Fine tuning chloroplast movements through physical interactions between phototropins

Phototropins are plant photoreceptors which regulate numerous responses to blue light, including chloroplast relocation. Weak blue light induces chloroplast accumulation, whereas strong light leads to an avoidance response. Two Arabidopsis phototropins are characterized by different light sensitivities. Under continuous light, both can elicit chloroplast accumulation, but the avoidance response is controlled solely by phot2. As well as continuous light, brief light pulses also induce chloroplast displacements. Pulses of 0.1s and 0.2s of fluence rate saturating the avoidance response lead to transient chloroplast accumulation. Longer pulses (up to 20s) trigger a biphasic response, namely transient avoidance followed by transient accumulation. This work presents a detailed study of transient chloroplast responses in Arabidopsis. Phototropin mutants display altered chloroplast movements as compared with the wild type: phot1 is characterized by weaker responses, while phot2 exhibits enhanced chloroplast accumulation, especially after 0.1s and 0.2s pulses. To determine the cause of these differences, the abundance and phosphorylation levels of both phototropins, as well as the interactions between phototropin molecules are examined. The formation of phototropin homo- and heterocomplexes is the most plausible explanation of the observed phenomena. The physiological consequences of this interplay are discussed, suggesting the universal character of this mechanism that fine-tunes plant reactions to blue light. Additionally, responses in mutants of different protein phosphatase 2A subunits are examined to assess the role of protein phosphorylation in signaling of chloroplast movements

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5

VC1 catalyzes a key step in the biosynthesis of vicine from GTP in faba bean

Faba bean is a widely adapted and high-yielding legume cultivated for its protein-rich seeds. However, the seeds accumulate the anti-nutritional pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in the 400 million individuals genetically predisposed by a deficiency in glucose-6-phosphate dehydrogenase. Here, we identify the first enzyme associated with vicine and convicine biosynthesis, which we name VC1. We show that VC1 co-locates with the major QTL for vicine and convicine content and that the expression of VC1 correlates highly with vicine content across tissues. We also show that low-vicine varieties express a version of VC1 carrying a small, frame-shift insertion, and that overexpression of wild-type VC1 leads to an increase in vicine levels. VC1 encodes a functional GTP cyclohydrolase II, an enzyme normally involved in riboflavin biosynthesis from the purine GTP. Through feeding studies, we demonstrate that GTP is a precursor of vicine both in faba bean and in the distantly related plant bitter gourd. Our results reveal an unexpected biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free from these anti-nutrients, providing a safe and sustainable source of dietary protein.Non peer reviewe

Eliminating vicine and convicine, the main anti-nutritional factors restricting faba bean usage

Background: Faba bean (Vicia faba L.) seeds are an excellent source of plant-based protein. In spite of the vast nutritional and environmental benefits provided by faba bean cultivation, its use as a food crop has been restricted, primarily due to the presence of the pyrimidine glycosides vicine and convicine (v-c). Ingestion of v-c can cause favism in individuals with a genetically inherited deficiency in glucose-6-phosphate dehydrogenase (G6PD). In monogastric animals, v-c can cause decreased feeding efficiency. The elimination of these glucosides is a goal of most faba bean breeding programs worldwide. Scope and approach: Our review focuses on the current genetic, molecular and biochemical knowledge surrounding the accumulation of v-c in faba beans. The gap between the current knowledge and what remains unknown is discussed. This review also explores historical and obscure information on v-c in faba bean. Key findings and conclusions: A low-v-c faba bean line was identified in the 1980s and this trait has been introduced into several modern cultivars. It has been shown that low-v-c faba beans are safe for G6PD-deficient individuals. A robust molecular marker is now available for marker-assisted breeding to reduce levels of v-c. The biosynthetic pathway of v-c is not yet understood and is currently under investigation. An international coordinated effort, led by the authors of this paper, is making progress towards full elucidation of the pathway. Further efforts in this direction could lead to lower levels of these compounds than the current low v-c genotypes offer, perhaps even complete elimination.Peer reviewe

VC1 catalyses a key step in the biosynthesis of vicine in faba bean

Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds1. However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals2. Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients

The giant diploid faba genome unlocks variation in a global protein crop

Publisher Copyright: © 2023, The Author(s).Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.Peer reviewe

Phototropin interaction studies using bimolecular fluorescence complementation and yeast two-hybrid system

Rośliny zależą od światła i ze względu na brak zdolności przemieszczania się muszą na bieżąco reagować i dostosowywać się do wahań jego ilości oraz jakości. Możliwe jest to dzięki wyspecjalizowanym fotoreceptorom absorbującym światło o ściśle określonej długości. Złożona sieć przekazu sygnału, na której początku znajdują się białka sensorowe, uruchamia i reguluje zmiany morfogenetyczne, umożliwia optymalizację procesu fotosyntezy oraz indukcję odpowiedzi na stres. U Arabidopsis thaliana fototropiny 1 i 2, będące receptorami światła niebieskiego, pośredniczą w fototropizmie, otwieraniu aparatów szparkowych, pozycjonowaniu liści oraz ruchu chloroplastów. Realizacja tych procesów możliwa jest dzięki oddziaływaniu fotoreceptorów z białkami i uruchomieniu kaskady przekazywaniu informacji o absorbowanym bodźcu – kwantach światła niebieskiego. Białka zaangażowane w kontrolę ruchów chloroplastów znajdujące się poniżej fototropin w ścieżce przekazywania sygnału nie są znane. Celem niniejszej pracy było zbadanie wzajemnych oddziaływań między fototropinami, oraz między tymi fotoreceptorami a kinazą At5g19150 o nieznanej funkcji. Do badania tych oddziaływań zastosowano technikę dwucząsteczkowej komplementacji fluorescencji. Oddziaływania pomiędzy fototropinami sprawdzono dodatkowo metodą dwuhybrydowego systemu drożdżowego. Opisano procedurę wykonania konstruktów fuzyjnych do obu analiz, tworzenia wektorów oraz testów interakcji pomiędzy białkami. Zademonstrowano tworzenie homo- i heterodimerów fototropin oraz oddziaływanie fototropiny 2 z białkiem At5G19150. Wykazano brak zależności procesu dimeryzacji receptorów od światła oraz zidentyfikowano lokalizację komórkową wszystkich badanych białek oraz tworzonych przez nie kompleksów.Plants depend on light. Due to the inability to move they have to react and adapt to fluctuations in the light quantity and quality in real time. This is enabled with specialized photoreceptors which absorb light at a well-defined wavelength. Complex network of signal transmission, with sensory proteins on the top, activates and regulates morphogenetic changes, optimizes photosynthesis and triggers the stress responses. In Arabidopsis thaliana blue light receptors called phototropins mediate phototropism, stomatal opening, leaf positioning and chloroplasts movement. Those processes are caused by photoreceptor interactions with other proteins, which launches stimulus transduction cascade triggered by the blue light quanta absorption. Proteins below phototropins in the signal transmission pathway involved in the control of chloroplast movements are not known. The aim of this study was to investigate the interactions between phototropins, and between phototropins and At5g19150 kinase of unknown function. To study these interactions bimolecular fluorescence complementation technique was used. Phototropin interactions were tested in the yeast two-hybrid system. The fusion constructs implementation, the test vectors creation and the protein interaction tests were described. Formation homo- and heterodimers of phototropins, and interaction of phototropin 2 and At5G19150 protein were shown. The results demonstrate no light-dependence of receptor dimerization process. Additionally, cellular localization of all studied proteins and complexes was identified

The Lotus japonicus AFB6 Gene Is Involved in the Auxin Dependent Root Developmental Program

Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N2-fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6. A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene