Summary: Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue‐specific regulation of the pathway remains mostly unknown. We enhance the high‐quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue‐specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock‐out of red betacyanins in natural accessions of amaranth. We reveal the tissue‐specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co‐option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.

The maintenance of Thioredoxin-1 (Trx-1) levels, and thus of cellular redox homeostasis, is vital for endothelial cells (ECs) to prevent senescence induction. One hallmark of EC functionality, their migratory capacity, which depends on intact mitochondria, is reduced in senescence. Caffeine improves the migratory capacity and mitochondrial functionality of ECs. However, the impact of caffeine on EC senescence has never been investigated. Moreover, a high-fat diet, which can induce EC senescence, results in approximately 1 ng/mL lipopolysaccharide (LPS) in the blood. Therefore, we investigated if low dose endotoxemia induces EC senescence and concomitantly reduces Trx-1 levels, and if caffeine prevents or even reverses senescence. We show that caffeine precludes H₂O₂-triggered senescence induction by maintaining endothelial NO synthase (eNOS) levels and preventing the elevation of p21. Notably, 1 ng/mL LPS also increases p21 levels and reduces eNOS and Trx-1 amounts. These effects are completely blocked by co-treatment with caffeine. This prevention of senescence induction is similarly accomplished by the permanent expression of mitochondrial p27, a downstream effector of caffeine. Most importantly, after senescence induction by LPS, a single bolus of caffeine inhibits the increase in p21. This treatment also blocks Trx-1 degradation, suggesting that the reversion of senescence is intimately associated with a normalized redox balance.

Summary: • Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. • We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. • Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2 . Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. • Our improved amaranth reference genome represents the most complete genome of amaranth to date and a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as possible reason for mutual exclusiveness of the two pigments.

Phytohormones are key players in the regulation of plant development and metabolism. The different phytohormone classes comprise numerous chemically very diverse compounds, which are often present at very low concentrations. The chemical properties of phytohormones range from acidic to basic and from polar to non-polar. Furthermore, concentration varies strongly among different phytohormones, between plant species, tissues and developmental stages. Challenges often arise when only small amounts of plant material are available and when plant species are investigated in which the phytohormone profile has not yet been characterized. To establish a method for comprehensive phytohormone analysis we addressed these challenges by choosing and optimizing a suitable extraction method followed by optimized HPLC separation. We compared the most widely-used mass spectrometric detection methods, multiple reaction monitoring (MRM) on a triple quad instrument with high-resolution mass spectrometry (HRMS) on a Q-TOF instrument, and discuss the advantages of both methods and their limitations. We compared various methods described in literature for the extraction of six phytohormone classes by liquid-liquid extraction and solid phase extraction purification and describe our optimizations to the selected method. We optimized HPLC separation for 50 different phytohormones. We evaluated the application of MRM and HRMS detection strategies.

The molecular framework underlying apoplastic root barrier formation has been unveiled in the model species Arabidopsis thaliana where establishment of Casparian strips occurs at an early stage of root development. In legumes, this region overlaps with the area where nitrogen-fixing bacteria can induce nodule formation, termed the susceptible zone. Moreover, while nodules themselves also contain an endodermis spanning their vascular bundles, it is current unknown if Casparian strips serve as a filter for transport across this specialized organ. Here we establish barrier mutants in the symbiosis model Lotus japonicus. We find that the while genetic network controlling Casparian strip formation is conserved in this legume species, formation of functional barriers is crucial for establishment of N-fixing nodules. By probing this in detail, we establish a model where the Casparian strip, via its linked Schengen pathway, converge with long distance N signaling and systemic regulation of nodulation. Moreover, this also reveal that the genetic system for barrier establishment in the root endodermis is shared in nodule vascular endodermis and required for nodule function. Combined, our findings uncover a novel role of apoplastic root barriers and establishes a mutant collection suitable to probe the role of root barriers in symbiotic plant-microbe relationships.