Quantitative proteomic, physiological and biochemical analysis of cotyledon, embryo, leaf and pod reveals the effects of high temperature and humidity stress on seed vigor formation in soybean.

BACKGROUND: Soybean developing seed is susceptible to high temperature and humidity (HTH) stress in the field, resulting in vigor reduction. Actually, the HTH in the field during soybean seed growth and development would also stress the whole plant, especially on leaf and pod, which in turn affect seed growth and development as well as vigor formation through nutrient supply and protection. RESULTS: In the present study, using a pair of pre-harvest seed deterioration-sensitive and -resistant cultivars Ningzhen No. 1 and Xiangdou No. 3, the comprehensive effects of HTH stress on seed vigor formation during physiological maturity were investigated by analyzing cotyledon, embryo, leaf, and pod at the levels of protein, ultrastructure, and physiology and biochemistry. There were 247, 179, and 517 differentially abundant proteins (DAPs) identified in cotyledon, embryo, and leaf of cv. Xiangdou No. 3 under HTH stress, while 235, 366, and 479 DAPs were identified in cotyledon, embryo, and leaf of cv. Ningzhen No. 1. Moreover, 120, 144, and 438 DAPs between the two cultivars were identified in cotyledon, embryo, and leaf under HTH stress, respectively. Moreover, 120, 144, and 438 DAPs between the two cultivars were identified in cotyledon, embryo, and leaf under HTH stress, respectively. Most of the DAPs identified were found to be involved in major metabolic pathways and cellular processes, including signal transduction, tricarboxylic acid cycle, fatty acid metabolism, photosynthesis, protein processing, folding and assembly, protein biosynthesis or degradation, plant-pathogen interaction, starch and sucrose metabolism, and oxidative stress response. The HTH stress had less negative effects on metabolic pathways, cell ultrastructure, and physiology and biochemistry in the four organs of Xiangdou No. 3 than in those of Ningzhen No. 1, leading to produce higher vigor seeds in the former. CONCLUSION: High seed vigor formation is enhanced by increasing protein biosynthesis and nutrient storage in cotyledon, stronger stability and viability in embryo, more powerful photosynthetic capacity and nutrient supply in leaf, and stronger protection in pod under HTH stress. These results provide comprehensive characteristics of leaf, pod and seed (cotyledon and embryo) under HTH stress, and some of them can be used as selection index in high seed vigor breeding program in soybean.

A transcriptomic analysis reveals soybean seed pre-harvest deterioration resistance pathways under high temperature and humidity stress.

Pre-harvest soybean seeds in the field are susceptible to high temperature and humidity (HTH) stress, leading to pre-harvest seed deterioration, which will result in a reduction in grain quality, yield, and seed vigor. To understand the gene expression involved in seed deterioration response under HTH stress, in this study, we conducted an RNA-Seq analysis using two previously screened soybean cultivars with contrasting seed deterioration resistance. HTH stress induced 1081 and 357 differentially expressed genes (DEGs) in the sensitive cultivar Ningzhen No. 1 and resistant cultivar Xiangdou No. 3, respectively. The majority of DEGs in the resistant cultivar were up-regulated, while down-regulated DEGs were predominant in the sensitive cultivar. KEGG pathway analysis revealed that metabolic pathways, biosynthesis of secondary metabolites, and protein processing in endoplasmic reticulum were the predominant pathways in both cultivars during seed deterioration under HTH stress. The genes involved in photosynthesis, carbohydrate metabolism, lipid metabolism, and heat shock proteins pathways might contribute to the different response to seed deterioration under HTH treatment in the two soybean cultivars. Our study extends the knowledge of gene expression in soybean seed under HTH stress and further provides insight into the molecular mechanism of seed deterioration as well as new strategies for breeding soybean with improved seed deterioration resistance.

CarNAC4, a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis.

KEY MESSAGE: CarNAC4 is a typical stress-responsive NAC transcription factor and enhances drought and salt stress tolerances in transgenic Arabidopsis. Chickpea (Cicer arietinum L.) is relatively vulnerable to abiotic stress conditions, but the tolerance mechanisms for such stresses in chickpea are largely unknown. To identify stress-related factors in chickpea, we previously constructed a cDNA library of chickpea leaves exposed to drought stress conditions. A cDNA encoding a putative NAC transcription factor (CarNAC4) was identified as a putative stress-responsive gene. Our study indicated that the transcript levels of CarNAC4 were enhanced in response to several abiotic stresses and phytohormones. Promoter analysis demonstrated that multiple stress-related cis-acting elements exist in promoter region of CarNAC4. CarNAC4 is localized in the nucleus and binds to the DNA sequence containing CGT[G/A], while the C-terminal region of CarNAC4 contains a transcriptional activation domain. Over-expression of CarNAC4 in Arabidopsis plants improved tolerance to drought and salt stresses. Transgenic plants exhibited greater reduced rates of water loss and more proline accumulation than Col-0 plants under drought stress and less MDA contents than Col-0 plants under salt stress. In addition, over-expression of CarNAC4 enhanced the expression of stress-responsive genes such as RD29A, ERD10, COR15A, COR47, KIN1 and DREB2A. These results indicated that CarNAC4 functions as a transcription factor involved in the regulation of drought and salt stress response.

[Adventitious Buds Induction of Mentha haplocalyx Under Variation Inducted by NaN3].

Objective: To establish the chemical mutagenesis in vitro system of Mentha haplocalyx,the effects of different plant hormone combination and Vc on the induction of mint stem adventitious buds and the mutation effects of different concentrations of NaN3 on their calli were studied. Methods: The internodes of mint used as the material,and based on the preliminary experiment, the effects of different concentrations of TDZ,6-BA,NAA and Vc on adventitious buds induction rate were researched. On the basis of screening the best induction formula, the Mentha haplocalyx calli were treated with different concentrations of NaN3（ 0,2,4,6,8,10,12,14,16 mg/L）. Results: The optimum medium for calli induction and adventitious buds formation was MS + 0. 1 mg / L TDZ + 0. 2 mg / L NAA + 1mg / L Vc + 30 g / L sucrose + 5. 5 g / L agar, the treatment concentration of NaN3 for LD50of calli induction was 14 mg / L for 10 d,or 12 mg / L for 20 d,or 10 mg / L for 30 d. Plantlet could differentiate from the calli treated with NaN3. By comparing to the regenerated plants,81 mutants had been selected. Conclusion: A chemical mutagenesis in vitro system for Mentha haplocalyx with NaN3 was preliminarily established.

GmSBH1, a homeobox transcription factor gene, relates to growth and development and involves in response to high temperature and humidity stress in soybean.

KEY MESSAGE: GmSBH1 involves in response to high temperature and humidity stress. Homeobox transcription factors are key switches that control plant development processes. Glycine max H1 Sbh1 (GmSBH1) was the first homeobox gene isolated from soybean. In the present study, the full ORF of GmSBH1 was isolated, and the encoded protein was found to be a typical class I KNOX homeobox transcription factor. Subcellular localization and transcriptional activation assays showed that GmSBH1 is a nuclear protein and possesses transcriptional activation activity in the homeodomain. The KNOX1 domain was found to play a clear role in suppressing the transcriptional activation activity of GmSBH1. GmSBH1 showed different expression levels among different soybean tissues and was involved in response to high temperature and humidity (HTH) stress in developing soybean seeds. The overexpression of GmSBH1 in Arabidopsis altered leaf and stoma phenotypes and enhanced seed tolerance to HTH stress. Overall, our results indicated that GmSBH1 is involved in growth, development, and enhances tolerance to pre-harvest seed deterioration caused by HTH stress in soybean.

Genome-Wide Identification and Expression Analysis of the Cucumber FKBP Gene Family in Response to Abiotic and Biotic Stresses.

The FKBP (FK506-binding protein) gene family is an important member of the PPlase protease family and plays a vital role during the processes of plant growth and development. However, no studies of the FKBP gene family have been reported in cucumber. In this study, 19 FKBP genes were identified in cucumber, which were located on chromosomes 1, 3, 4, 6, and 7. Phylogenetic analysis divided the cucumber FKBP genes into three subgroups. The FKBP genes in the same subgroup exhibited similar structures and conserved motifs. The cis-acting elements analysis revealed that the promoters of cucumber FKBP genes contained hormone-, stress-, and development-related cis-acting elements. Synteny analysis of the FKBP genes among cucumber, Arabidopsis, and rice showed that 12 kinds of syntenic relationships were detected between cucumber and Arabidopsis FKBP genes, and 3 kinds of syntenic relationships were observed between cucumber and rice FKBP genes. The tissue-specific expression analysis showed that some FKBP genes were expressed in all tissues, while others were only highly expressed in part of the 10 types of tissues. The expression profile analysis of cucumber FKBP genes under 13 types of stresses showed that the CsaV3_1G007080 gene was differentially expressed under abiotic stresses (high temperature, NaCl, silicon, and photoperiod) and biotic stresses (downy mildew, green mottle mosaic virus, Fusarium wilt, phytophthora capsica, angular leaf spot, and root-knot nematode), which indicated that the CsaV3_1G007080 gene plays an important role in the growth and development of cucumber. The interaction protein analysis showed that most of the proteins in the FKBP gene family interacted with each other. The results of this study will lay the foundation for further research on the molecular biological functions of the cucumber FKBP gene family.

Mapping of Two Major QTLs Controlling Flowering Time in Brassica napus Using a High-Density Genetic Map.

Research on the flowering habit of rapeseed is important for the selection of varieties adapted to specific ecological environments. Here, quantitative trait loci (QTL) for the days-to-flowering trait were identified using a doubled haploid population of 178 lines derived from a cross between the winter type SGDH284 and the semi-winter type 158A. A linkage map encompassing 3268.01 cM was constructed using 2777 bin markers obtained from next-generation sequencing. The preliminary mapping results revealed 56 QTLs for the days to flowering in the six replicates in the three environments. Twelve consensus QTLs were identified by a QTL meta-analysis, two of which (cqDTF-C02 and cqDTF-C06) were designated as major QTLs. Based on the micro-collinearity of the target regions between B. napus and Arabidopsis, four genes possibly related to flowering time were identified in the cqDTF-C02 interval, and only one gene possibly related to flowering time was identified in the cqDTF-C06 interval. A tightly linked insertion-deletion marker for the cqFT-C02 locus was developed. These findings will aid the breeding of early maturing B. napus varieties.

Molecular Dissection Unveiling Dwarfing Effects of Plant Growth Retardants on Pomegranate.

Dwarfed stature is a desired trait for modern orchard production systems. One effective strategy for dwarfing cultivation is exogenously applying plant growth retardants (PGRs) to plants. However, for many economic fruit trees, the current knowledge on the regulatory mechanisms underlying the dwarfing effect of PGRs is limited, which largely restricts the agricultural application of PGRs. In this study, we exogenously applied three kinds of PGRs [paclobutrazol, daminozide (B9), and mannitol] to the seedlings of pomegranate (Punica granatum L.) and performed comparative transcriptome analysis to elucidate the molecular features of PGR-induced dwarfing in pomegranates. Our results showed that all the three PGRs could significantly suppress plant growth of pomegranate. The inhibition of auxin biosynthetic processes, as well as auxin-mediated shoot development, may be considered as the main reason for the dwarfing. Besides that, different PGRs were also found to induce dwarfing via specific mechanisms, for example, cellular response to strigolactone was particularly suppressed by the application of paclobutrazol, while the level of carbohydrate homeostasis and metabolism were downregulated in conditions of either B9 or mannitol treatments. Furthermore, exogenous PGR application was supposed to cause adverse impacts on the normal physiological process of pomegranate seedlings, which may bring extra burden to pomegranate plants. These novel findings unveiled the genetic basis underlying the dwarfing in pomegranates, which provides deeper insights into PGR-mediated dwarfing cultivation of pomegranates.

Transcriptomic and photosynthetic responses to grafting of the Nod1 gene in nodulated and non-nodulated soybeans.

Legume plants form symbiotic relationships with rhizobia to convert N2 into ammonia, and the nodulation status can affect plant development including photosynthesis. However, the relationship between nitrogen fixation and photosynthesis during carbon and nitrogen metabolism remains unclear. This study was undertaken to unravel regulation of nodulation and photosynthesis using a spontaneous nonnodulated soybean mutant by grafting. The results of inheritance and gene mapping showed that the nonnodulated mutant was controlled by a recessive gene overlapped with the reported rj1 locus, and might be a new rj1 allele with 1 bp deletion in the fourth exon in comparison to the sequence of normal nodulation plants. According to grafting results, soybean nodulation is obviously determined by the roots, not the seedlings. Moreover, nitrogen content along with related metabolic enzyme activity, and photosynthetic capacity were enhanced by nonnodulated scions grafted with nodulated roots. Contrary results were obtained for nodulated scions grafted with nonnodulated roots. A total of 853 differentially expressed genes (DEGs) in the leaves and 1874 in the roots were identified by transcriptome analyses of the grafting treatments. We identified 285 differential gene ontology (GO) terms and 57 differential pathway terms identified in the leaves, while 856 differential GO terms and 207 differential pathway terms in the roots. Twenty DEGs interacting at translation level were selected, and the results of transcriptome analyses were verified by q-PCR. These findings indicated that the nodulation-related Nod allelic gene increases the nitrogen content of nonnodulated plants, which affects the enzymes involved in nitrogen metabolism, leading to changes in hormone levels and further regulation of photosynthesis and carbon metabolism.

[Establishment and optimization of systems for protoplasts isolation of soybean and chickpea that used in subcellular location].

Young leaves of Kabuli chickpea as well as soybean Xiangdou No.3, which are the current plants that studied in our laboratory were selected as materials. Effects on protoplasts yield and survival rate of different enzyme combination, concentration of D-Mannitol in enzyme combinations, pH of enzyme combinations and enzymolysis time are detected. The results showed that, the best condition for Xiangdou No.3 leaf protoplasts isolation is to rotate the cut materials for 6 hours in enyzme solution under temperature of 27 ℃ and rotate speed of 45 r/min for 6 h. Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%) in combination with Pectolyase Y-23 (0.4%) dissolving in CPW solution with MES (0.1%) and Mannitol (10%), pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.The best condition for protoplasts isolation of Kabuli chickpea is to put the cut materials into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27 ℃ and rotate speed of 45 r/min on water bath shaker, the optimum combination of enzyme consists of Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%), MES (0.1%) and Mannitol (10%) dissolved in CPW solution with pH 4.8. The protoplasts prepared with the methods above are used in subcellular location and the effects show well.

[Characterization and subcellular localization of two SBP genes and their response to abiotic stress in soybean (Glycine max (L.) Merr.)].

High temperature and humidity stress during seed growth and development of spring soybean can result in seed deterioration in South China. We isolated two genes (GmSBP and GmSBPL) encoding putative SBP proteins from soybean (Glycine max (L.) Merr.) to study their biological functions and response to abiotic stress,. The two SBP proteins are hydrophilic and incomplete membrane ones. Real-time quantitative (RT-PCR) analysis reveals that the expression of the two genes in the developing seeds of the seed deterioration resistant cultivar Xiangdou No. 3 and sensitive cultivar Ningzhen No. 1 was significantly affected by high temperature and humidity treatment. Meanwhile, the levels of sucrose and soluble sugar in the developing seeds of both cultivars were also affected under high temperature and humidity stress. During seed growth and development, the expression of the two genes as well as the levels of sucrose and soluble sugar reached the highest at 30 days after flower. GmSBP2 and GmSBPL were found to be differentially expressed in different soybean tissues. Sub-cellular localization indicated that two genes were located in cytoplasm and cell membrane. Our results indicate that GmSBP2 and GmSBPL might be involved in the response to abiotic stress, which will enrich our understanding of pre-harvest seed deterioration and resistance in soybean from one side.

[Effects of high temperature and humidity stress at the physiological maturity stage on seed vigor, main nutrients and coat structure of spring soybean].

A pot experiment was conducted to investigate the effects of high temperature and humidity stress [(40 +/- 2) degrees C/(30 +/- 2) degrees C, RH (95 +/- 5)%/(70 +/- 5)%, 10 h/14 h (day/night)] at the physiological maturity stage of two spring soybean cultivars (Xiangdou No. 3 and Ningzhen No. 1) on seed vigor indices, main nutritional components and coat anatomical structure. High temperature and humidity stress were found to cause the decrease of seed viability, germination potential, and germination percentage as well as the dehydrogenase and acid phosphatase activities, but increased the seed cell membrane permeability as well as H+, soluble sugar and leucine levels in the seed soaking liquid of each cultivar. Moreover, the stress led to irregular changes of seed oil and protein contents and alteration of anatomical structure of episperm and hilum in the two cultivars. A shortterm stress (less than 5 h) had no significant impact on seed vigor, but a long-term one (more than 48 h) caused rapid decrease of seed vigor indices. Xiangdou No. 3 showed less decreases in seed germination potential and enzyme activities, and less increase in extravasation content in the seed soaking liquid, had compact seed coat and intact hilum, suggesting it was more resistant to high temperature and humidity stress.

Comparative proteomics analysis reveals the mechanism of pre-harvest seed deterioration of soybean under high temperature and humidity stress.

High temperature and humidity (HTH) stress during soybean seed development and maturity in the field easily leads seed to pre-harvest deterioration. However, how proteins and their involved pathways in developing soybean seed systematically cause deterioration is still not largely understood. To reveal it, we compared the proteome composition of developing seed (R(7) period) of a pre-harvest seed deterioration sensitive soybean cultivar at different HTH stress time points (24, 96 and 168 h) with their corresponding controls by 2-DE. 42 protein spots were found to be differentially expressed and successfully identified by MALDI-TOF MS to match 31 diverse protein species. These proteins were involved in 13 cellular responses and metabolic processes including carbohydrate metabolism, signal transduction, protein biosynthesis, photosynthesis, protein folding and assembly, energy pathway, cell rescue and defense, cell cycle, nitrogen metabolism, lipid metabolism, amino acid metabolism, transcription regulation, and secondary metabolite biosynthesis. Based on these proteins' functions and involved pathways, together with ultrastructural, physical and chemical, and metabolomic data, a pre-harvest seed deterioration mechanism was proposed. Such a mechanism allows us to further understand the possible management strategy of cellular activities occurring in the HTH-stressed developing seeds and provides new insights into the HTH stress responses in crop developing seeds.

Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes.

Salinity is one of the major environmental constraints limiting yield of crop plants in many semi-arid and arid regions around the world. To understand responses in soybean seedling to salt stress at proteomic level, the extracted proteins from seedling leaves of salt-sensitive genotype Jackson and salt-tolerant genotype Lee 68 under 150 mM NaCl stress for 1, 12, 72 and 144 h, respectively, were analyzed by 2-DE. Approximately 800 protein spots were detected on 2-DE gels. Among them, 91 were found to be differently expressed, with 78 being successfully identified by MALDI-TOF-TOF. The identified proteins were involved in 14 metabolic pathways and cellular processes. Based on most of the 78 salt-responsive proteins, a salt stress-responsive protein network was proposed. This network consisted of several functional components, including balancing between ROS production and scavenging, accelerated proteolysis and reduced biosynthesis of proteins, impaired photosynthesis, abundant energy supply and enhanced biosynthesis of ethylene. Salt-tolerant genotype Lee 68 possessed the ability of higher ROS scavenging, more abundant energy supply and ethylene production, and stronger photosynthesis than salt-sensitive genotype Jackson under salt stress, which may be the major reasons why it is more salt-tolerant than Jackson.