Plant growth promotion under phosphate deficiency and improved phosphate acquisition by new fungal strain, Penicillium olsonii TLL1.

Microbiomes in soil ecosystems play a significant role in solubilizing insoluble inorganic and organic phosphate sources with low availability and mobility in the soil. They transfer the phosphate ion to plants, thereby promoting plant growth. In this study, we isolated an unidentified fungal strain, POT1 (Penicillium olsonii TLL1) from indoor dust samples, and confirmed its ability to promote root growth, especially under phosphate deficiency, as well as solubilizing activity for insoluble phosphates such as AlPO4, FePO4·4H2O, Ca3(PO4)2, and hydroxyapatite. Indeed, in vermiculite containing low and insoluble phosphate, the shoot fresh weight of Arabidopsis and leafy vegetables increased by 2-fold and 3-fold, respectively, with POT1 inoculation. We also conducted tests on crops in Singapore's local soil, which contains highly insoluble phosphate. We confirmed that with POT1, Bok Choy showed a 2-fold increase in shoot fresh weight, and Rice displayed a 2-fold increase in grain yield. Furthermore, we demonstrated that plant growth promotion and phosphate solubilizing activity of POT1 were more effective than those of four different Penicillium strains such as Penicillium bilaiae, Penicillium chrysogenum, Penicillium janthinellum, and Penicillium simplicissimum under phosphate-limiting conditions. Our findings uncover a new fungal strain, provide a better understanding of symbiotic plant-fungal interactions, and suggest the potential use of POT1 as a biofertilizer to improve phosphate uptake and use efficiency in phosphate-limiting conditions.

Effects of Storage Temperatures on Nitrogen Assimilation and Remobilization during Post-Harvest Senescence of Pak Choi.

In the agricultural industry, the post-harvest leafy vegetable quality and shelf life significantly influence market value and consumer acceptability. This study examined the effects of different storage temperatures on leaf senescence, nitrogen assimilation, and remobilization in Pak Choi (Brassica rapa subsp. chinensis). Mature Pak Choi plants were harvested and stored at two different temperatures, 4 °C and 25 °C. Senescence was tracked via chlorophyll content and leaf yellowing. Concurrently, alterations in the total nitrogen, nitrate, and protein content were quantified on days 0, 3, 6, and 9 in old, mid, and young leaves of Pak Choi plants. As expected, 4 °C alleviated chlorophyll degradation and delayed senescence of Pak Choi compared to 25 °C. Total nitrogen and protein contents were inversely correlated, while the nitrate content remained nearly constant across leaf groups at 25 °C. Additionally, the transcript levels of genes involved in nitrogen assimilation and remobilization revealed key candidate genes that were differentially expressed between 4 °C and 25 °C, which might be targeted to extend the shelf life of the leafy vegetables. Thus, this study provides pivotal insights into the molecular and physiological responses of Pak Choi to post-harvest storage conditions.

Comparative Transcriptome Analysis Reveals Coordinated Transcriptional Regulation of Central and Secondary Metabolism in the Trichomes of Cannabis Cultivars.

Cannabis is one of the few plant genera capable of producing cannabinoids, the effects of which are synergized by terpene interactions. As the biosynthesis of both metabolite classes requires the same intracellular feedstocks, this work describes the coordinated regulation of global metabolic pathways that allows for their joint copious production in vivo. To this end, a transcriptomics-based approach to characterize the glandular trichomes of five Cannabis cultivars was pursued. Besides revealing metabolic traits that enhanced and proportionated the supply of critical carbon precursors, in-depth analysis showed significantly increased gene expression of two particular enzymes to meet the huge nicotinamide adenine dinucleotide phosphate (NADPH) demand of secondary metabolite production. Furthermore, it led to a hypothesis that the methyl-d-erythritol 4-phosphate pathway might be utilized more than the mevalonic acid pathway in Cannabis trichomes. While both pathways were found to be activated in a modular and calibrated way that reflected their broad participation in physiological processes, the genes for hexanoate, cannabinoid, and terpene biosynthesis were, in contrast, up-regulated in an en bloc and multi-loci manner due to their specific roles in secondary metabolite production. In addition, three new terpene synthases were characterized based on both in silico and experimental assays. Altogether, the study enhances the current understanding of secondary metabolite production in Cannabis cultivars, which may assist in their characterization and development.

Identification and Functional Characterization of Tissue-Specific Terpene Synthases in Stevia rebaudiana.

In addition to the well-known diterpenoid steviol glycosides, Stevia rebaudiana (Stevia) produces many labdane-type diterpenoids and a wide range of mono- and sesquiterpenoids. However, biosynthesis of mono- and sesquiterpenoids in Stevia remains unknown. Here we analyzed the extracts of Stevia leaves, flowers, stems, and roots by Gas Chromatography-Mass Spectrometry and putatively identified a total of 69 volatile organic compounds, most of which were terpenoids with considerably varied quantities among the four tissues of Stevia. Using Stevia transcriptomes, we identified and functionally characterized five terpene synthases (TPSs) that produced major mono- and sesquiterpenoids in Stevia. Transcript levels of these Stevia TPSs and levels of corresponding terpenoids correlated well in Stevia tissues. Particularly, the root-specific SrTPS4 and SrTPS5 catalyzed the formation of γ-curcumene/zingiberene/ß-sesquiphellandrene and α-longipinene/ß-himachalene/himachalol as multifunctional sesqui-TPSs, respectively. Most of the SrTPSs were highly responsive to various environmental stresses in a tissue-specific manner. Taken together, our results provide new insights into how Stevia produces diverse terpenoids to confer differential responses to various environmental factors in each tissue.

Production of multiple terpenes of different chain lengths by subcellular targeting of multi-substrate terpene synthase in plants.

Multi-substrate terpene synthases (TPSs) are distinct from typical TPSs that react with a single substrate. Although in vitro activity of few multi-substrate TPSs have been reported, in vivo characterization has not been well investigated for most of them. Here, a new TPS from Cananga odorata, CoTPS5, belonging to TPS-f subfamily was functionally characterized in vitro as well as in vivo. CoTPS5 reacted with multiple prenyl-pyrophosphate substrates of various chain lengths as a multi-substrate TPS. It catalyzed the formation of (E)-ß-ocimene, (E,E)-α-farnesene and α-springene from geranyl pyrophosphate, (E,E)-farnesyl pyrophosphate and geranylgeranyl pyrophosphate, respectively. Upon transient expression in Nicotiana benthamiana, CoTPS5 localized to cytosol and produced only (E,E)-α-farnesene. However, expression of plastid-targeted CoTPS5 in N. benthamiana resulted in biosynthesis of all three compounds, (E)-ß-ocimene, (E,E)-α-farnesene and α-springene. Similarly, transgenic Arabidopsis plants overexpressing plastid-targeted CoTPS5 showed stable and sustainable production of (E)-ß-ocimene, (E,E)-α-farnesene and α-springene. Moreover, their production did not affect the growth and development of transgenic Arabidopsis plants. Our results demonstrate that redirecting multi-substrate TPS to a different intracellular compartment could be an effective way to prove in vivo activity of multi-substrate TPSs and thereby allowing for the production of multiple terpenoids simultaneously in plants.

CYP79D73 Participates in Biosynthesis of Floral Scent Compound 2-Phenylethanol in Plumeria rubra.

Plumeria (Plumeria rubra), well known for its brightly colored and fragrant flowers, emits a number of floral volatile organic compounds (VOCs). Plumeria flowers emit a total of 43 VOCs including nine phenylpropanoids/benzenoids, such as 2-phenylethanol (2PE), benzaldehyde, 2-phenylacetaldehyde (PAld), (E/Z)-phenylacetaldoxime (PAOx), benzyl nitrile (BN), and 2-phenylnitroethane (PN). To identify genes and pathways involved in the production of the major compound 2PE, we analyzed the plumeria floral transcriptome and found a highly expressed, flower-specific gene encoding a cytochrome P450 family 79D protein (PrCYP79D73), which catalyzed the formation of (E/Z)-PAOx. Feeding experiments with deuterated phenylalanine or deuterated (E/Z)-PAOx showed that (E/Z)-PAOx is an intermediate in the biosynthesis of 2PE, as are two nitrogen-containing volatiles, BN and PN, in plumeria flowers. Crude enzyme extracts from plumeria flowers converted l-phenylalanine to (E/Z)-PAOx, PAld, 2PE, BN, and PN. The biosynthesis of these compounds increased with addition of PrCYP79D73-enriched microsomes but was blocked by pretreatment with 4-phenylimidazole, an inhibitor of cytochrome P450 enzymes. Moreover, overexpression of PrCYP79D73 in Nicotiana benthamiana resulted in the emission of (E/Z)-PAOx as well as PAld, 2PE, BN, and PN, all of which were also found among plumeria floral VOCs. Taken together, our results demonstrate that PrCYP79D73 is a crucial player in the biosynthesis of the major floral VOC 2PE and other nitrogen-containing volatiles. These volatiles may be required for plant defense as well as to attract pollinators for the successful reproduction of plumeria.

Integrated metabolome and transcriptome analysis of Magnolia champaca identifies biosynthetic pathways for floral volatile organic compounds.

BACKGROUND: Magnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes. RESULTS: Volatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides. CONCLUSION: As our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent.

The floral transcriptome of ylang ylang (Cananga odorata var. fruticosa) uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase.

The pleasant fragrance of ylang ylang varieties (Cananga odorata) is mainly due to volatile organic compounds (VOCs) produced by the flowers. Floral scents are a key factor in plant-insect interactions and are vital for successful pollination. C. odorata var. fruticosa, or dwarf ylang ylang, is a variety of ylang ylang that is popularly grown in Southeast Asia as a small shrub with aromatic flowers. Here, we describe the combined use of bioinformatics and chemical analysis to discover genes for the VOC biosynthesis pathways and related genes. The scented flowers of C. odorata var. fruticosa were analysed by gas chromatography/mass spectrometry and a total of 49 VOCs were identified at four different stages of flower development. The bulk of these VOCs were terpenes, mainly sesquiterpenes. To identify the various terpene synthases (TPSs) involved in the production of these essential oils, we performed RNA sequencing on mature flowers. From the RNA sequencing data, four full-length TPSs were functionally characterized. In vitro assays showed that two of these TPSs were mono-TPSs. CoTPS1 synthesized four products corresponding to ß-thujene, sabinene, ß-pinene, and α-terpinene from geranyl pyrophosphate and CoTPS4 produced geraniol from geranyl pyrophosphate. The other two TPSs were identified as sesqui-TPSs. CoTPS3 catalysed the conversion of farnesyl pyrophosphate to α-bergamotene, whereas CoTPS2 was found to be a multifunctional and novel TPS that could catalyse the synthesis of three sesquiterpenes, ß-ylangene, ß-copaene, and ß-cubebene. Additionally, the activities of the two sesqui-TPSs were confirmed in planta by transient expression of these TPS genes in Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration.