated that a wide variety of vital biological processes were preferably targeted by lysine 2-hydroxyisobutyrylation, including glycolysis/gluconeogenesis, TCA cycle, starch biosynthesis, lipid metabolism, protein biosynthesis and processing. Our finding showed that 2-hydroxyisobutyrylated histone sites were conserved across plants, human, and mouse. A number of 2-hydroxyisobutyryl sites were shared with other lysine acylations in both histone and non-histone proteins. Comprehensive analysis of the lysine 2-hydroxyisobutyrylation sites illustrated that the modification sites were highly sequence specific with distinct motifs, and they had less surface accessibility than other lysine residues in the protein. Overall, our study provides the first systematic analysis of lysine 2-hydroxyisobutyrylation proteome in plants, and it serves as an important resource for future investigations of the regulatory mechanisms and functions of lysine 2-hydroxyisobutyrylation.