Interplay between silica deposition and viability during the life span of sorghum silica cells.

Silica cells are specialized epidermal cells found on both surfaces of grass leaves, with almost the entire lumen filled with solid silica. The mechanism precipitating silicic acid into silica is not known. Here we investigate this process in sorghum (Sorghum bicolor) leaves. Using fluorescent confocal microscopy, we followed silica cells' ontogeny, aiming to understand the fate of vacuoles and nuclei. Correlating the confocal and scanning electron microscopy, we timed the initiation of silica deposition in relation to cell's viability. Contrary to earlier reports, silica cells did not lose their nucleus before silica deposition. Vacuoles in silica cells did not concentrate silicic acid. Instead, postmaturation silicification initiated at the cell periphery in live cells. Less than 1% silica cells showed characteristics of programmed cell death in the cell maturation zone. In fully elongated mature leaves, 2.4% of silica cells were nonsilicified and 1.6% were partially silicified. Silica deposition occurs in the paramural space of live silica cells. The mineral does not kill the cells. Instead, silica cells are genetically programmed to undergo cell death independent of silicification. Fully silicified cells seem to have nonsilicified voids containing membrane remains after the completion of the cell death processes.

Sensitivity of sorghum pollen and pistil to high-temperature stress.

High temperature (HT) decreases seed set percentage in sorghum (Sorghum bicolor [L.] Moench). The relative sensitivity of pollen and particularly pistil and the mechanistic response that induces tolerance or susceptibility to HT are not well known and hence are the major objectives of this research. The male sterile (ATx399) and fertile (RTx430) lines were exposed to 30/20 °C (optimum temperature), 36/26 °C (HT1 ), and 39/29 °C (HT2 ) from the start of booting to seed set in a controlled environment. Similarly, in the field, HT stress was imposed using heat tents. HT stress decreased pollen germination. Relatively high levels of reactive oxygen species and decreased antioxidant enzyme activity and phospholipid unsaturation were observed in pollen compared to pistil under HT. The severe cell organelle damage was observed in pollen and pistil at 36/26 and 39/29 °C, respectively. The seed set percentage was higher in HT-stressed pistil pollinated with optimum-temperature pollen. Direct and reciprocal crosses indicate that pollen was more sensitive with larger decreases in seed set percentage than pistil under HT stress. The negative impact was greater in pollen than pistil at lower temperatures. Overall, pollen was more sensitive than pistil to HT stress because it is more susceptible to oxidative damage than pistil.

Host specificity in Sporisorium reilianum is determined by distinct mechanisms in maize and sorghum.

Smut fungi are biotrophic plant pathogens that exhibit a very narrow host range. The smut fungus Sporisorium reilianum exists in two host-adapted formae speciales: S. reilianum f. sp. reilianum (SRS), which causes head smut of sorghum, and S. reilianum f. sp. zeae (SRZ), which induces disease on maize. It is unknown why the two formae speciales cannot form spores on their respective non-favoured hosts. By fungal DNA quantification and fluorescence microscopy of stained plant samples, we followed the colonization behaviour of both SRS and SRZ on sorghum and maize. Both formae speciales were able to penetrate and multiply in the leaves of both hosts. In sorghum, the hyphae of SRS reached the apical meristems, whereas the hyphae of SRZ did not. SRZ strongly induced several defence responses in sorghum, such as the generation of H2 O2 , callose and phytoalexins, whereas the hyphae of SRS did not. In maize, both SRS and SRZ were able to spread through the plant to the apical meristem. Transcriptome analysis of colonized maize leaves revealed more genes induced by SRZ than by SRS, with many of them being involved in defence responses. Amongst the maize genes specifically induced by SRS were 11 pentatricopeptide repeat proteins. Together with the microscopic analysis, these data indicate that SRZ succumbs to plant defence after sorghum penetration, whereas SRS proliferates in a relatively undisturbed manner, but non-efficiently, on maize. This shows that host specificity is determined by distinct mechanisms in sorghum and maize.

In vivo monitoring of intracellular chloroplast movements in intact leaves of C4 plants using two-photon microscopy.

Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light-induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two-photon microscopy (TPM) system was adapted to monitor the intracellular 3-dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto-second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub-epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions.

Effects of extrusion conditions on physical and nutritional properties of extruded whole grain red sorghum (sorghum spp).

In order to analyze the effects of extrusion temperature (T: 164, 182, 200 °C) and grits moisture content (g/100 g sample) (%M: 14, 16.5, 19) on textural and physicochemical properties of red sorghum extrudates, whole grain flour was extruded according to a factorial experimental design. The higher values for specific mechanical energy consumption (1006.98 J/g) and expansion (3.36) were obtained at 164 °C-14%M and for sensorial hardness at 164 °C-19%M. While for specific volume, the highest value (10.41 cm³/g) was obtained at 200 °C-14%M. Water solubility and water absorption were directly related with T and inversely with M. Microscopic observation of the samples indicates that the greatest cooking degree was obtained at 200 °C-4%M and the lowest at 164 °C-19%M. Extrusion at 182 °C-14%M allows obtaining an expanded product with good properties. Proximal composition did not show statistically significant differences with raw sample. Extruded sample showed a 25.4% reduction of available lysine and a 31% increase in protein digestibility.

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum.

Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

[Impacts of drought stress on leaf osmotic adjustment and chloroplast ultrastructure of stay-green sorghum].

Taking stay-green sorghum (B35) and non-stay green sorghum (Sanchisan) as test materials, a pot experiment was conducted to study their leaf osmotic adjustment and chloroplast ultrastructure at flowering and filling stages under impacts of drought stress (45% -50% of maximum field capacity). For the two sorghum lines, drought stress caused the reduction of their leaf free water content and relative water content, and increased the leaf bound water content, water saturation deficit, and electrical conductivity, with the increment or dement being larger for Sanchisan than for B35. Drought stress increased the leaf soluble sugar content and proline content, with the increment of the soluble sugar content being larger for Sanchisan and the increment of the proline content being larger for B35, while decreased the leaf soluble protein content, with the decrement being larger for Sanchisan than for B35. The chloroplast ultrastructure of both B35 and Sanchisan under drought stress was damaged to some extent, but the damaged degree was obviously lower for B35 than for Sanchisan. The findings indicated that stay-green sorghum had a greater adaptation to drought stress through stronger osmotic adjustment.

Development of basal endosperm transfer cells in Sorghum bicolor (L.) Moench and its relationship with caryopsis growth.

During sorghum caryopsis development, endosperm epidermal cells near the basal main vascular bundle are specialized by depositing wall ingrowths, differentiating into basal endosperm transfer cells (BETCs). All the BETCs together compose the basal endosperm transfer layer (BETL). BETCs are the first cell type to become histologically differentiated during endosperm development. The initiation and subsequent development of BETCs shows the pattern of temporal and spatial gradient. The developmental process of BETL can be divided into four stages: initiation, differentiation, functional, and apoptosis stage. A placental sac full of nutrient solutions would emerge, enlarge, and eventually disappear between the outmost layer of BETL and nucellar cells during caryopsis development. BETCs have dense cytoplasm rich in mitochondria, lamellar rough endoplasmic reticulum, Golgi bodies, and their secretory vesicles. They show a series of typical characteristics of senescence such as nuclei distortion and subcellular organelle deterioration during their specialization. BETCs probably play an active role in nutrient transfer into the starchy endosperm and embryo. The occurrence, development, and apoptosis of BETCs are in close relation to the caryopsis growth and maturation especially the enrichment of endosperm and the growth of embryo. The timing when BETL is fully developed, composed of three to four layers in radial direction and 70 to 80 rows in tangential direction, consists with the timing when average daily gain of caryopsis dry weight reaches its maximum. It is conceivable that measures that delay the senescence and death of BETCs would help to increase the crop yield.

Effect of silver nanoparticles in crop plants Phaseolus radiatus and Sorghum bicolor: media effect on phytotoxicity.

Understanding some adverse effects of nanoparticles in edible crop plants is a matter of importance because nanoparticles are often released into soil environments. We investigated the phytotoxicity of silver nanoparticles (AgNPs) on the important crop plants, Phaseolus radiatus and Sorghum bicolor. The silver nanoparticles were selected for this study because of their OECD designation as a priority nanomaterial. The toxicity and bioavailability of AgNPs in the crop plant species P. radiatus and S. bicolor were evaluated in both agar and soil media. The seedling growth of test species was adversely affected by exposure to AgNPs. We found evidence of nanoparticle uptake by plants using electron microscopic studies. In the agar tests, P. radiatus and S. bicolor showed a concentration dependent-growth inhibition effect. Measurements of the growth rate of P. radiatus were not affected in the soil studies by impediment within the concentrations tested herein. Bioavailability of nanoparticles was reduced in the soil, and the dissolved silver ion effect also differed in the soil as compared to the agar. The properties of nanoparticles have been shown to change in soil, so this phenomenon has been attributed to the reduced toxicity of AgNPs to plants in soil medium. The application of nanoparticles in soil is a matter of great importance to elucidate the terrestrial toxicity of nanoparticles.

Microprobing the molecular spatial distribution and structural architecture of feed-type sorghum seed tissue (Sorghum Bicolor L.) using the synchrotron radiation infrared microspectroscopy technique.

Sorghum seed (Sorghum bicolor L.) has unique degradation and fermentation behaviours compared with other cereal grains such as wheat, barley and corn. This may be related to its cell and cell-wall architecture. The advanced synchrotron radiation infrared microspectroscopy (SR-IMS) technique enables the study of cell or living cell biochemistry within cellular dimensions. The objective of this study was to use the SR-IMS imaging technique to microprobe molecular spatial distribution and cell architecture of the sorghum seed tissue comprehensively. High-density mapping was carried out using SR-IMS on beamline U2B at the National Synchrotron Light Source (Brookhaven National Laboratory, NY, USA). Molecular images were systematically recorded from the outside to the inside of the seed tissue under various chemical functional groups and their ratios [peaks at ∼1725 (carbonyl C=O ester), 1650 (amide I), 1657 (protein secondary structure α-helix), 1628 (protein secondary structure ß-sheet), 1550 (amide II), 1515 (aromatic compounds of lignin), 1428, 1371, 1245 (cellulosic compounds in plant seed tissue), 1025 (non-structural CHO, starch granules), 1246 (cellulosic material), 1160 (CHO), 1150 (CHO), 1080 (CHO), 930 (CHO), 860 (CHO), 3350 (OH and NH stretching), 2960 (CH(3) anti-symmetric), 2929 (CH(2) anti-symmetric), 2877 (CH(3) symmetric) and 2848 cm(-1) (CH(2) asymmetric)]. The relative protein secondary structure α-helix to ß-sheet ratio image, protein amide I to starch granule ratio image, and anti-symmetric CH(3) to CH(2) ratio image were also investigated within the intact sorghum seed tissue. The results showed unique cell architecture, and the molecular spatial distribution and intensity in the sorghum seed tissue (which were analyzed through microprobe molecular imaging) were generated using SR-IMS. This imaging technique and methodology has high potential and could be used for scientists to develop specific cereal grain varieties with targeted food and feed quality, and can also be used to monitor the degree of grain maturity, grain damage, the fate of organic contaminants and the effect of chemical treatment on plant and grain seeds.

Structural and rheological characterisation of heteropolysaccharides produced by lactic acid bacteria in wheat and sorghum sourdough.

Hydrocolloids improve the volume, texture, and shelf life of bread. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough fermentation can replace hydrocolloids. It was the aim of this study to determine whether heteropolysaccharides (HePS) synthesized intracellularly from sugar nucleotides by glycosyltransferases are produced in wheat and gluten-free sorghum sourdough at effective levels. The HePS-producing strains Lactobacillus casei FUA3185, L. casei FUA3186, and Lactobacillus buchneri FUA3154 were used; Weissella cibaria 10M producing no EPS in the absence of sucrose served as control strain. Cell suspensions of L. buchneri in MRS showed the highest viscosity at low shear rate. Glycosyltransferase genes responsible of HePS formation in LAB were expressed in sorghum and wheat sourdough. However, only HePS produced by L. buchneri influenced the rheological properties of sorghum sourdoughs but not of wheat sourdoughs. Sorghum sourdough fermented with L. buchneri exhibited a low |G*| compared to the control, indicating a decrease in resistance to deformation. An increase in tan δ indicated decreased elasticity. The use of LAB producing HePS expands the diversity of EPS and increases the variety of cultures for use in baking.

The effects of four different pretreatments on enzymatic hydrolysis of sweet sorghum bagasse.

Four pretreatment processes including ionic liquids, steam explosion, lime, and dilute acid were used for enzymatic hydrolysis of sweet sorghum bagasse. Compared with the other three pretreatment approaches, steam-explosion pretreatment showed the greatest improvement on enzymatic hydrolysis of the bagasse. The maximum conversion of cellulose and the concentration of glucose obtained from enzymatic hydrolysis of steam explosion bagasse reached 70% and 25 g/L, respectively, which were both 2.5 times higher than those of the control (27% and 11 g/L). The results based on the analysis of SEM photos, FTIR, XRD and NMR detection suggested that both the reduction of crystallite size of cellulose and cellulose degradation from the Iα and Iß to the Fibril surface cellulose and amorphous cellulose were critical for enzymatic hydrolysis. These pretreatments disrupted the crystal structure of cellulose and increased the available surface area, which made the cellulose better accessible for enzymatic hydrolysis.

Immunodetection and subcellular localization of Mal de Río Cuarto virus P9-1 protein in infected plant and insect host cells.

Mal de Río Cuarto virus (MRCV), a member of the genus Fijivirus, family Reoviridae, has a genome consisting of 10 dsRNA segments. The segment 9 (S9) possesses two non-overlapping open reading frames (ORF-1 and ORF-2) encoding two putative proteins, MRCV P9-1 and MRCV P9-2, both of unknown function. The MRCV S9 ORF-1 was RT-PCR amplified, expressed in pET-15b vector, and the recombinant protein produced was used to raise an antiserum in rabbit. Western blot with the specific MRCV P9-1 antiserum detected a protein of about 39 kDa molecular weight present in crude protein extracts from infected plants and insects. However, no reaction was observed when this antiserum was tested against purified virus. In contrast, only virus particles were detected by a MRCV-coat antiserum used as a validation control. These results suggest that MRCV S9 ORF-1 encodes a non-structural protein of MRCV. Immunoelectron microscopy assays confirmed these results, and localized the MRCV P9-1 protein exclusively in electron-dense granular viroplasms within the cytoplasm of infected plants and insects cells. As viroplasms are believed to be the replication sites of reoviruses, the intracellular location of MRCV P9-1 protein suggests that it might be involved in the assembly process of MRCV particles.

Ethanol production from sorghum by a dilute ammonia pretreatment.

Sorghum fibers were pretreated with ammonium hydroxide and the effectiveness of the pretreatment evaluated by enzyme hydrolysis and ethanol production. The treatment was carried out by mixing sorghum fibers, ammonia, and water at a ratio of 1:0.14:8 at 160 degrees C for 1 h under 140-160 psi pressure. Approximately 44% lignin and 35% hemicellulose were removed during the process. Untreated and dilute-ammonia-treated fibers at 10% dry solids were hydrolyzed using combinations of commercially available enzymes, Spezyme CP and Novozyme 188. Enzyme combinations were tested at full strength (60 FPU Spezyme CP and 64 CBU Novozyme 188/g glucan) and at half strength (30 FPU Spezyme CP and 32 CBU Novozyme 188/g glucan). Biomass enzyme hydrolysis was conducted for 24 h. Saccharomyces cerevisiae D(5)A was added post hydrolysis for conversion of glucose to ethanol. Theoretical cellulose yields for treated biomass were 84% and 73%, and hemicellulose yields were 73% and 55% for full strength and half strength, respectively. Average cellulose yield was 38% and hemicellulose yield was 14.5% for untreated biomass. Ethanol yields were 25 g/100 g dry biomass and 21 g/100 g dry biomass for full strength and half strength enzyme concentrations, respectively. Controls averaged 10 g ethanol/100 g dry biomass.

Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis.

The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78-84% similarity, and yeasts 80-92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide.

Inheritance of inflorescence architecture in sorghum.

The grass inflorescence is the primary food source for humanity, and has been repeatedly shaped by human selection during the domestication of different cereal crops. Of all major cultivated cereals, sorghum [Sorghum bicolor (L.) Moench] shows the most striking variation in inflorescence architecture traits such as branch number and branch length, but the genetic basis of this variation is little understood. To study the inheritance of inflorescence architecture in sorghum, 119 recombinant inbred lines from an elite by exotic cross were grown in three environments and measured for 15 traits, including primary, secondary, and tertiary inflorescence branching. Eight characterized genes that are known to control inflorescence architecture in maize (Zea mays L.) and other grasses were mapped in sorghum. Two of these candidate genes, Dw3 and the sorghum ortholog of ramosa2, co-localized precisely with QTL of large effect for relevant traits. These results demonstrate the feasibility of using genomic and mutant resources from maize and rice (Oryza sativa L.) to investigate the inheritance of complex traits in related cereals.

In vitro culture methods in sorghum with shoot tip as the explant material.

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.