Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T₂ seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.     

Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.     

Do seed and microsite limitation interact with seed size in determining invasion patterns in flooding Pampa grasslands?

Additive influences of the invasion ability of species (invasiveness) and the characteristics of the habitat which make it invasible (invasibility) cannot fully explain grassland invasion patterns. We tested the hypothesis that different species assemblages of grassland communities may partly result from interactive influences between the relative invasiveness of available species and community invasibility. During 10 months, we evaluated seed and microsite limitation of seedling emergence, survival, and recruitment of plants belonging to species with different seed size (large-seeded species vs. small-seeded species), with in a two-phase community mosaic typical of semi-natural grasslands in the southern flooding Pampa of Argentina. Seeds of large versus small-seeded species were sown either in species-poor patches dominated by a tall tussock grass (“pajonal”) or in species-rich patches dominated by short grasses (“matrix”), subjected to different levels of canopy disturbance (cut vs. uncut). Seed addition promoted seedling emergence for 7 out of the 10 species sown, and this effect was higher for large than for small-seeded species. After seed limitations were removed, interactive effects among seed size, community state and canopy disturbance reflected a strong positive influence of seed size on plant recruitment only in cut pajonal patches. Therefore, according to the stage of invasion process, relative species success may depend on non-interactive (seed-size effect on seed limitation to seedling emergence) or interactive influences among species invasibility and community invasiveness (from seedling emergence to plant recruitment). As a general conclusion, different assemblages of species are expected to successfully colonize spatially close grassland patches, according to both the available invasible species (seed size) and the community state and stage (species composition and canopy disturbance).     

α- and β-Amylases in seed germination

During the first 24 h of germination of wheat seeds, starch is hydrolysed by free β-amylase. In the next 24 h, some amount of inactive form of β-amylase is converted into active form and this together with α-amylase synthesizedde novo brings about the hydrolysis of starch. The amount of α-amylase is greater in seeds with embryo intact than with embryo excised after 24 h hydration. However, at later stages of seed germination α-amylase becomes predominant and the activity of β-amylase steadily diminishes.     

Pollen availability,seed production and seed predator clutch size in a tephritid—thistle system

Summary We develop a simple model explaining clutch size behaviour ofOrellia ruficauda on its principle host in North America,Cirsium arvense. Offspring of flies feed solely on thistle seeds and seed production is pollen-limited. Thus, female flies risk reduced offspring fitness when committing large clutches to hosts (female flower heads) occurring in localities where male plants are locally absent. We therefore predict that attacked hosts will contain fewer eggs in such localities, a prediction that is consistent with data obtained in the field: large clutches are never laid in flower heads in low-pollination localities. However, larvae reared from such low-quality hosts are significantly smaller on average and will therefore carry smaller egg loads as adults. Small clutches in poor-quality hosts may thus be an expression of lower per-adult fecundity. Nevertheless, sufficient numbers of large, fecund flies are produced in low-pollination localities to make this last explanation less convincing.     

Is acid-induced extension in seed plants only protein-mediated?

Cell wall extensibility controls the rate of plant cell growth. It is determined by intrinsic mechanical properties of wall polymers and by wall proteins modifying these polymers and their interactions. Heat-inactivation of endogenous cell wall proteins inhibited acid-induced extension of onion epidermis peels transverse to the net cellulose alignment in the cell wall but not parallel to it. In the former case the acid-induced extension could be controlled by expansins and in the latter case by pectins restricting shear between microfibrils. Heat-inactivated cell walls stretched transversely to the net cellulose orientation extended faster at pH 5.7 and slower at pH 4.5 compared to native walls. Expansins seem to be inactive at pH 5.7, so that faster extension may result from heat-induced viscous flow of pectins and conformational changes in the cuticle of the epidermis. This stimulation of wall extension is not seen at pH 4.5 as it is outweighed by the inhibitory effect of expansin heat-inactivation. Thus, cell wall extension in higher plants might be controlled by a complex interplay between protein-dependent and protein-independent mechanisms, the result of which depends on pH and preferential orientation of main wall polymers.     

Are tortoises important seed dispersers in Amazonian forests?

According to most studies on seed dispersal in tropical forests, mammals and birds are considered the main dispersal agents and the role played by other animal groups remains poorly explored. We investigate qualitative and quantitative components of the role played by the tortoise Chelonoidis denticulata in seed dispersal in southeastern Amazon, and the influence of seasonal variation in tortoise movement patterns on resulting seed shadows. Seed shadows produced by this tortoise were estimated by combining information on seed passage times through their digestive tract, which varied from 3 to 17 days, with a robust dataset on movements obtained from 18 adult C. denticulata monitored with radio transmitters and spoon-and-line tracking devices. A total of 4,206 seeds were found in 94 collected feces, belonging to 50 seed morphotypes of, at least, 25 plant genera. Very low rates of damage to the external structure of the ingested seeds were observed. Additionally, results of germination trials suggested that passage of seeds through C. denticulata’s digestive tract does not seem to negatively affect seed germination. The estimated seed shadows are likely to contribute significantly to the dispersal of seeds away from parent plants. During the dry season seeds were dispersed, on average, 174.1 m away from the location of fruit ingestion; during the rainy season, this mean dispersal distance increased to 276.7 m. Our results suggest that C. denticulata plays an important role in seed dispersal in Amazonian forests and highlight the influence of seasonal changes in movements on the resulting seed shadows.     

Gene–gene and gene–environment interactions in ulcerative colitis

Genome-wide association studies (GWAS) have identified at least 133 ulcerative colitis (UC) associated loci. The role of genetic factors in clinical practice is not clearly defined. The relevance of genetic variants to disease pathogenesis is still uncertain because of not characterized gene–gene and gene–environment interactions. We examined the predictive value of combining the 133 UC risk loci with genetic interactions in an ongoing inflammatory bowel disease (IBD) GWAS. The Wellcome Trust Case–Control Consortium (WTCCC) IBD GWAS was used as a replication cohort. We applied logic regression (LR), a novel adaptive regression methodology, to search for high-order interactions. Exploratory genotype correlations with UC sub-phenotypes [extent of disease, need of surgery, age of onset, extra-intestinal manifestations and primary sclerosing cholangitis (PSC)] were conducted. The combination of 133 UC loci yielded good UC risk predictability [area under the curve (AUC) of 0.86]. A higher cumulative allele score predicted higher UC risk. Through LR, several lines of evidence for genetic interactions were identified and successfully replicated in the WTCCC cohort. The genetic interactions combined with the gene-smoking interaction significantly improved predictability in the model (AUC, from 0.86 to 0.89, P = 3.26E?05). Explained UC variance increased from 37 to 42 % after adding the interaction terms. A within case analysis found suggested genetic association with PSC. Our study demonstrates that the LR methodology allows the identification and replication of high-order genetic interactions in UC GWAS datasets. UC risk can be predicted by a 133 loci and improved by adding gene–gene and gene–environment interactions.     

Are subordinate ants the best seed dispersers? Linking dominance hierarchies and seed dispersal ability in myrmecochory interactions

True myrmecochory involves the dispersal of elaiosome-bearing seeds by ants. Between the guild of ants that are attracted to these seeds, only a few of them will act as effective dispersers, that is, transporting the seeds to suitable sites (the nests) for germination and plant establishment. Ant communities are known to be highly hierarchical, and subordinate ants quickly deliver resources to their nest rather than consuming it on-site, thereby avoiding encounters with more dominant species. As a result of a series of studies that were carried out during summer in semi-arid Northwest Argentina, we have found that the most important seed disperser of the myrmecochorous plant Jatropha excisa Griseb. (Euphorbiaceae), the ant Pogonomyrmex cunicularius pencosensis Forel, was the most subordinate species during interspecific interactions. The daily timing of release of the J. excisa seeds through ballistic dispersal increased their probability of being removed by the highly thermophilic P. cunicularius pencosensis. Foraging during the warmest hours of the day allowed P. cunicularius pencosensis ants to avoid the risk of interference competition with dominant species, which also behaved as elaiosome predators. As a conclusion, subordinance behaviour appears to be integral to successful myrmecochory, and also the timing of seed release plays a key role in shaping the dynamics of myrmecochorous interactions. Therefore, ant-dispersed plants should not only favour their discovery by subordinate ants, but also should present their seeds at those times of the day when the behaviourally dominant ants are less active.     

‘On-farm’ seed priming with zinc in chickpea and wheat in Pakistan

A series of on-station trials was implemented between 2002 and 2006 to assess the response of wheat (Triticum aestivum L.) and chickpea (Cicer arietinum) to zinc (Zn) added by soaking seeds (priming) in solutions of ZnSO₄ before sowing. Wheat seed was primed for 10 h in 0.3% Zn and chickpea for 6 h in 0.05% Zn. Seed treatments increased the seed concentration in wheat from 27 to 470 mg/kg and in chickpea from 49 to 780 mg/kg. Priming wheat seeds with 0.3% Zn significantly increased the mean shoot dry mass, Zn concentration and Zn uptake of 15-day-old seedlings relative to non-primed controls and seeds primed with water alone. Using 0.4% Zn further increased shoot Zn concentration but depressed shoot dry mass to the level of the non-primed control. In seven trials, mean grain yield of wheat was significantly increased from 2.28 to 2.42 t/ha (6%) by priming with water alone and to 2.61 t/ha (14%) by priming with 0.3% Zn. Mean grain yield of chickpea in seven trials was increased significantly from 1.39 to 1.65 t/ha (19%) by priming seeds with 0.05% Zn. The effect of priming chickpea seeds with water was intermediate (1.49 t/ha) and not statistically separable from the non-primed and zinc-primed treatments. Increased grain yield due to priming in both crops was associated with increases in total biomass but there was no significant effect of priming on harvest index. In addition to increasing yield, priming seeds with Zn also significantly increased grain zinc concentration, by 12% in wheat (mean of three trials) and by 29% in chickpea (one trial) and the total amount of Zn taken up by the grain (by 27% in wheat and by 130% in chickpea). Using ZnSO₄ to prime seeds was very cost-effective, with net benefit-to-cost ratios of 75 for wheat and 780 for chickpea. An erratum to this article can be found at     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Dα in soybean seed

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild‐type (WT) seeds stored for 33 months were non‐viable, 30%–50% of PLDα‐knockdown (PLD‐KD) soybean seeds stored for 33 months germinated. WT and PLD‐KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD‐KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long‐term storage.     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.