Integrated flow cytometric and proteomics analyses reveal the regulatory network underlying sugarcane protoplast responses to fusion.

BACKGROUND: Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion. METHOD: We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion. RESULTS: Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion. PURPOSE AND SIGNIFICANCE: Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis.

The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.

A high-throughput lipidomics and transcriptomic approach reveals novel compounds from sugarcane linked with promising therapeutic potential against COVID-19.

Sugarcane (Saccharum ssp., Poaceae) provides enormous metabolites such as sugars, lipid, and other dietary metabolites to humans. Among them, lipids are important metabolites that perform various functions and have promising pharmacological value. However, in sugarcane, few studies are focusing on lipidomics and few lipid compounds were reported, and their pharmacological values are not explored yet. The transcriptomic and widely targeted lipidomics approach quantified 134 lipid compounds from the rind of six sugarcane genotypes. These lipid compounds include 57 fatty acids, 30 lysophosphatidylcholines, 23 glycerol esters, 21 lysophosphatidylethanolamines, 2 phosphatidylcholines, and 1 sphingolipid. Among them, 119 compounds were first time reported in sugarcane rind. Seventeen lipids compounds including 12 fatty acids, 2 glycerol lipids, LysoPC 16:0, LysoPE 16:0, and choline alfoscerate were abundantly found in the rind of sugarcane genotypes. From metabolic and transcriptomic results, we have developed a comprehensive lipid metabolic pathway and highlighted key genes that are differentially expressed in sugarcane. Several genes associated with α-linolenic acid and linoleic acid biosynthesis pathways were highly expressed in the rind of the ROC22 genotype. ROC22 has a high level of α-linolenic acid (an essential fatty acid) followed by ROC16. Moreover, we have explored pharmacological values of lipid compounds and found that the 2-linoleoylglycerol and gingerglycolipid C have strong binding interactions with 3CLpro of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and these compounds can be utilized against SARS-CoV-2 as therapeutic agents. The transcriptome, metabolome, and bioinformatics analysis suggests that the sugarcane cultivars have a diversity of lipid compounds having promising therapeutic potential, and exploring the lipid metabolism will help to know more compounds that have promising cosmetic and pharmacological value.

Transcriptome Analysis of Sugarcane Young Leaves and Protoplasts after Enzymatic Digestion.

Sugarcane somatic cell hybridization can break through the barrier of genetic incompatibility between distantly related species in traditional breeding. However, the molecular mechanisms of sugarcane protoplast regeneration and the conditions for protoplast preparation remain largely unknown. In this study, young sugarcane (ROC22) leaves were enzymatically digested, and the viability of protoplasts reached more than 90% after enzymatic digestion (Enzymatic combination: 2% cellulase + 0.5% pectinase + 0.1% dissociative enzyme + 0.3% hemicellulase, pH = 5.8). Transcriptome sequencing was performed on young sugarcane leaves and protoplasts after enzymatic digestion to analyze the differences in gene expression in somatic cells before and after enzymatic digestion. A total of 117,411 unigenes and 43,460 differentially expressed genes were obtained, of which 21,123 were up-regulated and 22,337 down-regulated. The GO terms for the 43,460 differentially expressed genes (DEGs) were classified into three main categories: biological process, cellular component and molecular function, which related to developmental process, growth, cell proliferation, transcription regulator activity, signal transducer activity, antioxidant activity, oxidative stress, kinase activity, cell cycle, cell differentiation, plant hormone signal transduction, and so on. After enzymatic digestion of young sugarcane leaves, the expressions of GAUT, CESA, PSK, CyclinB, CyclinA, CyclinD3 and cdc2 genes associated with plant regeneration were significantly down-regulated to 65%, 47%, 2%, 18.60%, 21.32%, 52% and 45% of young leaves, respectively. After enzymatic digestion, Aux/IAA expression was up-regulated compared with young leaves, and Aux/IAA expression was 3.53 times higher than that of young leaves. Compared with young leaves, these key genes were significantly changed after enzymatic digestion. These results indicate that the process of somatic enzymatic digestion process may affect the regeneration of heterozygous cells to a certain extent.

Transcriptomic and Widely Targeted Metabolomic Approach Identified Diverse Group of Bioactive Compounds, Antiradical Activities, and Their Associated Genes in Six Sugarcane Varieties.

Sugarcane is cultivated mainly for its high sucrose content but it can also produce many metabolites with promising antioxidant potential. However, very few studies have been reported on the biosynthesis of metabolites in sugarcane to date. In this study, we have identified a wide range of amino acids and organic acids in the rind of six sugarcane varieties by the LC-MS/MS method. A total number of 72 amino acids and 55 organic acid compounds were characterized; among these, 100 were reported for the first time. Moreover, 13 amino acids and seven organic acids were abundantly distributed in all varieties tested and considered major amino acids and organic acids in sugarcane. The variety Taitang134 (F134) showed the highest content of total amino acids, whereas the varieties ROC16 and Yuetang93/159 (YT93/159) had maximum content of organic acids. The amino acids of the rind extract presented higher antioxidant capacity than the organic acids of the rind extract. In addition, the transcriptomic and metabolic integrated analysis highlighted some candidate genes associated with amino acid biosynthesis in sugarcane. We selected a transcription factor gene, MYB(t), and over-expressed it in Arabidopsis. The transgenic plants showed a higher accumulation of amino acids with higher antiradical activity compared with the wild-type Arabidopsis plants. Thus, we characterize a wide range of amino acids and organic acids and their antiradical activities in different sugarcane varieties and present candidate genes that can be potentially valuable for the genetic improvement of metabolites in sugarcane bagasse.

LC-MS/MS-based metabolomics approach revealed novel phytocompounds from sugarcane rind with promising pharmacological value.

BACKGROUND: Sugarcane provides many secondary metabolites for the pharmacological and cosmetic industries. Secondary metabolites, such as phenolic compounds, flavonoids, and anthocyanins, have been studied, but few reports focus on the identification of alkaloid and non-alkaloid phytocompounds in sugarcane. RESULTS: In this study, we identified 40 compounds in total from the rinds of cultivated sugarcane varieties (including eight alkaloids, 24 non-alkaloids, and eight others) by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. Among these compounds, 31 were novel and are reported for the first time in sugarcane. Some alkaloids such as 3-indoleacrylic acid, N,N-dimethyl-5-methoxytryptamine, tryptamine, 6-hydroxynicotinic acid, and 6-deoxyfagomine are identified the first time in sugarcane rind. Four alkaloids such as trigonelline, piperidine, 3-indoleacrylic acid, and 6-deoxyfagomine are found abundantly in sugarcane rind and these compounds have promising pharmaceutical value. Some phytocompounds such as choline and acetylcholine (non-alkaloid compounds) were most common in the rind of ROC22 and Yuetang93/159 (YT93/159). Hierarchical cluster analysis and principal component analysis revealed that the ROC22, Taitang172 (F172), and Yuetang71/210 (YT71/210) varieties were quite similar in alkaloid composition when compared with other sugarcane varieties. We have also characterized the biosynthesis pathway of sugarcane alkaloids. The rind of F172, ROC22, and YT71/210 showed the highest total alkaloid content, whereas the rind of ROC16 revealed a minimum level. Interestingly, the rind extract from YT71/210 and F172 showed maximum antioxidant activity, followed by ROC22. CONCLUSION: Our results showed the diversity of alkaloid and non-alkaloid compounds in the rind of six cultivated sugarcanes and highlighted the promising phytocompounds that can be extracted, isolated, and utilized by the pharmacological industry. © 2022 Society of Chemical Industry.