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.

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.

Nuclear factor-Y (NF-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB and NF-YC proteins. In plants, there are usually more than 10 genes for each family and their members have been identified to be key regulators in many developmental and physiological processes controlling gametogenesis, embryogenesis, nodule development, seed development, abscisic acid (ABA) signaling, flowering time, primary root elongation, blue light responses, endoplasmic reticulum (ER) stress response and drought tolerance. Taking the advantages of the recent soybean genome draft and information on functional characterizations of nuclear factor Y (NF-Y) transcription factor family in plants, we identified 21 GmNF-YA, 32 GmNF-YB, and 15 GmNF-YC genes in the soybean (Glycine max) genome. Phylogenetic analyses show that soybean’s proteins share strong homology to Arabidopsis and many of them are closely related to functionally characterized NF-Y in plants. Expression analysis in various tissues of flower, leaf, root, seeds of different developmental stages, root hairs under rhizobium inoculation, and drought-treated roots and leaves revealed that certain groups of soybean NF-Y are likely involved in specific developmental and stress responses. This study provides extensive evaluation of the soybean NF-Y family and is particularly useful for further functional characterization of GmNF-Y proteins in seed development, nodulation and drought adaptation of soybean.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-014-0978-2) contains supplementary material, which is available to authorized users.

Soybean [Glycine max (L.) Merr.] is a unique crop species because it has high levels of both protein and oil in its seed. Of the many quantitative trait loci (QTL) controlling soybean seed protein content, alleles of the cqSeed protein-003 QTL on chromosome 20 exert the greatest additive effect. The high-protein allele exists in both cultivated and wild soybean (Glycine soja Siebold & Zucc.) germplasm. Our objective was to fine map this QTL to enable positional-based cloning of its underlying causative gene(s). Fine mapping was achieved by developing and testing a series of populations in which the chromosomal region surrounding the segregating high-versus low-protein alleles was gradually narrowed, using marker-based detection of recombinant events. The resultant 77.8 kb interval was directly sequenced from a G. soja source and compared with the reference genome to identify structural and sequence polymorphisms. An insertion/deletion variant detected in Glyma.20G85100 was found to have near-perfect +/À concordance with high/low-protein allele genotypes inferred for this QTL in parents of published mapping populations. The indel structure was concordant with an evolutionarily recent insertion of a TIR transposon into the gene in the low-protein lineage. Seed protein was significantly greater in soybean expressing an RNAi hairpin downregulation element in two independent events relative to control null segregant lineages. We conclude that a transposon insertion within the CCT domain protein encoded by the Glyma.20G85100 gene accounts for the high/low seed protein alleles of the cqSeed protein-003 QTL.

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In Arabidopsis, NAC (NAM, ATAF and CUC) transcription factors have been found to promote lateral root number through the auxin signaling pathway. In the present study, the role of water stress–inducible soybean GmNAC003 and GmNAC004 genes in the enhancement of lateral root development under water deficit conditions was investigated. Both genes were highly expressed in roots, leaves and flowers of soybean and were strongly induced by water stress and moderately induced by a treatment with abscisic acid (ABA). They showed a slight response to treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The transgenic Arabidopsis plants overexpressing GmNAC004 showed an increase in lateral root number and length under non-stress conditions and maintained higher lateral root number and length under mild water stress conditions compared to the wild-type (WT), while the transgenic plants overexpressing GmNAC003 did not show any response. However, LR development of GmNAC004 transgenic Arabidopsis plants was not enhanced in the water-stressed compared to the well-watered treatment. In the treatment with ABA, LR density of the GmNAC004 transgenic Arabidopsis was less suppressed than that of the WT, suggesting that GmNAC004 counteracts ABA-induced inhibition of lateral root development. In the treatment with 2,4-D, lateral root density was enhanced in both GmNAC004 transgenic Arabidopsis and WT plants but the promotion was higher in the transgenic plants. Conversely, in the treatment with naphthylphthalamic acid (NPA), lateral root density was inhibited and there was no difference in the phenotype of the GmNAC004 transgenic Arabidopsis and WT plants, indicating that auxin is required for the action of GmNAC004. Transcript analysis for a number of known auxin and ABA related genes showed that GmNAC004's role may suppress ABA signaling but promote auxin signaling to increase lateral root development in the Arabidopsis heterologous system.

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.

Drought stress is the major limiting factor in agriculture. Wheat, which is the most widely grown crop in the world, is predominantly cultivated in drought-prone rainfed environments. Since roots play a critical role in water uptake, root response to water limitations is an important component for enhancing wheat adaptation. In an effort to discover novel genetic sources for improving wheat adaptation, we characterized a wheat translocation line with a chromosomal segment from Agropyron elongatum, a wild relative of wheat, which unlike common wheat maintains root growth under limited-water conditions. By exploring the root transcriptome data, we found that reduced transcript level of LATERAL ROOT DENSITY (LRD) gene under limited water in the Agropyron translocation line confers it the ability to maintain root growth. The Agropyron allele of LRD is down-regulated in response to water limitation in contrast with the wheat LRD allele, which is up-regulated by water deficit stress. Suppression of LRD expression in wheat RNAi plants confers the ability to maintain root growth under water limitation. We show that exogenous gibberellic acid (GA) promotes lateral root growth and present evidence for the role of GA in mediating the differential regulation of LRD between the common wheat and the Agropyron alleles under water stress. Suppression of LRD also had a positive pleiotropic effect on grain size and number under optimal growth conditions. Collectively, our findings suggest that LRD can be potentially useful for improving wheat response to water stress and altering yield components.