Trehalose 6-phosphate (Tre6P) is a signal metabolite that links carbon metabolism with plant development. Our current understanding of Tre6P metabolism and signalling is predominantly based on studies in Arabidopsis thaliana. Some features could be adapted to the specific physiology, anatomy, and life cycle of this C3 eudicot model species, and thus might not be representative of other angiosperms. To better understand Tre6P metabolism in monocot C4 species, we used Setaria viridis, which has been widely adopted as a model for the major C4 NADP-malic enzyme subtype crop species, such as maize (Zea mays), sorghum (Sorghum bicolor), and sugarcane (Saccharum officinarum). In this work, we analysed the levels of transcripts encoding Tre6P-related enzymes in different tissues and cell types from S. viridis. The TREHALOSE-6-PHOSPHATE SYNTHASE1 transcript, its encoded protein (TPS1, the enzyme responsible for Tre6P synthesis), and Tre6P were mainly located in bundle sheath cells of S. viridis. Our results show that Tre6P is predominately synthesized and located in bundle sheath and associated cells.

Trehalose 6-phosphate (Tre6P) is a signal metabolite that links carbon metabolism with plant development. Our current understanding of Tre6P metabolism and signalling is predominantly based on studies in Arabidopsis thaliana. Some features could be adapted to the specific physiology, anatomy, and life cycle of this C3 eudicot model species, and thus might not be representative of other angiosperms. To better understand the regulation of carbon metabolism by Tre6P in monocot C4 species we used Setaria viridis, which has been widely adopted as a model for the major C4 NADP-malic enzyme subtype crop species, such as maize (Zea mays), sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum). In this work, we analysed the levels of transcripts encoding Tre6P-related enzymes in different tissues and cell types from S. viridis. The TREHALOSE-6-PHOSPHATE SYNTHASE1 transcript, its encoded protein (TPS1, the enzyme responsible for Tre6P synthesis) and Tre6P were mainly located in bundle sheath cells of S. viridis. Our results show that Tre6P is predominately synthesized and located in bundle sheath and associated cells, where it could play a fundamental role in the regulation of sucrose levels by modulating phloem loading.

Sugar-alcohols are major photosynthates in plants from the Rosaceae family. Expression of the gene encoding aldose-6-phosphate reductase (Ald6PRase), the critical enzyme for glucitol synthesis in rosaceous species, is regulated by physiological and environmental cues. Additionally, Ald6PRase is inhibited by small molecules (hexose-phosphates and inorganic orthophosphate) and oxidizing compounds. This work demonstrates that Ald6PRase from peach leaves is phosphorylated in planta at the N-terminus. We also show in vitro phosphorylation of recombinant Ald6PRase by a partially purified kinase extract from peach leaves containing Ca2+-dependent protein kinases (CDPKs). Moreover, phosphorylation of recombinant Ald6PRase was inhibited by hexose-phosphates, phosphoenolpyruvate and pyrophosphate. We further show that phosphorylation of recombinant Ald6PRase was maximal using recombinant CDPKs. Overall, our results suggest that phosphorylation could fine-tune the activity of Ald6PRase.

Abstract Abstract. Data on protein post-translational modifications (PTMs) increased exponentially in the last years due to the refinement of mass spectrometry techniques and the development of databases to store and share datasets. Nevertheless, these data per se do not create comprehensive biochemical knowledge. Complementary studies on protein biochemistry are necessary to fully understand the function of these PTMs at the molecular level and beyond, for example, designing rational metabolic engineering strategies to improve crops. Phosphoenolpyruvate carboxykinases (PEPCKs) are critical enzymes for plant metabolism with diverse roles in plant development and growth. Multiple lines of evidence showed the complex regulation of PEPCKs, including PTMs. Herein, we present PEPCKs as an example of the integration of combined mechanisms modulating enzyme activity and metabolic pathways. PEPCK studies strongly advanced after the production of the recombinant enzyme and the establishment of standardized biochemical assays. Finally, we discuss emerging open questions for future research and the challenges in integrating all available data into functional biochemical models.

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. Our current understanding of Tre6P metabolism and signaling pathways in plants is based almost entirely on studies performed with Arabidopsis thaliana, a model plant that performs C3 photosynthesis. Conversely, our knowledge on the molecular mechanisms involved in Tre6P regulation of carbon partitioning and metabolism in C4 plants is scarce. This topic is especially relevant due to the agronomic importance of crops performing C4 photosynthesis, such as maize, sorghum and sugarcane. In this review, we focused our attention on recent developments related to Tre6P metabolism in C4 species and raised some open questions that should be addressed in the near future to improve the yield of economically important crops.

Plant metabolism is finely orchestrated to allow the occurrence of complementary and sometimes opposite metabolic pathways. In part this is achieved by the allosteric regulation of enzymes, which has been a cornerstone of plant research for many decades. The completion of the Arabidopsis genome and the development of the associated toolkits for Arabidopsis research moved the focus of many researchers to other fields. This is reflected by the increasing number of high-throughput proteomic studies, mainly focused on post-translational modifications. However, follow-up 'classical' biochemical studies to assess the functions and upstream signaling pathways responsible for such modifications have been scarce. In this work, we review the basic concepts of allosteric regulation of enzymes involved in plant carbon metabolism, comprising photosynthesis and photorespiration, starch and sucrose synthesis, glycolysis and gluconeogenesis, the oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Additionally, we revisit the latest results on the allosteric control of the enzymes involved in these pathways. To conclude, we elaborate on the current methods for studying protein-metabolite interactions, which we consider will become crucial for discoveries in the future.

During germination, seed reserves are mobilised to sustain the metabolic and energetic demands of plant growth. Mitochondrial respiration is presumably required to drive germination in several species, but only recently its role in this process has begun to be elucidated. Using Arabidopsis thaliana lines with changes in the levels of the respiratory chain component cytochrome c (CYTc), we investigated the role of this protein in germination and its relationship with hormonal pathways. Cytochrome c deficiency causes delayed seed germination, which correlates with decreased cyanide-sensitive respiration and ATP production at the onset of germination. In addition, CYTc affects the sensitivity of germination to abscisic acid (ABA), which negatively regulates the expression of CYTC-2, one of two CYTc-encoding genes in Arabidopsis. CYTC-2 acts downstream of the transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), which binds to a region of the CYTC-2 promoter required for repression by ABA and regulates its expression. The results show that CYTc is a main player during seed germination through its role in respiratory metabolism and energy production. In addition, the direct regulation of CYTC-2 by ABI4 and its effect on ABA-responsive germination establishes a link between mitochondrial and hormonal functions during this process.

Crassulacean acid metabolism (CAM) is a biochemical adaptation developed during evolution by many plants to perform photosynthetic carbon assimilation in highly arid and hostile environments. These plants (mostly succulents) are of particular importance after achieving high biomass productivity with very scarce climatic and background requisites. This chapter describes the distinctive anatomy and physiology characterizing CAM species. The metabolic process of carbon assimilation is analyzed exhaustively, considering the relationships between enzymatic reactions acting in coordination with physiological procedures developed during the day. The enzymes catalyzing critical reactions are detailed regarding their kinetic and regulatory characteristics, establishing a link of these features with the accurate operation of the CAM cycle during the light and dark periods. Besides, the stomata opening/closure and the temporary accumulation of malate in the vacuole are described as critical for the metabolism operation. The text emphasizes that many CAM species already have accurate commercial value as producers of food (as pineapple and Agave tequilana), or gardening and ornamental (orchids) products, while others are of high potential for emerging markets of biofuels and biorefineries.

Photosynthesis is the biological process that converts solar energy into chemical energy and reducing power, which serve to fix atmospheric CO2 in organic compounds. In higher plants, carbon photoassimilation comprises intra- and intercellular partitioning of critical intermediates. This chapter details such a process, particularly considering: (i) the interexchange between chloroplasts and the cytosol of green (source) cells, with the production of starch and sucrose as transitory reserve products; (ii) the transport of sucrose to non-photosynthetic (sink) tissues; and (iii) the distribution of sucrose among different routes within heterotrophic cells, to generate the main storage molecules in different plants. This chapter also describes the kinetic and regulatory properties of the enzymes involved in specific metabolic pathways, to better understand the function they play in plants. The analysis includes the routes of carbon fixation, glycolysis, and gluconeogenesis, as well as their connections with other metabolic pathways. Special attention is dedicated to the synthesis of sucrose, sugar alcohols, raffinose, starch, triacylglycerides, and proteins in crops that accumulate these major photosynthetic products.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 h post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Delta 19 and Delta 101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure as the wild type. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the.101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.

Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro, the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination.

AbstractPhosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis ofArabidopsis thalianaPEPCK1 (AthPEPCK1) in germinating seedlings. We found that expression ofAthPEPCK1 peaks at 24-48 hours post-imbibition. Concomitantly, we observed shorter versions ofAthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact ofAthMC9 cleavage on the kinetic and regulatory properties ofAthPEPCK1, we produced truncated mutants based on the reportedAthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants ofAthPEPCK1 showed similar kinetic parameters and the same quaternary structure than the WT. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis ofAthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.HighlightThis paper describes the effects of the N-terminal proteolytic cleavage on the kinetic and regulatory properties ofArabidopsis thalianaphosphoenolpyruvate carboxykinase-1.

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD(+), which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.