Artemisinin is currently the most effective ingredient in the treatment of malaria, which is thus of great significance to study the genetic regulation of Artemisia annua . Alternative splicing (AS) is a regulatory process that increases the complexity of transcriptome and proteome. The most common mechanism of alternative splicing (AS) in plant is intron retention (IR). However, little is known about whether the IR isoforms produced by light play roles in regulating biosynthetic pathways. In this work we would explore how the level of AS in A. annua responds to light regulation. We obtained a new dataset of AS by analyzing full-length transcripts using both Illumina- and single molecule real-time (SMRT)-based RNA-seq as well as analyzing AS on various tissues. A total of 5,854 IR isoforms were identified, with IR accounting for the highest proportion (48.48%), affirming that IR is the most common mechanism of AS. We found that the number of up-regulated IR isoforms (1534/1378, blue and red light, respectively) was more than twice that of down-regulated (636/682) after treatment of blue or red light. In the artemisinin biosynthetic pathway, 10 genes produced 16 differentially expressed IR isoforms. This work demonstrated that the differential expression of IR isoforms induced by light has the potential to regulate sesquiterpenoid biosynthesis. This study also provides high accuracy full-length transcripts, which can be a valuable genetic resource for further research of A. annua , including areas of development, breeding, and biosynthesis of active compounds.
Bisbenzylisoquinoline and aporphine alkaloids are the two main pharmacological compounds in the ancient sacred lotus (Nelumbo nucifera). Because bisbenzylisoquinoline alkaloids have been reported as potential therapeutic agents for COVID-19, the biosynthesis of bisbenzylisoquinoline and aporphine alkaloids has attracted extensive attention. Our study showed that NnCYP80A can catalyze C-O coupling in both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine to produce bisbenzylisoquinoline alkaloids with three different linkages. In addition, NnCYP80G catalyzed C-C coupling in aporphine alkaloids with extensive substrate selectivity, specifically using (R)-N-methylcoclaurine, (S)-N-methylcoclaurine, coclaurine and reticuline as substrates, but the synthesis of C-ring alkaloids without hydroxyl groups in the lotus remains to be elucidated. The key residues of NnCYP80G were also studied using the 3D structure of the protein predicted using Alphafold 2, and six key amino acids (G39, G69, A211, P288, R425 and C427) were identified. The R425A mutation significantly decreased the catalysis of (R)-N-methylcoclaurine and coclaurine inactivation, which might play important role in the biosynthesis of alkaloids with new configurations.
Tamarix ramosissima Ledeb., a major host plant for the parasitic angiosperm Cistanche tubulosa, and known for its unique drought tolerance, has significant ecological and economic benefits. However, the mechanisms of nitrogen acquisition by the T. ramosissima root system under drought have remained uncharacterized. Here, uptake of nitrate (NO3-) in various regions of the root system was measured in T. ramosissima using Non-invasive Micro-test Technology at the cellular level, and using a 15NO3--enrichment technique at the whole-root level. These results were compared with responses in the model system cotton (Gossypium hirsutum L.). Tamarix ramosissima had lower net NO3- influx and a significantly lower Km (the apparent Michalis-Menten constant; 8.5 μM) for NO3- uptake than cotton under normal conditions. Upon simulated drought conditions, using polyethylene glycol (PEG), NO3- flux in cotton switched from net influx to net efflux, with a substantive peak in the white zone (WZ) of the root. There were no significant NO3- influx signals observed in the WZ of T. ramosissima under control conditions, whereas PEG treatment significantly enhanced NO3- influx in the WZ of T. ramosissima. The effect of PEG application on NO3- fluxes was highly localized, and the increase in net NO3- influx in response to PEG stimulation was also found in C. tubulosa-inoculated T. ramosissima. Consistently, root nitrogen (N) content and root biomass were higher in T. ramosissima than in cotton under PEG treatment. Our study provides insights into NO3- uptake and the influence of C. tubulosa inoculation in T. ramosissima roots during acclimation to PEG-induced drought stress and provides guidelines for silvicultural practice and for breeding of T. ramosissima under coupled conditions of soil drought and N deficiency.
Tartary buckwheat sprouts have a high nutritional value and are gluten-free, and polyphenols are their main active constituents. However, information regarding the active constituents' difference of Tartary buckwheat sprouts grown from seeds with different morphology, at different developmental stages and environments is limited. Here, we developed a LC-MS-based targeted metabolomics approach to analyze polyphenols (46 flavonoids and 6 anthraquinones) in 40 Tartary buckwheat sprouts varieties. Both flavonoids and anthraquinones contributed to significant differences in sprouts grown from seed with different color or shape. Twenty-seven differential compounds were all at a higher level in 3-day-old sprouts, and the fold change from 3-day-old to 8-day-old sprouts was 1.42-6.64. A total of 25 differential compounds were all significantly upregulated upon UV-B radiation, especially for epicatechin. This study is valuable not only for better breeding cultivars of Tartary buckwheat sprouts, but also assessing their metabolic quality.
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