NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana.

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Genome-wide analysis of sucrose synthase family in soybean and their expression in response to abiotic stress and seed development.

The sucrose synthase (SS) is an important enzyme family which play a vital role in sugar metabolism to improve the fruit quality of the plants. In many plant species, the members of SS family have been investigated but the detailed information is not available in legumes particularly and Glycine max specifically. In the present study, we found thirteen SS members (GmSS1-GmSS13) in G. max genome. High conserved regions were present in the GmSS sequences that may due to the selection pressure during evolutionary events. The segmental duplication was the major factor to increase the number of GmSS family members. The identified thirteen GmSS genes were divided into Class I, Class II and Class III with variable numbers of genes in each class. The protein interaction of GmSS gave the co-expression of sucrose synthase with glucose-1-phosphate adenylyltransferase while SLAC and REL test found number of positive sites in the coding sequences of SS family members. All the GmSS family members except GmSS7 and few of class III members, were highly expressed in all the soybean tissues. The expression of the class I members decreased during seed development, whireas, the class II members expression increased during the seed developing, may involve in sugar metabolism during seed development. Solexa sequencing libraries of acidic condition (pH 4.2) stress samples showed that the expression of class I GmSS genes increased 1- to 2-folds in treated samples than control. The differential expression pattern was observed between the members of a paralogous. This study provides detailed genome-wide analysis of GmSS family in soybean that will provide new insights for future evolutionary and soybean breeding to improve the plant growth and development.

The Arabidopsis electron-transfer flavoprotein:ubiquinone oxidoreductase is required during normal seed development and germination.

The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.

Functional characterization of hydroxyproline-O-galactosyltransferases for Arabidopsis arabinogalactan-protein synthesis.

BACKGROUND: Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family. RESULTS: To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with ß-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to ß-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants. CONCLUSION: Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Nanoceria seed priming enhanced salt tolerance in rapeseed through modulating ROS homeostasis and α-amylase activities.

BACKGROUND: Salinity is a big threat to agriculture by limiting crop production. Nanopriming (seed priming with nanomaterials) is an emerged approach to improve plant stress tolerance; however, our knowledge about the underlying mechanisms is limited. RESULTS: Herein, we used cerium oxide nanoparticles (nanoceria) to prime rapeseeds and investigated the possible mechanisms behind nanoceria improved rapeseed salt tolerance. We synthesized and characterized polyacrylic acid coated nanoceria (PNC, 8.5 ± 0.2 nm, -43.3 ± 6.3 mV) and monitored its distribution in different tissues of the seed during the imbibition period (1, 3, 8 h priming). Our results showed that compared with the no nanoparticle control, PNC nanopriming improved germination rate (12%) and biomass (41%) in rapeseeds (Brassica napus) under salt stress (200 mM NaCl). During the priming hours, PNC were located mostly in the seed coat, nevertheless the intensity of PNC in cotyledon and radicle was increased alongside with the increase of priming hours. During the priming hours, the amount of the absorbed water (52%, 14%, 12% increase at 1, 3, 8 h priming, respectively) and the activities of α-amylase were significantly higher (175%, 309%, 295% increase at 1, 3, 8 h priming, respectively) in PNC treatment than the control. PNC primed rapeseeds showed significantly lower content of MDA, H2O2, and •O2- in both shoot and root than the control under salt stress. Also, under salt stress, PNC nanopriming enabled significantly higher K+ retention (29%) and significantly lower Na+ accumulation (18.5%) and Na+/K+ ratio (37%) than the control. CONCLUSIONS: Our results suggested that besides the more absorbed water and higher α-amylase activities, PNC nanopriming improves salt tolerance in rapeseeds through alleviating oxidative damage and maintaining Na+/K+ ratio. It adds more knowledge regarding the mechanisms underlying nanopriming improved plant salt tolerance.

Sphingadienine-1-phosphate levels are regulated by a novel glycoside hydrolase family 1 glucocerebrosidase widely distributed in seed plants.

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) ß-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice.

LPEATs Tailor Plant Phospholipid Composition through Adjusting Substrate Preferences to Temperature.

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms.

The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana.

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.

Sequential expression of raffinose synthase and stachyose synthase corresponds to successive accumulation of raffinose, stachyose and verbascose in developing seeds of Lens culinaris Medik.

Raffinose, stachyose and verbascose form the three major members of the raffinose family oligosaccharides (RFO) accumulated during seed development. Raffinose synthase (RS; EC 2.4.1.82) and stachyose synthase (STS; EC 2.4.1.67) have been associated with raffinose and stachyose synthesis, but the precise mechanism for verbascose synthesis is not well understood. In this study, full-length RS (2.7 kb) and STS (2.6 kb) clones were isolated by screening a cDNA library prepared from developing lentil seeds (18, 20, 22 and 24 days after flowering [DAF]) to understand the roles of RS and STS in RFO accumulation in developing lentil seeds. The nucleotide sequences of RS and STS genes were similar to those reported for Pisum sativum. Patterns of transcript accumulation, enzyme activities and RFO concentrations were also comparable to P. sativum. However, during lentil seed development raffinose, stachyose and verbascose accumulation corresponded to transcript accumulation for RS and STS, with peak transcript abundance occurring at about 22-24 DAF, generally followed by a sequential increase in raffinose, stachyose and verbascose concentrations followed by a steady level thereafter. Enzyme activities for RS, STS and verbascose synthase (VS) also indicated a sudden increase at around 24-26 DAF, but with an abrupt decline again coinciding with the subsequent steady state increase in the RFO. Galactan:galactan galactosyl transferase (GGT), the galactinol-independent pathway enzyme, however, exhibited steady increase in activity from 24 DAF onwards before abruptly decreasing at 34 DAF. Although GGT activity was detected, isolation of a GGT sequence from the cDNA library was not successful.

The Structural Characterization and Antipathogenic Activities of Quinoin, a Type 1 Ribosome-Inactivating Protein from Quinoa Seeds.

Quinoin is a type 1 ribosome-inactivating protein (RIP) we previously isolated from the seeds of pseudocereal quinoa (Chenopodium quinoa) and is known as a functional food for its beneficial effects on human health. As the presence of RIPs in edible plants could be potentially risky, here we further characterised biochemically the protein (complete amino acid sequence, homologies/differences with other RIPs and three-dimensional homology modeling) and explored its possible defensive role against pathogens. Quinoin consists of 254 amino acid residues, without cysteinyl residues. As demonstrated by similarities and homology modeling, quinoin preserves the amino acid residues of the active site (Tyr75, Tyr122, Glu177, Arg180, Phe181 and Trp206; quinoin numbering) and the RIP-fold characteristic of RIPs. The polypeptide chain of quinoin contains two N-glycosylation sites at Asn115 and Asp231, the second of which appears to be linked to sugars. Moreover, by comparative MALDI-TOF tryptic peptide mapping, two differently glycosylated forms of quinoin, named pre-quinoin-1 and pre-quinoin-2 (~0.11 mg/100 g and ~0.85 mg/100 g of seeds, respectively) were characterised. Finally, quinoin possesses: (i) strong antiviral activity, both in vitro and in vivo towards Tobacco Necrosis Virus (TNV); (ii) a growth inhibition effect on the bacterial pathogens of plants; and (iii) a slight antifungal effect against two Cryphonectria parasitica strains.

Verification of the versatility of the in vitro enzymatic reaction giving (+)-cis-12-Oxo-phytodienoic acid.

Jasmonic acid (JA) is a plant hormone involved in the defense response against insects and fungi. JA is synthesized from α-linolenic acid (LA) by the octadecanoid pathway in plants. 12-oxo-Phytodienoic acid (OPDA) is one of the biosynthetic intermediates in this pathway. The reported stereo selective total synthesis of cis-(+)-OPDA is not very efficient due to the many steps involved in the reaction as well as the use of water sensitive reactions. Therefore, we developed an enzymatic method for the synthesis of OPDA using acetone powder of flax seed and allene oxide cyclase (PpAOC2) from Physcomitrella patens. From this method, natural cis-(+)-OPDA can be synthesized in the high yield of approximately 40%. In this study, we investigated the substrate specificity of the enzymatic synthesis of other OPDA analogs with successions to afford OPDA amino acid conjugates, dinor-OPDA (dn-OPDA), and OPDA monoglyceride, and it was suggested that the biosynthetic pathway of arabidopsides could occur via MGDG.

Biochemical Characterization of Acyl-CoA: Lysophosphatidylcholine Acyltransferase (LPCAT) Enzyme from the Seeds of Salvia hispanica.

Salvia hispanica (chia) is the highest reported terrestrial plant source of alpha-linolenic acid (ALA, ~ 65%), an ω-3 polyunsaturated fatty acid with numerous health benefits. The molecular basis of high ALA accumulation in chia is yet to be understood. We have identified lysophosphatidylcholine acyltransferase (LPCAT) gene from the developing seed transcriptome data of chia and carried out its biochemical characterization through heterologous expression in Saccharomyces cerevisiae. Expression profiling showed that the enzyme was active throughout the seed development, indicating a pivotal role in oil biosynthesis. The enzyme could utilize both saturated and unsaturated lysophosphatidylcholine substrates at the same rate, to synthesize phosphatidylcholine (PC). The enzyme also exhibited lysophosphatidic acid acyltransferase (LPAAT) activity, by preferring lysophosphatidic acid substrate. Substrate specificity studies showed that the enzyme preferred both monounsaturated and polyunsaturated fatty acyl CoAs over saturated CoAs. This activity may play a key role in enriching the PC fraction with polyunsaturated fatty acids (PUFAs). PUFAs present on PC can be transferred to oil through the action of other acyltransferases. Our results describe a new LPCAT enzyme that can be used to biotechnologically alter oilseed crops to incorporate more PUFA in its seed oil.

TILLING-by-Sequencing+ Reveals the Role of Novel Fatty Acid Desaturases (GmFAD2-2s) in Increasing Soybean Seed Oleic Acid Content.

Soybean is the second largest source of oil worldwide. Developing soybean varieties with high levels of oleic acid is a primary goal of the soybean breeders and industry. Edible oils containing high level of oleic acid and low level of linoleic acid are considered with higher oxidative stability and can be used as a natural antioxidant in food stability. All developed high oleic acid soybeans carry two alleles; GmFAD2-1A and GmFAD2-1B. However, when planted in cold soil, a possible reduction in seed germination was reported when high seed oleic acid derived from GmFAD2-1 alleles were used. Besides the soybean fatty acid desaturase (GmFAD2-1) subfamily, the GmFAD2-2 subfamily is composed of five members, including GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E. Segmental duplication of GmFAD2-1A/GmFAD2-1B, GmFAD2-2A/GmFAD2-2C, GmFAD2-2A/GmFAD2-2D, and GmFAD2-2D/GmFAD2-2C have occurred about 10.65, 27.04, 100.81, and 106.55 Mya, respectively. Using TILLING-by-Sequencing+ technology, we successfully identified 12, 8, 10, 9, and 19 EMS mutants at the GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E genes, respectively. Functional analyses of newly identified mutants revealed unprecedented role of the five GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E members in controlling the seed oleic acid content. Most importantly, unlike GmFAD2-1 members, subcellular localization revealed that members of the GmFAD2-2 subfamily showed a cytoplasmic localization, which may suggest the presence of an alternative fatty acid desaturase pathway in soybean for converting oleic acid content without substantially altering the traditional plastidial/ER fatty acid production.

Lipase - The fascinating dynamics of enzyme in seed storage and germination - A real challenge to pearl millet.

Pearl millet is considered as 'nutri-cereal' because of high nutrient density of the seeds. The grain has limited use because of low keeping quality of the flour due to the activities of rancidity causing enzymes like lipase, lox, pox and PPO. Among all the enzymes, lipase is most notorious because of its robust nature and high activity under different conditions. we have identified 2180 putative transcripts showing homology with different variants of lipase precursor through transcriptome data mining (NCBI BioProject acc. no. PRJNA625418). Lipase plays dual role of facilitating the germination of seeds and deteriorating the quality of the pearl millet flour through hydrolytic rancidity. Different physiochemical methods like heat treatment, micro oven, hydrothermal, etc. have been developed to inhibit lipase activity in pearl millet flour. There is further need to develop improved processing technologies to inhibit the hydrolytic and oxidative rancidity in the floor with enhanced shelf-life.

Two ß-glucuronosyltransferases involved in the biosynthesis of type II arabinogalactans function in mucilage polysaccharide matrix organization in Arabidopsis thaliana.

BACKGROUND: Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of ß-glucuronosyltransferase genes/enzymes. RESULTS: Here, we have characterized two ß-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of ß-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size. CONCLUSIONS: These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

Identification and molecular characterization of rice bran-specific lipases.

KEY MESSAGE: Among the 113 lipases present in rice genome, bran and endosperm-specific lipases were identified and lipase activity for one of the selected lipase gene is demonstrated in yeast. Rice bran is nutritionally superior than endosperm as it has major reservoirs of various minerals, vitamins, essential mineral oils and other bioactive compounds, however it is often under-utilized as a food product due to bran instability after milling. Various hydrolytic enzymes, such as lipases, present in bran causes degradation of the lipids present and are responsible for the bran instability. Here, in this study, we have systematically analyzed the 113 lipase genes present in rice genome, and identified 21 seed-specific lipases. By analyzing the expression of these genes in different seed tissues during seed development, we have identified three bran-specific and three endosperm-specific lipases, and one lipase which expresses in both bran and endosperm tissues. Further analysis of these genes during seed maturation and seed germination revealed that their expression increases during seed maturation and decreases during seed germination. Finally, we have shown the lipase activity for one of the selected genes, LOC_Os05g30900, in heterologous system yeast. The bran-specific lipases identified in this study would be very valuable for engineering designer rice varieties having increased bran stability in post-milling.

Oilseed rape (Brassica napus): the importance of aminopeptidases in germination under normal and heavy metals stress conditions.

BACKGROUND: Oilseed rape is one of the most important oilseed crops worldwide, crucial in the food and feed industries. Different environment and climatic conditions can influence its sustainable cultivation and crop yield. Aminopeptidases are crucial enzymes in many physiological processes in all organisms, including humans, so it is important to learn their behavior in food and feed sources. This study presents, for the first time, a detailed discussion on the importance of aminopeptidases, during the oilseed rape germination process, under standard and stress conditions. RESULTS: During the germination of oilseed rape under standard conditions, a significant increase in aminopeptidases activity toward N-terminal amino acids - phenylalanine (Phe), alanine (Ala), glycine (Gly), leucine (Leu), proline (Pro), methionine (Met) - was observed. The change was substrate specific, with the highest increase being observed for Gly (3.2-fold), followed by Ala (2.9-fold), Pro (2.5-fold), Met (1.5-fold), and Phe (1.3-fold). Generally, N-terminal Phe was preferentially cleaved. Germination under stress conditions, caused by several heavy metal ions (e.g. divalent copper, zinc, cadmium, and lead ions), negatively influenced the plants' growth and quality, but significantly enhanced the expression of genes encoding aminopeptidases (or potentially activated aminopeptidases precursors), which was related to the dramatic increase of their activity. CONCLUSIONS: The activity/concentration of aminopeptidases in plants is adjusted to the needs at each stage of development and stress factors occurrence. The most significant increase of activity toward N-terminal Gly and Pro proved the key role of aminopeptidases in the defense mechanisms, by supplying the plants with osmoprotectants and organic nitrogen. The results provide new concepts of oilseed rape growth and cultivation under different conditions. © 2021 Society of Chemical Industry.