Integrated Transcriptomic and Metabolomic Analysis Reveal the Dynamic Process of Bama Hemp Seed Development and the Accumulation Mechanism of α-Linolenic Acid and Linoleic Acid.

Bama County is a world-famous longevity county in the Guangxi Province, China. Bama hemp is a traditional seed used in hemp cultivation in the Bama County. The seeds contain abundant unsaturated fatty acids, particularly linoleic acid (LA) and linolenic acid in the golden ratio. These two substances have been proven to be related to human health and the prevention of various diseases. However, the seed development and seed oil accumulation mechanisms remain unclear. This study employed a combined analysis of physiological, transcriptomic, and metabolomic parameters to elucidate the fatty acid formation patterns in Bama hemp seeds throughout development. We found that seed oil accumulated at a late stage in embryo development, with seed oil accumulation following an "S″-shaped growth curve, and positively correlated with seed size, sugar content, protein content, and starch content. Transcriptome analysis identified genes related to the metabolism of LA, α-linolenic acid (ALA), and jasmonic acid (JA). We found that the FAD2 gene was upregulated 165.26 folds and the FAD3 gene was downregulated 6.15 folds at day 21. Metabolomic changes in LA, ALA, and JA compounds suggested a competitive relationship among these substances. Our findings indicate that the peak period of substance accumulation and nutrient accumulation in Bama hemp seeds occurs during the midstage of seed development (day 21) rather than in the late stage (day 40). The results of this research will provide a theoretical basis for local cultivation and deep processing of Bama hemp.

Exogenous methyl jasmonate enhanced kenaf (Hibiscus cannabinus) tolerance against lead (Pb) toxicity by improving antioxidant capacity and osmoregulators.

In this study, the effects of exogenous methyl jasmonate (MeJA) on metal uptake and its ability to attenuate metal toxicity in kenaf plants under Pb stress were investigated. The experiment was conducted with five different MeJA concentrations (0, 40, 80, 160, and 320 µM) as a foilar application to kenaf plants exposed to 200 µM Pb stress. The results revealed that pretreatmen of MeJA significantly increased plant dry weight, plant height, and root architecture at all concentrations tested, with the most significant increase at 320 µM. Foliar application of MeJA at 160 µM and 320 µM increased the Pb concentrations in leaves and stems as well as the translocation factor (TF) from root to leaf. However, the bioaccumulation factor in the shoot initially decreased and then increased with increasing MeJA concentration. By increasing enzymatic (SOD, POD, and CAT) and non-enzymatic (AsA and non-protein thiols) antioxidants, MeJA pretreatment decreased lipid peroxidation, O2- and H2O2 accumulation and recovered photosynthetic pigment content under Pb stress. Increased osmolytes (proline, sugar, and starch) and protein content after MeJA pretreatment under Pb stress restore cellular homeostasis and improved kenaf tolerance. Our results suggest that MeJA pretreatment modifies the antioxidant machinery of kenaf and inhibits stress-related processes that cause lipid peroxidation, hence enhancing plant tolerance to Pb stress.

Morpho-physio-biochemical, molecular, and phytoremedial responses of plants to red, blue, and green light: a review.

Light is a basic requirement to drive carbon metabolism in plants and supports life on earth. Spectral quality greatly affects plant morphology, physiology, and metabolism of various biochemical pathways. Among visible light spectrum, red, blue, and green light wavelengths affect several mechanisms to contribute in plant growth and productivity. In addition, supplementation of red, blue, or green light with other wavelengths showed vivid effects on the plant biology. However, response of plants differs in different species and growing conditions. This review article provides a detailed view and interpretation of existing knowledge and clarifies underlying mechanisms that how red, blue, and green light spectra affect plant morpho-physiological, biochemical, and molecular parameters to make a significant contribution towards improved crop production, fruit quality, disease control, phytoremediation potential, and resource use efficiency.

OsGSTU34, a Bz2-like anthocyanin-related glutathione transferase transporter, is essential for rice (Oryza sativa L.) organs coloration.

Anthocyanins are a flavonoid compound known as one of the most important chromogenic substances. They play several functions, including health promotion and sustaining plants during adverse conditions. They are synthesized at the endoplasmic reticulum and sequestered in the vacuole. In this work, we generated knock-out lines of OsGSTU34, a glutathione transporter's tau gene family, with no transgene line and off-target through CRISPR/Cas9 mutagenesis and highlighted the loss of pigmentation in rice flowers, leaves, stems, shoots, and caryopsis. The anthocyanin quantification in the wild-type BLWT and mutant line BLG34-8 caryopsis showed that cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G) were almost undetectable in the mutant line. A tandem mass tag (TMT) labeling proteomic analysis was conducted to elucidate the proteomic changes in the BLWT and BLG34-8. The result revealed that 1175 proteins were altered, including 408 that were down-regulated and 767 that were upregulated. The accumulation of the OsGSTU34-related protein (Q8L576), along with several anthocyanin-related proteins, was down-regulated. The enrichment analysis showed that the down-regulated proteins were enriched in different pathways, among which the phenylpropanoid biosynthesis pathway, flavonoid biosynthesis metabolites, and anthocyanin biosynthesis pathway. Protein interaction network prediction revealed that glutathione-S-transferase (Q8L576) was connected to the proteins involved in the flavonoid and anthocyanin biosynthesis pathways, such as flavanone 3-dioxygenase 1 (Q7XM21), leucoanthocyanidin dioxygenase 1 (Q93VC3), 4-coumarate-CoA ligase 2 (Q42982), phenylalanine ammonia-lyase (P14717), chalcone synthase 1 (Q2R3A1), and 4-coumarate-CoA ligase 5 (Q6ZAC1). However, the expression of the most important anthocyanin biosynthesis gene was not altered, suggesting that only the transport mechanism was affected. Our findings highlight new insight into the anthocyanin pigmentation in black rice and provide new perspectives for future research.

Intercropping of kenaf and soybean affects plant growth, antioxidant capacity, and uptake of cadmium and lead in contaminated mining soil.

Intercropping is considered a sustainable agricultural practice that can reduce the environmental impacts on agriculture. Our study investigated the morphology, physiology, and cadmium (Cd) and lead (Pb) uptake of kenaf (Hibiscus cannabinus L.) and soybean (Glycine max L.) under intercropping in mining soil. Results showed that mutual intercropping is conducive to the growth and biomass accumulation of kenaf and soybean, compared to their respective monoculture. Intercropping increased the relative chlorophyll index in kenaf, while that in soybean had no significant effect. Furthermore, intercropping increased the antioxidant enzyme activity of kenaf, while that of soybean reduced or had no significant effect. The content of malondialdehyde (MDA) was decreased in both of the species. Compared to their respective monoculture, Cd content was increased in kenaf leaves and reduced in soybean roots. Moreover, intercropping decreased the Pb content in tissues of both the species, except that Pb content of kenaf roots was increased. At the same time, root, leaf, or stem bioconcentration factors also performed the same trend, and TF was less than 1. These results indicated that intercropping can increase the plant growth and decrease the metal content in plant tissues. Present findings could provide support for future research on kenaf and soybean cultivation in contaminated lands. In addition, the present study strengthens our understanding about the effectiveness of intercropping system on heavy metal-contaminated lands for sustainable agricultural production.

Physiological and DNA methylation analysis provides epigenetic insights into kenaf cadmium tolerance heterosis.

Soil heavy metal pollution is one of the most challenging problems. Kenaf is an important natural fiber crop with strong heterosis and a higher tolerance to heavy metals. However, little is known about the molecular mechanisms of kenaf heavy metal tolerance, especially the mechanism of genomic DNA methylation regulating heterosis. In this study, kenaf cultivars CP085, CP089, and their hybrid F1 seedlings were subjected to 300 µM cadmium stress and found obvious heterosis of cadmium resistance in morphology and antioxidant enzyme activity of F1 hybrid seedlings. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that the total DNA methylation level under cadmium decreased by 16.9 % in F1 and increased by 14.0 % and 3.0 % in parents CP085 and CP089, respectively. The hypomethylation rate was highest (21.84 %), but hypermethylation was lowest (17.24 %) in F1 compared to parent cultivars. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between F1 and its parents under cadmium. Furthermore, 21 differentially methylated DNA fragments (DMFs) were analyzed. Especially, the expression of NPF2.7, NADP-ME, NAC71, TPP-D, LRR-RLKs, and DHX51 genes were changed due to cadmium stress and related to cytosine methylation regulation. Finally, the knocked-down of the differentially methylated gene NPF2.7 by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under cadmium stress. It is speculated that low DNA methylation levels can regulate gene expression that led to the heterosis of cadmium tolerance in kenaf.

Comprehensive analysis reveals the underlying mechanism of arbuscular mycorrhizal fungi in kenaf cadmium stress alleviation.

Soil Cadmium (Cd) contamination has become a severe environmental problem around the world. Kenaf has great potential for utilization and phytoremediation of soil contaminated with heavy metal. Arbuscular mycorrhizal fungi (AMF) can help plants alleviate Cd stress, but the underlying mechanism remains completely unknown. In this study, kenaf was inoculated or not inoculated with AMF at cadmium concentrations of 10 mg kg-1 and 50 mg kg-1 from the seedling stage to the vigorous growth stage. The results showed that AMF symbionts improved nutrient transport efficiency and significantly improved plant growth. Additionally, AMF colonization increased cell wall polysaccharide content which help to bind Cd in the cell wall and reduced the transport of Cd to aboveground plant tissues. The increase in soil pH (6.9), total balcomycin and easily extractable balcomycin content facilitated the chelation of metal by mycorrhizal fungi and reduced the biological effectiveness of Cd. Furthermore, AMF upregulated the expression levels of key kenaf genes, such as Hc.GH3.1, Hc.AKR, and Hc.PHR1, which plays an important role in enhancing kenaf Cd tolerance. Our findings systematically revealed the mechanisms by which AMF responds to Cd stress in kenaf, inoculation of AMF with kenaf could be used to enhance the removal of Cd from soil pollution in mining areas by phytoremediation.

Integrated Methylome and Transcriptome Analysis Provides Insights into the DNA Methylation Underlying the Mechanism of Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.).

Cytoplasmic male sterility (CMS) is widely exploited in hybrid seed production. Kenaf is an important fiber crop with high heterosis. The molecular mechanism of kenaf CMS remains unclear, particularly in terms of DNA methylation. Here, using the anthers of a kenaf CMS line (P3A) and its maintainer line (P3B), comparative physiological, DNA methylation, and transcriptome analyses were performed. The results showed that P3A had considerably lower levels of IAA, ABA, photosynthetic products and ATP contents than P3B. DNA methylome analysis revealed 650 differentially methylated genes (DMGs) with 313 up- and 337 down methylated, and transcriptome analysis revealed 1788 differentially expressed genes (DEGs) with 558 up- and 1230 downregulated genes in P3A compared with P3B. Moreover, 45 genes were characterized as both DEGs and DMGs, including AUX,CYP, BGL3B, SUS6, AGL30 and MYB21. Many DEGs may be regulated by related DMGs based on methylome and transcriptome studies. These DEGs were involved in carbon metabolism, plant hormone signal transduction, the TCA cycle and the MAPK signaling pathway and were shown to be important for CMS in kenaf. These results provide new insights into the epigenetic mechanism of CMS in kenaf and other crops.

Integrated Methylome and Transcriptome Analyses Reveal the Molecular Mechanism by Which DNA Methylation Regulates Kenaf Flowering.

DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) (at concentrations of 0, 100, 200, 400, and 600 µM), and the results showed that pretreatment with 200 µM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 µM 5-azaC (5-azaC200) or with no 5-azaC (control conditions; 5-azaC0). Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 µM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.

5-azacytidine pre-treatment alters DNA methylation levels and induces genes responsive to salt stress in kenaf (Hibiscus cannabinus L.).

Soil salinization is becoming a major threat to the sustainable development of global agriculture. Kenaf is an industrial fiber crop with high tolerance to salt stress and could be used for soil phytoremediation. However, the molecular mechanism of kenaf salt tolerance remains largely unknown. DNA methylation is an important epigenetic modifications phenomena and plays a key role in gene expression regulation under abiotic stress condition. In the present study, the kenaf seedlings were pre-treated or not with 50 µM 5-azacytidine (5-azaC, a DNA methylation inhibitor) and then subjected to different concentrations of NaCl. Results showed that the biomass and antioxidant activities (superoxide dismutase, peroxidase and catalase) of kenaf seedlings pre-treated with 5-azaC were significantly increased, while the contents of superoxide anion (O2-) and malondialdehyde (MDA) were decreased, indicating that 5-azaC pre-treatment could significantly alleviate salt stress injury. Furthermore, the methylation-sensitive amplified polymorphism (MSAP) analysis revealed that DNA methylation level of keanf seedlings pre-treated with 5-azaC significantly decreased. The expression of seven differentially methylated genes responsing to salt stress was significantly changed from real-time fluorescent quantitative (qRT-PCR) analysis. Finally, knocked-down of the l-ascorbate oxidase (L-AAO) gene by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under salt stress. Overall, it was suggested that 5-azaC pre-treatment can significantly improve salt tolerance in kenaf by decreasing ROS content, raising anti-oxidant activities, and regulating DNA methylation and expression of stress-responsive genes.

Comparative transcriptomic analysis reveals key genes and pathways in two different cadmium tolerance kenaf (Hibiscus cannabinus L.) cultivars.

Soil cadmium (Cd) contamination has become a massive environmental problem. Kenaf is an industrial fiber crop with high tolerance to heavy metals and could be potentially used for soil phytoremediation. However, the molecular mechanism of Cd in kenaf tolerance remains largely unknown. In the present study, using two contrasting Cd sensitive kenaf (GH and YJ), the key factors accounting for differential Cd tolerance were investigated. GH has a stronger Cd transport and accumulation ability than YJ. In addition, physiological index investigation on malondialdehyde (MDA) contents and antioxidant enzyme (SOD, POD, and CAT) activities showed GH has a stronger detoxification capacity than YJ. Furthermore, the cell ultrastructure of GH is more stable than that of YJ under Cd stress. Transcriptome analysis revealed 2221 (689 up and 1532 down) and 3321 (2451 up and 870 down) genes were differentially expressed in GH and YJ, respectively. More DEGs (differentially expressed genes) were characterized as up-regulated in GH, indicating GH is inclined to activate gene expression to cope with cadmium stress. GO and KEGG analyses indicate that DEGs were assigned and enriched in different pathways. Plenty of critical Cd-induced DEGs such as SOD2, PODs, MT1, DTXs, NRT1, ABCs, CES, AP2/ERF, MYBs, NACs, and WRKYs were identified. The DEGs involved pathways, including antioxidant, heavy metal transport or detoxification, substance transport, plant hormone and calcium signals, ultrastructural component, and a wide range of transcription factors were suggested to play crucial roles in kenaf Cd tolerance, and accounting for the difference in Cd stress sensitivities.

A comprehensive integrated transcriptome and metabolome analyses to reveal key genes and essential metabolic pathways involved in CMS in kenaf.

KEY MESSAGE: Numbers of critical genes and pathways were found from the levels of transcriptome and metabolome, which were useful information for understanding of kenaf CMS mechanism. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants that leads to the inability to produce or release functional pollen. However, there is lack of comprehensive studies to reveal the molecular basis of CMS occurrence in kenaf. Herein, we performed transcriptome and UPLC-MS-based metabolome analyses in the anthers of a CMS (UG93A) and its maintainer (UG93B) to sort out essential genes and metabolites responding to CMS in kenaf. Transcriptome characterized 7769 differentially expressed genes (DEGs) between these two materials, and pathway enrichment analysis indicated that these DEGs were involved mainly in pentose and glucuronate interconversions, starch and sucrose metabolism, taurine and hypotaurine metabolism. In the metabolome assay, a total of 116 significantly different metabolites (SDMs) were identified between the CMS and its maintainer line, and these SDMs were involved in eight KEGG pathways, including flavone and flavonol biosynthesis, glycerophospholipid metabolism, flavonoid biosynthesis, glycosylphosphatidylinositol-anchor biosynthesi. Integrated analyses of transcriptome and metabolome showed that 50 genes had strong correlation coefficient values (R2 > 0.9) with ten metabolites enriched in six pathways; notably, most genes and metabolites of flavonoid biosynthesis pathways and flavone and flavonol biosynthesis pathways involved in flavonoids biosynthetic pathways were downregulated in CMS compared to those in maintainer. Taken together, the decreased accumulation of flavonoids resulted from the compromised biosynthesis pathways coupled with energy deficiency in the anthers may contribute largely to CMS in UG93A of kenaf.

The complete chloroplast genome sequence of Oryza eichingeri (Poaceae).

The complete chloroplast genome sequence of Oryza eichingeri (GenBank accession number: KX085496) was generated by de novo assembly with low-coverage whole-genome sequence data. The chloroplast genome is 134,821 bp in length and showed conserved typical chloroplast structure. The cpDNA contained four rRNA, 39 tRNA, and 79 unique protein-coding genes. Seventeen genes contain one intron, only ycf3 contains two introns; rps12 is trans-spliced, one of its exons is in the LSC region (5'_end) and the two reside in the IR regions (3'_end) separated. A pair of gene ndhH, due to the 5' part of ndhH which overlaps the IR/SSC junctions, was two unique genes. The AT content of O. eichingeri cp genome is 61%. Phylogenomic analysis showed that O. eichingeri is closely related to O. officinalis. The complete cpDNA of O. eichingeri provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for the genus Oryza.

The complete chloroplast genome sequence of Oryza rhizomatis (Poaceae).

The complete chloroplast genome sequence of Oryza rhizomatis (GenBank accession number: KX085497) was generated by de novo assembly with low-coverage whole-genome sequence data. The genome was 134,796 bp in length, containing a pair of inverted repeat (IRa and IRb) regions of 20,818 bp, which were separated by a large single-copy (LSC) region of 80,829 bp and a small single-copy (SSC) region of 12,330 bp, respectively. The genic regions account for 43.77% of whole cpDNA, and the AT content of the cpDNA was 60.99%. The O. rhizomatis cpDNA encodes 112 unigenes (79 protein-coding genes, four rRNA genes, and 29 tRNA genes). Eighteen genes contain introns, ycf3 contains two introns, and the rest of the gene contains one intron; rps12 is trans-spliced, one of its exons is in the LSC region (5'_end) and the two reside in the IR regions (3'_end) separated. A pair of gene ndhH, due to the 5' part of ndhH which overlaps the IR/SSC junctions, was two unique genes. The four rRNA genes are all located in the IR. Phylogenomic analysis showed that O. rhizomatis is closely related to O. officinalis. The new data will help to determine the phylogenetic placement of the genus Oryza and fill gaps in our understanding of Oryzae biology.