Sorghum Tissues Research Articles

Gene Editing in Sorghum Through Agrobacterium.

The application of CRISPR/Cas to introduce targeted genomic edits is powering research and discovery across the genetic frontier. Applying CRISPR/Cas in sorghum can facilitate the study of gene function and unlock our understanding of this robust crop that serves as a staple for some of the most food insecure regions on the planet. When paired with recent advances in sorghum tissue culture and Agrobacteria technology, CRISPR/Cas can be used to introduce desirable changes and natural genetic variations directly into agriculturally relevant sorghum lines facilitating product development. This chapter describes CRISPR/Cas gene editing and provides high-level strategies and expectations for applying this technology using Agrobacterium in sorghum.

Establishment of Metarhizium anisopliae (Metchnikoff) sorokin as endophyte in maize and sorghum

Glasshouse experiments were conducted to establish an indigenous soil-derived strain of Metarhizium anisopliae (ICAR-NBAIR Ma-35) as endophyte in maize and sorghum by artificial inoculation through foliar spray of conidial suspension. Colonization of M. anisopliae were studied at 15, 30, 45, 60, 75 and 90 Days After Treatment (DAT) in stem and leaf tissues of maize and sorghum by plating technique and PCR method. Metarhizium anisopliae showed variation with regard to the extent of colonization in stem and leaf tissues of maize and sorghum. In maize, colonization was observed at 30DAT in stem and 30-45DAT in leaf. In sorghum, colonization of M. anisopliae was observed during 15-60DAT in stem and 15-75DAT in leaf indicating longer persistence compared to maize. Colonization of M. anisopliae was observed in young growing stem tips and leaves (unsprayed) indicating the internal spread of M. anisopliae in maize and sorghum plants. M. anisopliae was not detected in the untreated stem and leaf tissues of maize and sorghum. The positive results of colonization of M. anisopliae in maize and sorghum stem/leaf tissues observed in plating technique were confirmed by the PCR amplification.

Method for Label-Free Quantitative Proteomics for Sorghum bicolor L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.

Alleviation of Al Toxicity by Si Is Associated with the Formation of Al-Si Complexes in Root Tissues of Sorghum.

Silicon is reported to reduce the toxic effects of Al on root elongation but the in planta mechanism by which this occurs remains unclear. Using seedlings of soybean (Glycine max) and sorghum (Sorghum bicolor), we examined the effect of up to 2 mM Si on root elongation rate (RER) in Al-toxic nutrient solutions. Synchrotron-based low energy X-ray fluorescence (LEXRF) was then used for the in situ examination of the distribution of Al and Si within cross-sections cut from the apical tissues of sorghum roots. The addition of Si potentially increased RER in Al-toxic solutions, with RER being up to ca. 0.3 mm h-1 (14%) higher for soybean and ca. 0.2 mm h-1 (17%) higher for sorghum relative to solutions without added Si. This improvement in RER could not be attributed to a change in Al-chemistry of the bulk nutrient solution, nor was it due to a change in the concentration of Al within the apical (0–10 mm) root tissues. Using LEXRF to examine sorghum, it was demonstrated that in roots exposed to both Al and Si, much of the Al was co-located with Si in the mucigel and outer apoplast. These observations suggest that Si reduces the toxicity of Al in planta through formation of Al–Si complexes in mucigel and outer cellular tissues, thereby decreasing the binding of Al to the cell wall where it is known to inhibit wall loosening as required for cell elongation.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.

Cs phytoremediation by Sorghum bicolor cultivated in soil and in hydroponic system

ABSTRACTCs accumulation characteristics by Sorghum bicolor were investigated in hydroponic system (Cs level at 50–1000 μmol/L) and in soil (Cs-spiked concentration was 100 and 400 mg/kg soil). Two varieties of S. bicolor Cowly and Nengsi 2# grown on pot soil during the entire growth period (100 days) did not show significant differences on the height, dry weight (DW), and Cs accumulation. S. bicolor showed the potential phytoextraction ability for Cs-contaminated soil with the bioaccumulation factor (BCF) and the translocation factor (TF) values usually higher than 1 in soil system and in hydroponic system. The aerial parts of S. bicolor contributed to 86–92% of the total removed amounts of Cs from soil. Cs level in solution at 100 μmol/L gave the highest BCF and TF values of S. bicolor. Cs at low level tended to transfer to the aerial parts, whereas Cs at high level decreased the transfer ratio from root to shoot. In soil, the plant grew well when Cs spiked level was 100 mg/kg soil, but was inhibited by Cs at 400 mg/kg soil with Cs content in sorghum reaching 1147 mg/kg (roots), 2473 mg/kg (stems), and 2939 mg/kg (leaves). In hydroponic system, average Cs level in sorghum reached 5270 mg/kg (roots) and 4513 mg/kg (aerial parts), without significant damages to its biomass at 30 days after starting Cs treatment. Cs accumulation in sorghum tissues was positively correlated with the metal concentration in medium.

MYB31/MYB42 Syntelogs Exhibit Divergent Regulation of Phenylpropanoid Genes in Maize, Sorghum and Rice.

ZmMYB31 and ZmMYB42 are R2R3-MYB transcription factors implicated in the regulation of phenylpropanoid genes in maize. Here, we tested the hypothesis that the regulatory function of MYB31 and MYB42 is conserved in other monocots, specifically in sorghum and rice. We demonstrate that syntelogs of MYB31 and MYB42 do bind to phenylpropanoid genes that function in all stages of the pathway and in different tissues along the developmental gradient of seedling leaves. We found that caffeic acid O-methyltransferase (COMT1) is a common target of MYB31 and MYB42 in the mature leaf tissues of maize, sorghum and rice, as evidenced by Chromatin immunoprecipitation (ChIP) experiments. In contrast, 4-coumarate-CoA ligase (4CL2), ferulate-5-hydroxylase (F5H), and caffeoyl shikimate esterase (CSE), were targeted by MYB31 or MYB42, but in a more species-specific fashion. Our results revealed MYB31 and MYB42 participation in auto- and cross-regulation in all three species. Apart from a limited conservation of regulatory modules, MYB31 and MYB42 syntelogs appear to have undergone subfunctionalization following gene duplication and divergence of maize, sorghum, and rice. Elucidating the different regulatory roles of these syntelogs in the context of positive transcriptional activators may help guide attempts to alter the flux of intermediates towards lignin production in biofuel grasses.

Map-based cloning and expression analysis of BMR-6 in sorghum.

Brown midrib mutants in sorghum are associated with reduced lignin content and increased cell wall digestibility. In this study, we characterized a bmr-6 sorghum mutant, which shows reddish pigment in the midrib and stem after the fifth-leaf stage. Compared to wild type, Kalson lignin content of bmr-6 is decreased significantly. We used histological analysis to determine that the mutant exhibited a modified pattern of lignin staining and found an increased polysaccharide content. We cloned BMR-6 gene, a gene encoded a cinnamyl alcohol dehydrogenase (CAD), using a map-based cloning approach. Genetic complementation confirmed that CAD is responsible for the BMR-6 phenotype. BMR-6 gene was expressed in all tested sorghum tissues, with the highest being in midrib and stem. Transient expression assays in Nicotiana benthamiana leaves demonstrated cytomplasmic localization of BMR-6. We found that the expression level of bmr-6 was significantly decreased in the mutant but expression of SbCAD3 and SbCAD5 were significantly increased. Our results indicate that BMR-6 not only affects the distribution of lignin but also the biosynthesis of lignin in sorghum.

Use of sewage sludge in bioenergy production—A case study on the effects on sorghum biomass production

The aim of the study was to determine the effect of different doses of municipal sewage sludge (0, 10, 20, 40, 60MgDMha−1) on sweet sorghum yields and quality as well as changes in physico-chemical and biological properties of the soil. In a three-year field experiment located on loamy clay on an area designated for a landfill site, three sorghum varieties (GK Csaba, Róna 1, Sucrosorgo 506) were tested in order to determine which one exploited the yielding potential of sewage sludge.It was found out that application of the highest doses of sewage sludge resulted in the highest yield of plants biomass. Among the tested cultivars of sorghum the most productive was Sucrosorgo 506, while the GK Csaba was characterized by the lowest productivity. What is more, the differences in yields of individual varieties were correlated with the level of sewage sludge fertilization. Sucrosorgo 506 generated the highest yields of biomass after the highest doses of sludge application, the best results for the GK Csaba variety were obtained after the application of 40MgDM sewage sludge per hectare, while Róna 1 gave the highest yields after applying the smallest doses of sludge. It was also observed that the content, uptake and index of bioaccumulation of macronutrients and heavy metals contained in the sludge increased along with the increasing dose of the applied biosolids, reaching the maximum at 60MgDMha−1. Sorghum biomass was characterized by favourable net and gross calorific values, which were the highest in objects with the lowest doses of sewage sludge. The highest energy value of biomass yield was obtained in the case of the Sucrosorgo 506 variety (on average 144GJMg−1ha−1), about 32% lower energy value was found in Róna 1, while the energy value of GK Csaba biomass was by half lower in comparison to Sucrosorgo 506. Sorghum tissues bioaccumulated nitrogen and cadmium intensively, zinc, copper, and nickel—at a medium level, and potassium, phosphorous, magnesium, chromium and lead were slightly accumulated. Introduction of the higher doses of municipal sewage sludge significantly affected the physico-chemical properties and enzymatic activity of soil, decreasing its pH but increasing hydrolytic acidity, total nitrogen as well as the concentration of available macronutrients but at the same time the heavy metals content. Municipal sewage sludge contributed to an increase in the organic carbon concentration, which varied primarily due to the different doses of sewage sludge. Sewage sludge introduction also resulted in a marked increase in enzymatic activity compared to the control objects, wherein the activity of dehydrogenases, acid and alkaline phosphatase, protease as well as urease increased progressively with increasing doses of sludge.

Distribution, structure and biosynthetic gene families of (1,3;1,4)-β-glucan in Sorghum bicolor.

In cereals, the presence of soluble polysaccharides including (1,3;1,4)-β-glucan has downstream implications for human health, animal feed and biofuel applications. Sorghum bicolor (L.) Moench is a versatile crop, but there are limited reports regarding the content of such soluble polysaccharides. Here, the amount of (1,3;1,4)-β-glucan present in sorghum tissues was measured using a Megazyme assay. Very low amounts were present in the grain, ranging from 0.16%-0.27% (w/w), while there was a greater quantity in vegetative tissues at 0.12-1.71% (w/w). The fine structure of (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl and cellotetraosyl residues, was assessed by high performance liquid chromatography (HPLC) and ranged from 2.6-3:1 in the grain, while ratios in vegetative tissues were lower at 2.1-2.6:1. The distribution of (1,3;1,4)-β-glucan was examined using a specific antibody and observed with fluorescence and transmission electron microscopy. Micrographs showed a variable distribution of (1,3;1,4)-β-glucan influenced by temporal and spatial factors. The sorghum orthologs of genes implicated in the synthesis of (1,3;1,4)-β-glucan in other cereals, such as the Cellulose synthase-like (Csl) F and H gene families were defined. Transcript profiling of these genes across sorghum tissues was carried out using real-time quantitative polymerase chain reaction, indicating that, as in other cereals, CslF6 transcripts dominated.

A robust tissue culture system for sorghum [Sorghum bicolor (L.) Moench

Sorghum tissue culture has been challenged by three predominant obstacles for decades, namely toxic pigments (phenolics), low regeneration frequencies and short duration of callus regenerability. Here, we report a robust tissue culture system for sorghum, which has minimized these major impediments. To optimize media, different concentrations of various plant growth regulators, such as 2,4-dichlorophenoxyacetic acid (2,4-D), N6-benzyladenine (BA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) were evaluated. Additional ingredients, including KH2PO4, CuSO4·5H2O, l-asparagine, l-proline and polyvinylpyrrolidone (PVP) were also assessed. Results showed that callus age had a conspicuous effect on its growth and regenerability, with callus weekly growth ratio and regenerability peaked at two weeks after induction. A callus induction rate up to 100% was achieved in inbred line Tx430, whereas regeneration rates up to 100% were obtained from SA281 and 91419R. This highly efficient system has been utilized for sorghum transformation for several years and has been proven to be reliable and reproducible.

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, “SDS-extracted” sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.

Development and function of caryopsis transport tissues in maize, sorghum and wheat

Cereal caryopsis transport tissues are essential channels via which nutrients are transported into the embryo and endosperm. There are differences and similarities between caryopsis transport tissues of maize, sorghum and wheat. Vascular bundle, endosperm transfer cells, endosperm conducting cells and embryo surrounding region are common in maize, sorghum and wheat. Placentochalaza is special in maize and sorghum, while chalaza and nucellar projection transfer cells are special in wheat. There is an obvious apoplastic cavity between maternal and filial tissues in sorghum and wheat caryopses, but there is no obvious apoplastic cavity in maize caryopsis. Based on the latest research, the development and function of the three cereal caryopsis transport tissues are discussed and investigated in this paper.

Suitability of Urban Wastes for Crop Production in Zaria, Northern Nigeria: Bioavailability, Phytotoxicity, and Fractions of Micronutrients

The suitability of two composted solid urban wastes for crop production was evaluated in a pot experiment with sorghum (Sorghum bicolor) that focused on the geochemical fractions, bioavailability, and phytotoxicity of copper (Cu), manganese (Mn), and zinc (Zn). Total concentrations of Cu, Mn, and Zn in soil increased with increasing waste application, ranging from 1.6 to 48.2 mg kg−1 for Cu, 84 to 474 mg kg−1 for Mn, and 13.8 to 597 mg kg−1 for Zn. Waste application significantly increased pH and electrical conductivity (EC) of the soil. Copper, Mn, and Zn in the waste-amended soil were speciated into mobile (F1), easily mobilizable (F2), occluded in Mn oxides (F3), organically bound (F4), occluded in amorphous Fe oxides (F5), occluded in crystalline Fe oxides (F6), and residual (F7) fractions to assess the lability of the metals. On the average, the F4 was the most dominant Cu and Zn fraction, accounting for between 37 and 60% of total Cu and from 14 to 40% of total Zn concentrations, whereas F3 was the dominant Mn fraction closely followed by F4. The concentrations of Cu, Mn, and Zn in sorghum dry matter (DM) decreased with increasing waste application, probably induced by osmotic stress and ionic toxicity. Tissue Zn (Y-Zn) and Mn (Y-Mn) correlated significantly with the F1 and F2 fractions, but pH was an overriding factor in predicting Cu and Zn bioconcentration. Used as soil amendments, the application rate for these Zaria urban wastes should be limited to ≤10% (w/w basis), as Zn in the sorghum tissue reached the toxic limit just from one application of the waste to soil.

Genomic inversion caused by gamma irradiation contributes to downregulation of a WBC11 homolog in bloomless sorghum

Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight-a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F2 bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.

Sorghum Allelopathy—From Ecosystem to Molecule

Sorghum allelopathy has been reported in a series of field experiments following sorghum establishment. In recent years, sorghum phytotoxicity and allelopathic interference also have been well-described in greenhouse and laboratory settings. Observations of allelopathy have occurred in diverse locations and with various sorghum plant parts. Phytotoxicity has been reported when sorghum was incorporated into the soil as a green manure, when residues remained on the soil surface in reduced tillage settings, or when sorghum was cultivated as a crop in managed fields. Allelochemicals present in sorghum tissues have varied with plant part, age, and cultivar evaluated. A diverse group of sorghum allelochemicals, including numerous phenolics, a cyanogenic glycoside (dhurrin), and a hydrophobic p-benzoquinone (sorgoleone) have been isolated and identified in recent years from sorghum shoots, roots, and root exudates, as our capacity to analyze and identify complex secondary products in trace quantities in the plant and in the soil rhizosphere has improved. These allelochemicals, particularly sorgoleone, have been widely investigated in terms of their mode(s) of action, specific activity and selectivity, release into the rhizosphere, and uptake and translocation into sensitive indicator species. Both genetics and environment have been shown to influence sorgoleone production and expression of genes involved in sorgoleone biosynthesis. In the soil rhizosphere, sorgoleone is released continuously by living root hairs where it accumulates in significant concentrations around its roots. Further experimentation designed to study the regulation of sorgoleone production by living sorghum root hairs may result in increased capacity to utilize sorghum cover crops more effectively for suppression of germinating weed seedlings, in a manner similar to that of soil-applied preemergent herbicides like trifluralin.

Quantitative RT-PCR analysis of 20 transcription factor genes of MADS, ARF, HAP2, MBF and HB families in moisture stressed shoot and root tissues of sorghum

Transcription factors (TFs) are an important target in understanding the regulation of plant responses to environmental stress including moisture stress. Members of the same TF family may differ in their response to moisture stress. The expression pattern could vary between shoot and root tissues depending on level of moisture stress. A set of five rarely studied TF families viz., MADS-box (MCM1, AGAMOUS, DEFICIENS and SRF), Auxin Responsive Factor (ARF), Heme Activator Protein 2 (HAP2), Multiprotein Bridging Factor (MBF) and Homeobox (HB) together having 20 members in sorghum, were expression analyzed through quantitative real-time PCR (qRT-PCR) in well watered and moisture stressed shoot and root tissues of sorghum using SYBR Green® to quantify dsDNA synthesis. Fluorescence values were used to calculate PCR efficiency by using LinRegPCR. The PTSb00029.1 and PTSb00033.1 of ARF family and PTSb00174.1 and PTSb00175.1 of HB family recorded 2 to 5, PTSb00221.1 and PTSb00208.1 of MADS family and PTSb00128.1 of HAP2 family recorded 5 to 10 fold up-regulation under moisture stress regimes. The PTSb00128.1, a HAP2 family member, recorded 15 fold up-regulation in mild moisture stressed root tissues. TF genes such as PTSb00218.1, PTSb00220.1, PTSb00031.1, PTSb00032.1, PTSb00034.1 and PTSb00223.1 were found down regulating in both tissues types under moisture stress condition. However, the PTSb00128.1, PTSb00221.1, PTSb00029.1, PTSb00033.1 and PTSb00174.1 TFs were found up-regulating to varied levels in mild and severe moisture stressed root tissues only. Verification of qRT-PCR results was done by in situ hybridization (ISH) of randomly selected two TF genes in shoot and root tissues of sorghum. Taken together, moisture stress triggered up-regulation of more genes in root tissue compared to shoot tissue in sorghum.

Management effects on bioenergy sorghum growth, yield and nutrient uptake

Bioenergy sorghum (Sorghum bicolor L. Moench.) productivity and nutrient uptake may be affected by cropping sequence complexity, fertilization, and residue removal. The goal of this research was to optimize the efficiency of crop management in high biomass (bioenergy) sorghum systems. The two-year field study was conducted in two diverse locations near College Station and Weslaco, Texas. The study utilized a complete factorial design with four replications of the following factors: Rotation: continuous biomass sorghum vs. biannual rotation with corn (Zea mays L.); Stover Return: 0, 25, 50% of the sorghum biomass and all corn stover; and N Rate: 0 vs. non-limiting N. The bioenergy sorghum used was a high-yield photoperiod-sensitive hybrid. Other inputs and practices were those commonly used in the respective areas. Sorghum was harvested for yield, and C, N, P and K, were determined. Rotation, fertilization, and residue return affected yields, plant growth, and nutrient uptake (p < 0.05). Total yields, C, N, P, and K uptake in sorghum were significantly increased by rotation and N fertilization both years, while 25% residue return increased sorghum yield and N uptake at College Station the first year. Uptake of C, N, P and K were increased by N fertilization. Sorghum tissue concentrations of N, P and K declined from the first to the second year although mean yield increased, possibly indicating decreased soil nutrient availability after only two years. A regression equation was developed relating biomass yield and site, rotation, nitrogen rate, and plant physical traits (R2 = 0.67).

Homolog of the maize -glucosidase aggregating factor from sorghum is a jacalin-related GalNAc-specific lectin but lacks protein aggregating activity

Recently, we identified the maize beta-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for beta-glucosidase aggregation. By searching for BGAF homologs in sorghum, we identified and obtained an EST clone and determined its complete sequence. The predicted protein had the same modular structure as maize BGAF, shared 67% sequence identity with it, and revealed the presence of two potential carbohydrate-binding sites (GG...ATYLQ, site I and GG...GVVLD, site II). Maize BGAF1 is the only lectin from a class of modular JRLs containing an N-terminal dirigent and a C-terminal JRL domain, whose sugar specificity and beta-glucosidase aggregating activity have been studied in detail. We purified to homogeneity a BGAF homolog designated as SL (Sorghum lectin) from sorghum and expressed its recombinant version in Escherichia coli. The native protein had a molecular mass of 32 kD and was monomeric. Both native and recombinant SL-agglutinated rabbit erythrocytes, and inhibition assays indicated that SL is a GalNAc-specific lectin. Exchanging the GG...GVVLD motif in SL with that of maize BGAF1 (GG...GIAVT) had no effect on GalNAc-binding, whereas binding to Man was abolished. Substitution of Thr(293) and Gln(296) in site I to corresponding residues (Val(294) and Asp(297)) of maize BGAF1 resulted in the loss of GalNAc-binding, indicating that site I is responsible for generating GalNAc specificity in SL. Gel-shift and pull-down assays after incubating SL with maize and sorghum beta-glucosidases showed no evidence of interaction nor were any SL-protein complexes detected in sorghum tissue extracts, suggesting that the sorghum homolog does not participate in protein-protein interactions.

Tissue specific response of Agrobacterium tumefaciens attachment to Sorghum bicolor (L) Moench

Agrobacterium mediated genetic transformation of plants have advantages over other methods, especially for making single copy transgenic plants with reduced chances of gene silencing and instability. However, monocotyledonous plant species could not utilize the full potential of this system because of possible limitations in Agrobacterium interaction with monocot plant cells. Agrobacterium attachment as a factor in genetic transformation was studied in the leaf, shoot apex, and leaf derived callus of sorghum (Sorghum bicolor (L) Moench). Pre-induction of Agrobacterium with acetosyringone was found necessary for Agrobacterium attachment to sorghum tissues. All the explants responded positively, with preferential Agrobacterium attachment and colonization around the tissues having actively dividing cells. Callus proved to be the best explant for Agrobacterium attachment as observed in scanning electron microscopy and transient GUS expression. Loss of Agrobacterium attachment was observed with an increase in the degree of tissue differentiation.