To Örebro University

Plant systems have gained increased attention as an alternative platform for producing heterologous proteins, particularly for industrially relevant proteins. The Cynara cardunculus L. flower extract is traditionally used in cheese production across Mediterranean countries due to its milk-clotting properties. To address the growing demand for plant-based milk-clotting enzymes, we investigated the heterologous production of cyprosin B (CYPB), a key milk-clotting enzyme, in Nicotiana benthamiana. We also examined the role of its plant-specific insert (PSI) domain in enzymatic activity, protein yield, and subcellular localization. Full-length CYPB and a PSI domain-deleted variant (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using agroinfiltration. Proteins were purified nine days post-infiltration, yielding ~81 mg/kg (CYPB) and ~60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed higher proteolytic activity (~168 IU/mg) than CYPB (~57 IU/mg) and exhibited faster milk clotting times, suggesting that PSI removal may contribute to enhanced enzymatic efficiency. However, additional factors such as altered glycosylation or localization may also play a role. Subcellular localization indicated that CYPB and its PSI domain targeted the vacuole and endocytic vesicles, while CYPBΔPSI predominantly localized to the endoplasmic reticulum and tonoplast. This suggests that the PSI domain’s vital role in vacuolar targeting and membrane permeabilization ultimately influences protein yield. Our study shows N. benthamiana as a scalable platform for producing recombinant CYPB variants with improved enzymatic activity. It highlights the PSI domain's role in vacuolar sorting without impairing 21 function. These findings contribute to the development of plant-based systems for milk-clotting 22 enzymes for cheese-making.

BACKGROUND: Proso millet (Panicum miliaceum L.), one of the oldest domesticated crop, remains an underexploited resource with significant potential for nutrition and yield. With evolving breeding perspectives, genomic knowledge is increasingly vital for developing new crop varieties. However, the limited genomic information on indigenous proso millet hinders its full utilization. This study addresses this gap by compiling chloroplast genome (cp. genome) data for the native variety ATL1 and its mutant derivative TNPmPEM 001, aiming to facilitate the development of new varieties.

RESULTS: Both Panicum miliaceum cv. ATL1 and TNPmPEM 001 chloroplast genomes exhibited the characteristic quadripartite structure. While they shared identical total lengths (139 837 bp), small single-copy (SSC: 12 795 bp), large single-copy (LSC: 84 522 bp), and inverted repeat (IR: 20 560 bp) regions, these metrics diverged from the reference genome, which displayed a total length of 139 826 bp, with distinct SSC (12 574 bp), LSC (81 682 bp), and IR (22 785 bp) regions. While soybean, cotton, sunflower, and pea constituted outgroups, the phylogenetic analysis showed a tight link between ATL1, TNPmPEM 001 and reference cp. genome as well as with little millet. The identification of protein-coding genes regulating photosynthesis components (photosystems I and II, NADH dehydrogenase, cytochrome complexes, rubisco, and ribosomal/tRNA/rRNA genes) in both investigated cp. genomes provides critical insights into the genomic basis of photosynthesis efficiency in underutilized C4 crops like proso millet, a key trait for improving stress-resilient sustainable agriculture. Additionally, 11 unique simple sequence repeat (SSR) markers, exclusively identified in the mutant derivative, offer novel tools for marker-assisted breeding programs targeting agronomic trait enhancement.

CONCLUSIONS: These findings address critical gaps in proso millet genomics, particularly the limited molecular resources for Indian landraces. The mutant-derived SSRs and structural variants offer actionable targets for enhancing yield stability under variable photoperiods, a priority for climate-resilient proso millet breeding in marginal agroecosystems.

Coconut haustorium is a tropical delicacy that mobilizes nutrients from the endosperm to nourish the germinating embryo. This study profiles coconut haustorium for sugars, organic acids, phenolics, and flavonoids using advanced chromatographic techniques. Fructose (62.70%) and glucose (18.21%) were the major sugars with negligible lactose, suggesting potential use in baby food and for lactose-intolerant individuals. Malic acid (71.79%), citric acid (12.80%), and shikimic acid (7.59%) were the prominent organic acids, contributing to the haustorium's unique taste and refreshing flavor. Ferulic acid (60.96%) and p-coumaric acid (22.05%) were the prominent phenolic acids, while naringenin (35.36%) and catechin (29.68%) were the primary flavonoids. Total phenols, flavonoids, and antioxidant activity (61.18%) were also recorded, confirming the presence of bioactive compounds. The study reveals coconut haustorium as a rich source of readily available natural sugars, organic acids, phenols, and flavonoids offering potential for developing innovative nutraceuticals and functional foods.

Purple-fleshed dragon fruit is gaining popularity worldwide due to its distinctive characteristics and health benefits. This climbing cactus, introduced to humid tropical climates, presents challenges in assessing fruit quality. The dynamic transformations in fruit color, bioactive compounds, and textural attributes across 11 developmental stages from 10 to 32 days after flowering under humid tropical conditions were studied. Color analysis revealed significant intensification of red-violet hues, with L* values decreasing by 14.74% and a* values increasing from -8.14 to 32.96. The color transformation is initiated in the pulp at 25 days and the peel at 27 days after flowering. Betalain synthesis commenced after 20 days with rapid accumulation between 25 and 32 days, correlating with color development. Antioxidant activity increased from 79.38% at 10 days to 86.76% at 20 days, followed by a steady decline. Phenolic content peaked at 121.40 mg gallic acid equivalent per 100 g at 25 days before declining, while the flavonoid content decreased with the advancement of fruit development. Concurrent reduction in peel thickness and fruit firmness was also observed. These findings show that purple-fleshed dragon fruit can adapt well to humid tropical conditions, with a 32-day developmental cycle, offering vital insights into quality and maturation phases.

In recent years, environmental stresses viz., drought and high-temperature negatively impacts the tomato growth, yield and quality. The effects of combined drought and high-temperature (HT) stresses during the flowering stage were investigated. The main objective was to assess the effects of foliar spray of melatonin under both individual and combined drought and HT stresses at the flowering stage. Drought stress was imposed by withholding irrigation, whereas HT stress was imposed by exposing the plants to an ambient temperature (AT)+5°C temperature. The drought+HT stress was imposed by exposing the plants to drought first, followed by exposure to AT+5°C temperature. The duration of individual and combined drought or HT stress was 10 days. The results showed that drought+HT stress had a significant negative effect compared with individual drought or HT stress alone. However, spraying 100 µM melatonin on the plants challenged with individual or combined drought and HT stress showed a significant increase in total chlorophyll content [drought: 16%, HT: 14%, and drought+HT: 11%], Fv/Fm [drought: 16%, HT: 15%, and drought+HT: 13%], relative water content [drought: 10%, HT: 2%, and drought+HT: 8%], and proline [drought: 26%, HT: 17%, and drought+HT: 14%] compared with their respective stress control. Additionally, melatonin positively influenced the stomatal and trichome characteristics compared with stress control plants. Also, the osmotic adjustment was found to be significantly increased in the melatonin-sprayed plants, which, in turn, resulted in an increased number of fruits, fruit set percentage, and fruit yield. Moreover, melatonin spray also enhanced the quality of fruits through increased lycopene content, carotenoid content, titratable acidity, and ascorbic acid content, compared with the stress control. Overall, this study highlights the usefulness of melatonin in effectively mitigating the negative effects of drought, HT, and drought+HT stress, thus leading to an increased drought and HT stress tolerance in tomato.

Yellow mosaic disease (YMD) with typical symptoms of alternating bright yellow to green patches associated with stunting, downward cupping, and wrinkling has been observed in mung bean on agricultural farms in Coimbatore, Tamil Nadu, India. PCR using gene-specific primers indicated the presence of the yellow mosaic virus in symptomatic plants. Rolling circle amplification (RCA) followed by restriction digestion detected ~2.7 kb of DNA-A and DNA-B, allowing the identification of a bipartite genome. The full-length genome sequences were deposited in NCBI GenBank with the accession numbers MK317961 (DNA-A) and MK317962 (DNA-B). Sequence analysis of DNA-A showed the highest sequence identity of 98.39% to the DNA-A of mungbean yellow mosaic virus (MYMV)-Vigna radiata (MW736047), while DNA-B exhibited the highest level of identity (98.21%) to the MYMV-Vigna aconitifolia isolate (DQ865203) reported from Tamil Nadu. Recombinant analysis revealed distinct evidence of recombinant breakpoints of DNA-A within the region encoding the open reading frame (ORF) AC2 (transcription activation protein), with the major parent identified as MYMV-PA1 (KC9111717) and the potential minor parent as MYMV-Namakkal (DQ86520.1). Interestingly, a recombination event in the common region (CR) of DNA-B, which encodes the nuclear shuttle protein and the movement protein, was detected. MYMIV-M120 (FM202447) and MYMV-Vigna (AJ132574) were identified as the event's major and minor parents, respectively. This large variation in DNA-B led us to suspect a recombination in DNA-B. Dimeric MYMV infectious clones were constructed, and the infectivity was confirmed through agroinoculation. In future prospects, unless relying on screening using whiteflies, breeders and plant pathologists can readily use this agroinoculation procedure to identify resistant and susceptible cultivars to YMD.