This study examines how post-transcriptional gene silencing of STARCH BRANCHING ENZYME 1 (SBE1) and GLUCAN WATER DIKINASE 1 (GWD1) affects the structure and properties of potato tuber starch. Silencing of either gene individually or simultaneously altered starch chemistry physical properties. Repression of StGWD1 reduced phosphate content, while repression of StSBE1 increased it. The phosphate content of starch isolated from plants where both genes were repressed was increased compared to StGWD1 repressed lines, but lower than both the SBE1 repressed lines and the untransformed control. Constituent chain lengths of starches from all lines were altered, and amylose content was increased in the gwd1 and sbe1/gwd1 double repressed lines, which also accumulated small numbers of lobed starch granules. Pasting properties were also affected, with starch from StSBE1-repressed lines demonstrating increased peak and trough viscosities and gwd1 lines showing decreased peak and trough viscosities, compared with the control. Peak and trough viscosities were lowest in the sbe1/gwd1 repressed lines. We believe that these data demonstrate that alterations in starch phosphate influence the degree of branching within starch and offer a novel in planta strategy for optimizing the industrial properties of potato storage starch.

Lumichrome, a novel bacterial molecule derived from Sinorhizobium meliloti, plays a crucial role in enhancing plant growth, particularly by increasing photosynthesis and influencing carbon metabolism. Through its impact on gene expression, it has been found to promote turgor-driven growth and ethylene-related delay leaf senescence. In line with previous studies, the current study revealed that treating Arabidopsis thaliana with lumichrome suggested the activation of jasmonate-related signals that delayed leaf senescence. Decreased AOC2 levels, together with the high levels of ABR protein, suggested delayed light and dark-induced leaf senescence. This, along with an increase in chlorophyll content, may possibly be attributed to the enhancement of optimal light absorption, energy conversion, and electron transfer capacity of active PSII reactions. In addition, the increased expression of MPH2 and ATPD subunits might have contributed to a better PSII repair, maintenance of proper photosynthetic function under changing light, and increased ATP synthase activity, ultimately resulting in higher photosynthesis efficiency. Moreover, the increased expression of TRX-M1, TRX-M4, plastid PRX-IIE, and cytosolic APX1 proteins could have improved the ability of treated plants to counteract ROS at PSII and PSI, leading to a better CO2 assimilation rate and plant growth. Subsequently, increased FBA8 could have directed the fixed carbon partitioning into the cytosolic glycolysis pathway. Altogether, these changes may have contributed to increased growth through higher levels of sucrose and reducing sugars.

COPYRIGHT © 2023 Lloyd, Wilhelm, Sharma, Kossmann and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 30 January 2023 DOI 10.3389/fpls.2023.1147930

Abstract Trehalose is a non‐reducing disaccharide widely distributed in nature. The trehalose biosynthetic intermediate, trehalose 6‐phosphate (Tre6P) is an essential regulatory and signaling molecule involved in both regulation of carbon metabolism and photosynthesis. To investigate the effect of altered trehalose synthesis on sucrose accumulation in sugarcane (Saccharum spp. hybrid), we independently overexpressed the Escherichia coli otsA (trehalose‐6‐phosphate synthase; TPS) and otsB (trehalose‐6‐phosphate phosphatase; TPP) genes and additionally partially silenced native TPS expression. In mature cane, sucrose levels in the otsA transgenic plants were lowered, whereas sucrose levels in the otsB transgenic plants were increased. Partial silencing of TPS expression in sugarcane transformed with a TPS‐targeted microRNA recombinant construct was confirmed in leaf and mature internode tissue of transgenic plants. Most of the silencing transgenic lines accumulated trehalose at lower levels than the wild‐type (WT) plants. The immature stalk tissue of these transgenic lines had lower levels of glucose and fructose, whereas the mature internode tissue had lower sucrose and glucose levels, when compared with the WT. Furthermore, various minor metabolites and sugars were detected in the sugarcane plants, which mostly decreased as the stalk tissue of the cane matured. The results demonstrate that manipulation of Tre6P/trehalose metabolism has the potential to modify the profile of soluble sugars accumulated in sugarcane stems.

SPECIALTY GRAND CHALLENGE article Front. Plant Sci., 06 September 2021 | https://doi.org/10.3389/fpls.2021.728328

The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation - glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) - were disrupted, we examined if endogenous polyglucan degradation affects cell size. It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ΔmalP mutant cells did, however, accumulate increased DnaA amounts at late growth stages indicating a potential effect on DNA replication. Replication run-out experiments demonstrated that this was indeed the case, and that DNA replication was also affected in the other mutants. Bacteria with a disruption in glgX accumulated glycogen and protein inclusion bodies that coincided with each other at inter-nucleoid and polar regions.

Ice particle activation and evolution have important atmospheric implications for cloud formation, initiation of precipitation and radiative interactions. The initial formation of atmospheric ice by heterogeneous ice nucleation requires the presence of a nucleating seed, an ice-nucleating particle (INP), to facilitate its first emergence. Unfortunately, only a few long-term measurements of INPs exist, and as a result, knowledge about geographic and seasonal variations of INP concentrations is sparse. Here we present data from nearly 2 years of INP measurements from four stations in different regions of the world: the Amazon (Brazil), the Caribbean (Martinique), central Europe (Germany) and the Arctic (Svalbard). The sites feature diverse geographical climates and ecosystems that are associated with dissimilar transport patterns, aerosol characteristics and levels of anthropogenic impact (ranging from near pristine to mostly rural). Interestingly, observed INP concentrations, which represent measurements in the deposition and condensation freezing modes, do not differ greatly from site to site but usually fall well within the same order of magnitude. Moreover, short-term variability overwhelms all long-term trends and/or seasonality in the INP concentration at all locations. An analysis of the frequency distributions of INP concentrations suggests that INPs tend to be well mixed and reflective of large-scale air mass movements. No universal physical or chemical parameter could be identified to be a causal link driving INP climatology, highlighting the complex nature of the ice nucleation process. Amazonian INP concentrations were mostly unaffected by the biomass burning season, even though aerosol concentrations increase by a factor of 10 from the wet to dry season. Caribbean INPs were positively correlated to parameters related to transported mineral dust, which is known to increase during the Northern Hemisphere summer. A wind sector analysis revealed the absence of an anthropogenic impact on average INP concentrations at the site in central Europe. Likewise, no Arctic haze influence was observed on INPs at the Arctic site, where low concentrations were generally measured. We consider the collected data to be a unique resource for the community that illustrates some of the challenges and knowledge gaps of the field in general, while specifically highlighting the need for more long-term observations of INPs worldwide.

ObjectiveCurrent global trends on natural therapeutics suggest an increasing market interest toward the use and discovery of new plant-derived therapeutic compounds, often referred to as traditional medicine (TM). The Cannabis industry is currently one such focal area receiving attention, owing to the occurrence of phytocannabinoids (pCBs) which have shown promise in health-promotion and disease prevention. However, the occurrence of pCBs in other plant species are often overlooked and rarely studied.Leonotis leonurus (L.) R. Br. is endemic to South Africa with a rich history of use in TM practices amongst indigenous people and, has been recorded to induce mild psychoactive effects akin to Cannabis. While the leaves have been well-reported to contain therapeutic phytochemicals, little information exists on the flowers. Consequently, as part of a larger research venture, we targeted the flowers of L. leonurus for the identification of potential pCB or pCB-like compounds.ResultsFlower extracts were separated and analyzed using high performance thin layer chromatography (HPTLC). A single pCB candidate was isolated from HPTLC plates and, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we could successfully group this compound as a fatty amide and tentatively identified as 7,10,13,16-Docosatetraenoylethanolamine (adrenoyl-EA), a known bioactive compound.

Background: Fruits and vegetables contain significant amounts of biologically active phytochemicals (such as polyphenols, glucosinolates, phytoestrogens, and carotenoids, amongst others), which have associated with human health and nutrition. Numerous bio-fortification strategies are employed to enhance the nutritional profile of plant-based foods to address and minimize the severe outcomes of malnutrition.Methods: Using an established high light-induced bio-fortification strategy, we aimed to augment the accumulation of health-promoting phytochemicals in a selection of Brassica micro-greens (kale and radish). High throughput tandem mass spectrometry was used to identify the differential accumulation of phytochemicals and subsequently determined their antioxidant capacity. Using a classical DNA protection assay, we demonstrated that human genomic DNA could be protected from oxidative stress.Results: We report here on the potential link between the increased phytochemicals, total antioxidant, capacity and potential consequent role in human DNA protection.Conclusion: Bio-fortification implemented as a future strategy could enhance the phytochemical profile and consequent antioxidant potential for the development of functional foods and food supplements.Keywords: antioxidant, bio-fortification, Brassica, DNA protection, high light, micro-greens, phytochemicals

The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.

Starch is a plant storage polyglucan that accumulates in plastids. It is composed of two polymers, amylose and amylopectin, with different structures and plays several roles in helping to determine plant yield. In leaves, it acts as a buffer for night time carbon starvation. Genetically altered plants that cannot synthesize or degrade starch efficiently often grow poorly. There have been a number of successful approaches to manipulate leaf starch metabolism that has resulted in increased growth and yield. Its degradation is also a source of sugars that can help alleviate abiotic stress. In edible parts of plants, starch often makes up the majority of the dry weight constituting much of the calorific value of food and feed. Increasing starch in these organs can increase this as well as increasing yield. Enzymes involved in starch metabolism are well known, and there has been much research analyzing their functions in starch synthesis and degradation, as well as genetic and posttranslational regulatory mechanisms affecting them. In this mini review, we examine work on this topic and discuss future directions that could be used to manipulate this metabolite for improved yield.

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.

Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

Drought is a serious agronomic problem, and urgent attention to overcome drought stress is vital to eradicate or minimize its effects on crop production. Random induction of genomic mutation is a technique that can enhance genetic diversity leading to useful traits such as enhanced drought tolerance. In this study, sugarcane callus was exposed to different concentrations of the chemical mutagen, ethyl methanesulfonate (EMS). Concentrations of 20 mM and lower were identified as useful to induce genomic mutations without compromising in vitro sugarcane plant regeneration abilities. Furthermore, sugarcane callus was exposed to varying concentrations of polyethylene glycol (PEG) for different time periods in order to identify a suitable in vitro osmotic selection regime to simulate drought stress in vitro. The optimal in vitro osmotic selection treatment was identified as callus exposed to 20% (w/v) PEG6000 for 8 weeks, followed by a 2 weeks osmotic recovery period without PEG and ending with a further 8 week PEG selection period during somatic embryo regenerations. Sugarcane callus from the NCo310 cultivar was subsequently mutagenized with 16 mM EMS and in vitro selected on 20% (w/v) PEG6000, which resulted in the survival of 18 plantlets. These in vitro selected lines were subjected to preliminary greenhouse pot trials to confirm drought tolerance. Pot trials identified seven lines that outlived NCo310 control plants. In addition, when re-watered after the drought stress period, plants from one mutant line recovered and were able to form new shoots. The results from this study indicate, therefore, that EMS mutagenesis and in vitro selection for osmotic pressure using PEG can be successfully applied to cultivate sugarcane plants with improved morphological and physiological responses to water stress.

Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4weeks to select herbicide resistant calli clones on 10mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil.

Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis.

Background : The term ‘functional food’ received recognition by established sustenance entities in Japan, Europe and the United States (FOSHU, FUFOSE and FDA respectively) despite its existence since the 20 th century (Columbus, Captain’s log, Santa Maria). These entities acknowledge that functional foods are pivotal in promoting human health, beyond basic nutritional requirements, provided it is grounded by scientific and medical credibility. Globally, non-communicable diseases (cancer, diabetes, cardiovascular-, respiratory- and malnutrition-related diseases) are resulting in increased mortality statistics, often a result of inadequate intake of dietary nutrients (malnutrition). Functional foods contain significant amounts of bio-active compounds (such as polyphenols, glucosinolates, phytoestrogens and carotenoids, amongst others) which have been associated with non-communicable disease prevention/treatment. Collectively termed phytochemicals, these bioactive compounds form part of a plant’s secondary metabolite profile and usually accumulate as part of global stress response (both abiotic and biotic) mechanisms. Brassica vegetables and leafy greens (broccoli, brussel sprouts, rocket, kale and mustard, amongst others) are consumed globally, as mature plants/plant parts, due to their association with health promoting benefits and are generally accepted as functional foods. Although plants accumulate phytochemicals ubiquitously, micro-greens (immature plants with 2-4 true leaves) are considered nutritionally superior as they hyper-accumulate phytochemicals (up to 1000 times more than mature plants). We believe that Brassica micro-greens are a potential target for bio-fortification (enhancement of the phytochemical profile) through the use of abiotic environmental stress stimuli. Consequently, human health beneficial micro-green based products could be developed in the functional food sector for their use in prevention of non-communicable diseases. Objective : Establishing a high light-induced bio-fortification strategy to augment the accumulation of bio-active compounds in Brassica micro-greens (rocket, mustard and kale), purposed toward development of a ‘designer’ micro-green melange (functional food product) containing a diverse range of bio-active (disease preventative) compounds. Methods : A selection of Brassica leafy greens (wild rocket, mustard greens and kale) were propagated under high light to micro-green stage (two to four true leaves). Micro-greens were harvested and analysed as either (i) fresh or (ii) lyophilized material. Analyses of extracted phenolic compounds were conducted with high-throughput LC-MS/MS to quantify and/or identify accumulated compounds. Further, antioxidant capacity was determined using the Cu + -reducing CUPRAC method. Extracts from both fresh and lyophilized micro-green tissue were assiduously tested on human breast cancer cell lines (MCF-7) to derive individual and combined anti-cancer potential. Results : The application of high light stimulated micro-greens to accumulate significant amounts of known phytochemicals (quercetin, isorhamnetin) when compared to those grown under a normal light regime. Further, previously undocumented phytochemicals (resveratrol, catechin, epicatechin and kaempferol, amongst others) also accumulated in micro-greens to adequate concentrations required for anti-cancer activity. Plant extracts from bio-fortified micro-greens (derived from either fresh or lyophilized tissue) displayed increased anti-oxidant capacity (up to 3-fold, when compared to normal), a key component in cancer cell apoptosis. Conclusion : Innovative strategies to enhance phytochemical profiles of plants is currently a major focus of the world health organization (WHO), given the significant mortality rates imparted by malnutrition. We describe here a simple method of achieving this goal by environmentally manipulating (using high light) the phytochemical accumulation in a range of well-known Brassica species, at micro-green stage. Fresh produce is not always easily accessible and/or difficult to distribute. We therefore aim to further this idea into product development (using lyophilized material), which could preserve the health beneficial compounds and be readily distributable. Results described here are either for fresh micro-greens or lyophilized material. Outcomes thus far have indicated (i) unambiguous increases in polyphenolic content by high light-stimulation, (ii) accumulation of previously undocumented polyphenolics and, (iii) consequent increases in antioxidant capacity. Lyophilisation did not alter the antioxidant potential. Ongoing in vitro investigations are being conducted to determine the chemoprotective effects of lyophilized microgreen extracts on a range of cancer cell lines, and we propose that this system of phytochemical enhancement could potentially generate a non-genetically modified, bio-fortified product (either in fresh or lyophilized form) to be used as a functional food/food product featuring in preventative strategies regarding non-communicable diseases. Keywords : Brassica, Micro-greens, Non-communicable diseases, Phytochemicals How to cite this abstract: Loedolff B., Stander M., Peters S., Kossmann J.; FFC’s 22 nd International Conference; Boston, MA, USA; Organized by FFC and BIDMC/ Harvard Medical School Teaching Hospital; Volume 1; Supplement 1: 176-179