Incorporation of nitrogen in antinutritional Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids.

A NAC triad modulates plant immunity by negatively regulating N-hydroxy pipecolic acid biosynthesis.

N-hydroxy pipecolic acid (NHP) plays an important role in plant immunity. In contrast to its biosynthesis, our current knowledge with respect to the transcriptional regulation of the NHP pathway is limited. This study commences with the engineering of Arabidopsis plants that constitutively produce high NHP levels and display enhanced immunity. Label-free proteomics reveals a NAC-type transcription factor (NAC90) that is strongly induced in these plants. We find that NAC90 is a target gene of SAR DEFICIENT 1 (SARD1) and induced by pathogen, salicylic acid (SA), and NHP. NAC90 knockout mutants exhibit constitutive immune activation, earlier senescence, higher levels of NHP and SA, as well as increased expression of NHP and SA biosynthetic genes. In contrast, NAC90 overexpression lines are compromised in disease resistance and accumulated reduced levels of NHP and SA. NAC90 could interact with NAC61 and NAC36 which are also induced by pathogen, SA, and NHP. We next discover that this protein triad directly represses expression of the NHP and SA biosynthetic genes AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1), FLAVIN MONOOXYGENASE 1 (FMO1), and ISOCHORISMATE SYNTHASE 1 (ICS1). Constitutive immune response in nac90 is abolished once blocking NHP biosynthesis in the fmo1 background, signifying that NAC90 negative regulation of immunity is mediated via NHP biosynthesis. Our findings expand the currently documented NHP regulatory network suggesting a model that together with NHP glycosylation, NAC repressors take part in a 'gas-and-brake' transcriptional mechanism to control NHP production and the plant growth and defense trade-off.

Multigenerational inheritance drives symbiotic interactions of the bacterium Bacillus subtilis with its plant host.

Bacillus subtilis is a beneficial bacterium that supports plant growth and protects plants from bacterial, fungal, and viral infections. Using a simplified system of B. subtilis and Arabidopsis thaliana interactions, we studied the fitness and transcriptome of bacteria detached from the root over generations of growth in LB medium. We found that bacteria previously associated with the root or exposed to its secretions had greater stress tolerance and were more competitive in root colonization than bacteria not previously exposed to the root. Furthermore, our transcriptome results provide evidence that plant secretions induce a microbial stress response and fundamentally alter signaling by the cyclic nucleotide c-di-AMP, a signature maintained by their descendants. The changes in cellular physiology due to exposure to plant exudates were multigenerational, as they allowed not only the bacterial cells that colonized a new plant but also their descendants to have an advance over naive competitors of the same species, while the overall plasticity of gene expression and rapid adaptation were maintained. These changes were hereditary but not permanent. Our work demonstrates a bacterial memory manifested by multigenerational reversible adaptation to plant hosts in the form of activation of the stressosome, which confers an advantage to symbiotic bacteria during competition.

The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction.

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.

Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory.

Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

Carotenoid retention during post-harvest storage of Capsicum annuum: the role of the fruit surface structure.

In this study, a chilli pepper (Capsicum annuum) panel for post-harvest carotenoid retention was studied to elucidate underlying mechanisms associated with this commercial trait of interest. Following drying and storage, some lines within the panel had an increase in carotenoids approaching 50% compared with the initial content at the fresh fruit stage. Other lines displayed a 25% loss of carotenoids. The quantitative determination of carotenoid pigments with concurrent cellular analysis indicated that in most cases, pepper fruit with thicker (up to 4-fold) lipid exocarp layers and smooth surfaces exhibit improved carotenoid retention properties. Total cutin monomer content increased in medium/high carotenoid retention fruits and subepidermal cutin deposits were responsible for the difference in exocarp thickness. Cutin biosynthesis and cuticle precursor transport genes were differentially expressed between medium/high and low carotenoid retention genotypes, and this supports the hypothesis that the fruit cuticle can contribute to carotenoid retention. Enzymatic degradation of the cuticle and cell wall suggests that in Capsicum the carotenoids (capsanthin and its esters) are embedded in the lipidic exocarp layer. This was not the case in tomato. Collectively, the data suggest that the fruit cuticle could provide an exploitable resource for the enhancement of fruit quality.