Forgotten Gems: Exploring the Untapped Benefits of Underutilized Legumes in Agriculture, Nutrition, and Environmental Sustainability.

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.

Starch phosphorylation regulates starch granule morphological homogeneity in Arabidopsis thaliana.

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.

Gradual Analytics of Starch-Interacting Proteins Revealed the Involvement of Starch-Phosphorylating Enzymes during Synthesis of Storage Starch in Potato (Solanum tuberosum L.) Tubers.

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.

Glucan, water dikinase (GWD) penetrates the starch granule surface and introduces C6 phosphate in the vicinity of branching points.

Starch phosphorylation mediated by α-glucan, water dikinase is an integral part of starch metabolism. So far however, it is not fully understood. For getting deeper insights, several in vitro assays and intensive mass spectrometry analyses were performed. Such analyses allowed us to determine the phosphorylation position within the amylopectin in detail. Thus, unique features of the starch structure and GWD action were correlated. Therefore, recombinant potato GWD (Solanum tuberosum L.; StGWD) was used for detailed analyses of the phosphorylation pattern of various starches. Additionally, oil palm (Elaeis guineensis Jacq.; EgGWD) GWD was cloned and characterized, representing the first characterization of GWD of a monocot species. The distribution patterns of single phosphorylated glucan chains catalyzed by both GWDs were compared. The phosphorylation distribution patterns of both GWDs varied for different starches. It was proven that GWD phosphorylates different positions within the amylopectin of native starch granules. GWD enters the starch granule surface and phosphorylates the glucosyl units in the proximity of branching points to convert the highly ordered glucan chains into a less ordered state and to render them accessible for the downstream acting hydrolases. This enables deciphering the GWD actions and the related structural properties of starch granules.

Customizing Starch Properties: A Review of Starch Modifications and Their Applications.

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.

Transcriptomic analysis of mesocarp tissue during fruit development of the oil palm revealed specific isozymes related to starch metabolism that control oil yield.

The oil palm (Elaeis guineensis Jacq.) produces a large amount of oil from the fruit. However, increasing the oil production in this fruit is still challenging. A recent study has shown that starch metabolism is essential for oil synthesis in fruit-producing species. Therefore, the transcriptomic analysis by RNA-seq was performed to observe gene expression alteration related to starch metabolism genes throughout the maturity stages of oil palm fruit with different oil yields. Gene expression profiles were examined with three different oil yields group (low, medium, and high) at six fruit development phases (4, 8, 12, 16, 20, and 22 weeks after pollination). We successfully identified and analyzed differentially expressed genes in oil palm mesocarps during development. The results showed that the transcriptome profile for each developmental phase was unique. Sucrose flux to the mesocarp tissue, rapid starch turnover, and high glycolytic activity have been identified as critical factors for oil production in oil palms. For starch metabolism and the glycolytic pathway, we identified specific gene expressions of enzyme isoforms (isozymes) that correlated with oil production, which may determine the oil content. This study provides valuable information for creating new high-oil-yielding palm varieties via breeding programs or genome editing approaches.

Nutritional and pharmacological potentials of orphan legumes: Subfamily faboideae.

Legumes are a major food crop in many developing nations. However, orphan or underutilized legumes are domesticated legumes that have valuable properties but are less significant than main legumes due to use and supply restrictions. Compared to other major legumes, they are better suited to harsh soil and climate conditions, and their great tolerance to abiotic environmental circumstances like drought can help to lessen the strains brought on by climate change. Despite this, their economic significance in international markets is relatively minimal. This article is aimed at carrying out a comprehensive review of the nutritional and pharmacological benefits of orphan legumes from eight genera in the sub-family Faboidea, namely Psophocarpus Neck. ex DC., Tylosema (Schweinf.) Torre Hillc., Vigna Savi., Vicia L., Baphia Afzel. ex G. Lodd., Mucuna Adans, Indigofera L. and Macrotyloma (Wight & Arn.) Verdc, and the phytoconstituents that have been isolated and characterized from these plants. A literature search was conducted using PubMed, Google Scholar, and Science Direct for articles that have previously reported the relevance of underutilized legumes. The International Union for Conservation of Nature (IUCN) red list of threatened species was also conducted for the status of the species. References were scrutinized and citation searches were performed on the study. The review showed that many underutilized legumes have a lot of untapped potential in terms of their nutritional and pharmacological activities. The phytoconstituents from plants in the subfamily Faboideae could serve as lead compounds for drug discovery for the treatment of a variety of disorders, indicating the need to explore these plant species.

More than the main structural genes: Regulation of resistant starch formation in rice endosperm and its potential application.

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.

The Plastidial Glucan Phosphorylase Affects the Maltooligosaccharide Metabolism in Parenchyma Cells of Potato (Solanum tuberosum L.) Tuber Discs.

Maltodextrin metabolism is thought to be involved in both starch initiation and degradation. In this study, potato tuber discs from transgenic lines containing antisense constructs against the plastidial and cytosolic isoforms of α-glucan phosphorylase and phosphoglucomutase were used to evaluate their influences on the conversion of externally supplied glucose-1-phosphate into soluble maltodextrins, as compared to wild-type potato tubers (Solanum tuberosum L. cv. Desiree). Relative maltodextrin amounts analyzed by capillary electrophoresis with laser-induced fluorescence revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30, as opposed to tubers repressing the plastidial glucan phosphorylase. The results presented here support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate-independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucosyl polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.

Starch parameters and short-term temperature fluctuations - Important but not yet in focus?

Plants are regularly challenged by unfavorable environmental conditions. As climate change continues, adverse situations such as drought, heat, and cold are expected to increase and become more severe. Most starchy crops are affected by such stresses. In recent years, researchers have made many new discoveries about starch metabolism in general and also on granule structure, including effects on starch following longer-term temperature stresses. However, in this study, we focus on short-term temperature stress on storage starch granule properties. Here our knowledge is less and it is likely that also short-term temperature stresses can affect various starch parameters. Therefore, we see a need for this type of analysis and discuss the matter in more detail and we conclude that a deeper knowledge particularly of starch granule parameters could allow targeted breeding of cultivars that exhibit different starch characteristics as a result of short-term stress. For these reasons, we are convinced that more comprehensive research on the effects of short-term temperature stress on starch granule characteristics is important, necessary, and timely.

Dpe2/phs1 revealed unique starch metabolism with three distinct phases characterized by different starch granule numbers per chloroplast, allowing insights into the control mechanism of granule number regulation by gene co-regulation and metabolic profiling.

An Arabidopsis mutant lacking both the cytosolic Disproportionating enzyme 2 (DPE2) and the plastidial glucan Phosphorylase 1 (PHS1) revealed a unique starch metabolism. Dpe2/phs1 has been reported to have only one starch granule number per chloroplast when grown under diurnal rhythm. For this study, we analyzed dpe2/phs1 in details following the mutant development, and found that it showed three distinct periods of granule numbers per chloroplast, while there was no obvious change observed in Col-0. In young plants, the starch granule number was similar to that in Col-0 at first, and then decreased significantly, down to one or no granule per chloroplast, followed by an increase in the granule number. Thus, in dpe2/phs1, control over the starch granule number is impaired, but it is not defective in starch granule initiation. The data also indicate that the granule number is not fixed, and is regulated throughout plant growth. Furthermore, the chloroplasts revealed alterations during these three periods, with a partially strong aberrant morphology in the middle phase. Interestingly, the unique metabolism was perpetuated when starch degradation was further impaired through an additional lack of Isoamylase 3 (ISA3) or Starch excess 4 (SEX4). Transcriptomic studies and metabolic profiling revealed the co-regulation of starch metabolism-related genes and a clear metabolic separation between the periods. Most senescence-induced genes were found to be up-regulated more than twice in the starch-less mature leaves. Thus, dpe2/phs1 is a unique plant material source, with which we may study starch granule number regulation to obtain a more detailed understanding.

LIKE EARLY STARVATION 1 alters the glucan structures at the starch granule surface and thereby influences the action of both starch-synthesizing and starch-degrading enzymes.

For starch metabolism to take place correctly, various enzymes and proteins acting on the starch granule surface are crucial. Recently, two non-catalytic starch-binding proteins, pivotal for normal starch turnover in Arabidopsis leaves, namely, EARLY STARVATION 1 (ESV1) and its homolog LIKE EARLY STARVATION 1 (LESV), have been identified. Both share nearly 38% sequence homology. As ESV1 has been found to influence glucan phosphorylation via two starch-related dikinases, α-glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD), through modulating the surface glucan structures of the starch granules and thus affecting starch degradation, we assess the impact of its homolog LESV on starch metabolism. Thus, the 65-kDa recombinant protein LESV and the 50-kDa ESV1 were analyzed regarding their influence on the action of GWD and PWD on the surface of the starch granules. We included starches from various sources and additionally assessed the effect of these non-enzymatic proteins on other starch-related enzymes, such as starch synthases (SSI and SSIII), starch phosphorylases (PHS1), isoamylase and ß-amylase. The data obtained indicate that starch phosphorylation, hydrolyses and synthesis were affected by LESV and ESV1. Furthermore, incubation with LESV and ESV1 together exerted an additive effect on starch phosphorylation. In addition, a stable alteration of the glucan structures at the starch granule surface following treatment with LESV and ESV1 was observed. Here, we discuss all the observed changes that point to modifications in the glucan structures at the surface of the native starch granules and present a model to explain the existing processes.

Indication that starch and sucrose are biomarkers for oil yield in oil palm (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis Jacq.) is the most productive oil-producing crop per hectare of land. The oil that accumulates in the mesocarp tissue of the fruit is the highest observed among fruit-producing plants. A comparative analysis between high-, medium-, and low-yielding oil palms, particularly during fruit development, revealed unique characteristics. Metabolomics analysis was able to distinguish accumulation patterns defining of the various developmental stages and oil yield. Interestingly, high- and medium-yielding oil palms exhibited substantially increased sucrose levels compared to low-yielding palms. In addition, parameters such as starch granule morphology, granule size, total starch content, and starch chain length distribution (CLD) differed significantly among the oil yield categories with a clear correlation between oil yield and various starch parameters. These results provide new insights into carbohydrate and starch metabolism for biosynthesis of oil palm fruits, indicating that starch and sucrose can be used as novel, easy-to-analyze, and reliable biomarker for oil yield.

Carbon pathways during transitory starch degradation in Arabidopsis differentially affect the starch granule number and morphology in the dpe2/phs1 mutant background.

The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation.

A review of starch, a unique biopolymer - Structure, metabolism and in planta modifications.

Starch is a complex carbohydrate polymer produced by plants and especially by crops in huge amounts. It consists of amylose and amylopectin, which have α-1,4- and α-1,6-linked glucose units. Despite this simple chemistry, the entire starch metabolism is complex, containing various (iso)enzymes/proteins. However, whose interplay is still not yet fully understood. Starch is essential for humans and animals as a source of nutrition and energy. Nowadays, starch is also commonly used in non-food industrial sectors for a variety of purposes. However, native starches do not always satisfy the needs of a wide range of (industrial) applications. This review summarizes the structural properties of starch, analytical methods for starch characterization, and in planta starch modifications.

Starch Granule Size and Morphology of Arabidopsis thaliana Starch-Related Mutants Analyzed during Diurnal Rhythm and Development.

Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis.

Effect of Short-Term Cold Treatment on Carbohydrate Metabolism in Potato Leaves.

Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 °C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3-6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the α-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.

Starch Granules in Arabidopsis thaliana Mesophyll and Guard Cells Show Similar Morphology but Differences in Size and Number.

Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.

Starch granule initiation in Arabidopsis thaliana chloroplasts.

The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.

Starch-A complex and undeciphered biopolymer.

Starch is a natural storage carbohydrate in plants and algae. It consists of two relatively simple homo-biopolymers, amylopectin and amylose, with only α-1,4 and α-1,6 linked glucosyl units. Starch is an essential source of nutrition and animal food, as well as an important raw material for industry. However, despite increasing knowledge, detailed information about its structure and turnover are largely lacking. In the last decades, most data were generated using bulk experiments, a method which obviously presents limitations regarding a deeper understanding of the starch metabolism. Here, we discuss some unavoidable questions arising from the existing data. We focus on a few examples related to starch biosynthesis, degradation, and structure - where these limitations strongly emerge. Closing these knowledge gaps will also be extremely important for taking the necessary steps in order to set up starch-providing crops for the challenges of the ongoing climate changes, as well as for increasing the usability of starches for industrial applications by biotechnology.