Effects of microwave heat treatment on fungal growth, functional properties, total phenolic content, and antioxidant activity of sorghum (Sorghum bicolor L.) grain.

Sorghum grains were subjected to microwave heating at different power levels 350 and 500 W for application times of 15, 30, and 45 s. The effect of microwave heating on fungal growth, protein content, in vitro digestibility, protein solubility, and functional and antioxidant properties of sorghum grain was investigated. The microwave heating at 350 and 500 W significantly reduced fungal incidence in the grain up to 26.2 and 33.4%. No significant changes were found in the crude protein and digestibility of protein, water holding capacity, and oil holding capacity of sorghum. However, application of microwave energy at 500 W for 30 & 45 s caused a sharp reduction on the protein solubility (8.2-7.6%), foaming capacity (6.47-0.98%), emulsion capacity (0.43-0.32 mL/g) and the emulsion stability (2.2-1.6%) of sorghum grain, respectively. Conversely, a significant increment of grain total phenolic content up to 47.1 and 50.8 mg GAE/g and the antioxidant activity up to 40.9 and 59.1% after microwave heat treatment at 350 and 500 W for 45 s, respectively, was observed. These findings revealed that sorghum grain should be treated with microwave at 350 and 500 W for 45 and 15 s, respectively, in order to maintained and enhanced its functional and nutritional properties. Accordingly, microwave heating, particularly at low power, may be an effective emerging method for improving the physicochemical and nutritional properties of sorghum grain.

Phenotypic, Phytochemical, and Transcriptomic Analysis of Black Sorghum (Sorghum bicolor L.) Pericarp in Response to Light Quality.

Black sorghum [Sorghum bicolor (L.) Moench] is characterized by the black appearance of the pericarp and production of 3-deoxyanthocyanidins (3-DOA), which are valued for their cytotoxicity to cancer cells and as natural food colorants and antioxidant additives. The black pericarp phenotype is not fully penetrant in all environments, which implicates the light spectrum and/or photoperiod as the critical factor for trait expression. In this study, black- or red-pericarp genotypes were grown under regimes of visible light, visible light supplemented with UVA or supplemented with UVA plus UVB (or dark control). Pericarp 3-DOAs and pericarp pigmentation were maximized in the black genotype exposed to a light regime supplemented with UVB. Changes in gene expression during black pericarp development revealed that ultraviolet light activates genes related to plant defense, reactive oxygen species, and secondary metabolism, suggesting that 3-DOA accumulation is associated with activation of flavonoid biosynthesis and several overlapping defense and stress signaling pathways.

Development of a transposon-based marker system for mutation breeding in sorghum (Sorghum bicolor L.).

Under certain circumstances, transposable elements (TE) can create or reverse mutations and alter the genome size of a cell. Sorghum (Sorghum bicolor L.) is promising for plant transposon tagging due to its small genome size and its low content of repetitive DNA. We developed a marker system based on targeted region amplification polymorphisms (TE-TRAP) that uses the terminal inverted repeats (TIRs) of transposons. A total of 3816 class 2 transposons belonging to the PIF/Harbinger family were identified from the whole sorghum genome that produced five primers, including eight types of TIRs. To define the applicability and utilization of TE-TRAP, we used 21 individuals that had been bred after ɤ-ray irradiation. In total, 31 TE-TRAP, 16 TD, and 21 AFLP primer combinations generated 1133, 223, and 555 amplicons, respectively. The percent polymorphic marker was 62.8, 51.1, and 59.3% for the TE-TRAP, TD, and AFLP markers, respectively. Phylogenetic and principal component analyses revealed that TE-TRAP divided the 21 individuals into three groups. Analysis of molecular variance suggested that TE-TRAP had a higher level of genetic diversity than the other two marker systems. After verifying the efficiency of TE-TRAP, 189 sorghum individuals were used to investigate the associations between the markers and the ɤ-ray doses. Two significant associations were found among the polymorphic markers. This TE-based method provides a useful marker resource for mutation breeding research.

Differential characteristics of photochemical acclimation to cold in two contrasting sweet sorghum hybrids.

Sweet sorghum has a photosynthetic system which is highly sensitive to cold stress and hence strongly limits its development in temperate environments; therefore, the identification of key exploitable cold tolerance traits is imperative. From a preliminary field trial, two dissimilar sweet sorghum hybrids (ICSSH31 and Bulldozer), in terms of early vigor and productivity, were selected for a controlled-environment trial aiming at identifying useful traits related to acclimation mechanisms to cold stress. The higher cold tolerance of Bulldozer was partially related to a more efficient photochemical regulation mechanism of the incoming light energy: the higher tolerance of photosystem II (PSII) to photo-inactivation was because of a more effective dissipation capacity of the excess of energy and to a more balanced diversion of the absorbed energy into alternative energy sinks. ICSSH31 increased the dissipation and accumulation of a large amount of xanthophylls, as in Bulldozer, but, at the same time, inactivated the oxygen evolving complex and the re-synthesis of chlorophyll (Chl) a and b, thus, leading to an overproduction of CO2 fixation enzymes after re-warming. In summary, in Bulldozer, the acclimation adjustments of the photosynthetic apparatus occurred through an efficient control of energy transfer toward the reaction centers, and this likely allowed a more successful seedling establishment; ICSSH31, conversely, exhibited a fast re-synthesis of Chl pigments, which appears to divert photosynthates from dry matter accumulation. Such broad acclimation traits may constitute a source for selecting higher genetic gain traits relevant for enlarging the growing season of promising biomass sorghum ideotypes in temperate climates.

Energy sorghum--a genetic model for the design of C4 grass bioenergy crops.

Sorghum is emerging as an excellent genetic model for the design of C4 grass bioenergy crops. Annual energy Sorghum hybrids also serve as a source of biomass for bioenergy production. Elucidation of Sorghum's flowering time gene regulatory network, and identification of complementary alleles for photoperiod sensitivity, enabled large-scale generation of energy Sorghum hybrids for testing and commercial use. Energy Sorghum hybrids with long vegetative growth phases were found to accumulate more than twice as much biomass as grain Sorghum, owing to extended growing seasons, greater light interception, and higher radiation use efficiency. High biomass yield, efficient nitrogen recycling, and preferential accumulation of stem biomass with low nitrogen content contributed to energy Sorghum's elevated nitrogen use efficiency. Sorghum's integrated genetics-genomics-breeding platform, diverse germplasm, and the opportunity for annual testing of new genetic designs in controlled environments and in multiple field locations is aiding fundamental discovery, and accelerating the improvement of biomass yield and optimization of composition for biofuels production. Recent advances in wide hybridization between Sorghum and other C4 grasses could allow the deployment of improved genetic designs of annual energy Sorghums in the form of wide-hybrid perennial crops. The current trajectory of energy Sorghum genetic improvement indicates that it will be possible to sustainably produce biofuels from C4 grass bioenergy crops that are cost competitive with petroleum-based transportation fuels.

Effects of mutual shading on the regulation of photosynthesis in field-grown sorghum.

In the field, close planting inevitably causes mutual shading and depression of leaf photosynthesis. To clarify the regulative mechanisms of photosynthesis under these conditions, the effects of planting density on leaf structure, gas exchange and proteomics were carefully studied in field-grown sorghum. In the absence of mineral deficiency, (1) close planting induced a significant decrease in light intensity within populations, which further resulted in much lower stomatal density and other anatomical characteristics associated with shaded leaves; (2) sorghum grown at high planting density had a lower net photosynthetic rate and stomatal conductance than those grown at low planting density; (3) approximately 62 protein spots changed their expression levels under the high planting density conditions, and 22 proteins associated with photosynthesis were identified by mass spectrometry. Further analysis revealed the depression of photosynthesis caused by mutual shading involves the regulation of leaf structure, absorption and transportation of CO2, photosynthetic electron transport, production of assimilatory power, and levels of enzymes related to the Calvin cycle. Additionally, heat shock protein and oxygen-evolving enhancer protein play important roles in photoprotection in field-grown sorghum. A model for the regulation of photosynthesis under mutual shading was suggested based on our results.

Unravelling the genetic complexity of sorghum seedling development under low-temperature conditions.

Sorghum is a promising alternative to maize for bioenergy production in Europe; however, its use is currently limited by poor adaptation to low temperatures during and after germination. We collected multi-trait phenotype data under optimal and suboptimal temperatures in a genetically diverse recombinant inbred line (RIL) mapping population showing contrasting segregation patterns for pre- and post-emergence chilling tolerance. Germination, emergence, seedling development, root architecture and seedling survival were assessed in two different seedlots. Emergence and root establishment were found to be the key determinants of development and survival under chilling stress. Highly interactive epistatic quantitative trait loci (QTL) hotspots, including a previously unknown QTL on Sb06 with a significant effect on prolonged chilling survival, were found to regulate different physiological mechanisms contributing to maintenance of growth and development despite the chilling temperatures. The major QTL regions harbour promising candidate genes with known roles in abiotic stress tolerance. Identification of loci in the QTL hotspot regions conferring maintenance of cell division and growth under early chilling stress represents a promising step towards breeding for successful establishment of sorghum in temperate climates.

Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum.

Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27kDa γ-, 24kDa α-A1 and the 22kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals.

A Common histone modification code on C4 genes in maize and its conservation in Sorghum and Setaria italica.

C4 photosynthesis evolved more than 60 times independently in different plant lineages. Each time, multiple genes were recruited into C4 metabolism. The corresponding promoters acquired new regulatory features such as high expression, light induction, or cell type-specific expression in mesophyll or bundle sheath cells. We have previously shown that histone modifications contribute to the regulation of the model C4 phosphoenolpyruvate carboxylase (C4-Pepc) promoter in maize (Zea mays). We here tested the light- and cell type-specific responses of three selected histone acetylations and two histone methylations on five additional C4 genes (C4-Ca, C4-Ppdk, C4-Me, C4-Pepck, and C4-RbcS2) in maize. Histone acetylation and nucleosome occupancy assays indicated extended promoter regions with regulatory upstream regions more than 1,000 bp from the transcription initiation site for most of these genes. Despite any detectable homology of the promoters on the primary sequence level, histone modification patterns were highly coregulated. Specifically, H3K9ac was regulated by illumination, whereas H3K4me3 was regulated in a cell type-specific manner. We further compared histone modifications on the C4-Pepc and C4-Me genes from maize and the homologous genes from sorghum (Sorghum bicolor) and Setaria italica. Whereas sorghum and maize share a common C4 origin, C4 metabolism evolved independently in S. italica. The distribution of histone modifications over the promoters differed between the species, but differential regulation of light-induced histone acetylation and cell type-specific histone methylation were evident in all three species. We propose that a preexisting histone code was recruited into C4 promoter control during the evolution of C4 metabolism.

The changes of GA level and signaling are involved in the regulation of mesocotyl elongation during blue light mediated de-etiolation in Sorghum bicolor.

De-etiolation during seedling development is antagonistically regulated by blue light (BL) and gibberellins (GAs). The crosstalk between blue light (BL) and GA metabolism and signaling remains unclear. Using the mutant har1 which is specifically hypersensitive to BL in de-etiolation, the involvement possibility of the GA metabolism, GA signaling in the inhibition of mesocotyl elongation of the sorghum (Sorghum bicolor L. var. R111) seeding under BL was investigated. The inhibition of mesocotyl and cell elongation by BL was restored by application of exogenous GA(3) in har1. The endogenous GA(3) level correspondingly decreased in har1 mesocotyl especially from 1 to 4 h after BL irradiation. Putative genes of GA metabolism enzymes SbGA20ox, SbGA3ox and SbGA2ox were detected by Real-Time PCR and the results showed that one of the SbGA2ox homologs appeared significantly higher transcript level in har1 than in R111 at 2 h after BL irradiation. Putative homologous genes of DELLAs increased after BL irradiation and were higher in har1 among the three homologs. Remarkable increase of the DELLA expression was observed responding to exogenous paclobutrazol (PAC). Our research provided evidence in monocot sorghum, that the changes of a set of the GA metabolism and signaling genes might be involved in BL-induced inhibition of cell elongation.

Elevated CO2 increases water use efficiency by sustaining photosynthesis of water-limited maize and sorghum.

Maize and grain sorghum seeds were sown in pots and grown for 39 days in sunlit controlled-environment chambers at 360 (ambient) and 720 (double-ambient, elevated)µmol mol(-1) carbon dioxide concentrations [CO(2)]. Canopy net photosynthesis (PS) and evapotranspiration (TR) was measured throughout and summarized daily from 08:00 to 17:00h Eastern Standard Time. Irrigation was withheld from matched pairs of treatments starting on 26 days after sowing (DAS). By 35 DAS, cumulative PS of drought-stress maize, compared to well-watered plants, was 41% lower under ambient [CO(2)] but only 13% lower under elevated [CO(2)]. In contrast, by 35 DAS, cumulative PS of drought-stress grain sorghum, compared to well-watered plants, was only 9% lower under ambient [CO(2)] and 7% lower under elevated [CO(2)]. During the 27-35 DAS drought period, water use efficiency (WUE, mol CO(2)Kmol(-1)H(2)O), was 3.99, 3.88, 5.50, and 8.65 for maize and 3.75, 4.43, 5.26, and 9.94 for grain sorghum, for ambient-[CO(2)] well-watered, ambient-[CO(2)] stressed, elevated-[CO(2)] well-watered and elevated-[CO(2)] stressed plants, respectively. Young plants of maize and sorghum used water more efficiently at elevated [CO(2)] than at ambient [CO(2)], especially under drought. Reductions in biomass by drought for young maize and grain sorghum plants were 42 and 36% at ambient [CO(2)], compared to 18 and 14% at elevated [CO(2)], respectively. Results of our water stress experiment demonstrated that maintenance of relatively high canopy photosynthetic rates in the face of decreased transpiration rates enhanced WUE in plants grown at elevated [CO(2)]. This confirms experimental evidence and conceptual models that suggest that an increase of intercellular [CO(2)] (or a sustained intercellular [CO(2)]) in the face of decreased stomatal conductance results in relative increases of growth of C(4) plants. In short, drought stress in C(4) crop plants can be ameliorated at elevated [CO(2)] as a result of lower stomatal conductance and sustaining intercellular [CO(2)]. Furthermore, less water might be required for C(4) crops in future higher CO(2) atmospheres, assuming weather and climate similar to present conditions.

Systemic regulation of leaf anatomical structure, photosynthetic performance, and high-light tolerance in sorghum.

Leaf anatomy of C3 plants is mainly regulated by a systemic irradiance signal. Since the anatomical features of C4 plants are different from that of C3 plants, we investigated whether the systemic irradiance signal regulates leaf anatomical structure and photosynthetic performance in sorghum (Sorghum bicolor), a C4 plant. Compared with growth under ambient conditions (A), no significant changes in anatomical structure were observed in newly developed leaves by shading young leaves alone (YS). Shading mature leaves (MS) or whole plants (S), on the other hand, caused shade-leaf anatomy in newly developed leaves. By contrast, chloroplast ultrastructure in developing leaves depended only on their local light conditions. Functionally, shading young leaves alone had little effect on their net photosynthetic capacity and stomatal conductance, but shading mature leaves or whole plants significantly decreased these two parameters in newly developed leaves. Specifically, the net photosynthetic rate in newly developed leaves exhibited a positive linear correlation with that of mature leaves, as did stomatal conductance. In MS and S treatments, newly developed leaves exhibited severe photoinhibition under high light. By contrast, newly developed leaves in A and YS treatments were more resistant to high light relative to those in MS- and S-treated seedlings. We suggest that (1) leaf anatomical structure, photosynthetic capacity, and high-light tolerance in newly developed sorghum leaves were regulated by a systemic irradiance signal from mature leaves; and (2) chloroplast ultrastructure only weakly influenced the development of photosynthetic capacity and high-light tolerance. The potential significance of the regulation by a systemic irradiance signal is discussed.

Co-regulation of dark and light reactions in three biochemical subtypes of C(4) species.

Regulation of light harvesting in response to changes in light intensity, CO(2) and O(2) concentration was studied in C(4) species representing three different metabolic subtypes: Sorghum bicolor (NADP-malic enzyme), Amaranthus edulis (NAD-malic enzyme), and Panicum texanum (PEP-carboxykinase). Several photosynthetic parameters were measured on the intact leaf level including CO(2) assimilation rates, O(2) evolution, photosystem II activities, thylakoid proton circuit and dissipation of excitation energy. Gross rates of O(2) evolution (J(O)2'), measured by analysis of chlorophyll fluorescence), net rates of O(2) evolution and CO(2) assimilation responded in parallel to changes in light and CO(2) levels. The C(4) subtypes had similar energy requirements for photosynthesis since there were no significant differences in maximal quantum efficiencies for gross rates of O(2) evolution (average value = 0.072 O(2)/quanta absorbed, approximately 14 quanta per O(2) evolved). At saturating actinic light intensities, when photosynthesis was suppressed by decreasing CO(2), ATP synthase proton conductivity (g (H) (+)) responded strongly to changes in electron flow, decreasing linearly with J(O)2', which was previously observed in C(3) plants. It is proposed that g (H) (+) is controlled at the substrate level by inorganic phosphate availability. The results suggest development of nonphotochemical quenching in C(4) plants is controlled by a decrease in g (H) (+), which causes an increase in proton motive force by restricting proton efflux from the lumen, rather than by cyclic or pseudocyclic electron flow.

Alteration of DNA methylation level and pattern in sorghum (Sorghum bicolor L.) pure-lines and inter-line F1 hybrids following low-dose laser irradiation.

Low-dose laser irradiation can stimulate a number of biological processes and has been widely used in various fields including producing useful mutants in crop improvement. Nonetheless, the molecular and genetic basis for the mutagenic property of low-dose laser irradiation has not been elucidated. DNA cytosine methylation is sensitive and responsive to both intrinsic perturbations and environmental cues. This study was aimed to probe the possible effect of low-dose laser irradiation on stability of DNA methylation in sorghum pure-lines and intraspecific F1 hybrids. For this purpose, a pair of Sorghum bicolor L. reciprocal F1 hybrids and their parental pure-lines was used, and their germinating seeds were treated by low-dose laser irradiation. The level and pattern of DNA methylation in the plants were analyzed by methylation-sensitive amplified polymorphism (MSAP). Results showed that low-dose laser irradiation induced low-frequency but significant alterations in DNA methylation level and pattern in sorghum plants, demonstrating the treatment is epigenetically mutagenic in plants. In addition, we observed that the alteration frequency in the inter-line F1 hybrids was higher than that of their pure-line parents, suggesting an interaction of hybridity and the laser irradiation. We propose that the combined use of intraspecific hybridization and an epigenetically mutagenic treatment like low-dose laser irradiation might be a useful means to generate heritable epigenetic variations in plants.

Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase.

The photosynthetic phosphoenolpyruvate carboxylase (C(4)-PEPC) is regulated by phosphorylation by a phosphoenolpyruvate carboxylase kinase (PEPC-k). In Digitaria sanguinalis mesophyll protoplasts, this light-mediated transduction cascade principally requires a phosphoinositide-specific phospholipase C (PI-PLC) and a Ca(2+)-dependent step. The present study investigates the cascade components at the higher integrated level of Sorghum bicolor leaf discs and leaves. PEPC-k up-regulation required light and photosynthetic electron transport. However, the PI-PLC inhibitor U-73122 and inhibitors of calcium release from intracellular stores only partially blocked this process. Analysis of [(32)P]phosphate-labelled phospholipids showed a light-dependent increase in phospholipase D (PLD) activity. Treatment of leaf discs with n-butanol, which decreases the formation of phosphatidic acid (PA) by PLD, led to the partial inhibition of the C(4)-PEPC phosphorylation, suggesting the participation of PLD/PA in the signalling cascade. PPCK1 gene expression was strictly light-dependent. Addition of neomycin or n-butanol decreased, and a combination of both inhibitors markedly reduced PPCK1 expression and the concomitant rise in PEPC-k activity. The calcium/calmodulin antagonist W7 blocked the light-dependent up-regulation of PEPC-k, pointing to a Ca(2+)-dependent protein kinase (CDPK) integrating both second messengers, calcium and PA, which were shown to increase the activity of sorghum CDPK.

Synthesis and phytotoxic activity of ozonides.

The [4 + 3] cycloaddition of the proper furans with the oxyallyl cation, generated in situ from 2,4-dibromopentan-3-one, produced a series of 8-oxabicyclo [3.2.1]oct-6-en-3-ones. Exposure of the oxabicycles to ozone afforded the corresponding 8,9,10,11-tetraoxatricyclo[5.2.1.1 (2,6)]undecan-4-ones in variable yields (7-100%). The phytotoxic properties of these ozonides (or 1,2,4-trioxolanes) and their oxabicycle precursors were evaluated as the ability to interfere with the growth of Sorghum bicolor and Cucumis sativus seedlings. Among oxabicycles, the highest inhibitory activity was shown by compounds possessing a alpha,beta-unsaturated carbonyl moiety. A differential sensitivity of the two crops was evident with ozonides. The most active compounds were also tested against the weed species Ipomoea grandifolia and Brachiaria decumbens. To the best of our knowledge, this is the first article describing ozonides as potential herbicides.

Pollen-mediated transformation of Sorghum bicolor plants.

Pollen from sorghum (Sorghum bicolor L. Moench) were transformed by a novel genetic transformation approach using mild ultrasonication. The plasmid pBI121 contained the NPT II gene encoding NPT II (neomycin phosphotransferase II) and a GUS (beta-glucuronidase) reporter gene. Plasmid DNA and pollen from sorghum A(2)V4B were submerged in a 0.3 mol/l sucrose solution and then subjected to ultrasonication. The treated pollen was then used for pollination of stigmas from the male sterile line A(2)V4A. Detection of transient and stable GUS activity confirmed that the GUS gene was expressed in transgenic plants. PCR amplification and Southern-blot analysis of transgenic plants confirmed that the NPT II gene had been integrated into the sorghum genome. These results indicate that direct gene transfer to pollen can be mediated by mild ultrasonication.

phyB-1 sorghum maintains responsiveness to simulated shade, irradiance and red light: far-red light.

The sorghum [Sorghum bicolor (L.) Moench] phyB-1 mutant exhibits a constitutive shade-avoidance phenotype including excessive shoot elongation. It was previously shown that this mutant also overproduces ethylene. Although phytochrome B (phyB) is assumed to be the pigment most important in sensing and transducing shade signals, the sorghum phyB-1 mutant still responds to light signals characteristic of shade. Specifically, it was determined that the leaf blade : leaf sheath elongation of phyB-1 is responsive to red : far red (R : FR), but this response is opposite that of wild type (WT). Reducing the photosynthetic photon flux density (PPFD) strongly reduced the leaf blade : leaf sheath of WT but did not affect phyB-1, demonstrating a role for phyB in sensing PPFD. Using light-emitting diode (LED) lighting, it was found that WT ethylene production was increased with low R : FR while PPFD had no effect. Conversely, phyB-1 ethylene production increased only with high PPFD, high R : FR which was the treatment resulting in the least ethylene production by WT. Elevated ethylene production inhibits shoot elongation, but may contribute to shade avoidance by reducing leaf blade : leaf sheath elongation. Ethylene responses to light treatments designed to promote or reduce phytochrome A (phyA) activity, and the analysis of PHYA levels in the two cultivars suggests that phyA could be involved in transducing shade signals in light-grown sorghum. Responses potentially tranduced by phyA are elevated in phyB-1 which also over-expresses PHYA.