Comparative Ubiquitination Proteomics Revealed the Salt Tolerance Mechanism in Sugar Beet Monomeric Additional Line M14.

Post-translational modifications (PTMs) are important molecular processes that regulate organismal responses to different stresses. Ubiquitination modification is not only involved in human health but also plays crucial roles in plant growth, development, and responses to environmental stresses. In this study, we investigated the ubiquitination proteome changes in the salt-tolerant sugar beet monomeric additional line M14 under salt stress treatments. Based on the expression of the key genes of the ubiquitination system and the ubiquitination-modified proteins before and after salt stress, 30 min of 200 mM NaCl treatment and 6 h of 400 mM NaCl treatment were selected as time points. Through label-free proteomics, 4711 and 3607 proteins were identified in plants treated with 200 mM NaCl and 400 mM NaCl, respectively. Among them, 611 and 380 proteins were ubiquitinated, with 1085 and 625 ubiquitination sites, in the two salt stress conditions, respectively. A quantitative analysis revealed that 70 ubiquitinated proteins increased and 47 ubiquitinated proteins decreased. At the total protein level, 42 were induced and 20 were repressed with 200 mM NaCl, while 28 were induced and 27 were repressed with 400 mM NaCl. Gene ontology, KEGG pathway, protein interaction, and PTM crosstalk analyses were performed using the differentially ubiquitinated proteins. The differentially ubiquitinated proteins were mainly involved in cellular transcription and translation processes, signal transduction, metabolic pathways, and the ubiquitin/26S proteasome pathway. The uncovered ubiquitinated proteins constitute an important resource of the plant stress ubiquitinome, and they provide a theoretical basis for the marker-based molecular breeding of crops for enhanced stress tolerance.

Functional Characterization of a Sugar Beet BvbHLH93 Transcription Factor in Salt Stress Tolerance.

The basic/helix-loop-helix (bHLH) transcription factor (TF) plays an important role for plant growth, development, and stress responses. Previously, proteomics of NaCl treated sugar beet leaves revealed that a bHLH TF, BvbHLH93, was significantly increased under salt stress. The BvbHLH93 protein localized in the nucleus and exhibited activation activity. The expression of BvbHLH93 was significantly up-regulated in roots and leaves by salt stress, and the highest expression level in roots and leaves was 24 and 48 h after salt stress, respectively. Furthermore, constitutive expression of BvbHLH93 conferred enhanced salt tolerance in Arabidopsis, as indicated by longer roots and higher content of chlorophyll than wild type. Additionally, the ectopic expression lines accumulated less Na+ and MDA, but more K+ than the WT. Overexpression of the BvBHLH93 enhanced the activities of antioxidant enzymes by positively regulating the expression of antioxidant genes SOD and POD. Compared to WT, the overexpression plants also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. These results suggest that BvbHLH93 plays a key role in enhancing salt stress tolerance by enhancing antioxidant enzymes and decreasing ROS generation.

Mechanisms of Sugar Beet Response to Biotic and Abiotic Stresses.

Sugar beet is used not only in the sugar production, but also in a wide range of industries including the production of bioethanol as a source of renewable energy, extraction of pectin and production of molasses. The red beetroot has attracted much attention as health-promoting and disease-preventing functional food. The negative effects of environmental stresses, including abiotic and biotic ones, significantly decrease the cash crop sugar beet productivity. In this paper, we outline the mechanisms of sugar beet response to biotic and abiotic stresses at the levels of physiological change, the genes' functions, transcription and translation. Regarding the physiological changes, most research has been carried out on salt and drought stress. The functions of genes from sugar beet in response to salt, cold and heavy metal stresses were mainly investigated by transgenic technologies. At the transcriptional level, the transcriptome analysis of sugar beet in response to salt, cold and biotic stresses were conducted by RNA-Seq or SSH methods. At the translational level, more than 800 differentially expressed proteins in response to salt, K+/Na+ ratio, iron deficiency and resupply and heavy metal (zinc) stress were identified by quantitative proteomics techniques. Understanding how sugar beet respond and tolerate biotic and abiotic stresses is important for boosting sugar beet productivity under these challenging conditions. In order to minimize the negative impact of these stresses, studying how the sugar beet has evolved stress coping mechanisms will provide new insights and lead to novel strategies for improving the breeding of stress-resistant sugar beet and other crops.

Overexpression of a S-Adenosylmethionine Decarboxylase from Sugar Beet M14 Increased Araidopsis Salt Tolerance.

Polyamines play an important role in plant growth and development, and response to abiotic stresses. Previously, differentially expressed proteins in sugar beet M14 (BvM14) under salt stress were identified by iTRAQ-based quantitative proteomics. One of the proteins was an S-adenosylmethionine decarboxylase (SAMDC), a key rate-limiting enzyme involved in the biosynthesis of polyamines. In this study, the BvM14-SAMDC gene was cloned from the sugar beet M14. The full-length BvM14-SAMDC was 1960 bp, and its ORF contained 1119 bp encoding the SAMDC of 372 amino acids. In addition, we expressed the coding sequence of BvM14-SAMDC in Escherichia coli and purified the ~40 kD BvM14-SAMDC with high enzymatic activity. Quantitative real-time PCR analysis revealed that the BvM14-SAMDC was up-regulated in the BvM14 roots and leaves under salt stress. To investigate the functions of the BvM14-SAMDC, it was constitutively expressed in Arabidopsis thaliana. The transgenic plants exhibited greater salt stress tolerance, as evidenced by longer root length and higher fresh weight and chlorophyll content than wild type (WT) under salt treatment. The levels of spermidine (Spd) and spermin (Spm) concentrations were increased in the transgenic plants as compared with the WT. Furthermore, the overexpression plants showed higher activities of antioxidant enzymes and decreased cell membrane damage. Compared with WT, they also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. Together, these results suggest that the BvM14-SAMDC mediated biosynthesis of Spm and Spd contributes to plant salt stress tolerance through enhancing antioxidant enzymes and decreasing ROS generation.

Quantitative proteomics and phosphoproteomics of sugar beet monosomic addition line M14 in response to salt stress.

UNLABELLED: Salinity is a major abiotic stress affecting plant growth, development and agriculture productivity. Understanding the molecular mechanisms of salt stress tolerance will provide valuable information for effective crop engineering and breeding. Sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. and Beta corolliflora Zoss exhibits tolerance to salt stress. In this study, the changes in the M14 proteome and phosphoproteome induced by salt stress were analyzed. We report the characteristics of the M14 plants under 0, 200, and 400mM NaCl using label-free quantitative proteomics approaches. Protein samples were subjected to total proteome profiling using LC-MS/MS and phosphopeptide enrichment to identify phosphopeptides and phosphoproteins. A total of 2182 proteins were identified and 114 proteins showed differential levels under salt stress. Interestingly, 189 phosphoproteins exhibited significant changes at the phosphorylation level under salt stress. Several signaling components associated with salt stress were found, e.g. 14-3-3 and mitogen-activated protein kinases (MAPK). Fifteen differential phosphoproteins and proteins involved in signal transduction were tested at the transcriptional level. The results revealed the short-term salt responsive mechanisms of the special sugar beet M14 line using label-free quantitative phosphoproteomics. BIOLOGICAL SIGNIFICANCE: Sugar beet monosomic addition line M14 is a special germplasm with salt stress tolerance. Analysis of the M14 proteome and phosphoproteome under salt stress has provided insight into specific response mechanisms underlying salt stress tolerance. Reversible protein phosphorylation regulates a wide range of cellular processes such as transmembrane signaling, intracellular amplification of signals, and cell-cycle control. This study has identified significantly changed proteins and phosphoproteins, and determined their potential relevance to salt stress response. The knowledge gained can be potentially applied to improving crop salt tolerance.

Salt stress response of membrane proteome of sugar beet monosomic addition line M14.

Understanding how plants respond to and tolerate salt stress is important for engineering and breeding effort to boost plant productivity and bioenergy in an ever challenging environment. Sugar beet M14 line is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and it exhibits tolerance to salt stress. Here we report the changes in membrane proteome of the M14 plants in response to salt stress (0, 200, 400mM NaCl) using an iTRAQ two-dimensional LC-MS/MS technology for quantitative proteomic analysis. In total, 274 proteins, mostly membrane proteins, were identified, and 50 proteins exhibited differential protein level changes, with 40 proteins increased and 10 decreased. The proteins were mainly involved in transport, metabolism, protein synthesis, photosynthesis, protein folding and degradation, signal transduction, stress and defense, energy, and cell structure. These results have revealed interesting mechanisms underlying the M14 response and tolerance to salt stress. BIOLOGICAL SIGNIFICANCE: Sugar beet monosomic addition line M14 is a special variety with salt stress tolerance. Analysis of the M14 membrane proteome under salt stress may provide useful information regarding specific adaptive mechanisms underlying salt stress tolerance. Membrane proteins are known to play critical roles in salt stress signaling and adaptation. The purpose of this study was to identify significantly changed membrane proteins and determine their possible relevance to salt tolerance. The proteomic analysis of the M14 line revealed important molecular mechanisms that can be potentially applied to improving crop salt tolerance. This article is part of a Special Issue entitled: Proteomics in India.

Proteomic analysis of salt tolerance in sugar beet monosomic addition line M14.

Understanding the mechanisms of plant salinity tolerance can facilitate plant engineering for enhanced salt stress tolerance. Sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. and Beta corolliflora Zoss exhibits tolerance to salt stress. Here we report the salt-responsive characteristics of the M14 plants under 0, 200, and 400 mM NaCl conditions using quantitative proteomics approaches. Proteins from control and the salt treated M14 plants were separated using 2D-DIGE. Eighty-six protein spots representing 67 unique proteins in leaves and 22 protein spots representing 22 unique proteins in roots were identified. In addition, iTRAQ LC-MS/MS was employed to identify and quantify differentially expressed proteins under salt stress. Seventy-five differentially expressed proteins in leaves and 43 differentially expressed proteins in roots were identified. The proteins were mainly involved in photosynthesis, energy, metabolism, protein folding and degradation, and stress and defense. Moreover, gene transcription data obtained from the same samples were compared to the corresponding protein data. Thirteen proteins in leaves and 12 in roots showed significant correlation in gene expression and protein levels. These results suggest the important processes for the M14 tolerance to salt stress include enhancement of photosynthesis and energy metabolism, accumulation of osmolyte and antioxidant enzymes, and regulation of methionine metabolism and ion uptake/exclusion.

Sugar beet M14 glyoxalase I gene can enhance plant tolerance to abiotic stresses.

Glyoxalase I is the first enzyme of the glyoxalase system that can detoxify methylglyoxal, a cytotoxic compound increased rapidly under stress conditions. Here we report cloning and characterization of a glyoxalase I from sugar beet M14 line (an interspecific hybrid between a wild species Beta corolliflora Zoss and a cultivated species B. vulgaris L). The full-length gene BvM14-glyoxalase I has 1,449 bp in length with an open reading frame of 1,065 bp encoding 354 amino acids. Sequence analysis shows the conserved glyoxalase I domains, metal and glutathione binding sites and secondary structure (α-helixes and ß-sheets). The BvM14-glyoxalase I gene was ubiquitously expressed in different tissues of sugar beet M14 line and up-regulated in response to salt, mannitol and oxidative stresses. Heterologous expression of BvM14-glyoxalase I could increase E. coli tolerance to methylglyoxal. Transgenic tobacco plants constitutively expressing BvM14-glyoxalase I were generated. Both leaf discs and seedlings showed significant tolerance to methylglyoxal, salt, mannitol and H2O2. These results suggest an important role of BvM14-glyoxalase I in cellular detoxification and tolerance to abiotic stresses.

Salt stress induced proteome and transcriptome changes in sugar beet monosomic addition line M14.

Sugar beet monosomic addition line M14 displays interesting phenotypes such as apomixis and salt stress tolerance. Here we reported proteomic and transcriptomic analysis of M14 leaves and roots under 500mM NaCl treatment for seven days. Proteins from control and treated samples were extracted and separated using two-dimensional difference gel electrophoresis (2D-DIGE). A total of 40 protein spots from leaf gels and 36 protein spots from root gels exhibited significant changes. Using mass spectrometry and database searching, 38 unique proteins in leaves and 29 unique proteins in roots were identified. The proteins included those involved in metabolism, protein folding, photosynthesis, and protein degradation. In addition, cDNA libraries of differentially expressed genes were constructed using suppression subtractive hybridization (SSH). Fifty-eight unigenes including 14 singletons and 44 contigs were obtained. Some salt-responsive genes were identified to function in metabolism, photosynthesis, stress and defense, energy, protein synthesis and protein degradation. This research has revealed candidate genes and proteins for detailed functional characterization, and set the stage for further investigation of the salt tolerance mechanisms in sugar beet.

Identification of a sugar beet BvM14-MADS box gene through differential gene expression analysis of monosomic addition line M14.

Monosomic addition line M14 carrying an additional chromosome 9 from Beta corolliflora Zosimovic ex Buttler was obtained through hybridization between the wild species B. corolliflora and a cultivated species Beta vulgaris L. var Saccharifera Alef. The M14 line showed diplosporic reproduction and stress tolerance. To identify differentially expressed genes in M14, a subtractive cDNA library was prepared by suppression subtractive hybridization (SSH) between M14 (2n=18+1) and B. vulgaris (2n=18). A total of 190 unique sequences were identified in the library and their putative functions were analyzed using Gene Ontology (GO). One of the genes, designated as BvM14-MADS box, encodes a MADS box transcription factor. It was cloned from M14 and over-expressed in transgenic tobacco plants. Interestingly, this gene was located on chromosome 2 of B. vulgaris, not on the additional chromosome 9. Overexpression of BvM14-MADS box led to significant phenotypic changes in tobacco. The differential expression of BvM14-MADS box gene in M14 may be caused by the interaction between the additional chromosome 9 from B. corolliflora and the B. vulgaris chromosomes in M14.

Proteomic analysis of sugar beet apomictic monosomic addition line M14.

Apomixis in plants holds great promise for agriculture because of its vigor associated with heterozygosity and superior genotype. Despite the significance of apomictic reproductive process, our knowledge of proteins and their functions in apomictic development is limited. Here we report a comparative proteomic and transcriptomic analysis of sexual and apomictic processes in sugar beet. A total of 71 differentially expressed protein spots were successfully identified in the course of apomictic reproductive development using high-resolution 2-DE and MS analysis. The differentially expressed proteins were involved in several processes that might work cooperatively to lead to apomictic reproduction. This study has generated potential protein markers important for apomictic development.