Sorghum Genes Research Articles

Genome-wide identification of the sorghum OVATE gene family and revelation of its expression characteristics in sorghum seeds and leaves

The OVATE gene family plays an important role in regulating the development of plant organs and resisting stress, but its expression characteristics and functions in sorghum have not been revealed. In this study, we identified 26 OVATE genes in the sorghum BTx623 genome, which were divided into four groups and distributed unevenly across 9 chromosomes. Evolutionary analysis showed that after differentiation between sorghum and Arabidopsis, the OVATE gene family may have experienced unique expansion events, and all OVATE family members were negatively selected. Transcriptome sequencing and RT-qPCR results showed that OVATE genes in sorghum showed diverse expression characteristics, such as gene SORBl_3001G468900 and SORBl_3009G173400 were significantly expressed in seeds, while SORBI_3005G042700 and SORBI_3002G417700 were only highly expressed in L1. Meantime, in the promoter region, a large number of hormone-associated cis-acting elements were identified, and these results suggest that members of the OVATE gene family may be involved in regulating specific development of sorghum leaves and seeds. This study improves the understanding of the OVATE gene family of sorghum and provides important clues for further exploration of the function of the OVATE gene family.

Genome-wide screening and characterization of phospholipase A (PLA)-like genes in sorghum (Sorghum bicolor L.).

The characterisation of PLA genes in the sorghum genome using in-silico methods revealed their essential roles in cellular processes, providing a foundation for further detailed studies. Sorghum bicolor (L.) Moench is the fifth most cultivated crop worldwide, and it is used in many ways, but it has always gained less popularity due to the yield, pest, and environmental constraints. Improving genetic background and developing better varieties is crucial for better sorghum production in semi-arid tropical regions. This study focuses on the phospholipase A (PLA) family within sorghum, comprehensively characterising PLA genes and their expression across different tissues. The investigation identified 32 PLA genes in the sorghum genome, offering insights into their chromosomal localization, molecular weight, isoelectric point, and subcellular distribution through bioinformatics tools. PLA-like family genes are classified into three groups, namely patatin-related phospholipase A (pPLA), phospholipase A1 (PLA1), and phospholipase A2 (PLA2). In-silico chromosome localization studies revealed that these genes are unevenly distributed in the sorghum genome. Cis-motif analysis revealed the presence of several developmental, tissue and hormone-specific elements in the promoter regions of the PLA genes. Expression studies in different tissues such as leaf, root, seedling, mature seed, immature seed, anther, and pollen showed differential expression patterns. Taken together, genome-wide analysis studies of PLA genes provide a better understanding and critical role of this gene family considering the metabolic processes involved in plant growth, defence and stress response.

Revealing critical mechanisms in determining sorghum resistance to drought and salt using mRNA, small RNA and degradome sequencing

BackgroundPlant growth and development are severely threatened by drought and salt stresses. Compared with structural genes, transcription factors (TFs) play more pivotal roles in plant growth and stress adaptation. However, the underlying mechanisms of sorghum adapting to drought and salt are insufficient, and systematic analysis of TFs in response to the above stresses is lacking.ResultsIn this study, TFs were identified in sorghum and model plants (Arabidopsis thaliana and rice), and gene number and conserved domain were compared between sorghum and model plants. According to syntenic analysis, the expansion of sorghum and rice TFs may be due to whole-genome duplications. Between sorghum and model plants TFs, specific conserved domains were identified and they may be related to functional diversification of TFs. Forty-five key genes in sorghum, including four TFs, were likely responsible for drought adaption based on differently expression analysis. MiR5072 and its target gene (Sobic.001G449600) may refer to the determination of sorghum drought resistance according to small RNA and degradome analysis. Six genes were associated with drought adaptation of sorghum based on weighted gene co-expression network analysis (WGCNA). Similarly, the core genes in response to salt were also characterized using the above methods. Finally, 15 candidate genes, particularly two TFs (Sobic.004G300300, HD-ZIP; Sobic.003G244100, bZIP), involved in combined drought and salt resistance of sorghum were identified.ConclusionsIn summary, the findings in this study help clarify the molecular mechanisms of sorghum responding to drought and salt. We identified candidate genes and provide important genetic resource for potential development of drought-tolerant and salt-tolerant sorghum plants.

Association between Reactive Oxygen Species, Transcription Factors, and Candidate Genes in Drought-Resistant Sorghum.

Drought stress is one of the most severe natural disasters in terms of its frequency, length, impact intensity, and associated losses, making it a significant threat to agricultural productivity. Sorghum (Sorghum bicolor), a C4 plant, shows a wide range of morphological, physiological, and biochemical adaptations in response to drought stress, paving the way for it to endure harsh environments. In arid environments, sorghum exhibits enhanced water uptake and reduced dissipation through its morphological activity, allowing it to withstand drought stress. Sorghum exhibits physiological and biochemical resistance to drought, primarily by adjusting its osmotic potential, scavenging reactive oxygen species, and changing the activities of its antioxidant enzymes. In addition, certain sorghum genes exhibit downregulation capabilities in response to drought stress. Therefore, in the current review, we explore drought tolerance in sorghum, encompassing its morphological characteristics and physiological mechanisms and the identification and selection of its functional genes. The use of modern biotechnological and molecular biological approaches to improving sorghum resistance is critical for selecting and breeding drought-tolerant sorghum varieties.

Transcriptome-wide expression landscape and starch synthesis pathway co-expression network in sorghum.

The gene expression landscape across different tissues and developmental stages reflects their biological functions and evolutionary patterns. Integrative and comprehensive analyses of all transcriptomic data in an organism are instrumental to obtaining a comprehensive picture of gene expression landscape. Such studies are still very limited in sorghum, which limits the discovery of the genetic basis underlying complex agricultural traits in sorghum. We characterized the genome-wide expression landscape for sorghum using 873 RNA-sequencing (RNA-seq) datasets representing 19 tissues. Our integrative analysis of these RNA-seq data provides the most comprehensive transcriptomic atlas for sorghum, which will be valuable for the sorghum research community for functional characterizations of sorghum genes. Based on the transcriptome atlas, we identified 595 housekeeping genes (HKGs) and 2080 tissue-specific expression genes (TEGs) for the 19 tissues. We identified different gene features between HKGs and TEGs, and we found that HKGs have experienced stronger selective constraints than TEGs. Furthermore, we built a transcriptome-wide co-expression network (TW-CEN) comprising 35 modules with each module enriched in specific Gene Ontology terms. High-connectivity genes in TW-CEN tend to express at high levels while undergoing intensive selective pressure. We also built global and seed-preferential co-expression networks of starch synthesis pathways, which indicated that photosynthesis and microtubule-based movement play important roles in starch synthesis. The global transcriptome atlas of sorghum generated by this study provides an important functional genomics resource for trait discovery and insight into starch synthesis regulation in sorghum.

The Role of the ADF Gene Family in Maize Response to Abiotic Stresses

The highly conserved actin depolymerizing factor (ADF) plays an important role in plant growth, development and responses to biotic and abiotic stresses. A total of 72 ADF genes in Arabidopsis, wheat, rice and sorghum can be divided into four groups. The multicollinearity analysis revealed that the maize ADF gene family exhibited more collinearity events with closely related gramineous plants. Fifteen ADF genes in maize were screened from the latest database, and bioinformatics analysis showed that these ADF genes were distributed across seven chromosomes in maize. The gene structure of the ADF gene family in maize exhibits significant conservation and cluster consistency. The promoter region contains rich regulatory elements that are involved in various regulations related to growth, development and adverse stresses. The drought-tolerant ZmADF5 gene in maize was further studied, and it was found that the allelic variations in ZmADF5 were mainly concentrated in its promoter region. A superior haplotype, with drought tolerance, was identified by candidate-gene association analysis of 115 inbred lines. By comparing the phenotypes of anthesis silking interval, grain yield and ear height, it was found that Hap2 performed better than Hap1 under drought stress. This study provides a theoretical reference for understanding the function of the ADF gene family and proposes further investigation into the role of ZmADF5 in abiotic-stress tolerance.

Genome-Wide Identification and Characterization of the Strigolactone (SL) Pathway and Associated Genes in Sorghum

Genome-Wide Identification and Characterization of the Strigolactone (SL) Pathway and Associated Genes in Sorghum

A large sequenced mutant library - valuable reverse genetic resource that covers 98% of sorghum genes.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutationswithin the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

Structural and functional characteristics and expression profile of the 20S proteasome gene family in Sorghum under abiotic stress

The 26S proteasome is a molecular machine that catalyzes and degrades protein intracellularly with the help of its core complex called 20S proteasome. The 20S proteasomes degrade and cleave denatured, cytotoxic, damaged, and unwanted proteins via proteolysis and impart biotic and abiotic stress tolerance in model plants. This study identified 20 genes, namely, 10 SbPA and 10 SbPB that encode for α- and β-subunits of the 20S proteasome in Sorghum bicolor (L.) Moench (2n= 20). These genes have been found distributed on the 1st, 2nd, 3rd, 4th, 5th, 7th, and 10th chromosomes. These sorghum genes were orthologous to corresponding rice. Phylogenetic analysis clustered these genes into seven clades, each with one of the seven α-subunits (1 to 7) and one of the seven β-subunits (1 to 7). In silico gene expression analysis suggested that nine genes were involved in abiotic stress response (cold, drought, and abscisic acid hormone). The expression of these proteasomal genes was studied in shoots and roots exposed to different abiotic stresses (cold, drought, and abscisic acid) by quantitative real-time polymerase chain reaction. A significant increase in the relative fold expression of SbPBA1, SbPAA1, SbPBG1, SbPBE1, and SbPAG1 genes under ABA and drought stress provides an insight into its involvement in abiotic stress. No expression was observed for cold stress of these genes indicating their non-involvement. It is believed that additional investigation into the SbPA/SbPB genes would aid in the creation of S. bicolor cultivars that are resistant to climate change.

A genome‑wide approach to the systematic and comprehensive analysis of LIM gene family in sorghum (Sorghum bicolor L.).

The LIM domain-containing proteins are dominantly found in plants and play a significant role in various biological processes such as gene transcription as well as actin cytoskeletal organization. Nevertheless, genome-wide identification as well as functional analysis of the LIM gene family have not yet been reported in the economically important plant sorghum (Sorghum bicolor L.). Therefore, we conducted an in silico identification and characterization of LIM genes in S. bicolor genome using integrated bioinformatics approaches. Based on phylogenetic tree analysis and conserved domain, we identified five LIM genes in S. bicolor (SbLIM) genome corresponding to Arabidopsis LIM (AtLIM) genes. The conserved domain, motif as well as gene structure analyses of the SbLIM gene family showed the similarity within the SbLIM and AtLIM members. The gene ontology (GO) enrichment study revealed that the candidate LIM genes are directly involved in cytoskeletal organization and various other important biological as well as molecular pathways. Some important families of regulating transcription factors such as ERF, MYB, WRKY, NAC, bZIP, C2H2, Dof, and G2-like were detected by analyzing their interaction network with identified SbLIM genes. The cis-acting regulatory elements related to predicted SbLIM genes were identified as responsive to light, hormones, stress, and other functions. The present study will provide valuable useful information about LIM genes in sorghum which would pave the way for the future study of functional pathways of candidate SbLIM genes as well as their regulatory factors in wet-lab experiments.

Structural Similarities and Overlapping Activities among Dihydroflavonol 4-Reductase, Flavanone 4-Reductase, and Anthocyanidin Reductase Offer Metabolic Flexibility in the Flavonoid Pathway.

Flavonoids are potent antioxidants that play a role in defense against pathogens, UV-radiation, and the detoxification of reactive oxygen species. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) reduce dihydroflavonols and flavanones, respectively, using NAD(P)H to produce flavan-(3)-4-(di)ols in flavonoid biosynthesis. Anthocyanidin reductase (ANR) reduces anthocyanidins to flavan-3-ols. In addition to their sequences, the 3D structures of recombinant DFR, FNR and ANR from sorghum and switchgrass showed a high level of similarity. The catalytic mechanism, substrate-specificity and key residues of three reductases were deduced from crystal structures, site-directed mutagenesis, molecular docking, kinetics, and thermodynamic ana-lyses. Although DFR displayed its highest activity against dihydroflavonols, it also showed activity against flavanones and anthocyanidins. It was inhibited by the flavonol quercetin and high concentrations of dihydroflavonols/flavonones. SbFNR1 and SbFNR2 did not show any activity against dihydroflavonols. However, SbFNR1 displayed activity against flavanones and ANR activity against two anthocyanidins, cyanidin and pelargonidin. Therefore, SbFNR1 and SbFNR2 could be specific ANR isozymes without delphinidin activity. Sorghum has high concentrations of 3-deoxyanthocyanidins in vivo, supporting the observed high activity of SbDFR against flavonols. Mining of expression data indicated substantial induction of these three reductase genes in both switchgrass and sorghum in response to biotic stress. Key signature sequences for proper DFR/ANR classification are proposed and could form the basis for future metabolic engineering of flavonoid metabolism.

Plant growth-promoting bacteria modulate gene expression and induce antioxidant tolerance to alleviate synergistic toxicity from combined microplastic and Cd pollution in sorghum

Microplastics (MPs) can act as carriers for environmental pollutants; therefore, MPs combined with heavy metal pollution are attracting increasing attention from researchers. In this study, the potential of the plant growth-promoting bacterium Bacillus sp. SL-413 to mitigate the stress caused by exposure to both MPs and cadmium (Cd) in sorghum plants was investigated. The effects of inoculation on sorghum biomass were investigated using hydroponic experiments, and evaluation of Cd accumulation and enzyme activity changes and transcriptomics approaches were used to analyze its effect on sorghum gene expression. The results showed that combined polyethylene (PE) and Cd pollution reduced the length and the fresh and dry weights of sorghum plants and thus exerted a synergistic toxic effect. However, inoculation with the strains alleviated the stress caused by the combined pollution and significantly increased the biomass. Inoculation increased the dry weights of the aboveground and belowground parts by 11.5–44.6% and 14.9–38.4%, respectively. Plant physiological measurements indicated that inoculation reduced the reactive oxygen species (ROS) content of sorghum by 10.5–27.2% and thereby alleviated oxidative stress. Transcriptome sequencing showed that exposure to combined Cd+MP contamination induced downregulation of gene expression, particularly that of genes related to amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, and plant hormone signal transduction, in sorghum. However, inoculation with Bacillus sp. SL-413 resulted in an increase in the proportion of upregulated genes involved in signal transduction, antioxidant defense, cell wall biology, and other metabolic pathways, which included the phenylpropanoid biosynthesis, photosynthesis, flavonoid biosynthesis, and MAPK signaling pathways. The upregulation of these genes promoted the tolerance of sorghum under combined Cd+MP pollution stress and alleviated the stress induced by these conditions. This study provides the first demonstration that plant growth-promoting bacteria can alleviate the stress caused by combined pollution with MPs and Cd by regulating plant gene expression. These findings provide a reference for the combined plant-microbial remediation of MPs and Cd.

Identification of ARF transcription factor gene family and its defense responses to bacterial infection and salicylic acid treatment in sugarcane.

Auxin response factor (ARF) is a critical regulator in the auxin signaling pathway, involved in a variety of plant biological processes. Here, gene members of 24 SpapARFs and 39 SpnpARFs were identified in two genomes of Saccharum spontaneum clones AP85-441 and Np-X, respectively. Phylogenetic analysis showed that all ARF genes were clustered into four clades, which is identical to those ARF genes in maize (Zea mays) and sorghum (Sorghum bicolor). The gene structure and domain composition of this ARF family are conserved to a large degree across plant species. The SpapARF and SpnpARF genes were unevenly distributed on chromosomes 1-8 and 1-10 in the two genomes of AP85-441 and Np-X, respectively. Segmental duplication events may also contribute to this gene family expansion in S. spontaneum. The post-transcriptional regulation of ARF genes likely involves sugarcane against various stressors through a miRNA-medicated pathway. Expression levels of six representative ShARF genes were analyzed by qRT-PCR assays on two sugarcane cultivars [LCP85-384 (resistant to leaf scald) and ROC20 (susceptible to leaf scald)] triggered by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections and salicylic acid (SA) treatment. ShARF04 functioned as a positive regulator under Xa and Aaa stress, whereas it was a negative regulator under SA treatment. ShARF07/17 genes played positive roles against both pathogenic bacteria and SA stresses. Additionally, ShARF22 was negatively modulated by Xa and Aaa stimuli in both cultivars, particularly LCP85-384. These findings imply that sugarcane ARFs exhibit functional redundancy and divergence against stressful conditions. This work lays the foundation for further research on ARF gene functions in sugarcane against diverse environmental stressors.

A streamlined guide RNA screening system for genome editing in Sorghum bicolor

BackgroundGenome editing tools derived from clustered regularly interspaced short palindromic repeats (CRISPR) systems have been developed for generating targeted mutations in plants. Although these tools hold promise for rapid crop improvement, target-specific guide RNAs exhibit variable activity. To improve genome editing, a rapid and precise method for evaluating their efficiency is necessary.ResultsHere we report an efficient system for screening single guide RNAs (sgRNAs) for genome editing in sorghum using a transient protoplast transfection assay. Protoplasts were isolated from leaves from sorghum plants cultivated under three different conditions. Cultivation for three days of continuous darkness following seven days with a 16-h light and 8-h dark photoperiod resulted in the highest yield of viable protoplasts and the highest protoplast transfection efficiency. We tested both plasmid-mediated and ribonucleoprotein-based delivery to protoplasts, via polyethylene glycol-mediated transfection, of CRISPR components targeting the sorghum genome. The frequencies of small insertions and deletions induced by a set of sgRNAs targeting four endogenous sorghum genes were analyzed via targeted deep sequencing. Our screening system induced indels in sorghum protoplasts at frequencies of up to 77.8% (plasmid) and 18.5% (RNP). The entire screening system was completed within 16 days.ConclusionsThe screening system optimized in this study for predicting sgRNA activity for genome editing in sorghum is efficient and straightforward. This system will reduce the time and effort needed for sorghum genome editing.

Analysis of plant expression profiles revealed that aphid attack triggered dynamic defense responses in sorghum plant.

Sorghum [Sorghum bicolor (L.) Moench] is one of the most important cereal crops grown worldwide but is often attacked by greenbug (aphid). In response to aphid attack, host plant initiates a large transcriptional reorganization, leading to activation of the host defense genes in aphid-attacked plants. In this study, our objective was to analyze defensive responses of sorghum against aphid and identify aphid resistance genes in sorghum. For the experiments, seedlings developed from an aphid resistant germplasm line (PI 550607) were divided into two groups, then, one group was infested with greenbug ((Schizaphis graminum Rondani) and the other group was used as control (un-infested). In addition, seedlings of sorghum cultivar Tx 7000, a susceptible genotype, prepared under the same conditions, were used as a genetic control. Those plant samples were used to develop transcriptional profiles using the microarray method, from which 26.1% of the 1,761 cDNA sequences spotted on the microarray showed altered expression between two treatments at 4days after infestation. Sequence annotation and molecular analysis revealed that many differentially expressed genes (DEGs) were related to direct host defense or signal transduction pathways, which regulate host defense. In addition to common responsive genes, unique transcripts were identified in response to greenbug infestation specifically. Later, a similar transcriptional profiling was conducted using the RNA-seq method, resulted in the identification of 2,856 DEGs in the resistant line with a comparison between infested and non-infested at 4days and 4,354 DEGs in the resistant genotype compared to the susceptible genotype at 4days. Based on the comparative analysis, the data of RNA-seq provided a support for the results from the microarray study as it was noticed that many of the DEGs are common in both platforms. Analysis of the two differential expression profiles indicate that aphid triggered dynamic defense responses in sorghum plants and sorghum plant defense against aphid is a complex process involving both general defense systems and specific resistance mechanisms. Finally, the results of the study provide new insights into the mechanisms underlying host plant defense against aphids and will help us design better strategies for effectively controlling aphid pest.

CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase (CCD) Genes in Sorghum Alters Strigolactone Biosynthesis and Plant Biotic Interactions

Strigolactones are a group of small molecules that play critical roles in plant developmental processes and root biotic interactions. Strigolactones are agronomically important due to their role as a signal for the germination of a parasitic weed ( Striga spp.) that reduces yields of cereal crops worldwide. To identify the genes encoding strigolactones in sorghum and their function, we characterized two CRISPR/Cas9-mediated gene knockouts of carotenoid cleavage dioxygenase 8 ( CCD8) genes ( SbCCD8a and SbCCD8b), which have been shown in other plant species to be involved in strigolactone biosynthesis. Although strigolactones are important for the parasitization of sorghum in Africa, the functions of members of the CCD8 family have not been characterized. The impact of the knockouts on strigolactone production, plant growth and development, resistance to the parasitic weed Striga, and the root-associated microbiomes were investigated in this study. The results revealed that knockout of SbCCD8 genes in sorghum significantly reduced orobanchol production and Striga germination. Strigolactone deficiency altered the shoot and root architecture and reduced grain yield of sorghum. The knockout of the SbCCD8b gene significantly affected the rhizosphere bacterial diversity and community composition at sorghum plant grain-fill stage due to the abolition of orobanchol exudation from roots. Reduced amounts of orobanchol in root exudates also influenced root-associated fungal taxa abundance. Our findings provide new insights into potentially sustainable approaches for the recruitment of beneficial microbes and for parasitic weed control through manipulation of strigolactone production in sorghum.

Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum.

Phosphoenolpyruvate carboxylase (PEPC) plays central roles in photosynthesis, respiration, amino acid synthesis, and seed development. PEPC is regulated by different post-translational modifications. Between them, the phosphorylation by PEPC-kinase (PEPCk) is widely documented. In this work, we simultaneously silenced the three sorghum genes encoding PEPCk (SbPPCK1-3) by RNAi interference, obtaining 12 independent transgenic lines (Ppck1-12 lines), showing different degrees of SbPPCK1-3 silencing. Among them, two T2 homozygous lines (Ppck-2 and Ppck-4) were selected for further evaluation. Expression of SbPPCK1 was reduced by 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. Expression of SbPPCK2 was higher in roots and decreased by 50% in Ppck-2 and Ppck-4 in this tissue. Expression of SbPPCK3 was low and highly variable. Despite the incomplete gene silencing, it decreased the degree of phosphorylation of PEPC in illuminated leaves, P-deficient plants, and NaCl-treated plants. Both leaves and seeds of Ppck lines had altered metabolic profiles and a general decrease in amino acid content. In addition, Ppck lines showed delayed flowering, and 20% of Ppck-4 plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in Ppck-2 and Ppck-4 lines, respectively. The quality of seeds was lower in Ppck lines: lower amino acid content, including Lys, and higher phytate content. These data confirm the relevance of the phosphorylation of PEPC in sorghum development, stress responses, yield, and quality of seeds.

Two Sweet Sorghum (Sorghum bicolor L.) WRKY Transcription Factors Promote Aluminum Tolerance via the Reduction in Callose Deposition.

Aluminum (Al) toxicity is a primary limiting factor for crop production in acidic soils. The WRKY transcription factors play important roles in regulating plant growth and stress resistance. In this study, we identified and characterized two WRKY transcription factors, SbWRKY22 and SbWRKY65, in sweet sorghum (Sorghum bicolor L.). Al induced the transcription of SbWRKY22 and SbWRKY65 in the root apices of sweet sorghum. These two WRKY proteins were localized in the nucleus and exhibited transcriptional activity. SbWRKY22 showed the significant transcriptional regulation of SbMATE, SbGlu1, SbSTAR1, SbSTAR2a, and SbSTAR2b, which are major known Al tolerance genes in sorghum. Interestingly, SbWRKY65 had almost no effect on the aforementioned genes, but it significantly regulated the transcription of SbWRKY22. Therefore, it is speculated that SbWRKY65 might indirectly regulate Al-tolerance genes mediated by SbWRKY22. The heterologous expression of SbWRKY22 and SbWRKY65 greatly improved the Al tolerance of transgenic plants. The enhanced Al tolerance phenotype of transgenic plants is associated with reduced callose deposition in their roots. These findings suggest the existence of SbWRKY22- and SbWRKY65-mediated Al tolerance regulation pathways in sweet sorghum. This study extends our understanding of the complex regulatory mechanisms of WRKY transcription factors in response to Al toxicity.

Resolving intergenotypic Striga resistance in sorghum.

Genetic underpinnings of host-pathogen interactions in the parasitic plant Striga hermonthica, a root parasitic plant that ravages cereals in sub-Saharan Africa, are unclear. We performed a comparative transcriptome study on five genotypes of sorghum exhibiting diverse resistance responses to S. hermonthica using weighted gene co-expression network analysis (WGCNA). We found that S. hermonthica elicits both basal and effector-triggered immunity-like a bona fide pathogen. The resistance response was genotype specific. Some resistance responses followed the salicylic acid-dependent signaling pathway for systemic acquired resistance characterized by cell wall reinforcements, lignification, and callose deposition, while in others the WRKY-dependent signaling pathway was activated, leading to a hypersensitive response. In some genotypes, both modes of resistance were activated, while in others either mode dominated the resistance response. Cell wall-based resistance was common to all sorghum genotypes but strongest in IS2814, while a hypersensitive response was specific to N13, IS9830, and IS41724. WGCNA further allowed for pinpointing of S. hermonthica resistance causative genes in sorghum, including glucan synthase-like 10 gene, a pathogenesis-related thaumatin-like family gene, and a phosphoinositide phosphatase gene. Such candidate genes will form a good basis for subsequent functional validation and possibly future resistance breeding.

New methods for sorghum transformation in temperate climates.

Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens-mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.