Auxin receptors as integrators of developmental and hormonal signals during reproductive development in pea.

Auxins regulate many aspects of plant growth and development. In pea, three of the five TIR1/AFB members (PsTIR1a, PsTIR1b, and PsAFB2) have been implicated in auxin-related responses during fruit/seed development; however, the roles of PsAFB4 and PsAFB6 in these processes are unknown. Using yeast two-hybrid assays, we found that all five pea TIR1/AFB receptor proteins interacted with the pea AUX/IAAs PsIAA6 and/or PsIAA7 in an auxin-dependent manner, a requirement for functional auxin receptors. All five auxin receptors are expressed in young ovaries (pericarps) and rapidly developing seeds, with overlapping and unique developmental and hormone-regulated gene expression patterns. Pericarp PsAFB6 expression was suppressed by seeds and increased in response to deseeding, and exogenous hormone treatments suggest that seed-derived auxin and deseeding-induced ethylene are involved in these responses, respectively. Ethylene-induced elevation of pericarp PsAFB6 expression was associated with 4-Cl-IAA-specific reduction in ethylene responsiveness. In developing seeds, expression of PsTAR2 and PsYUC10 auxin biosynthesis genes was associated with high auxin levels in seed coat and cotyledon tissues, and PsAFB2 dominated the seed tissue transcript pool. Overall, auxin receptors had overlapping and unique developmental and hormone-regulated gene expression patterns during fruit/seed development, suggesting mediation of diverse responses to auxin, with PsAFB6 linking auxin and ethylene signaling.

Balancing of hormonal biosynthesis and catabolism pathways, a strategy to ameliorate the negative effects of heat stress on reproductive growth.

In pea (Pisum sativum L.), moderate heat stress during early flowering/fruit set increased seed/ovule abortion, and concomitantly produced fruits with reduced ovary (pericarp) length, and fewer seeds at maturity. Plant hormonal networks coordinate seed and pericarp growth and development. To determine if these hormonal networks are modulated in response to heat stress, we analyzed the gene expression patterns and associated these patterns with precursors, and bioactive and inactive metabolites of the auxin, gibberellin (GA), abscisic acid (ABA), and ethylene biosynthesis/catabolism pathways in young developing seeds and pericarps of non-stressed and 4-day heat-stressed fruits. Our data suggest that within the developing seeds heat stress decreased bioactive GA levels reducing GA growth-related processes, and that increased ethylene levels may have promoted this inhibitory response. In contrast, heat stress increased auxin biosynthesis gene expression and auxin levels in the seeds and pericarps, and seed ABA levels, both effects can increase seed sink strength. We hypothesize that seeds with higher auxin- and ABA-induced sink strength and adequate bioactive GA levels will set and continue to grow, while the seeds with lower sink strength (low auxin, ABA, and GA levels) will become more sensitive to heat stress-induced ethylene leading to ovule/seed abortion.

A transferase interactome that may facilitate channeling of polyunsaturated fatty acid moieties from phosphatidylcholine to triacylglycerol.

Polyunsaturated fatty acids (PUFAs) such as α-linolenic acid (ALA, 18:3Δ9cis,12cis,15cis ) have high nutritional and industrial values. In oilseed crops, PUFAs are synthesized on phosphatidylcholine (PC) and accumulated in triacylglycerol (TAG). Therefore, exploring the mechanisms that route PC-derived PUFA to TAG is essential for understanding and improving PUFA production. The seed oil of flax (Linum usitatissimum) is enriched in ALA, and this plant has many lipid biosynthetic enzymes that prefer ALA-containing substrates. In this study, using membrane yeast two-hybrid and bimolecular fluorescence complementation assays, we probed recombinant flax transferase enzymes, previously shown to contribute to PUFA enrichment of TAG, for physical interactions with each other under in vivo conditions. We found that diacylglycerol acyltransferases, which catalyze the final reaction in acyl-CoA-dependent TAG biosynthesis, interact with the acyl-editing enzymes phosphatidylcholine: diacylglycerol cholinephosphotransferase, and lysophosphatidylcholine acyltransferase. Physical interactions among the acyl-editing enzymes were also identified. These findings reveal the presence of an assembly of interacting transferases that may facilitate the channeling of PUFA from PC to TAG in flax and possibly also in other oleaginous plants that produce seeds enriched in PC-modified fatty acids.

Seed-specific down-regulation of Arabidopsis CELLULOSE SYNTHASE 1 or 9 reduces seed cellulose content and differentially affects carbon partitioning.

KEY MESSAGE: Seed-specific down-regulation of AtCESA1 and AtCESA9, which encode cellulose synthase subunits, differentially affects seed storage compound accumulation in Arabidopsis. High amounts of cellulose can negatively affect crop seed quality, and, therefore, diverting carbon partitioning from cellulose to oil, protein and/or starch via molecular breeding may improve seed quality. To determine the effect of seed cellulose content reduction on levels of storage compounds, Arabidopsis thaliana CELLULOSE SYNTHASE1 (AtCESA1) and AtCESA9 genes, which both encode cellulose synthase subunits, were individually down-regulated using seed-specific intron-spliced hairpin RNA (hpRNAi) constructs. The selected seed-specific AtCESA1 and AtCESA9 Arabidopsis RNAi lines displayed reduced cellulose contents in seeds, and exhibited no obvious visual phenotypic growth defects with the exception of a minor effect on early root development in AtCESA1 RNAi seedlings and early hypocotyl elongation in the dark in both types of RNAi line. The seed-specific down-regulation of AtCESA9 resulted in a reduction in seed weight compared to empty vector controls, which was not observed in AtCESA1 RNAi lines. In terms of effects on carbon partitioning, AtCESA1 and AtCESA9 RNAi lines exhibited distinct effects. The down-regulation of AtCESA1 led to a ~ 3% relative increase in seed protein content (P = 0.04) and a ~ 3% relative decrease in oil content (P = 0.02), but caused no alteration in soluble glucose levels. On the contrary, AtCESA9 RNAi lines did not display a significant reduction in seed oil, protein or soluble glucose content. Taken together, our results indicate that the seed-specific down-regulation of AtCESA1 causes alterations in seed storage compound accumulation, while the effect of AtCESA9 on carbon partitioning is absent or minor in Arabidopsis.

Engineering Arabidopsis long-chain acyl-CoA synthetase 9 variants with enhanced enzyme activity.

Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple amino acid residue substitutions were identified with enhanced enzyme activity. To explore the effect of each amino acid residue substitution, single-site mutants were generated and the amino acid substitutions C207F and D238E were found to be primarily responsible for the increased activity of the two variants. Furthermore, evolutionary analysis revealed that the beneficial amino acid site C207 is conserved among LACS9 from plant eudicots, whereas the other beneficial amino acid site D238 might be under positive selection. Together, our results provide valuable information for the production of LACS variants for applications in the metabolic engineering of lipid biosynthesis in oleaginous organisms.

TIR1 auxin receptors are implicated in the differential response to 4-Cl-IAA and IAA in developing pea fruit.

The auxins indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA) occur naturally in pea (Pisum sativum); however, only 4-Cl-IAA mimics the presence of seeds in stimulating pericarp growth. To examine if this differential auxin effect is mediated through TIR1/AFB auxin receptors, pea TIR1 and AFB2 homologs were functionally characterized in Arabidopsis, and receptor expression, and auxin distribution and action were profiled in developing pea fruits. PsTIR1a, PsTIR1b, and PsAFB2 restored the auxin-sensitive root growth response to the mutant Arabidopsis seedlings Attir1-10 and/or Attir1-10 afb2-3. Expression of PsTIR1 or AtTIR1 in Attir1-10 afb2-3 mutants also restored the greater root inhibitory response of 4-Cl-IAA compared to that of IAA, implicating TIR1 receptors in this response. The ability of 4-Cl-IAA to stimulate a stronger DR5::GUS auxin response than IAA at the same concentration in pea pericarps was associated with its ability to enrich the auxin-receptor transcript pool with PsTIR1a and PsAFB2 by decreasing the transcript abundance of PsTIR1b (mimicking results in pericarps with developing seeds). Therefore, the markedly different effect of IAA and 4-Cl-IAA on pea fruit growth may at least partially involve TIR1/AFB receptors and the differential modulation of their population, resulting in specific Aux/IAA protein degradation that leads to an auxin-specific tissue response.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid.

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis ), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis ) or linoleic acid (18:2Δ9cis,12cis ). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1.

The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield (e.g. in canola-type Brassica napus). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.

Heat stress differentially modifies ethylene biosynthesis and signaling in pea floral and fruit tissues.

KEY MESSAGE: Ethylene biosynthesis is regulated in reproductive tissues in response to heat stress in a manner to optimize resource allocation to pollinated fruits with developing seeds. High temperatures during reproductive development are particularly detrimental to crop fruit/seed production. Ethylene plays vital roles in plant development and abiotic stress responses; however, little is known about ethylene's role in reproductive tissues during development under heat stress. We assessed ethylene biosynthesis and signaling regulation within the reproductive and associated tissues of pea during the developmental phase that sets the stage for fruit-set and seed development under normal and heat-stress conditions. The transcript abundance profiles of PsACS [encode enzymes that convert S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid (ACC)] and PsACO (encode enzymes that convert ACC to ethylene), and ethylene evolution were developmentally, environmentally, and tissue-specifically regulated in the floral/fruit/pedicel tissues of pea. Higher transcript abundance of PsACS and PsACO in the ovaries, and PsACO in the pedicels was correlated with higher ethylene evolution and ovary senescence and pedicel abscission in fruits that were not pollinated under control temperature conditions. Under heat-stress conditions, up-regulation of ethylene biosynthesis gene expression in pre-pollinated ovaries was also associated with higher ethylene evolution and lower retention of these fruits. Following successful pollination and ovule fertilization, heat-stress modified PsACS and PsACO transcript profiles in a manner that suppressed ovary ethylene evolution. The normal ethylene burst in the stigma/style and petals following pollination was also suppressed by heat-stress. Transcript abundance profiles of ethylene receptor and signaling-related genes acted as qualitative markers of tissue ethylene signaling events. These data support the hypothesis that ethylene biosynthesis is regulated in reproductive tissues in response to heat stress to modulate resource allocation dynamics.

Hormonal regulation of reproductive growth under normal and heat-stress conditions in legume and other model crop species.

Legume crops are grown throughout the world and provide an excellent food source of digestible protein and starch, as well as dietary fibre, vitamins, minerals, and flavonoids. Fruit and seeds from legumes are also an important source of vegetables for a well-balanced diet. A trend in elevated temperature as a result of climate change increases the risk of a heat stress-induced reduction in legume crop yield. High temperatures during the crop reproductive development phase are particularly detrimental to fruit/seed production because the growth and development of the reproductive tissues are sensitive to small changes in temperature. Hormones are signalling molecules that play important roles in a plant's ability to integrate different environmental inputs and modify their developmental processes to optimize growth, survival, and reproduction. This review focuses on the hormonal regulation of reproductive development and heat stress-induced alteration of this regulation during (i) pollination, (ii) early fruit set, and (iii) seed development that affects fruit/seed yield in legume and other model crops. Further understanding of hormone-regulated reproductive growth under non-stress and heat-stress conditions can aid in trait selection and the development of gene modification strategies and cultural practices to improve heat tolerance in legume crops contributing to improved food security.

Regulation of ethylene-related gene expression by indole-3-acetic acid and 4-chloroindole-3-acetic acid in relation to pea fruit and seed development.

In pea, the auxins 4-chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-acetic acid (IAA) occur naturally; however, only 4-Cl-IAA stimulates pericarp growth and gibberellin (GA) biosynthesis, and inhibits the ethylene response in deseeded ovaries (pericarps), mimicking the presence of seeds. Expression of ovary ethylene biosynthesis genes was regulated similarly in most cases by the presence of 4-Cl-IAA or seeds. PsACS1 [which encodes an enzyme that synthesizes 1-aminocyclopropane-1-carboxylic acid (ACC)] transcript abundance was high in pericarp tissue adjacent to developing seeds following pollination. ACC accumulation in 4-Cl-IAA-treated deseeded pericarps was driven by high PsASC1 expression (1800-fold). 4-Cl-IAA, but not IAA, also suppressed the pericarp transcript levels of PsACS4. 4-Cl-IAA increased PsACO1 and decreased PsACO2 and PsACO3 expression (enzymes that convert ACC to ethylene) but did not change ACO enzyme activity. Increased ethylene was countered by a 4-Cl-IAA-specific decrease in ethylene responsiveness potentially via modulation of pericarp ethylene receptor and signaling gene expression. This pattern did not occur in IAA-treated pericarps. Overall, the effect of 4-Cl-IAA and IAA on ethylene biosynthesis gene expression generally explains the ethylene evolution patterns, and their effects on GA biosynthesis and ethylene signaling gene expression explain the tissue response patterns in young pea ovaries.

Characterization of proanthocyanidin metabolism in pea (Pisum sativum) seeds.

BACKGROUND: Proanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied. RESULTS: PAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. 'Courier' and 'Solido' PAs were primarily prodelphinidin-types, whereas the PAs from 'LAN3017' were mainly the procyanidin-type. The mean degree of polymerization of 'LAN3017' PAs was also higher than those from 'Courier' and 'Solido'. Next-generation sequencing of 'Courier' seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis. CONCLUSION: Significant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR.

Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3ß-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.

Impact of Susceptibility on Plant Hormonal Composition during Clubroot Disease Development in Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease of crucifers associated with the formation of large root galls. This root enlargement suggests modulation of plant hormonal networks by the pathogen, stimulating cell division and elongation and influencing host defense. We studied physiological changes in two Brassica napus cultivars, including plant hormone profiles-salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), the auxin indole-3-acetic acid (IAA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)-along with their selected derivatives following inoculation with virulent and avirulent P. brassicae pathotypes. In susceptible plants, water uptake declined from the initial appearance of root galls by 21 days after inoculation, but did not have a significant effect on photosynthetic rate, stomatal conductance, or leaf chlorophyll levels. Nonetheless, a strong increase in ABA levels indicated that hormonal mechanisms were triggered to cope with water stress due to the declining water uptake. The free SA level in the roots increased strongly in resistant interactions, compared with a relatively minor increase during susceptible interactions. The ratio of conjugated SA to free SA was higher in susceptible interactions, indicating that resistant interactions are linked to the plant's ability to maintain higher levels of bioactive free SA. In contrast, JA and its biologically active form JA-Ile declined up to 7-fold in susceptible interactions, while they were maintained during resistant interactions. The ACC level increased in the roots of inoculated plants by 21 days, irrespective of clubroot susceptibility, indicating a role of ethylene in response to pathogen interactions that is independent of disease severity. IAA levels at early and later infection stages were lower only in susceptible plants, suggesting a modulation of auxin homeostasis by the pathogen relative to the host defense system.

Tissue-specific regulation of gibberellin biosynthesis in developing pea seeds.

Previous work suggests that gibberellins (GAs) play an important role in early seed development. To more fully understand the roles of GAs throughout seed development, tissue-specific transcription profiles of GA metabolism genes and quantitative profiles of key GAs were determined in pea (Pisum sativum) seeds during the seed-filling development period (8-20 d after anthesis [DAA]). These profiles were correlated with seed photoassimilate acquisition and storage as well as morphological development. Seed coat growth (8-12 DAA) and the subsequent dramatic expansion of branched parenchyma cells were correlated with both transcript abundance of GA biosynthesis genes and the concentration of the growth effector GA, GA(1). These results suggest GA(1) involvement in determining the rate of seed coat growth and sink strength. The endosperm's PsGA20ox transcript abundance and the concentration of GA(20) increased markedly as the endosperm reached its maximum volume (12 DAA), thus providing ample GA(20) substrate for the GA 3-oxidases present in both the embryo and seed coat. Furthermore, PsGA3ox transcript profiles and trends in GA(1) levels in embryos at 10 to 16 DAA and also in embryo axes at 18 DAA suggest localized GA(1)-induced growth in these tissues. A shift from synthesis of GA(1) to that of GA(8) occurred after 18 DAA in the embryo axis, suggesting that deactivation of GA(1) to GA(8) is a likely mechanism to limit embryo axis growth and allow embryo maturation to proceed. We hypothesize that GA biosynthesis and catabolism are tightly regulated to bring about the unique developmental events that occur during seed growth, development, and maturation.

Assessment of the mechanisms exerting glucose-lowering effects of dried peas in glucose-intolerant rats.

The present study compared the effects of feeding uncooked pea fractions (embryo v. seed coat) on glucose homeostasis in glucose-intolerant rats and examined potential mechanisms influencing glucose homeostasis. Rats were made glucose intolerant by high-fat feeding, after which diets containing both high-fat and pea fractions were fed for 4 weeks. Rats fed diets containing uncooked pea seed coats low (non-coloured seed coat; NSC) or high (coloured seed coat; CSC) in proanthocyanidins but not embryos had improved oral glucose tolerance (P < 0·05). NSC also lowered fasting and glucose-stimulated insulin secretion (P < 0·05), decreased ß-cell mass by 50 % (P < 0·05) and lowered levels of malondialdehyde, a marker of oxidative stress. Furthermore, NSC decreased the mucosal thickness of the colon by 25 % (P < 0·05), which might affect fibre fermentation and other gut functions. Small but statistically significant (P < 0·05) effects consistent with enhanced glucose transport or metabolism were observed in the skeletal muscle of rats fed NSC or CSC, for example, increased levels of AMP-dependent kinase or akt. We conclude that pea seed coats are the fraction exerting beneficial effects on glucose tolerance. Most of the changes were small in amplitude, suggesting that additive effects on multiple tissues may be important. NSC content appeared to have the most beneficial effects in improving glucose homeostasis but our ability to detect the effect of flavonoids may have been limited by their low concentration in the diet.

The Potential of Genome Editing for Improving Seed Oil Content and Fatty Acid Composition in Oilseed Crops.

A continuous rise in demand for vegetable oils, which comprise mainly the storage lipid triacylglycerol, is fueling a surge in research efforts to increase seed oil content and improve fatty acid composition in oilseed crops. Progress in this area has been achieved using both conventional breeding and transgenic approaches to date. However, further advancements using traditional breeding methods will be complicated by the polyploid nature of many oilseed crops and associated time constraints, while public perception and the prohibitive cost of regulatory processes hinders the commercialization of transgenic oilseed crops. As such, genome editing using CRISPR/Cas is emerging as a breakthrough breeding tool that could provide a platform to keep pace with escalating demand while potentially minimizing regulatory burden. In this review, we discuss the technology itself and progress that has been made thus far with respect to its use in oilseed crops to improve seed oil content and quality. Furthermore, we examine a number of genes that may provide ideal targets for genome editing in this context, as well as new CRISPR-related tools that have the potential to be applied to oilseed plants and may allow additional gains to be made in the future.

Pea polyphenolics and hydrolysis processing alter microbial community structure and early pathogen colonization in mice.

Health benefits associated with pea consumption have been attributed to the fiber and polyphenolic content concentrated within the pea seed coat. However, the amount of pea polyphenols can vary between cultivars, and it has yet to be studied whether pea polyphenols impact the intestinal microbiota. We hypothesized that pea polyphenols promote a healthy microbiome that supports intestinal integrity and pathogen colonization resistance. To investigate the effects of pea polyphenols, pea cultivars rich and poor in proanthocyanidins were supplemented in raw or acid hydrolyzed form to an isocaloric diet in mice. Acid hydrolysis increases the absorption of pea polyphenols by cleaving polymeric proanthocyanidins to their readily absorbable anthocyanidin monomers. After 3 weeks of diet, mice were challenged with Citrobacter rodentium and pathogen colonization and inflammation were assessed. Counter to our hypothesis, pea seed coat fraction supplementation, especially the non-hydrolyzed proanthocyanidin-rich fraction diet adversely increased C. rodentium pathogen load and inflammation. Ileal, cecal and colon microbial communities were notably distinct between pea seed cultivar and hydrolysis processing. The consumption of intact proanthocyanidins decreased microbial diversity indicating that proanthocyanidins have antimicrobial properties. Together our results indicate supplementation of raw pea seed coat rich in proanthocyanidins adversely affect intestinal integrity. However, acid hydrolysis processing restored community structure and colonization resistance, and the anthocyanidin-rich fractions reduced weight gain on a high fat diet. Establishing a clear understanding of the effects of pea fiber and polyphenolic form on health will help to develop research-based pea products and dietary recommendations.

Properties and Biotechnological Applications of Acyl-CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae.

Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty-acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl-CoA-dependent or acyl-CoA-independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl-CoA-dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane-bound and soluble forms of DGAT have been identified with very different amino-acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl-CoA-independent pathways via the catalytic action of membrane-bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty-acid composition of TAG is also discussed.