Development of 18 microsatellite markers for Atractylodes japonica.

PREMISE: Atractylodes japonica (Asteraceae) is endemic to East Asia, where its rhizomes are used in traditional medicine. To investigate the genetic diversity of this species, we developed polymorphic microsatellite markers. METHODS AND RESULTS: We obtained a total of 175,825 simple sequence repeat (SSR) loci using the Illumina HiSeq 2500 system. Eighteen polymorphic SSR primer pairs were selected to determine heterozygosity levels and allele numbers in 80 individuals from four A. japonica populations. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.133 to 0.892, respectively. Cross-amplification in the related species A. macrocephala and A. lancea was successful in 15 and 14 of the 18 markers, respectively. CONCLUSIONS: These microsatellite markers will be useful for future studies involving A. japonica population genetics and breeding.

Leaf proteome characterization in the context of physiological and morphological changes in response to copper stress in sorghum.

Copper (Cu) is an essential micronutrient required for normal growth and development of plants; however, at elevated concentrations in soil, copper is also generally considered to be one of the most toxic metals to plant cells due to its inhibitory effects against many physiological and biochemical processes. In spite of its potential physiological and economical significance, molecular mechanisms under Cu stress has so far been grossly overlooked in sorghum. To explore the molecular alterations that occur in response to copper stress, the present study was performed in ten-day-old Cu-exposed leaves of sorghum seedlings. The growth characteristics were markedly inhibited, and ionic alterations were prominently observed in the leaves when the seedlings were exposed to different concentrations (0, 100, and 150 µM) of CuSO4. Using two-dimensional gels with silver staining, 643 differentially expressed protein spots (≥1.5-fold) were identified as either significantly increased or reduced in abundance. Of these spots, a total of 24 protein spots (≥1.5-fold) from Cu-exposed sorghum leaves were successfully analyzed by MALDI-TOF-TOF mass spectrometry. Of the 24 differentially expressed proteins from Cu-exposed sorghum leaves, 13 proteins were up-regulated, and 11 proteins were down-regulated. The abundance of most identified protein species, which function in carbohydrate metabolism, stress defense and protein translation, was significantly enhanced, while that of another protein species involved in energy metabolism, photosynthesis and growth and development were severely reduced. The resulting differences in protein expression patterns together with related morpho-physiological processes suggested that these results could help to elucidate plant adaptation to Cu stress and provide insights into the molecular mechanisms of Cu responses in C4 plants.

Morpho-Physiological and Proteome Level Responses to Cadmium Stress in Sorghum.

Cadmium (Cd) stress may cause serious morphological and physiological abnormalities in addition to altering the proteome in plants. The present study was performed to explore Cd-induced morpho-physiological alterations and their potential associated mechanisms in Sorghum bicolor leaves at the protein level. Ten-day-old sorghum seedlings were exposed to different concentrations (0, 100, and 150 µM) of CdCl2, and different morpho-physiological responses were recorded. The effects of Cd exposure on protein expression patterns in S. bicolor were investigated using two-dimensional gel electrophoresis (2-DE) in samples derived from the leaves of both control and Cd-treated seedlings. The observed morphological changes revealed that the plants treated with Cd displayed dramatically altered shoot lengths, fresh weights and relative water content. In addition, the concentration of Cd was markedly increased by treatment with Cd, and the amount of Cd taken up by the shoots was significantly and directly correlated with the applied concentration of Cd. Using the 2-DE method, a total of 33 differentially expressed protein spots were analyzed using MALDI-TOF/TOF MS. Of these, treatment with Cd resulted in significant increases in 15 proteins and decreases in 18 proteins. Major changes were absorbed in the levels of proteins known to be involved in carbohydrate metabolism, transcriptional regulation, translation and stress responses. Proteomic results revealed that Cd stress had an inhibitory effect on carbon fixation, ATP production and the regulation of protein synthesis. Our study provides insights into the integrated molecular mechanisms involved in responses to Cd and the effects of Cd on the growth and physiological characteristics of sorghum seedlings. We have aimed to provide a reference describing the mechanisms involved in heavy metal damage to plants.

Gel-free/label-free proteomic analysis of root tip of soybean over time under flooding and drought stresses.

Growth in the early stage of soybean is markedly inhibited under flooding and drought stresses. To explore the responsive mechanisms of soybean, temporal protein profiles of root tip under flooding and drought stresses were analyzed using gel-free/label-free proteomic technique. Root tip was analyzed because it was the most sensitive organ against flooding, and it was beneficial to root penetration under drought. UDP glucose: glycoprotein glucosyltransferase was decreased and increased in soybean root under flooding and drought, respectively. Temporal protein profiles indicated that fermentation and protein synthesis/degradation were essential in root tip under flooding and drought, respectively. In silico protein-protein interaction analysis revealed that the inductive and suppressive interactions between S-adenosylmethionine synthetase family protein and B-S glucosidase 44 under flooding and drought, respectively, which are related to carbohydrate metabolism. Furthermore, biotin/lipoyl attachment domain containing protein and Class II aminoacyl tRNA/biotin synthetases superfamily protein were repressed in the root tip during time-course stresses. These results suggest that biotin and biotinylation might be involved in energy management to cope with flooding and drought in early stage of soybean-root tip.

Characterization of proteins in soybean roots under flooding and drought stresses.

Flooding and drought affect soybean growth because soybean is a stress-sensitive crop. In 2-day-old plants exposed to 2-day flooding or drought, the fresh weight of roots was markedly suppressed, although the root morphology clearly differed between two conditions. To understand the response mechanisms of soybean to flooding and drought stresses, a gel-free proteomic technique was used. A total of 97 and 48 proteins were significantly changed in response to flooding and drought stresses, respectively. Proteins involved in protein synthesis were decreased by flooding stress and increased by drought. Glycolysis-related proteins were increased in roots by both flooding and drought stresses. Fermentation, stress, and cell wall-related proteins were increased in response to flooding stress, whereas cell organization and redox-related proteins were increased under drought stress. Among the identified proteins, three S-adenosylmethionine synthetases were commonly decreased and increased in response to flooding and drought stresses, respectively. The mRNA expression levels of S-adenosylmethionine synthetase genes displayed a similar tendency to the changes in protein abundance. These results suggest that S-adenosylmethionine synthetase is involved in the regulation of stress response because it was changed in response to flooding and drought stresses. BIOLOGICAL SIGNIFICANCE: This study reported on the response mechanisms of soybean to flooding and drought stresses using the gel-free proteomic technique. Proteins involved in protein synthesis were decreased by flooding stress and increased by drought. Glycolysis-related proteins were increased in roots by both flooding and drought stresses. Fermentation, stress, and cell wall-related proteins were increased in response to flooding stress, whereas cell organization and redox-related proteins were increased under drought stress. Among the identified proteins, three S-adenosylmethionine synthetases were commonly decreased and increased in response to flooding and drought stresses, respectively. The mRNA expression levels of S-adenosylmethionine synthetase genes displayed a similar tendency to the changes in protein abundance. These results suggest that S-adenosylmethionine synthetase is involved in the regulation of stress response because it was changed in response to flooding and drought stresses.

Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions. To better understand the flooding-responsive mechanisms of soybean, the effect of exogenous calcium on flooding-stressed soybeans was analyzed using proteomic technique. An increase in exogenous calcium levels enhanced soybean root elongation and suppressed the cell death of root tip under flooding stress. Proteins were extracted from the roots of 4-day-old soybean seedlings exposed to flooding stress without or with calcium for 2 days and analyzed using gel-free proteomic technique. Proteins involved in protein degradation/synthesis/posttranslational modification, hormone/cell wall metabolisms, and DNA synthesis were decreased by flooding stress; however, their reductions were recovered by calcium treatment. Development, lipid metabolism, and signaling-related proteins were increased in soybean roots when calcium was supplied under flooding stress. Fermentation and glycolysis-related proteins were increased in response to flooding; however, these proteins were not affected by calcium supplementation. Furthermore, urease and copper chaperone proteins exhibited similar profiles in 4-day-old untreated soybeans and 4-day-old soybeans exposed to flooding for 2 days in the presence of calcium. These results suggest that calcium might affect the cell wall/hormone metabolisms, protein degradation/synthesis, and DNA synthesis in soybean roots under flooding stress.

Analysis of response mechanism in soybean under low oxygen and flooding stresses using gel-base proteomics technique.

A proteomics approach was used to analyze the response mechanism in soybean seedlings under low oxygen and flooding stresses. Three-day-old soybean seedlings were subjected to low oxygen and flooding stresses. Growth of root was suppressed in both stresses with more extent of suppression in flooded seedlings at 3 and 6 days following the treatments. Proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. Of total 1,233 protein spots, 27 protein spots were commonly changed under low oxygen and flooding stresses; while the differential change in 4 and 18 protein spots was specific to low oxygen and flooding stresses, respectively. Proteins related to metabolism and energy were increased; while protein destination/storage related proteins were decreased commonly under low oxygen and flooding stresses. Protein specie, TCP domain class transcription factor was decreased specifically under low oxygen stress; while decrease of nine proteins related to metabolism, protein destination/storage and disease/defense was specific in flooded seedlings. The decrease in majority of the proteins related to protein destination/storage specifically in flooded seedlings implies the misfolding of proteins resulting in flooded injuries in an independent way of oxygen deprivation. These results suggest that decrease in proteins related to protein destination/storage and disease/defense causes more growth suppression in soybean seedlings under flooding stress compared to low oxygen stress.