Transitory Starch Research Articles

Integration of transcriptomic and proteomic analyses reveals several levels of metabolic regulation in the excess starch and early senescent leaf mutant lses1 in rice

BackgroundThe normal metabolism of transitory starch in leaves plays an important role in ensuring photosynthesis, delaying senescence and maintaining high yield in crops. OsCKI1 (casein kinase I1) plays crucial regulatory roles in multiple important physiological processes, including root development, hormonal signaling and low temperature-treatment adaptive growth in rice; however, its potential role in regulating temporary starch metabolism or premature leaf senescence remains unclear. To reveal the molecular regulatory mechanism of OsCKI1 in rice leaves, physiological, transcriptomic and proteomic analyses of leaves of osckI1 allele mutant lses1 (leaf starch excess and senescence 1) and its wild-type varieties (WT) were performed.ResultsPhenotypic identification and physiological measurements showed that the lses1 mutant exhibited starch excess in the leaves and an obvious leaf tip withering phenotype as well as high ROS and MDA contents, low chlorophyll content and protective enzyme activities compared to WT. The correlation analyses between protein and mRNA abundance are weak or limited. However, the changes of several important genes related to carbohydrate metabolism and apoptosis at the mRNA and protein levels were consistent. The protein-protein interaction (PPI) network might play accessory roles in promoting premature senescence of lses1 leaves. Comprehensive transcriptomic and proteomic analysis indicated that multiple key genes/proteins related to starch and sugar metabolism, apoptosis and ABA signaling exhibited significant differential expression. Abnormal increase in temporary starch was highly correlated with the expression of starch biosynthesis-related genes, which might be the main factor that causes premature leaf senescence and changes in multiple metabolic levels in leaves of lses1. In addition, four proteins associated with ABA accumulation and signaling, and three CKI potential target proteins related to starch biosynthesis were up-regulated in the lses1 mutant, suggesting that LSES1 may affect temporary starch accumulation and premature leaf senescence through phosphorylation crosstalk ABA signaling and starch anabolic pathways.ConclusionThe current study established the high correlation between the changes in physiological characteristics and mRNA and protein expression profiles in lses1 leaves, and emphasized the positive effect of excessive starch on accelerating premature leaf senescence. The expression patterns of genes/proteins related to starch biosynthesis and ABA signaling were analyzed via transcriptomes and proteomes, which provided a novel direction and research basis for the subsequent exploration of the regulation mechanism of temporary starch and apoptosis via LSES1/OsCKI1 in rice.

How Stress Affects Your Budget-Stress Impacts on Starch Metabolism.

Starch is a polysaccharide that is stored to be used in different timescales. Transitory starch is used during nighttime when photosynthesis is unavailable. Long-term starch is stored to support vegetative or reproductive growth, reproduction, or stress responses. Starch is not just a reserve of energy for most plants but also has many other roles, such as promoting rapid stomatal opening, making osmoprotectants, cryoprotectants, scavengers of free radicals and signals, and reverting embolised vessels. Biotic and abiotic stress vary according to their nature, strength, duration, developmental stage of the plant, time of the day, and how gradually they develop. The impact of stress on starch metabolism depends on many factors: how the stress impacts the rate of photosynthesis, the affected organs, how the stress impacts carbon allocation, and the energy requirements involved in response to stress. Under abiotic stresses, starch degradation is usually activated, but starch accumulation may also be observed when growth is inhibited more than photosynthesis. Under biotic stresses, starch is usually accumulated, but the molecular mechanisms involved are largely unknown. In this mini-review, we explore what has been learned about starch metabolism and plant stress responses and discuss the current obstacles to fully understanding their interactions.

BETA-AMYLASE9 is a plastidial nonenzymatic regulator of leaf starch degradation.

β-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the BAM gene family diversified, giving rise to isoforms with different compartmentation and biological activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features including their plastidial localization and lack of measurable α-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants already impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpression of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant’s starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.

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.

Glucan, Water-Dikinase 1 (GWD1), an ideal biotechnological target for potential improving yield and quality in rice.

SummaryThe source–sink relationship determines the overall agronomic performance of rice. Cloning and characterizing key genes involved in the regulation of source and sink dynamics is imperative for improving rice yield. However, few source genes with potential application in rice have been identified. Glucan, Water‐Dikinase 1 (GWD1) is an essential enzyme that plays a pivotal role in the first step of transitory starch degradation in source tissues. In the present study, we successfully generated gwd1 weak mutants by promoter editing using CRISPR/Cas9 system, and also leaf‐dominant overexpression lines of GWD1 driven by Osl2 promoter. Analysis of the gwd1 plants indicated that promoter editing mediated down‐regulation of GWD1 caused no observable effects on rice growth and development, but only mildly modified its grain transparency and seed germination. However, the transgenic pOsl2::GWD1 overexpression lines showed improvements in multiple key traits, including rice yield, grain shape, rice quality, seed germination and stress tolerance. Therefore, our study shows that GWD1 is not only involved in transitory starch degradation in source tissues, but also plays key roles in the seeds, which is a sink tissue. In conclusion, we find that GWD1 is an ideal biotechnological target with promising potential for the breeding of elite rice cultivars via genetic engineering.

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.

Sucrose transport and metabolism control carbon partitioning between stem and grain in rice.

Source-sink relationships are key to overall crop performance. Detailed understanding of the factors that determine source-sink dynamics is imperative for the balance of biomass and grain yield in crop plants. We investigated the differences in source-sink relationships between a cultivated rice, Oryza sativa cv. Nipponbare, and a wild rice, Oryza australiensis, which show striking differences in biomass and grain yield. Oryza australiensis, which accumulates a higher biomass, not only showed higher photosynthesis per unit leaf area but also exported more sucrose from leaves compared with Nipponbare. However, grain features and sugar content suggested limited sucrose mobilization to grains in the wild rice due to vasculature and sucrose transporter functions. Low cell wall invertase activity and high sucrose synthase cleavage activity followed by higher expression of cellulose synthase genes in O. australiensis stem indicated that it utilized photosynthates preferentially for the synthesis of structural carbohydrates, resulting in high biomass. In contrast, source-sink relationships favored high grain yield in Nipponbare via accumulation of transitory starch in the stem, due to higher expression of starch biosynthetic genes, which is mobilized to panicles at the grain filling stage. Thus, vascular features, sucrose transport, and functions of sugar metabolic enzymes explained the differences in source-sink relationships between Nipponbare and O. australiensis.

Diurnal variation of transitory starch metabolism is regulated by plastid proteins WXR1/WXR3 in Arabidopsis young seedlings.

Transitory starch is the portion of starch that is synthesized during the day in the chloroplast and usually used for plant growth overnight. Here, we report altered metabolism of transitory starch in the wxr1/wxr3 (weak auxin response 1/3) mutants of Arabidopsis. WXR1/WXR3 were previously reported to regulate root growth of young seedlings and affect the auxin response mediated by auxin polar transport in Arabidopsis. In this study the wxr1/wxr3 mutants accumulated transitory starch in cotyledon, young leaf, and hypocotyl at the end of night. WXR1/WXR3 expression showed diurnal variation. Grafting experiments indicated that the WXRs in root were necessary for proper starch metabolism and plant growth. We also found that photosynthesis was inhibited and the transcription level of DIN1/DIN6 (Dark-Inducible 1/6) was reduced in wxr1/wxr3. The mutants also showed a defect in the ionic equilibrium of Na+ and K+, consistent with our bioinformatics data that genes related to ionic equilibrium were misregulated in wxr1. Loss of function of WXR1 also resulted in abnormal trafficking of membrane lipids and proteins. This study reveals that the plastid proteins WXR1/WXR3 play important roles in promoting transitory starch degradation for plant growth over night, possibly through regulating ionic equilibrium in the root.

Impact of Drought on Soluble Sugars and Free Proline Content in Selected Arabidopsis Mutants.

Simple SummaryDrought has severe effects on plants, negatively impacting economic, agricultural and environmental processes. Depending on the duration and the strength of water stress conditions, plants adjust a series of physiological, cellular, and molecular mechanisms aimed at providing a correct stress response and, if possible, at establishing stress tolerance. The model plant Arabidopsis thaliana is one of the best tools for analyzing the involvement of specific genes in the response to drought. Thanks to this tool, the role of two genes encoding enzymes involved in sugars metabolism, and one gene encoding an enzyme involved in proline synthesis, have been investigated. In addition to suggesting an interaction between the metabolism of proline and that of soluble sugars, results broaden our understanding of the predominant role played by the accumulation of soluble sugars in counteracting mild osmotic stress.Water shortage is an increasing problem affecting crop yield. Accumulation of compatible osmolytes is a typical plant response to overcome water stress. Sucrose synthase 1 (SUS1), and glucan, water dikinase 2 (GWD2) and δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) are members of small protein families whose role in the response of Arabidopsis thaliana plants to mild osmotic stress has been studied in this work. Comparative analysis between wild-type and single loss-of-function T-DNA plants at increasing times following exposure to drought showed no differences in the content of water-insoluble carbohydrate (i.e., transitory starch and cell wall carbohydrates) and in the total amount of amino acids. On the contrary, water-soluble sugars and proline contents were significantly reduced compared to wild-type plants regardless of the metabolic pathway affected by the mutation. The present results contribute to assigning a physiological role to GWD2, the least studied member of the GWD family; strengthening the involvement of SUS1 in the response to osmotic stress; showing a greater contribution of soluble sugars than proline in osmotic adjustment of Arabidopsis in response to drought. Finally, an interaction between proline and soluble sugars emerged, albeit its nature remains speculative and further investigations will be required for complete comprehension.

Correlation between expression and activity of ADP glucose pyrophosphorylase and starch synthase and their role in starch accumulation during grain filling under drought stress in rice

ADP glucose pyrophosphorylase (AGPase, EC 2.7.7.27) and starch synthase (SS, EC 2.4.1.21) are key regulatory enzymes involved in the starch biosynthesis. Comprehensive analysis of transcription levels of ADP-glucose pyrophosphorylase and starch synthase genes was performed in leaves, roots, and developing grains of drought susceptible (IR64) and drought-tolerant (N22) cultivars under applied water deficit stress (WDS). AGPase and SS genes are differentially regulated in leaves, roots, and grains under the drought stress. The expression pattern of SS and AGPase genes was correlated with the activity of both AGPase, SS, and starch content of developing grains under the drought. Drought stress reduced transitory starch in leaves and enhanced storage starch in developing grains. An increase in the activity of AGPase in developing grains was due to induced expression of ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in N22 and both ADP glucose pyrophosphorylase small subunit 2 (AGPS2) & ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in IR64 and a positive correlation was established with starch content. Similarly, an increase in the SS activity in developing grains was due to induced expression of soluble starch synthase (SSIIB, SSIVA, and SSIVB) in N22 and SSIVB in IR64.

Differential Evolution of α-Glucan Water Dikinase (GWD) in Plants.

The alpha-glucan water dikinase (GWD) enzyme catalyzes starch phosphorylation, an integral step in transitory starch degradation. The high phosphate content in stored starch has great industrial value, due to its physio–chemical properties making it more versatile, although the phosphate content of stored starch varies depending on the botanical source. In this study, we used various computational approaches to gain insights into the evolution of the GWD protein in 48 plant species with possible roles in enzyme function and alteration of phosphate content in their stored starch. Our analyses identified deleterious mutations, particularly in the highly conserved 5 aromatic amino acid residues in the dual tandem carbohydrate binding modules (CBM-45) of GWD protein in C. zofingiensis, G. hirsutum, A. protothecoides, P. miliaceum, and C. reinhardtii. These findings will inform experimental designs for simultaneous repression of genes coding for GWD and the predicted interacting proteins to elucidate the role this enzyme plays in starch degradation. Our results reveal significant diversity in the evolution of GWD enzyme across plant species, which may be evolutionarily advantageous according to the varying needs for phosphorylated stored starch between plants and environments.

Role of transitory starch on growth, development and metal accumulation of Triticum aestivum cultivars grown under textile effluent fertilization.

Accumulation of transitory starch in leaves is an environment-dependent multifaceted process affected through stress caused by nutrient deficiency or excess of heavy metals in growing medium. On the other hand, textile effluent is one of the major pollution causing industrial waste due to the presence of heavy metal and organic contaminants. Besides the presence of higher pollution load, this effluent also contains some minerals essential for plant growth and metabolism and can serve as source of nutrients to plants. In presented experiment, a mesocosm study was conducted to evaluate the phenotypic, biochemical performance and trace element status of Triticum aestivum (cv. LOK-101 and GW-496) cultivars in response to transitory starch activity grown under textile effluent fertilization. Improved activity of transitory starch under textile effluent fertilization deals with plant growth by providing carbon in the form of soluble sugar. Study also finds a strong correlation of photosynthetic pigments, carbohydrates and plant biomass to transitory starch. As expected, the elemental concentration (Zn, Cu, Mn, Fe, Co, Pb, Cd, and As) in plants increased with increasing dose of textile effluent. The study concluded that the transitory starch is one of the key components in plant leaves that regulate plant growth under stress condition. Furthermore, the study also concluded that the lower dose of textile effluent significantly favours growth and nutrient status of plants without any negative impact. Therefore, the application of lower concentration of textile effluent as basal dose in agriculture may serve as source of nutrient/micronutrient to plants and also can be a sustainable way for effluent management.

Defining the Regulatory Roles of Glucan Phosphatases in Transitory Starch Degradation

Plants generate starch during the day and metabolize it at night to store and use for cellular energy. The glucan phosphatase Starch EXcess4 (SEX4) plays a crucial role in transitory starch degradation via its involvement in the reversible starch phosphorylation mechanism. Previous structural studies have shown that SEX4 preferentially binds to and dephosphorylates starch at the C6 position of glucosyl residues, but there remains little data on how SEX4 navigates and interacts with the complex starch granule. Further, enzyme kinetics and regulatory mechanisms of SEX4 are currently not well understood. We employed both generic and substrate specific phosphatase assays to determine kinetics and substrate binding mechanisms of SEX4. Upon use of a generic phosphatase substrate, p‐nitro phenyl phosphate (p‐NPP), SEX4 followed Michaelis‐Menten kinetics. A sigmoidal curve observed for the physiological substrate amylopectin suggests a non‐ Michaelis‐Menten behavior for SEX4 and provides experimental evidence for a possible allosteric regulatory mechanism of SEX4. The enhanced dephosphorylation rates obtained for generic phosphatase assays with SEX4 preincubated with different domains of starch structure strongly suggest a conformational change within the active site of SEX4 upon glucan binding. Collectively, our finding suggests a novel posttranslational regulatory mechanism of SEX4 that is necessary for the regulation of transitory starch degradation at night.

Identifying Post‐translational Regulatory Strategies of Glucan Phosphatases in Transitory Starch Metabolism

Plant glucan phosphatases remove phosphates from the outer most layers of the starch granule, which is already partially solubilized and phosphorylated by glucan dikinases. Thus, the activity of glucan phosphatase is at an interface between semi‐crystalline starch structure and soluble chains of amylopectin. Recent structural studies of glucan phosphatases bound to a soluble glucans have provided important insights into how glucan phosphatase family of enzymes engage with the linear portion of the amylopectin chains of the complex starch structure. However, our understanding of how these enzymes interact with the insoluble or partially soluble semi‐crystalline starch granule is not clear. Using enzymatic assays coupled with Differential Scanning Fluorometry (DSF) and X‐ray crystallographic studies, we have identified the physical mechanisms glucan phosphatases utilize to bind, activate and regulate their enzymatic activity at the starch granule. In this work, we also investigate how glucan phosphatases respond to the metabolic and energy changes during day and night. The findings explore novel posttranslational regulatory mechanisms of glucan phosphatase family of enzymes in transitory starch metabolism.

Flux balance analysis of metabolism during growth by osmotic cell expansion and its application to tomato fruits

Cell expansion is a significant contributor to organ growth and is driven by the accumulation of osmolytes to increase cell turgor pressure. Metabolic modelling has the potential to provide insights into the processes that underpin osmolyte synthesis and transport, but the main computational approach for predicting metabolic network fluxes, flux balance analysis, often uses biomass composition as the main output constraint and ignores potential changes in cell volume. Here we present growth-by-osmotic-expansion flux balance analysis (GrOE-FBA), a framework that accounts for both the metabolic and ionic contributions to the osmotica that drive cell expansion, as well as the synthesis of protein, cell wall and cell membrane components required for cell enlargement. Using GrOE-FBA, the metabolic fluxes in dividing and expanding cells were analysed, and the energetic costs for metabolite biosynthesis and accumulation in the two scenarios were found to be surprisingly similar. The expansion phase of tomato fruit growth was also modelled using a multiphase single-optimization GrOE-FBA model and this approach gave accurate predictions of the major metabolite levels throughout fruit development, as well as revealing a role for transitory starch accumulation in ensuring optimal fruit development.

Genetic engineering of transitory starch accumulation by knockdown of OsSEX4 in rice plants for enhanced bioethanol production.

Rice straw, a common agricultural waste, is used as a potential feedstock for bioethanol production. Currently, bioethanol is made mostly from the microbial fermentation of starch-containing raw materials. Therefore, genetically engineered starch-excess rice straw through interference of starch degradation as a potential strategy to enhance bioethanol production was evaluated in this study. Arabidopsis Starch Excess 4 (SEX4) encodes a chloroplast-localized glucan phosphatase and plays a role in transitory starch degradation. Despite the identification of a SEX4 homolog in rice, OsSEX4, its biological function remains uncertain. Ectopic expression of OsSEX4 complementary DNA complemented the leaf starch-excess phenotype of the Arabidopsis sex4-4 mutant. OsSEX4-knockdown transgenic rice plants were generated using the RNA interference approach. Starch accumulation was higher in OsSEX4-knockdown suspension-cultured cells, leaves, and rice straw compared with the wild type, suggesting that OsSEX4 plays an important role in degradation of transitory starch. The OsSEX4-knockdown rice plants showed normal plant growth and no yield penalty. Starch-excess OsSEX4-knockdown rice straw used as feedstock for fermentation resulted in improved bioethanol yield, with a 50% increase in ethanol production in a vertical mass-flow type bioreactor, compared with that of the wild-type straw.

Faster Removal of 2-Phosphoglycolate through Photorespiration Improves Abiotic Stress Tolerance of Arabidopsis.

Photorespiration metabolizes 2-phosphoglyolate (2-PG) to avoid inhibition of carbon assimilation and allocation. In addition to 2-PG removal, photorespiration has been shown to play a role in stress protection. Here, we studied the impact of faster 2-PG degradation through overexpression of 2-PG phosphatase (PGLP) on the abiotic stress-response of Arabidopsis thaliana (Arabidopsis). Two transgenic lines and the wild type were subjected to short-time high light and elevated temperature stress during gas exchange measurements. Furthermore, the same lines were exposed to long-term water shortage and elevated temperature stresses. Faster 2-PG degradation allowed maintenance of photosynthesis at combined light and temperatures stress and under water-limiting conditions. The PGLP-overexpressing lines also showed higher photosynthesis compared to the wild type if grown in high temperatures, which also led to increased starch accumulation and shifts in soluble sugar contents. However, only minor effects were detected on amino and organic acid levels. The wild type responded to elevated temperatures with elevated mRNA and protein levels of photorespiratory enzymes, while the transgenic lines displayed only minor changes. Collectively, these results strengthen our previous hypothesis that a faster photorespiratory metabolism improves tolerance against unfavorable environmental conditions, such as high light intensity and temperature as well as drought. In case of PGLP, the likely mechanism is alleviation of inhibitory feedback of 2-PG onto the Calvin–Benson cycle, facilitating carbon assimilation and accumulation of transitory starch.

Phosphorus Enhances Photosynthetic Storage Starch Production in a Green Microalga (Chlorophyta) Tetraselmis subcordiformis in Nitrogen Starvation Conditions.

Microalgae are potential starch producers as alternatives to agricultural crops. This study disclosed the effects and mechanism of phosphorus availability exerted on storage starch production in a starch-producing microalga Tetraselmis subcordiformis in nitrogen starvation conditions. Excessive phosphorus supply facilitated starch production, which differed from the conventional cognition that phosphorus would inhibit transitory starch biosynthesis in plants. Phosphorus enhanced energy utilization efficiency for biomass and storage starch production. ADP-glucose pyrophosphorylase (AGPase), conventionally known to be critical for starch biosynthesis, was negatively correlated to storage starch biosynthesis. Excessive phosphorus supply maintained large cell volumes, enhanced activities of starch phosphorylases (SPs) along with branching enzymes and isoamylases, and increased phosphoenolpyruvate and trehalose-6-phosphate levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. This work highlighted the importance of phosphorus in the production of microalgal starch and provided further evidence for the SP-based storage starch biosynthesis pathway.

Redox Regulation of Starch Metabolism

Metabolism of starch is a major biological integrator of plant growth supporting nocturnal energy dynamics by transitory starch degradation as well as periods of dormancy, re-growth, and reproduction by utilization of storage starch. Especially, the extraordinarily well-tuned and coordinated rate of transient starch biosynthesis and degradation suggests the presence of very sophisticated regulatory mechanisms. Together with the circadian clock, land plants (being autotrophic and sessile organisms) need to monitor, sense, and recognize the photosynthetic rate, soil mineral availability as well as various abiotic and biotic stress factors. Currently it is widely accepted that post-translational modifications are the main way by which the diel periodic activity of enzymes of transient starch metabolism are regulated. Among these mechanisms, thiol-based redox regulation is suggested to be of fundamental importance and in chloroplasts, thioredoxins (Trx) are tightly linked up to photosynthesis and mediate light/dark regulation of metabolism. Also, light independent NADP-thioredoxin reductase C (NTRC) plays a major role in reactive oxygen species scavenging. Moreover, Trx and NTRC systems are interconnected at several levels and strongly influence each other. Most enzymes involved in starch metabolism are demonstrated to be redox-sensitive in vitro. However, to what extent their redox sensitivity is physiologically relevant in synchronizing starch metabolism with photosynthesis, heterotrophic energy demands, and oxidative protection is still unclear. For example, many hydrolases are activated under reducing (light) conditions and the strict separation between light and dark metabolic pathways is now challenged by data suggesting degradation of starch during the light period.