Wild Relatives of Maize, Rice, Cotton, and Soybean: Treasure Troves for Tolerance to Biotic and Abiotic Stresses.

Global food demand is expected to nearly double by 2050 due to an increase in the world's population. The Green Revolution has played a key role in the past century by increasing agricultural productivity worldwide, however, limited availability and continued depletion of natural resources such as arable land and water will continue to pose a serious challenge for global food security in the coming decades. High yielding varieties with proven tolerance to biotic and abiotic stresses, superior nutritional profiles, and the ability to adapt to the changing environment are needed for continued agricultural sustainability. The narrow genetic base of modern cultivars is becoming a major bottleneck for crop improvement efforts and, therefore, the use of crop wild relatives (CWRs) is a promising approach to enhance genetic diversity of cultivated crops. This article provides a review of the efforts to date on the exploration of CWRs as a source of tolerance to multiple biotic and abiotic stresses in four global crops of importance; maize, rice, cotton, and soybean. In addition to the overview of the repertoire and geographical spread of CWRs in each of the respective crops, we have provided a comprehensive discussion on the morphological and/or genetic basis of the traits along with some examples, when available, of the research in the transfer of traits from CWRs to cultivated varieties. The emergence of modern molecular and genomic technologies has not only accelerated the pace of dissecting the genetics underlying the traits found in CWRs, but also enabled rapid and efficient trait transfer and genome manipulation. The potential and promise of these technologies has also been highlighted in this review.

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach.

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean.

Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include functional genes and regulatory transcription factors. The Arabidopsis thaliana DREB1D transcription factor driven by the constitutive and ABA-inducible promoters was introduced into soybean through Agrobacterium tumefaciens-mediated gene transfer. Several transgenic lines were generated and molecular analysis was performed to confirm transgene integration. Transgenic plants with an ABA-inducible promoter showed a 1.5- to two-fold increase of transgene expression under severe stress conditions. Under well-watered conditions, transgenic plants with constitutive and ABA-inducible promoters showed reduced total leaf area and shoot biomass compared to non-transgenic plants. No significant differences in root length or root biomass were observed between transgenic and non-transgenic plants under non-stress conditions. When subjected to gradual water deficit, transgenic plants maintained higher relative water content because the transgenic lines used water more slowly as a result of reduced total leaf area. This caused them to wilt slower than non-transgenic plants. Transgenic plants showed differential drought tolerance responses with a significantly higher survival rate compared to non-transgenic plants when subjected to comparable severe water-deficit conditions. Moreover, the transgenic plants also showed improved drought tolerance by maintaining 17-24 % greater leaf cell membrane stability compared to non-transgenic plants. The results demonstrate the feasibility of engineering soybean for enhanced drought tolerance by expressing stress-responsive genes.

Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions.

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases.

BACKGROUND: Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. RESULTS: We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development. CONCLUSIONS: The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction.

Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis.

Sterol C24 methyltransferase (SMT2) genes governing the pattern of phytosterols synthesized in higher plants have been studied in Glycine seedlings and wild-type and engineered Arabidopsis thaliana plants. The SMT2 genes of soybean (SMT2-1 and SMT2-2) previously cloned and characterized (Neelakandan et al. 2009) were shown to complement the SMT deficient cvp1 mutant Arabidopsis plants, consistent with their role in regulation of 24-alkyl sterol-controlled plant physiology. Further analysis of these genes showed that environmental cues, including dehydration, cold, and abscisic acid induced differential changes in transcript levels of the SMT2 during soybean seedling growth. Sterol analyses of transgenic Arabidopsis seeds originating in variant constructs of AtHMGR1, GmSMT1, and GmSMT2 engineered in seeds showed relevant modifications in the ratio of 24-methyl to 24-ethyl sterol in the direction of sitosterol formation. To provide insight into the structural features of the sterol gene that affects transcript regulation, the upstream promoter sequences of soybean SMT2 genes were cloned and characterized. Sequence analysis revealed several important cis-elements and transcription factor binding sites. The analysis of promoter-GUS fusions in transgenic Arabidopsis plants revealed shared and distinct expression features in different developmental stages and tissues. The data are interpreted to imply that SMT2 is an important contributor to normal plant growth and development.

Differential expression of isoflavone biosynthetic genes in soybean during water deficits.

Numerous environmental factors influence isoflavone accumulation and have long hampered their genetic dissection. Temperature and water regimes are two of the most significant abiotic factors. However, while the effects of temperature have been widely studied, little is known about how water scarcity might affect isoflavone concentration in seeds. Studies have shown that accumulation of isoflavones is promoted by well-watered conditions, but the molecular basis remains elusive. The length and severity of the water stress required to induce changes are also still unknown. In the present work, several intensities of water stress were evaluated at various critical stages for soybean [Glycine max (L.) Merr.] seed development, in both field and controlled environments. The results suggested that only long-term progressive drought, spanning most of the seed developmental stages, significantly decreased isoflavone content in seeds. The reduction is proportional to the intensity of the stress and appears to occur in a genotype-dependent manner. However, regardless of water regime, isoflavone compounds were mainly accumulated in the later seed developmental stages. Transcripts of the most important genes for isoflavone biosynthesis were also quantified from samples collected at key seed developmental stages under well-watered and long-term water deficit conditions. Expression of CHS7, CHS8 and IFS2 correlated with isoflavone accumulation under well-watered conditions. Interestingly, we found that the two isoflavone synthase genes in soybean (IFS1 and IFS2) showed different patterns of expression. The abundance of IFS1 transcripts was maintained at a constant rate, whereas IFS2 was down-regulated and highly correlated with isoflavone accumulation under both water deficit and well-watered conditions, suggesting IFS2 as a main contributor to isoflavone diminution under drought.

Physiological and molecular approaches to improve drought resistance in soybean.

Drought stress is a major constraint to the production and yield stability of soybean [Glycine max (L.) Merr.]. For developing high yielding varieties under drought conditions, the most widely employed criterion has traditionally been direct selection for yield stability over multiple locations. However, this approach is time consuming and labor intensive, because yield is a highly quantitative trait with low heritability, and influenced by differences arising from soil heterogeneity and environmental factors. The alternative strategy of indirect selection using secondary traits has succeeded only in a few crops, due to problems with repeatability and lack of phenotyping strategies, especially for root-related traits. Considerable efforts have been directed towards identifying traits associated with drought resistance in soybean. With the availability of the whole genome sequence, physical maps, genetics and functional genomics tools, integrated approaches using molecular breeding and genetic engineering offer new opportunities for improving drought resistance in soybean. Genetic engineering for drought resistance with candidate genes has been reported in the major food crops, and efforts for developing drought-resistant soybean lines are in progress. The objective of this review is to consolidate the current knowledge of physiology, molecular breeding and functional genomics which may be influential in integrating breeding and genetic engineering approaches for drought resistance in soybean.

Molecular characterization of stress-inducible GmNAC genes in soybean.

Drought is detrimental to plant growth and development, and often results in significant losses to the yields of economically important crops such as soybeans (Glycine max L.). NAC transcription factors (TFs), which consist of a large family of plant-specific TFs, have been reported to enhance drought tolerance in a number of plants. In this study, 31 unigenes that contain the complete open reading frames encoding GmNAC proteins were identified and cloned from soybean. Analysis of C-terminal regulatory domain using yeast one-hybrid system indicated that among 31 GmNAC proteins, 28 have transcriptional activation activity. Expression analysis of these GmNAC genes showed that they are differentially expressed in different organs, suggesting that they have diverse functions during plant growth and development. To search for the drought-inducible GmNAC genes, we prescreened and re-confirmed by quantitative real-time PCR analysis that nine GmNAC genes are induced by dehydration stress with differential induction levels in both shoot and root. The expression profiles of these nine GmNAC genes were also examined under other stresses such as high salinity, cold and with abscisic acid hormone treatments. Phylogenetic analysis of the GmNAC proteins with previously reported drought-inducible NAC proteins of Arabidopsis and rice revealed that the nine drought-inducible GmNAC proteins belong to the "stress-inducible" NAC group. The results of this systematic analysis of the GmNAC family will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars.