Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

Ensuring Global Food Security by Improving Protein Content in Major Grain Legumes Using Breeding and 'Omics' Tools.

Grain legumes are a rich source of dietary protein for millions of people globally and thus a key driver for securing global food security. Legume plant-based 'dietary protein' biofortification is an economic strategy for alleviating the menace of rising malnutrition-related problems and hidden hunger. Malnutrition from protein deficiency is predominant in human populations with an insufficient daily intake of animal protein/dietary protein due to economic limitations, especially in developing countries. Therefore, enhancing grain legume protein content will help eradicate protein-related malnutrition problems in low-income and underprivileged countries. Here, we review the exploitable genetic variability for grain protein content in various major grain legumes for improving the protein content of high-yielding, low-protein genotypes. We highlight classical genetics-based inheritance of protein content in various legumes and discuss advances in molecular marker technology that have enabled us to underpin various quantitative trait loci controlling seed protein content (SPC) in biparental-based mapping populations and genome-wide association studies. We also review the progress of functional genomics in deciphering the underlying candidate gene(s) controlling SPC in various grain legumes and the role of proteomics and metabolomics in shedding light on the accumulation of various novel proteins and metabolites in high-protein legume genotypes. Lastly, we detail the scope of genomic selection, high-throughput phenotyping, emerging genome editing tools, and speed breeding protocols for enhancing SPC in grain legumes to achieve legume-based dietary protein security and thus reduce the global hunger risk.

Combined application of biochar and nitrogen fertilizer promotes the activity of starch metabolism enzymes and the expression of related genes in rice in a dual cropping system.

BACKGROUND: Overuse of chemical fertilizer highly influences grain filling rate and quality of rice grain. Biochar is well known for improving plant growth and grain yield under lower chemical fertilization. Therefore field trials were conducted in the early and late seasons of 2019 at Guangxi University, China to investigate the effects of combined biochar (B) and nitrogen (N) application on rice yield and yield components. There were a total of eight treatments: N1B0, 135 kg N ha- 1+ 0 t B ha- 1; N2B0,180 kg N ha- 1+ 0 t B ha- 1; N1B1,135 kg N ha- 1+ 10 t B ha- 1; N1B2,135kg N ha- 1+ 20 t B ha- 1; N1B3,135 kg N ha- 1+ 30 t B ha- 1; N2B1,180 kg N ha- 1+ 10 t B ha- 1; N2B2,180 kg N ha- 1+ 20 t B ha- 1; and N2B3,180 kg N ha- 1+ 30 t B ha- 1. RESULTS: Biochar application at 30 t ha- 1combined with low N application (135 kg ha- 1) increased the activity of starch-metabolizing enzymes (SMEs) during the early and late seasons compared with treatments without biochar. The grain yield, amylose concentration, and starch content of rice were increased in plots treated with 30 t B ha-1and low N. RT-qPCR analysis showed that biochar addition combined with N fertilizer application increased the expression of AGPS2b, SSS1, GBSS1, and GBSE11b, which increased the activity of SMEs during the grain-filling period. CONCLUSION: Our results suggest that the use of 20 to 30 t B ha- 1coupled with 135 kg N ha- 1 is optimal for improving the grain yield and quality of rice.

Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2.

Here, we aimed to investigate the diagnostic value of a serological assay using the nucleocapsid protein developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection and evaluated its performance using three commercial enzyme-linked immunosorbent assays (ELISAs), namely, Standard E 2019 novel coronavirus disease (COVID-19) total antibody (Ab) ELISA (SD Biosensor), and EDI novel coronavirus COVID-19 IgG and IgM ELISA. A recombinant nucleocapsid protein (rNP) was expressed from plants and Escherichia coli for the detection of serum total Ab. We prospectively collected 141 serum samples from 32 patients with reverse transcription-PCR (RT-PCR)-confirmed COVID-19 and determined the sensitivity and dynamics of their total Ab response. Specificity was evaluated using 158 prepandemic samples. To validate the assays, we evaluated the performance using two different cutoff values. The sensitivity and specificity for each assay were as follows: 92.91% and 94.30% (plant-rNP), 83.69% and 98.73% (SD Biosensor), 75.89% and 98.10% (E. coli-rNP), 76.47% and 100% (EDI-IgG), and 80.39% and 80% (EDI-IgM). The plant-based rNP showed the highest sensitivity and area under the receiver operating characteristic (ROC) curve (0.980) among all the assays (P < 0.05). The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs. After 2 weeks of PSO, the seroconversion rates were >80% and 100% for EDI-IgM and EDI-IgG ELISA, respectively. Seroconversion occurred earlier with rNP plant-based ELISA (5 days PSO) compared with E. coli-based (7 days PSO) and SD Biosensor (8 days PSO) ELISA. We determined that rNP produced in plants enables the robust detection of SARS-CoV-2 total Abs. The assay can be used for serosurvey and complementary diagnosis of COVID-19. IMPORTANCE At present, the principal diagnostic methods for COVID-19 comprise the identification of viral nucleic acid by genetic approaches, including PCR-based techniques or next-generation sequencing. However, there is an urgent need for validated serological assays which are crucial for the understanding of immune responses against SARS-CoV-2. In this study, a highly sensitive and specific serological antibody assay was developed for the detection of SARS-CoV-2 with an overall accuracy of 93.56% using a recombinant nucleoprotein expressed from plants.

Large-scale integration of meta-QTL and genome-wide association study discovers the genomic regions and candidate genes for yield and yield-related traits in bread wheat.

KEY MESSAGE: Based on the large-scale integration of meta-QTL and Genome-Wide  Association Study, 76 high-confidence MQTL regions and 237 candidate genes that affected wheat yield and yield-related traits were discovered. Improving yield and yield-related traits are key goals in wheat breeding program. The integration of accumulated wheat genetic resources provides an opportunity to uncover important genomic regions and candidate genes that affect wheat yield. Here, a comprehensive meta-QTL analysis was conducted on 2230 QTL of yield-related traits obtained from 119 QTL studies. These QTL were refined into 145 meta-QTL (MQTL), and 89 MQTL were verified by GWAS with different natural populations. The average confidence interval (CI) of these MQTL was 2.92 times less than that of the initial QTL. Furthermore, 76 core MQTL regions with a physical distance less than 25 Mb were detected. Based on the homology analysis and expression patterns, 237 candidate genes in the MQTL involved in photoperiod response, grain development, multiple plant growth regulator pathways, carbon and nitrogen metabolism and spike and flower organ development were determined. A novel candidate gene TaKAO-4A was confirmed to be significantly associated with grain size, and a CAPS marker was developed based on its dominant haplotype. In summary, this study clarified a method based on the integration of meta-QTL, GWAS and homology comparison to reveal the genomic regions and candidate genes that affect important yield-related traits in wheat. This work will help to lay a foundation for the identification, transfer and aggregation of these important QTL or candidate genes in wheat high-yield breeding.

ROS/RNS Balancing, Aerobic Fermentation Regulation and Cell Cycle Control - a Complex Early Trait ('CoV-MAC-TED') for Combating SARS-CoV-2-Induced Cell Reprogramming.

In a perspective entitled 'From plant survival under severe stress to anti-viral human defense' we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named 'ReprogVirus' was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the 'ReprogVirus platform' was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to 'RegroVirus' complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called 'CoV-MAC-TED'. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target 'CoV-MAC-TED' in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that 'de-stressing' disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system.

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.

Caffeoylquinic acids: chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity.

Caffeoylquinic acids (CQAs) are specialized plant metabolites we encounter in our daily life. Humans consume CQAs in mg-to-gram quantities through dietary consumption of plant products. CQAs are considered beneficial for human health, mainly due to their anti-inflammatory and antioxidant properties. Recently, new biosynthetic pathways via a peroxidase-type p-coumaric acid 3-hydroxylase enzyme were discovered. More recently, a new GDSL lipase-like enzyme able to transform monoCQAs into diCQA was identified in Ipomoea batatas. CQAs were recently linked to memory improvement; they seem to be strong indirect antioxidants via Nrf2 activation. However, there is a prevalent confusion in the designation and nomenclature of different CQA isomers. Such inconsistencies are critical and complicate bioactivity assessment since different isomers differ in bioactivity and potency. A detailed explanation regarding the origin of such confusion is provided, and a recommendation to unify nomenclature is suggested. Furthermore, for studies on CQA bioactivity, plant-based laboratory animal diets contain CQAs, which makes it difficult to include proper control groups for comparison. Therefore, a synthetic diet free of CQAs is advised to avoid interferences since some CQAs may produce bioactivity even at nanomolar levels. Biotransformation of CQAs by gut microbiota, the discovery of new enzymatic biosynthetic and metabolic pathways, dietary assessment, and assessment of biological properties with potential for drug development are areas of active, ongoing research. This review is focused on the chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity recently reported for mono-, di-, tri-, and tetraCQAs.