Starch granule initiation involves the extension of maltooligosaccharide primers by glucosyltransferases. STARCH SYNTHASE 4 (SS4) plays a central role in almost all examined plant species, while the plastidial PHOSPHORYLASE 1 (Pho1) also plays an important role in some species, including rice and wheat. In Arabidopsis, an additional enzymatically inactive homolog of SS4, STARCH SYNTHASE 5 (SS5) contributes to starch granule initiation. To elucidate the mechanism of starch granule initiation in potato tubers, we used CRISPR/Cas9 to generate ss4, ss5, and pho1a knockout mutants in the commercial tetraploid 'Clearwater Russet', to systematically investigate their contribution to granule initiation. In ss4 and ss5 tubers, starch granule size and morphology were unaltered relative to the wild type, suggesting that SS4 and SS5 are dispensable for normal granule initiation in potato tubers. In contrast, pho1a tubers had compound starch granules that arose from multiple initiations, greatly reduced granule size, and highly variable granule morphologies. Affinity pull-down to find Pho1a interaction partners identified LIKE EARLY STARVATION (LESV), although yeast 2-hybrid assays did not show direct protein-protein binding. When expressed alone in Nicotiana benthamiana leaves, Pho1a located to the chloroplast stroma, but when expressed alongside LESV, both proteins co-located on starch granules. This co-localisation, alongside the similar accumulation of small starch granules when LESV is knocked out in tubers, suggest a possible functional interaction in planta. These findings position Pho1a as the central glucosyltransferase in starch granule initiation in Clearwater Russet tubers, where it acts together with LESV.

The phosphorylation of the storage carbohydrates, starch and glycogen, is a process that is fundamental to their physicochemical properties and their turnover. Therefore, the interest utilising phosphorylation as a biotechnological tool to customize polysaccharides has risen permanently. Today, the phosphoesterification of both carbohydrate forms is much better understood. In recent years, important new insights have been gained into the molecular mechanism of starch phosphoesterification and its effects. In the following, the current state of knowledge on starch phosphorylation is briefly summarized. In addition, protein structure predictions for GWD are presented and considered for the first time in the context of recently published analyses of starch phosphorylation, which have opened up novel perspectives on this process. Therefore, we focus on a detailed discussion of the molecular events that occur at the surface of starch granules and enable a revised and in-depth understanding of starch granule phosphorylation.

Digestive enzymes play a crucial role in carbohydrate hydrolysis and subsequent glucose absorption, and their inhibition can contribute to improved glycemic regulation. Legumes, with their inherent enzyme-inhibitory properties, offer a natural approach for achieving this. In this study, accessions of M. uniflorum (Lam.) Verdc, an underutilized legume, were evaluated in vitro for their α-amylase and α-glucosidase inhibitory activities, as well as their protein, amylose, and resistant starch contents. The results revealed significant variation among the accessions. PI 174827 01 SD (IC50 = 23.29 ± 0.01 µg/mL) and PI 173901 01 SD (IC50 = 24.60 ± 0.01 µg/mL) demonstrated strong inhibition of α-amylase and α-glucosidase, respectively. Protein content ranged from 13.81 to 27.08%w/w d.w., with PI 180437 01 SD showing the highest percentage. Total starch content ranged from 27.48 to 54.70%w/w d.w., amylose from 27.05 to 48.13%w/w d.w., and resistant starch from 5.89% to 7.09%w/w d.w., with PI 174827 01 SD exhibiting both higher amylose and resistant starch contents. These findings suggest that M. uniflorum accessions possess enzyme-inhibitory and nutritional components that could be harnessed to develop functional foods, nutraceuticals, and pharmaceuticals for the management of diabetes and obesity.

The widespread colonization of diverse habitats by plants is attributed to their ability to adapt to changing environments through environmental phenotypic plasticity. This flexibility, particularly in carbon turnover, allows plants to adjust their physiology and development. Plants store carbon reserves as a metabolic strategy to overcome adversity, with a variety of isozymes evolving to enhance metabolic plasticity. Among these isoforms, some with entirely new functions have emerged, involved in novel metabolic pathways for carbon storage. Here, we discuss the role of these carbon stores, their impact on plant plasticity, methods by which such metabolic plasticity can be analyzed, and evolutionary aspects that have led to well-characterized as well as less well-known molecular mechanisms underlying carbon storage.

Osmotic shock is the first step of high salt or drought action that involves biochemical and molecular changes during plant response to these unfavorable conditions. Indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp) is the main amide conjugate of auxin in pea (Pisum sativum L.) tissues. Although the exact molecular mechanism of the IAA-Asp action is unknown, this conjugate’s indole-3-acetic acid (IAA)-independent biological activity has been observed during physiological and stress conditions. In this work, we investigated the effect of IAA-Asp alone, as well as in combination with NaCl or polyethylene glycol (PEG) (osmotic shock) on reduced/oxidized glutathione (GSH/GSSG) ratio, activities of enzymes modulating glutathione concentration, protein S-glutathionylation, and IAA homeostasis. We did not observe the hydrolysis of IAA-Asp to IAA in pea seedlings, which, together with other results, suggests that IAA-Asp modulates plant response to abiotic stimuli independently of IAA. Moreover, despite the effect of IAA-Asp on the enzymes responsible for IAA conjugation, no changes in this phytohormone level were visible. Furthermore, 3h plant treatment with IAA-Asp increased the activity of glutathione reductase (GR), which correlates with an elevated GSH/GSSG ratio. On the contrary, more extended (48h) incubation with IAA-Asp diminished the GSH/GSSG ratio and increased the activity of glutathione peroxidase (GPX). IAA-Asp reduced GR activity during salt treatment but did not affect the GSH/GSSG ratio. Similarly, under plant incubation with PEG, IAA-Asp did not change the GSH/GSSG ratio but increased glutathione S-transferase (GST) activity. We also analyzed the effect of IAA-Asp on pea protein S-glutathionylation. Increased S-glutathionylation of heat shock 70 kDa protein (HSP70) was observed after plant treatment with IAA-Asp, PEG, or IAA-Asp combined with PEG. The proteomic analysis also revealed that IAA-Asp diminished S-glutathionylation of lipoxygenase during plant incubation with PEG. Thus, we suggest that IAA-Asp modulates redox status in pea during oxidative stress and under normal physiological conditions.

The plastidial phosphorylase (Pho1 or Phs1; E.C. 2.4.1.1) is a ubiquitous enzyme among plants that catalyzes the formation and degradation of glucans. Although the first report connecting Pho1 with starch metabolism came out > 80 years ago, its precise role is still a matter of debate. In this article, we evaluate the catalytic and regulatory mechanisms of Pho1 in the context of known mechanisms in its animal, fungal, and bacteria homologs. We further discuss recent breakthroughs in understanding Pho1's function in initiating starch granule formation. This role is relevant to both photosynthetic and nonphotosynthetic tissues, as loss of Pho1 affects the regulation of the number of transitory starch granules in Arabidopsis leaves under various metabolic contexts, as well as the number of storage starch granules and/or starch granule morphology in wheat endosperm and potato tubers. Our comparison of phosphorylases across kingdoms reveals several regulatory mechanisms that require further investigation in plants. We also discuss emerging research on Pho1 protein interactions that give rise to other metabolic processes, such as photosynthesis. Overall, these multiple emerging roles of phosphorylase emphasize its importance in plant metabolism and its broad potential as a target for crop improvement.

The plastidial α-glucan phosphorylase (PHS1) can catalyze the elongation and degradation of glucans, but its exact physiological role in plants is not completely deciphered. Many studies have indicated that PHS1 is involved in transitory starch turnover both in photosynthetic tissues and reserve starch accumulation in sink organs, by exerting its effects on the plastidial maltodextrin pools. Recent studies have also established its role in the mobilization of short maltooligosaccharides, thereby assisting in starch granule initiation. Here we report findings from studying four constitutive double knockout mutants related to plastidial maltodextrin metabolism, namely phs1dpe1, phs1ptst2, phs1pgm1, and phs1isa3 of Arabidopsis thaliana. We observed different effects on carbon partitioning in these double mutants. Carbon allocation between starch and sucrose in different double mutants varied with respect to time and light conditions, with significant overall changes in phs1dpe1. Furthermore, we uncovered a potential time-specific function of PHS1 in maltodextrin metabolism. Changes in maltodextrin turnover exerted effects on the starch granule number and size in the double mutants, especially phs1dpe1. We further assessed the double mutants in terms of photosynthetic efficiency and starch parameters, such as internal structure and morphology, in detail. We found that the different photosynthetic parameters in pgm1 and its corresponding double mutant were affected relative to the wild type and phs1. However, other double mutants were not impaired in terms of photosynthetic efficiency despite alterations in their maltooligosaccharide levels.

Starch metabolism in plants involves a complex network of interacting proteins that work together to ensure the efficient synthesis and degradation of starch. These interactions are crucial for regulating the balance between energy storage and release, adapting to the plant's developmental stage and environmental conditions. Several studies have been performed to investigate protein-protein interactions (PPIs) in starch metabolism complexes, yet it remains impossible to unveil all of the PPIs in this highly regulated process. This study uses yeast-two-hybrid (Y2H) screening against the Arabidopsis leaf cDNA library to explore PPIs, focusing on the starch-granule-initiating protein named Protein Targeting to Starch 2 (PTST2, At1g27070) and the protein involved in starch and maltodextrin metabolism, namely, plastidial phosphorylase 1 (PHS1, EC 2.4.1.1). More than 100 positive interactions were sequenced, and we found chloroplastidial proteins to be putative interacting partners of PTST2 and PHS1. Among them, photosynthetic proteins were discovered. These novel interactions could reveal new roles of PTST2 and PHS1 in the connection between starch metabolism and photosynthesis. This dynamic interplay between starch metabolism and other chloroplast functions highlights the importance of starch as both an energy reservoir and a regulatory component in the broader context of plant physiology and adaptation.

In an era dominated by conventional agricultural practices, underutilized legumes termed “Forgotten Gems” represent a reservoir of untapped benefits with the unique opportunity to diversify agricultural landscapes and enhance global food systems. Underutilized crops are resistant to abiotic environmental conditions such as drought and adapt better to harsh soil and climatic conditions. Underutilized legumes are high in protein and secondary metabolites, highlighting their role in providing critical nutrients and correcting nutritional inadequacies. Their ability to increase dietary variety and food security emerges as a critical component of their importance. Compared to mainstream crops, underutilized legumes have been shown to reduce the environmental impact of climate change. Their capacity for nitrogen fixation and positive impact on soil health make them sustainable contributors to biodiversity conservation and environmental balance. This paper identifies challenges and proposes strategic solutions, showcasing the transformative impact of underutilized legumes on agriculture, nutrition, and sustainability. These “Forgotten Gems” should be recognized, integrated into mainstream agricultural practices, and celebrated for their potential to revolutionize global food production while promoting environmental sustainability.

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities.

Glucan phosphorylase is present in plants in two isozymes, namely, a plastidial isoform (PHO1) and a cytosolic isoform (PHO2), and is involved in starch-related carbohydrate metabolism. The aim of this study was to determine whether mutations in the genes encoding glucan phosphorylase caused these plants to have increased resistance to short-term drought. One of the strategies plants use to defend themselves against drought stress is to change their starch content, which may be due to changes in glucan phosphorylase activity. In our greenhouse pot experiment, we used potato leaves from wild-type plants and transgenic mutant lines with reduced expression of genes encoding both PHO isozymes. The plants were exposed to drought or were grown under optimal conditions. A lack of water strongly affected the water saturation deficit (WSD) and leaf protein content. The activity of the plastidial glucan phosphorylase isoform (PHO1) in mutant plants increased under drought stress, in contrast to its activity in wild-type plants. After analyzing several physiological parameters, we found that suppressed expression of the gene encoding one of the subunits of plastidial glucan phosphorylase, PHO1a, resulted in increased tolerance to drought in potatoes.

Previously, in Arabidopsis thaliana, we found atypical spherical starch granules in dpe2ss4 and dpe2phs1ss4. However, the mechanism of such abnormal morphogenesis is still obscure. By tracking starch granule length and thickness with leaf ageing, we reported that the starch granules in dpe2phs1ss4 gradually change to a spherical shape over time. In comparison, Col-0 and the parental line ss4 did not exhibit macroscopic morphological alteration. In this study, firstly, we specify that the additional lack of DPE2 resulted in the gradual alteration of starch granule morphology over time. Similar gradual morphological alterations were also found in dpe2, mex1, and sex4 but not in the other starch degradation-related mutants, such as sex1-8, pwd, and bam3. The gradual alteration of starch morphology can be eliminated by omitting the dark phase, suggesting that the particular impaired starch degradation in dpe2- and mex1-related mutants influences starch morphology. Secondly, we observed that spherical starch morphology generation was accompanied by prominent elevated short glucan chains of amylopectin and an increased amylose proportion. Thirdly, the interplay between soluble starch synthase 2 and branching enzymes was affected and resulted in the formation of spherical starch granules. The resulting spherical starch granules allow for elevated starch synthesis efficiency. Fourthly, the starch phosphate content at the granule surface correlated with the morphology alteration of the starch granules. Herewith, we propose a model that spherical starch granules, accumulated in mutants with a misbalance of the starch degradation pathway, are result of elevated starch synthesis to cope with overloaded carbohydrates.

Carbohydrates are significant components of legumes, and their profiling can provide information about their nutritional value and potential health benefits. This study explores the proximate composition and carbohydrate profiles of accessions of three underutilized legumes, namely, Vigna radiata, Vigna mungo, and Macrotyloma uniflorum. The total starch and soluble sugar are determined. Starch granule morphology and diameter are determined using scanning electron microscopy and chain length distribution of amylopectin is analyzed using Capillary Electrophoresis. All accessions of the legumes vary both in their proximate composition and carbohydrate components. Accessions of M. uniflorum (PI-658594-01-SD) have the highest carbohydrate component (7.6%) while V. radiata (TVR-42) has the least (4.2%). Macrotyloma uniflorum accensions (PI-658594-01-SD, and PI-180437-01-SD) have the highest starch and sucrose contents, while V. mungo accensions (TVM-13, and TVM-11) have the lowest. The glucose and fructose contents are lower than sucrose in all the accessions of the legumes. Macrotyloma uniflorum accensions exhibit larger granules while accessions of Vigna species have the smallest granules. All species accessions exhibit similar amylopectin chain length distribution profiles although accessions of V. mungo slightly differ in their proportion of long and short glucan chains. The three species of underutilized legumes exhibit unique characteristics which make them suitable for consumption and may be exploited as a source of nutraceuticals and pharmaceuticals.

Starch granule morphological homogeneity presents a gap in starch research. Transitory starch granules in wild-type plants are discoid, regardless of species. Notably, while the shape of starch granules can differ among mutants, it typically remains homogeneous within a genotype. We found an Arabidopsis thaliana mutant, dpe2sex4, lacking both the cytosolic disproportionating enzyme 2 (DPE2) and glucan phosphatase SEX4, showing an unprecedented bimodal starch granule diameter distribution when grown under a light/dark rhythm. dpe2sex4 contained 2 types of starch granules: large granules and small granules. In contrast to the double starch initiation in wheat (Triticum aestivum) endosperm, where A-type granules are initiated first and B-type granules are initiated later, dpe2sex4 small and large granules developed simultaneously in the same chloroplast. Compared with the large granules, the small granules had more branched amylopectin and less surface starch-phosphate, thus having a more compact structure that may hinder starch synthesis. During plant aging, the small granules barely grew. In in vitro experiments, fewer glucosyl residues were incorporated in small granules. Under continuous light, dpe2sex4 starch granules were morphologically homogeneous. Omitting the dark phase after a 2-wk light/dark cycle by moving plants into continuous light also reduced morphological variance between these 2 types of granules. These data shed light on the impact of starch phosphorylation on starch granule morphology homogeneity.

The widespread colonization of diverse habitats by plants is attributed to their ability to adapt to changing environments through environmental phenotypic plasticity. This flexibility, particularly in carbon turnover, allows plants to adjust their physiology and development. Plants store carbon reserves as a metabolic strategy to overcome adversity, with a variety of isozymes evolving to enhance metabolic plasticity. Among these isoforms, some with entirely new functions have emerged, involved in novel metabolic pathways for carbon storage. Here, we discuss the role of these carbon stores, their impact on plant plasticity, methods by which such metabolic plasticity can be analyzed, and evolutionary aspects that have led to well-characterized as well as less well-known molecular mechanisms underlying carbon storage.

Starch has been a convenient, economically important polymer with substantial applications in the food and processing industry. However, native starches present restricted applications, which hinder their industrial usage. Therefore, modification of starch is carried out to augment the positive characteristics and eliminate the limitations of the native starches. Modifications of starch can result in generating novel polymers with numerous functional and value-added properties that suit the needs of the industry. Here, we summarize the possible starch modifications in planta and outside the plant system (physical, chemical, and enzymatic) and their corresponding applications. In addition, this review will highlight the implications of each starch property adjustment.

In the past decade, research on resistant starch has evoked interest due to the prevention and inhibition of chronic human diseases, such as diabetes, cancer, and obesity. Increasing the amylose content (AC) and resistant starch (RS) has been pivotal in improving the nutritional benefit of rice. However, the exact mechanism of RS formation is complex due to interconnected genetic factors regulating amylose-amylopectin variation. In this review, we discussed the regulatory factors influencing the RS formation centered on the transcription, post-transcriptional, and post-translational processes. Furthermore, we described the developments in RS and AC levels in rice compared with other high RS cereals. Briefly, we enumerated potential applications of high RS mutants in health, medical, and other industries. We contest that the information captured herein can be deployed for marker-assisted breeding and precision breeding techniques through genome editing to improve rice varieties with enhanced RS content.

Underutilized legumes are common crops in developing countries with superior dietary potentials that could be useful sources of protein as well as some phytoconstituents. They are more tolerant of abiotic environmental conditions like drought than the major legumes. This makes them more adapted to harsh soil and climatic conditions, which helps to minimize the pressure brought on by climate change. However, despite their potential, underutilized legumes have been greatly overlooked compared to the major legumes due to supply constraints. Underutilized legumes in the subfamily Caesalpinioideae are better suited for use as animal feeds with little or no value as food for humans, and the extracts and infusions of the different parts of plant species in this subfamily are traditionally used for the treatment of different diseases. In addition, underutilized legumes in this subfamily contain phytoconstituents that are of pharmacological relevance, some of which have been isolated, characterized and evaluated for use in the treatment of a variety of disorders. Therefore, this review describes the medicinal activities of some selected underutilized legumes from five genera in the subfamily Caesalpinioideae as well as their phytoconstituents, which could be exploited as lead compounds for drug discovery.

The complete mechanism behind starch regulation has not been fully characterized. However, significant progress can be achieved through proteomic approaches. In this work, we aimed to characterize the starch-interacting proteins in potato (Solanum tuberosum L. cv. Desiree) tubers under variable circumstances. Starch-interacting proteins were extracted from developing tubers of wild type and transgenic lines containing antisense inhibition of glucan phosphorylases. Further, proteins were separated by SDS-PAGE and characterized through mass spectrometry. Additionally, starch-interacting proteins were analyzed in potato tubers stored at different temperatures. Most of the proteins strongly interacting with the potato starch granules corresponded to proteins involved in starch metabolism. GWD and PWD, two dikinases associated with starch degradation, were consistently found bound to the starch granules. This indicates that their activity is not only restricted to degradation but is also essential during storage starch synthesis. We confirmed the presence of protease inhibitors interacting with the potato starch surface as previously revealed by other authors. Starch interacting protein profiles of transgenic tubers appeared differently from wild type when tubers were stored under different temperatures, indicating a differential expression in response to changing environmental conditions.