Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response.

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.

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.

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.

Functional analysis of water stress-responsive soybean GmNAC003 and GmNAC004 transcription factors in lateral root development in arabidopsis.

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.

Molecular characterization of Glycine max squalene synthase genes in seed phytosterol biosynthesis.

The reaction catalyzed by squalene synthase (EC.2.5.1.21) that converts two molecules of farnesyl pyrophosphate to squalene represents a crucial branch point of the isoprenoid pathway in diverting carbon flux towards the biosynthesis of sterols. In the present study two soybean squalene synthase genes, GmSQS1 and GmSQS2, were identified in the soybean genome and functionally characterized for their roles in sterol biosynthesis. Both genes encode a deduced protein of 413 amino acids. Complementation assays showed that the two genes were able to convert yeast sterol auxotrophy erg9 mutant to sterol prototrophy. Expression of GmSQS1 and GmSQS2 was ubiquitous in roots, stem, leaves, flower and young seeds of soybean, however GmSQS1 transcript was preferential in roots while GmSQS2 transcript was more in leaves. Their expression was lower in response to dehydration treatments suggesting they might be negative regulators of water stress adaptation. Transgenic Arabidopsis plants overexpressing GmSQS1 driven by either constitutive or seed-specific promoters showed increases in the major end product sterols: campesterol, sitosterol and stigmasterol, which resulted in up to 50% increase in total sterol content in the seeds. The increase in the end product sterols by GmSQS1 overexpression was at the level achievable by previously reported overexpression of individual or combination of other key enzymes in the sterol pathway. Together the data demonstrate that soybean SQS genes play an important role in diverting carbon flux to the biosynthesis of the end product sterols in the seeds.

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.