MsWRKY33 increases alfalfa (Medicago sativa L.) salt stress tolerance through altering the ROS scavenger via activating MsERF5 transcription.

Alfalfa (Medicago sativa L.) is considered to be the most important forage crop on a global scale. Nevertheless, soil salinity significantly decreases productivity, seriously threatening food security worldwide. One viable strategy is to explore salt stress-responsive factors and elucidate their underlying molecular mechanism, and utilize them in further alfalfa breeding. In the present study, we designated MsWRKY33 as a representative salt stress-responsive factor preferentially expressed in alfalfa roots and leaves. Subsequently, it was demonstrated that MsWRKY33 was localized in the cell nucleus, and functioned as a transcriptional activator of the W-box element. Transgenic alfalfa overexpressing MsWRKY33 displayed enhanced salt stress tolerance and antioxidant activities with no significant difference in other agronomic traits. Transcriptome profiling of MsWRKY33 transgenic alfalfa under control and salt treatment unveiled significantly altered expression of reactive oxygen species (ROS) scavenger genes in transgenic alfalfa. Subsequent examination revealed that MsWRKY33 binded to the promoter of MsERF5, activating its expression and consequently fine-tuning the ROS-scavenging enzyme activity. Furthermore, MsWRKY33 interacted with the functional fragment of MsCaMBP25, which participates in Ca2+ signaling transduction. Collectively, this research offers new insight into the molecular mechanism of alfalfa salt stress tolerance and highlights the potential utility of MsWRKY33 in alfalfa breeding.

Genome-Wide Identification and Characterization of Growth Regulatory Factor Family Genes in Medicago.

Growth Regulatory Factors (GRF) are plant-specific transcription factors that play critical roles in plant growth and development as well as plant tolerance against stress. In this study, a total of 16 GRF genes were identified from the genomes of Medicago truncatula and Medicago sativa. Multiple sequence alignment analysis showed that all these members contain conserved QLQ and WRC domains. Phylogenetic analysis suggested that these GRF proteins could be classified into five clusters. The GRF genes showed similar exon-intron organizations and similar architectures in their conserved motifs. Many stress-related cis-acting elements were found in their promoter region, and most of them were related to drought and defense response. In addition, analyses on microarray and transcriptome data indicated that these GRF genes exhibited distinct expression patterns in various tissues or in response to drought and salt treatments. In particular, qPCR results showed that the expression levels of gene pairs MtGRF2-MsGRF2 and MtGRF6-MsGRF6 were significantly increased under NaCl and mannitol treatments, indicating that they are most likely involved in salt and drought stress tolerance. Collectively, our study is valuable for further investigation on the function of GRF genes in Medicago and for the exploration of GRF genes in the molecular breeding of highly resistant M. sativa.

Inheritance and expression of Bt cry1Ba3 gene in progeny from transformed cabbage plants.

Stable inheritance and expression of transgene are important parameters for successful use of a transgenic crop. We previously transformed a Bt cry1Ba3 gene into cabbage inbred line CA21-3. To evaluate the stability of our Bt cabbage lineages, transgene inheritance and expression were examined in four successive generations under greenhouse conditions. In our study, T1, T2 and T3 progenies of the three independent transgenic lineages (YA-1, YA-2 and YA-3) were generated and then the inheritance and expression of cry1Ba3 were analyzed in sexually derived progeny. Segregation ratio of 2.81:1, 3.27:1 and 3.07:1 was found in T1 progeny of lineages YA-1, YA-2 and YA-3, respectively. Chi-square analysis indicated that these segregation ratios of corresponding population fit the 3:1 ratio. Segregation ratios of the transgene in T2 progeny showed either 3:1 or all expression of cry1Ba3. These data suggest that cry1Ba3 in CA21-3 can be inherited in a Mendelian manner. ELISA analysis of transgenic plants from four generations demonstrated that cry1Ba3 had been stably transmitted to the T3 progeny. Additionally, under artificial infestation conditions, the homozygous T3-YA-1-2-1 line exhibited excellent resistance to Plutella xylostella as compared with un-transformed CA21-3. All these results imply that the three cabbage lineages are genetically stable and can be used to inhibit damage on cabbage caused by P. xylostella.

Development of glyphosate-resistant alfalfa (Medicago sativa L.) upon transformation with the GR79Ms gene encoding 5-enolpyruvylshikimate-3-phosphate synthase.

MAIN CONCLUSION: The glyphosate-resistant gene, GR79Ms, was successfully introduced into the genome of alfalfa. The transgenic events may serve as novel germplasm resources in alfalfa breeding. Weed competition can reduce the alfalfa yield, generating new alfalfa germplasm with herbicide resistance is essential. To obtain transgenic alfalfa lines with glyphosate resistance, a new synthetic glyphosate-resistant gene GR79Ms encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was introduced into alfalfa germplasm by Agrobacterium tumefaciens-mediated transformation. In total, 67 transformants were obtained. PCR and Southern blot analyses confirmed that GR79Ms was successfully inserted into the genome of alfalfa. Reverse transcription-PCR and western blot analyses further demonstrated the expression of GR79Ms and its product, GR79Ms EPSPS. Moreover, two homozygous transgenic lines were developed in the T2 generation by means of molecular-assisted selection. Herbicide tolerance spray tests showed that the transgenic plants T0-GR1, T0-GR2, T0-GR3 and two homozygous lines were able to tolerate fourfold higher commercial usage of glyphosate than non-transgenic plants.

Influences of Cry1Ac broccoli on larval survival and oviposition of diamondback moth.

Larval survival and oviposition behavior of three genotypes of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), (homozygous Cry1Ac-susceptibile, Cry1Ac-resistant, and their F1 hybrids), on transgenic Bacillus thuringiensis (Bt) broccoli expressing different levels of Cry1Ac protein were evaluated in laboratory. These Bt broccoli lines were designated as relative low, medium, and high, respectively, according to the Cry1Ac content. Untransformed brocccoli plants were used as control. Larval survival of diamondback moth on non-Bt leaves was not significantly different among the three genotypes. The Cry1Ac-resistant larvae could survive on the low level of Bt broccoli plants, while Cry1Ac-susceptible and F1 larvae could not survive on them. The three genotypes of P. xylostella larvae could not survive on medium and high levels of Bt broccoli. In oviposition choice tests, there was no significant difference in the number of eggs laid by the three P. xylostella genotypes among different Bt broccoli plants. The development of Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella on intact Bt plants was also tested in greenhouse. All susceptible P. xylostella larvae died on all Bt plants, while resistant larvae could survive on broccoli, which expresses low Cry1Ac protein under greenhouse conditions. The results of the greenhouse trials were similar to that of laboratory tests. This study indicated that high dose of Bt toxins in broccoli cultivars or germplasm lines is required for effective resistance management.

Overexpression of MsNIP2 improves salinity tolerance in Medicago sativa.

Alfalfa (Medicago sativa) is one of the most widely cultivated forage crops in the world. However, alfalfa yield and quality are adversely affected by salinity stress. Nodulin 26-like intrinsic proteins (NIPs) play essential roles in water and small molecules transport and response to salt stress. Here, we isolated a salt stress responsive MsNIP2 gene and demonstrated its functions by overexpression in alfalfa. The open reading frame of MsNIP2 is 816 bp in length, and it encodes 272 amino acids. It has six transmembrane domains and two NPA motifs. MsNIP2 showed high identity to other known NIP proteins, and its tertiary model was similar to the crystal structure of OsNIP2-1 (7cjs) tetramer. Subcellular localization analysis showed that MsNIP2 protein fused with green fluorescent protein (GFP) was localized to the plasma membrane. Transgenic alfalfa lines overexpressing MsNIP2 showed significantly higher height and branch number compared with the non-transgenic control. The POD and CAT activity of the transgenic alfalfa lines was significantly increased and their MDA content was notably reduced compared with the control group under the treatment of NaCl. The transgenic lines showed higher capability in scavenging oxygen radicals with lighter NBT staining than the control under salt stress. The transgenic lines showed relative lower water loss rate and electrolyte leakage, but relatively higher Na+ content than the control line under salt stress. The relative expression levels of abiotic-stress-related genes (MsHSP23, MsCOR47, MsATPase, and MsRD2) in three transgenic lines were compared with the control, among them, only the expression of MsCOR47 was up-regulated. Consequently, this study offers a novel perspective for exploring the function of MsNIP2 in improving salt tolerance of alfalfa.

Genome-wide analysis and expression of the aquaporin gene family in Avena sativa L.

Background: Oat (Avena sativa L.) belongs to the early maturity grass subfamily of the Gramineae subfamily oats (Avena) and has excellent characteristics, such as tolerance to barrenness, salt, cold, and drought. Aquaporin (AQP) proteins belong to the major intrinsic protein (MIP) superfamily, are widely involved in plant growth and development, and play an important role in abiotic stress responses. To date, previous studies have not identified or analyzed the AsAQP gene family system, and functional studies of oat AQP genes in response to drought, cold, and salt stress have not been performed. Methods: In this study, AQP genes (AsAQP) were identified from the oat genome, and various bioinformatics data on the AQP gene family, gene structure, gene replication, promoters and regulatory networks were analyzed. Quantitative real-time PCR technology was used to verify the expression patterns of the AQP gene family in different oat tissues under different abiotic stresses. Results: In this study, a total of 45 AQP genes (AsAQP) were identified from the oat reference genome. According to a phylogenetic analysis, 45 AsAQP were divided into 4 subfamilies (PIP, SIP, NIP, and TIP). Among the 45 AsAQP, 23 proteins had interactions, and among these, 5AG0000633.1 had the largest number of interacting proteins. The 20 AsAQP genes were expressed in all tissues, and their expression varied greatly among different tissues and organs. All 20 AsAQP genes responded to salt, drought and cold stress. The NIP subfamily 6Ag0000836.1 gene was significantly upregulated under different abiotic stresses and could be further verified as a key candidate gene. Conclusion: The findings of this study provide a comprehensive list of members and their sequence characteristics of the AsAQP protein family, laying a solid theoretical foundation for further functional analysis of AsAQP in oats. This research also offers valuable reference for the creation of stress-tolerant oat varieties through genetic engineering techniques.

Analyses on Flavonoids and Transcriptome Reveals Key MYB Gene for Proanthocyanidins Regulation in Onobrychis Viciifolia.

Onobrychis viciifolia (sainfoin) is one of the most high-quality legume forages, which is rich in proanthocyanidins that is beneficial for the health and production of animals. In this study, proanthocyanidins and total flavonoids in leaves of 46 different sainfoin germplasm resources were evaluated, and it showed that soluble proanthocyanidin contents varied greatly in these sainfoin germplasm resources, but total flavonoids did not show significant difference. Transcriptome sequencing with high and low proanthocyanidins sainfoin resulted in the identification of totally 52,926 unigenes in sainfoin, and they were classed into different GOC categories. Among them, 1,608 unigenes were differentially expressed in high and low proanthocyanidins sainfoin samples, including 1,160 genes that were upregulated and 448 genes that were downregulated. Analysis on gene enrichment via KEGG annotation revealed that the differentially expressed genes were mainly enriched in the phenylpropanoid biosynthetic pathway and the secondary metabolism pathway. We also analyzed the expression levels of structural genes of the proanthocyanidin/flavonoid pathway in roots, stems, and leaves in the high proanthocyanidin sainfoin via RT-qPCR and found that these genes were differentially expressed in these tissues. Among them, the expression levels of F3'5'H and ANR were higher in leaves than in roots or stems, which is consistent with proanthocyanidins content in these tissues. Among MYB genes that were differentially expressed, the expression of OvMYBPA2 was relatively high in high proanthocyanidin sainfoin. Over-expression level of OvMYBPA2 in alfalfa hairy roots resulted in decreased anthocyanin content but increased proanthocyanidin content. Our study provided transcriptome information for further functional characterization of proanthocyanidin biosynthesis-related genes in sainfoin and candidate key MYB genes for bioengineering of proanthocyanidins in plants.

The complete chloroplast genome sequence of Lotus corniculatus L.

Lotus corniculatus L., a member of the Fabaceae family, is considered one of the most agriculturally important forage plants, owing to its anti-bloating properties; its ability to grow in low-fertility, acidic, and high-salinity soils; and high nutritional value. In this study, we obtained the complete chloroplast genome of L. corniculatus by Illumina sequencing and GetOrganelle assembly pipeline. The whole chloroplast genome of L. corniculatus is 150,700 bp in length, and has a typical circular structure with four parts: a large single-copy region (LSC 82,117 bp), a small single-copy region (SSC 18,275 bp), and a pair of inverted repeat regions (25,154 bp for both IRa and IRb). The overall GC content is 36.03%. The plastome has 109 unique genes, consisting of 78 protein-coding genes, 27 unique tRNA gene, and 4 unique rRNA genes. Based on the protein-coding gene sequences from 17 species, we reconstructed a maximum likelihood (ML) tree. The phylogenetic result shows that L. corniculatus has a closer relationship with Lotus japonicas.

The complete chloroplast genome of Galega officinalis L.

Galega officinalis L. is a perennial herb of the Fabaceae family. The flowers of G. officinalis L. are colorful and suitable for ornamental purposes. It can be used as a food complement for animals and humans, and it could promote lactation in animals and humans. In this study, we obtained the complete chloroplast genome of G. officinalis L. and found it is 125,086 bp in length. The GC content of this genome is 34.18%. Among the 112 unique genes in the chloroplast genome of G. officinalis L., 30 tRNA, 4 rRNA and 78 protein-coding genes were successfully annotated. We constructed the maximum likelihood (ML) tree with 26 species, and concluded that G. officinalis is phylogenetically closely related to the genus of Cicer, Glycine and Desmodium.

The complete chloroplast genome of Desmodium uncinatum (Fabaceae).

Desmodium uncinatum is one of the most important legume forage which distributes in tropical and subtropical regions of the world. In our study, we obtained the complete chloroplast genome of D. uncinatum with a length of 148,853 bp, including a large single copy region of 84,019 bp, small single copy region of 18,223 bp, and a pair of inverted repeat regions of 20,672 bp. The GC content in the whole chloroplast genome of D. uncinatum is 35.16%. Among the 133 unique genes in the circular genome, 37 tRNA, 12 rRNA and 84 protein-coding genes were successfully annotated. We constructed the Maximum likelihood (ML) tree with 11 species, and came to the conclusion that D. uncinatum was phylogenetically closely related to the genus of Glycine and Trifolium.

The complete chloroplast genome sequence of Sainfoin (Onobrychis viciifolia).

Sainfoin (Onobrychis viciifolia) is one of the dominant legume forages distributed in Northern China. In our study, we assembled and annotated the structure of the complete chloroplast genome of sainfoin. The length of the circular genome is 122,102 bp. It contains 115 genes, including 79 protein-coding genes (68.7%), 31 tRNA genes (26.96%) and 5 rRNA genes (4.35%). The GC content of the total chloroplast genome of sainfoin is 34.58%. We construct the phylogenetic relationships between the chloroplast genome of sainfoin and the other 16 species by the Maximum likelihood (ML), and found sainfoin is most closely related to Hedysarum petrovii and Hedysarum taipeicum.

Genome-Wide Identification, Expression Analysis and Functional Study of CCT Gene Family in Medicago truncatula.

The control of flowering time has an important impact on biomass and the environmental adaption of legumes. The CCT (CO, COL and TOC1) gene family was elucidated to participate in the molecular regulation of flowering in plants. We identified 36 CCT genes in the M. truncatula genome and they were classified into three distinct subfamilies, PRR (7), COL (11) and CMF (18). Synteny and phylogenetic analyses revealed that CCT genes occurred before the differentiation of monocot and dicot, and CCT orthologous genes might have diversified among plants. The diverse spatial-temporal expression profiles indicated that MtCCT genes could be key regulators in flowering time, as well as in the development of seeds and nodules in M. truncatula. Notably, 22 MtCCT genes with typical circadian rhythmic variations suggested their different responses to light. The response to various hormones of MtCCT genes demonstrated that they participate in plant growth and development via varied hormones dependent pathways. Moreover, six MtCCT genes were dramatically induced by salinity and dehydration treatments, illustrating their vital roles in the prevention of abiotic injury. Collectively, our study provides valuable information for the in-depth investigation of the molecular mechanism of flowering time in M. truncatula, and it also provides candidate genes for alfalfa molecular breeding with ideal flowering time.

Retraction Note: Doubled haploid production in alfalfa (Medicago sativa L.) through isolated microspore culture.

This paper has been retracted.

Doubled haploid production in alfalfa (Medicago sativa L.) through isolated microspore culture.

Rapid production of doubled haploids (DHs) through isolated microspore culture is an important and promising method for genetic study of alfalfa. To induce embryogenesis in alfalfa, isolated microspores were submitted to abiotic stresses during their initial culture, in order to stimulate them to form embryos and plantlets. 'Baoding' and 'Zhongmu No 1' alfalfa cultivars supported reproducible and reliable proliferation response irrespective of any stress treatment of microspores. The microspore developmental stage for isolated microspore culture was studied and we found that uninucleate microspores were best to initiate culture. Exposure of microspores to appropriate low temperature or heat shock stresses were able to increase the efficiency of embryogenesis. The most effective low-temperature treatment was 4 °C for 24 h and the frequency of plantlets induction was 20.0%. The most effective heat shock treatment was 32 °C for 2 d and the frequency of plantlets induction was 14.17%. The analysis of ploidy level performed by flow cytometer revealed that the majority of 278 regenerated plantlets were haploid (65.83%) or doubled haploid (33.81%). This is the first report of haploid production in alfalfa through isolated microspore culture.

Effects of Bt cabbage pollen on the honeybee Apis mellifera L.

Honeybees may be exposed to insecticidal proteins from transgenic plants via pollen during their foraging activity. Assessing effects of such exposures on honeybees is an essential part of the risk assessment process for transgenic Bacillus thuringiensis (Bt) cabbage. Feeding trials were conducted in a laboratory setting to test for possible effects of Cry1Ba3 cabbage pollen on Italian-derived honeybees Apis mellifera L. Newly emerged A. mellifera were fed transgenic pollen, activated Cry1Ba3 toxin, pure sugar syrup (60% w/v sucrose solution), and non-transgenic cabbage pollen, respectively. Then the effects on survival, pollen consumption, weight, detoxification enzyme activity and midgut enzyme activity of A. mellifera were monitored. The results showed that there were no significant differences in survival, pollen consumption, weight, detoxification enzyme activity among all treatments. No significant differences in the activities of total proteolytic enzyme, active alkaline trypsin-like enzyme and weak alkaline trypsin-like enzyme were observed among all treatments. These results indicate that the side-effects of the Cry1Ba3 cabbage pollen on A. mellifera L. are unlikely.

High resistance of transgenic cabbage plants with a synthetic cry1Ia8 gene from Bacillus thuringiensis against two lepidopteran species under field conditions.

BACKGROUND: Plutella xylostella (Linnaeus) has become the most destructive pest in cabbage throughout the world. Cry1Ia8 cabbage has been developed to reduce pest attacks. To gain a better understanding of the efficacy of Cry1Ia8 cabbage, a homozygous Cry1Ia8 cabbage line A14-5 was produced, and its resistance to P. xylostella, Pieris rapae (Linnaeus) and other lepidopteran pests was evaluated in the field in 2011, 2012 and 2013. RESULTS: Under natural infestation conditions, the homozygous transgenic line was highly resistant against P. xylostella and P. rapae as compared with the untransformed control and susceptible to Mamestra brassicae (Linnaeus) and Spodoptera exigua (Hübner). The homozygous transgenic plants showed slight symptoms of damaged leaves by lepidopteran species, while the untransformed plants exhibited serious damage symptoms throughout the cabbage growing season. CONCLUSION: Compared with the control, the homozygous transgenic cabbage line showed great potential for protecting cabbage from attack by P. xylostella and P. rapae in the field.