MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter.

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.

Genome-wide investigation and expression analyses of the pentatricopeptide repeat protein gene family in foxtail millet.

BACKGROUND: Pentatricopeptide repeat (PPR) proteins are encoded by a large gene family of approximately 450 members in Arabidopsis and 477 in rice, which characterized by tandem repetitions of a degenerate 35 amino acid characteristic sequence motifs. A large majority of the PPR genes in the higher plants are localized in organelles. Their functions remain as yet largely unknown. The majority of characterized PPR proteins have been found to function in modulating the expression plastid and mitochondrial genes in plants. RESULTS: Here, a genome-wide identification and comparison of the PPR genes from 5 organisms was performed, including the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, the eudicot Arabidopsis, and the monocots rice and foxtail millet. It appears that the expansion of this gene family prior to the divergence of the euphyllophytes and the lycophytes in land plants. The duplication and divergence rates of the foxtail millet PPR genes (SiPPRs) showed that the expansion period of this gene family around 400 Mya, and indicated that genome segmental duplication was very likely the primary mechanism underlying the expansion of the PPR gene family in vascular plants. An analysis of a complete set of SiPPR genes/proteins that included classification, chromosomal location, orthologous relationships, duplication analysis, and auxiliary motifs is presented. Expression analysis of the SiPPR genes under stress conditions revealed that the expression of 24 SiPPR genes was responsive to abiotic stress. Subcellular localization analysis of 11 PPR proteins indicated that 5 proteins were localized to chloroplasts, that 4 were localized to mitochondria, and that 2 were localized to the cytoplasm. CONCLUSIONS: Our results contribute to a more comprehensive understanding the roles of PPR proteins and will be useful in the prioritization of particular PPR proteins for subsequent functional validation studies in foxtail millet.

Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis.

Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation.

Alfalfa MsbHLH115 confers tolerance to cadmium stress through activating the iron deficiency response in Arabidopsis thaliana.

Cadmium (Cd) pollution severely affects plant growth and development, posing risks to human health throughout the food chain. Improved iron (Fe) nutrients could mitigate Cd toxicity in plants, but the regulatory network involving Cd and Fe interplay remains unresolved. Here, a transcription factor gene of alfalfa, MsbHLH115 was verified to respond to iron deficiency and Cd stress. Overexpression of MsbHLH115 enhanced tolerance to Cd stress, showing better growth and less ROS accumulation in Arabidopsis thaliana. Overexpression of MsbHLH115 significantly enhanced Fe and Zn accumulation and did not affect Cd, Mn, and Cu concentration in Arabidopsis. Further investigations revealed that MsbHLH115 up-regulated iron homeostasis regulation genes, ROS-related genes, and metal chelation and detoxification genes, contributing to attenuating Cd toxicity. Y1H, EMSA, and LUC assays confirmed the physical interaction between MsbHLH115 and E-box, which is present in the promoter regions of most of the above-mentioned iron homeostasis regulatory genes. The transient expression experiment showed that MsbHLH115 interacted with MsbHLH121pro. The results suggest that MsbHLH115 may directly regulate the iron-deficiency response system and indirectly regulate the metal detoxification response mechanism, thereby enhancing plant Cd tolerance. In summary, enhancing iron accumulation through transcription factor regulation holds promise for improving plant tolerance to Cd toxicity, and MsbHLH115 is a potential candidate for addressing Cd toxicity issues.

MsYSL6, A Metal Transporter Gene of Alfalfa, Increases Iron Accumulation and Benefits Cadmium Resistance.

Iron (Fe) is necessary for plant growth and development. The mechanism of uptake and translocation in Cadmium (Cd) is similar to iron, which shares iron transporters. Yellow stripe-like transporter (YSL) plays a pivotal role in transporting iron and other metal ions in plants. In this study, MsYSL6 and its promoter were cloned from leguminous forage alfalfa. The transient expression of MsYSL6-GFP indicated that MsYSL6 was localized to the plasma membrane and cytoplasm. The expression of MsYSL6 was induced in alfalfa by iron deficiency and Cd stress, which was further proved by GUS activity driven by the MsYSL6 promoter. To further identify the function of MsYSL6, it was heterologously overexpressed in tobacco. MsYSL6-overexpressed tobacco showed better growth and less oxidative damage than WT under Cd stress. MsYSL6 overexpression elevated Fe and Cd contents and induced a relatively high Fe translocation rate in tobacco under Cd stress. The results suggest that MsYSL6 might have a dual function in the absorption of Fe and Cd, playing a role in the competitive absorption between Fe and Cd. MsYSL6 might be a regulatory factor in plants to counter Cd stress. This study provides a novel gene for application in heavy metal enrichment or phytoremediation and new insights into plant tolerance to toxic metals.

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis.

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.

A systemic proteomic analysis of Populus chloroplast by using shotgun method.

The chloroplast is one of the most important organelles in plants. Proteomic investigations of chloroplasts have been undertaken for many herb plant species, but to date no such investigation has been reported for woody plant chloroplasts. In the present study we initiated a systematic proteomic study of Populus chloroplasts using a shotgun proteomic method. After isolation of chloroplasts and tryptic digestion of the proteins, the protein fragments were separated via HPLC using an SCX column, and the peptides were analyzed by LC-MS/MS; 119 proteins were successfully identified. Based on annotation information in the UniProtKB/Swiss-Prot database, these proteins were identified as being localized in the chloroplast thylakoid membrane, chloroplast stroma, chloroplast thylakoid lumen, and plastoglobules. Over 50% of all identified proteins were confirmed as chloroplast thylakoid proteins, and 85 are encoded by the chloroplast genome with the remaining proteins encoded by the nuclear genome. Based on functional annotation, these proteins were classified into four functional categories, including photosynthesis, redox regulation and stress, primary and secondary metabolism, transport and signaling. These data provide a valuable basis for further studies on photosynthesis in poplar species.

De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress.

Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen species and malondialdehyde and a decreased content of chlorophyll, indicating that anti-oxidation and detoxification play an important role in response to saline-alkaline stress. Overall, the transcriptome analysis provided novel insights into the saline-alkaline stress tolerance response mechanisms in alfalfa.

The E-Subgroup Pentatricopeptide Repeat Protein Family in Arabidopsis thaliana and Confirmation of the Responsiveness PPR96 to Abiotic Stresses.

Pentatricopeptide repeat (PPR) proteins are extensive in all eukaryotes. Their functions remain as yet largely unknown. Mining potential stress responsive PPRs, and checking whether known PPR editing factors are affected in the stress treatments. It is beneficial to elucidate the regulation mechanism of PPRs involved in biotic and abiotic stress. Here, we explored the characteristics and origin of the 105 E subgroup PPRs in Arabidopsis thaliana. Phylogenetic analysis categorized the E subgroup PPRs into five discrete groups (Cluster I to V), and they may have a common origin in both A. thaliana and rice. An in silico expression analysis of the 105 E subgroup PPRs in A. thaliana was performed using available microarray data. Thirty-four PPRs were differentially expressed during A. thaliana seed imbibition, seed development stage(s), and flowers development processes. To explore potential stress responsive PPRs, differential expression of 92 PPRs was observed in A. thaliana seedlings subjected to different abiotic stresses. qPCR data of E subgroup PPRs under stress conditions revealed that the expression of 5 PPRs was responsive to abiotic stresses. In addition, PPR96 is involved in plant responses to salt, abscisic acid (ABA), and oxidative stress. The T-DNA insertion mutation inactivating PPR96 expression results in plant insensitivity to salt, ABA, and oxidative stress. The PPR96 protein is localized in the mitochondria, and altered transcription levels of several stress-responsive genes under abiotic stress treatments. Our results suggest that PPR96 may important function in a role connecting the regulation of oxidative respiration and environmental responses in A. thaliana.

Variations in a hotspot region of chloroplast DNAs among common wheat and Aegilops revealed by nucleotide sequence analysis.

The second largest BamHI fragment (B2) of the chloroplast DNA in Triticum (wheat) and Aegilops contains a highly variable region (a hotspot), resulting in four types of B2 of different size, i.e. B2l (10.5kb), B2m (10.2kb), B2 (9.6kb) and B2s (9.4kb). In order to gain a better understanding of the molecular nature of the variations in length and explain unexpected identity among B2 of Ae. ovata, Ae. speltoides and common wheat (T. aestivum), the nucleotide sequence between a stop codon of rbcL and a HindIII site in cemA in the hotspot was determined for Ae. ovata, Ae. speltoides, Ae. caudata and Ae. mutica. The total number of nucleotides in the region was 2808, 2810, 3302, and 3594 bp, for Ae. speltoides, Ae. ovata, Ae. caudata and Ae. mutica, respectively, and the sequences were compared with the corresponding ones of Ae. crassa 4x, T. aestivum and Ae. squarrosa. Compared with the largest B2l fragment of Ae. mutica, a 791bp and a 793 bp deletion were found in Ae. speltoides and Ae. ovata, respectively, and the possible site of deletion in the two species is the same as that of T. aestivum. However, a deleted segment in Ae. ovata is 2 bp longer than that of Ae. speltoides (and T. aestivum), demonstrating that recurrent deletions had occurred in the chloroplast genomes of both species. Comparison of the sequences from Ae. caudata and Ae. crassa 4x with that of Ae. mutica revealed a 289 bp and a 61 bp deletion at the same site in Ae. caudata and Ae. crassa 4x, respectively. Sequence comparison using wild Aegilops plants showed that the large length variations in a hotspot are fixed to each species. A considerable number of polymorphisms are observed in a loop in the 3' of rbcL. The study reveals the relative importance of the large and small indels and minute inversions to account for variations in the chloroplast genomes among closely related species.

[Inducing chromosome translocation and deletions by Chinese Spring-Agilops 2C disomic addition x Chinese Spring-Elytriga 5E disomic addition].

Crossing between two disomic addition lines, Chinese Spring-E. elongata and Chinese Sping with two gametocidal chromosomes 2C (from Ae. cylindric), was carried out to investigate the function of gametocidal chromosome. After scrutinizing the meiosis of pollen mother cells (PMCs) in F1 hybrids, several results were concluded: (1) In seven of the crossing combinations, the number of univalents exceeded the expected and some trivalents and tetravalents appeared also in MI; lagging, breakage and bridge of chromosomes were observed in anaphase and telophase; considerable micronuclei formed in telophase and tetrads. These were mainly caused by the gametocidal chromosome 2C. (2) Chromosomes 6E and 7E were more susceptible to the effect of the gametocidal chromosome 2C. (3) The gametocidal chromosome 2C functioned in prophase viz. the period of forming synaptonemal complex. Four F1 lines, 5-14, 5-37, 5-67 and 5-71, were identified to be T5ES 4AST5EL 2BS, T5EL 3AS, T5ES 5BS translocation respectively by using C-banding and genome in situ hybrydization(GISH) analysis. Deletion was detected in line 5-17 (short arm of chromosome 2A), 5-27(6B), 5-18(4B and 5B), 5-72(4A) and 5-4(4B) by C-banding analysis. The statistic data showed that gametocidal chromosome could induce translocation with a high frequency of 5% and reacted on group B more efficiently than on groups A and D since translocation involving chromosome 4A, 2B, 3A, 5B and deletion involving chromosome 6B, 5B, 4B, 4A 2A according to Endo's work.

An RGD-Modified Endostatin Peptide Expressed at E. coli Shows Anti-Tumor Activity In vivo.

Tumor vasculatures express high levels of α(V)ß(3)/α(V)ß(5) and α(5)ß(1) integrins. Peptide containing the RGD (Arg-Gly-Asp) sequence, which is present in ligands of integrins, is effective in targeting therapeutic reagents to tumor vascular endothelium. In this study, we investigated whether the biological activity of endostatin 27 peptides can be enhanced by the addition of an integrin targeting sequence. RGDRGD and GGGRGD sequence were added to the carboxyl terminus of endostatin 27 and 25 peptides, respectively. Modification of endostatin 27 peptides with the RGD motif showed specific and increased binding to endothelial cells and the increased binding is consistent with improved antiangiogenic property. RGD-modified endostatin 27 peptides was more effective than human endostatin and endostatin 27 peptides in inhibiting liver cancer growth in athymic mice. These finding indicates that addition of a vascular targeting sequence can enhance the biological activity of an antiangiogenic peptides molecule.

Insulin-like growth factor-1 fused with thrombopoietin mimetic peptide effectively increase platelets count in vivo.

OBJECTIVE: To design the expression of fusion proteins containing one or 2 thrombopoietin mimetic peptide (TMP). METHODS: This study was conducted at Harbin Pharmaceutical Group Research and Development Center, Harbin, China from June 2009 to January 2010. We designed the protein that was fused to the C-terminus of insulin-like growth factors (IGF-1) by a flexible peptide linker by Dami cell proliferation assay, colony-forming assay, and analysis of platelet in mice to prove our hypothesis. The total number of mice used was 48 in all 4 groups. RESULTS: The fusion proteins were produced in Escherichia coli BL21 (DE3) at up to 26% of the total cell proteins. Subsequent biological activity assays showed that the fusion proteins exhibited higher potency than recombinant human thrombopoietin (TPO). Our results showed that the fusion proteins IGF-1-TMP exhibited higher biological activities than TMP in Dami cell proliferation, human cord blood cell colony-forming assays, and in experiments on acute myeloid radiation sickness mice, which can effectively increase the number of platelets. CONCLUSION: Experiments in mice and biology activity assay, which can effectively increase the number of platelets, indicated that it has a potential role in pharmaceutical applications for the treatment of thrombocytopenia.

Shotgun proteomics analysis on maize chloroplast thylakoid membrane.

In this study we initiated a proteomic investigation of the maize thylakoid membrane by using a shotgun proteomic approach based on LC-MS(E). A total of 34 maize thylakoid membrane proteins were identified, the majority of which are primarily involved in photosynthesis, including the light-reaction and carbon assimilation. It is noteworthy that all of the core subunits of the Photosystem II were identified in our search. Proteins involved in other processes, such as iron storage, were also detected in our study. The quantity of each identified protein was also determined. Of interest, we discovered that the amount of the three ATP synthase subunits were not equivalent, suggesting that these proteins perform other functions in addition to ATP synthesis. To our knowledge this is the first extensive proteomic investigation of the maize thylakoid membrane, and will likely enable further study of maize photosynthesis and chloroplast development.