Tyrosylprotein Sulfotransferase Positively Regulates Symbiotic Nodulation and Root Growth.

Posttranslational tyrosine sulfation of peptides and proteins is catalysed by tyrosylprotein sulfotransferases (TPSTs). In Arabidopsis, tyrosine sulfation is essential for the activities of peptide hormones, such as phytosulfokine (PSK) and root meristem growth factor (RGF). Here, we identified a TPST-encoding gene, MtTPST, from model legume Medicago truncatula. MtTPST expression was detected in all organs, with the highest level in root nodules. A promoter:GUS assay revealed that MtTPST was highly expressed in the root apical meristem, nodule primordium and nodule apical meristem. The loss-of-function mutant mttpst exhibited a stunted phenotype with short roots and reduced nodule number and size. Application of both of the sulfated peptides PSK and RGF3 partially restored the defective root length of mttpst. The reduction in symbiotic nodulation in mttpst was partially recovered by treatment with sulfated PSK peptide. MtTPST-PSK module functions downstream of the Nod factor signalling to promote nodule initiation via regulating accumulation and/or signalling of cytokinin and auxin. Additionally, the small-nodule phenotype of mttpst, which resulted from decreased apical meristematic activity, was partially complemented by sulfated RGF3 treatment. Together, these results demonstrate that MtTPST, through its substrates PSK, RGF3 and other sulfated peptide(s), positively regulates nodule development and root growth.

Molecular oxygen levels regulate Sinorhizobium meliloti cell division through a FixJ-dependent transcription control mechanism.

Rhizobia infect the roots of host legumes and induce formation of nitrogen-fixing nodules, where nitrogenase genes are inducibly expressed by micro-aerobic signals. FixL/FixJ is an oxygen signal sensing system that is unique to rhizobia. FixL monitors molecular oxygen levels and phosphorylates the response regulator FixJ, thereby regulating downstream gene expression. The cell division of rhizobia is regulated by a phosphorylation relaying cascade that includes the transcription factors CtrA, GcrA, and DnaA. In Sinorhizobium meliloti the expression of these proteins is regulated by NtrX, which affects cell division. In the present work, by analyzing the cell division phenotypes and gene expression patterns of S. meliloti fixJ and ntrX mutants, we found that S. meliloti cell division is regulated by oxygen gas levels. Under normal conditions, FixJ induced NtrX and DnaA expression, but repressed CtrA and GcrA expression. In contrast, under hypoxic conditions, phosphorylated FixJ specifically bound to gene promoter regions to directly induce CtrA and GcrA expression, but to repress DnaA expression. Our findings reveal that molecular oxygen levels regulate S. meliloti cell division by a FixJ-dependent transcription control mechanism.

A legume-specific novel type of phytosulfokine, PSK-δ, promotes nodulation by enhancing nodule organogenesis.

Phytosulfokine-α (PSK-α), a tyrosine-sulfated pentapeptide with the sequence YSO3IYSO3TQ, is widely distributed across the plant kingdom and plays multiple roles in plant growth, development, and immune response. Here, we report a novel type of phytosulfokine, PSK-δ, and its precursor proteins (MtPSKδ, LjPSKδ, and GmPSKδ1), specifically from legume species. The sequence YSO3IYSO3TN of sulfated PSK-δ peptide is different from PSK-α at the last amino acid. Expression pattern analysis revealed PSK-δ-encoding precursor genes to be expressed primarily in legume root nodules. Specifically, in Medicago truncatula, MtPSKδ expression was detected in root cortical cells undergoing nodule organogenesis, in nodule primordia and young nodules, and in the apical region of mature nodules. Accumulation of sulfated PSK-δ peptide in M. truncatula nodules was detected by LC/MS. Application of synthetic PSK-δ peptide significantly increased nodule number in legumes. Similarly, overexpression of MtPSKδ in transgenic M. truncatula markedly promoted symbiotic nodulation. This increase in nodule number was attributed to enhanced nodule organogenesis induced by PSK-δ. Additional genetic evidence from the MtPSKδ mutant and RNA interference assays suggested that the PSK-δ and PSK-α peptides function redundantly in regulating nodule organogenesis. These results suggest that PSK-δ, a legume-specific novel type of phytosulfokine, promotes symbiotic nodulation by enhancing nodule organogenesis.

The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula.

Leguminous root nodules specifically induced by rhizobium species fix nitrogen gas to gain nitrogen sources, which is important in sustainable agriculture and ecological balance. Several peptide signals are reported to be involved in regulation of legume nodule number and development. There are fifteen genes coding Root Meristem Growth Factor (RGF) peptide in Medicago truncatula, herein we find the expression of MtRGF3 is significantly induced by Sinorhizobium meliloti with production of Nod factors. The gene promoter is active in nodule primordia, young nodules and the meristem region of mature nodules. Knock-down (RNAi) roots of the gene (MtRGF3-RNAi) formed more root nodules than the empty vector control, and the nodule number decreased in MtRGF3-overexpressing (MtRGF3-OX) roots. Exogenous addition of the synthesized peptide significantly promoted primary root growth and inhibited lateral root emergence, in addition, the peptide application reduced the number of infection threads, nodule primordia and root nodules of M. truncatula. We also found that tyrosine sulfation determines the biological activity of MtRGF3 functioning in nodulation process, and MtRGF3 peptide negatively regulates nodulation in a dosage manner. These results demonstrate that the MtRGF3 peptide is a novel regulator during nodulation of Medicago trucatula.

Expression of a novel PSK-encoding gene from soybean improves seed growth and yield in transgenic plants.

MAIN CONCLUSION: Expression of GmPSKγ1 , a novel PSK-encoding gene from soybean, increases seed size and yield in transgenic plants by promoting cell expansion. Phytosulfokine-α (PSK-α), a sulfated pentapeptide hormone with the sequence YIYTQ, plays important roles in many aspects of plant growth and development. In this study, we identified a pair of putative precursor genes in soybean, GmPSKγ1 and -2, encoding a PSK-like peptide: PSK-γ. Similar to PSK-α in amino acid composition, the sequence of PSK-γ is YVYTQ, and the tyrosines undergo sulfonylation. Treatment of Arabidopsis seedlings with synthetic sulfated PSK-γ significantly enhanced root elongation, indicating that PSK-γ might be a functional analog of PSK-α. Expression pattern analysis revealed that the two GmPSKγ genes, especially GmPSKγ1, are primarily expressed in developing soybean seeds. Heterologous expression of GmPSKγ1 under the control of a seed-specific promoter markedly increased seed size and weight in Arabidopsis, and this promoting effect of PSK-γ on seed growth was further confirmed in transgenic tobacco constitutively expressing GmPSKγ1. Cytological analysis of transgenic Arabidopsis seeds revealed that PSK-γ promotes seed growth by inducing embryo cell expansion. In addition, expression analysis of downstream candidate genes suggested that PSK-γ signaling might regulate cell wall loosening to promote cell expansion in Arabidopsis seeds. Overall, our results shed light on the mechanism by which PSK-γ promotes seed growth, paving the way for the use of this new peptide for biotechnological improvement of crop seed/grain size and yield.

The LsrB Protein Is Required for Agrobacterium tumefaciens Interaction with Host Plants.

Agrobacterium tumefaciens infects and causes crown galls in dicot plants by transferring T-DNA from the Ti plasmid to the host plant via a type IV secretion system. This process requires appropriate environmental conditions, certain plant secretions, and bacterial regulators. In our previous work, a member of the LysR family of transcriptional regulators (LsrB) in Sinorhizobium meliloti was found to modulate its symbiotic interactions with the host plant alfalfa. However, the function of its homolog in A. tumefaciens remains unclear. In this study, we show that the LsrB protein of A. tumefaciens is required for efficient transformation of host plants. A lsrB deletion mutant of A. tumefaciens exhibits a number of defects, including in succinoglycan production, attachment, and resistance to oxidative stress and iron limitation. RNA-sequencing analysis indicated that 465 genes were significantly differentially expressed (upregulation of 162 genes and downregulation of 303 genes) in the mutant, compared with the wild-type strain, including those involved in succinoglycan production, iron transporter, and detoxification enzymes for oxidative stress. Moreover, expression of the lsrB gene from S. meliloti, Brucella abortus, or A. tumefaciens rescued the defects observed in the S. meliloti or A. tumefaciens lsrB deletion mutant. Our findings suggest that a conserved mechanism of LsrB function exists in symbiotic and pathogenic bacteria of the family Rhizobiaceae.

Wound-induced polypeptides improve resistance against Pseudomonas syringae pv. tomato DC3000 in Arabidopsis.

Wound-induced polypeptides (WIPs) are a novel class of polypeptides with the length less than 100 amino acids. Our previous research has identified a number of WIP genes in soybean (Glycine max) root nodules. However, functions of WIPs in planta remains largely unknown. Here, we identified five WIP-encoding genes, AtWIP1-5, in Arabidopsis. Among them, AtWIP1 and -2 are ubiquitously expressed in a partially overlapping pattern as revealed by both qRT-PCR and promoter:GUS assays. Subcellular localization analyses reveal that both AtWIP1 and -2 are localized at the plasma membrane while AtWIP1 shows a punctate distribution pattern. AtWIP1, -2 are transcriptionally induced by flg22 treatment, but repressed by effector(s) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Heterologous overexpression of GmWIP genes enhances resistance of Arabidopsis to Pst DC3000 at the cost of growth inhibition. Moreover, overexpression of GmWIP genes promotes pattern-triggered immunity (PTI) evidenced by increased expressions of flg22-inducible genes and enhanced seedling growth inhibition under flg22 treatment. Taken together, our results indicate that WIPs positively regulate plant resistance against Pst DC3000 by enhancing PTI responses.

An auxin-induced ß-type endo-1,4-ß-glucanase in poplar is involved in cell expansion and lateral root formation.

MAIN CONCLUSION: PtrGH9A7, a poplar ß-type endo-1,4-ß-glucanase gene induced by auxin, promotes both plant growth and lateral root development by enhancing cell expansion. Endo-1,4-ß-glucanase (EGase) family genes function in multiple aspects of plant growth and development. Our previous study found that PtrCel9A6, a poplar EGase gene of the ß subfamily, is specifically expressed in xylem tissue and is involved in the cellulose biosynthesis required for secondary cell wall formation (Yu et al. in Mol Plant 6:1904-1917, 2013). To further explore the functions and regulatory mechanism of ß-subfamily EGases, we cloned and characterized another poplar ß-type EGase gene PtrGH9A7, a close homolog of PtrCel9A6. In contrast to PtrCel9A6, PtrGH9A7 is predominantly expressed in parenchyma tissues of the above-ground part; in roots, PtrGH9A7 expression is specifically restricted to lateral root primordia at all stages from initiation to emergence and is strongly induced by auxin application. Heterologous overexpression of PtrGH9A7 promotes plant growth by enhancing cell expansion, suggesting a conserved role for ß-type EGases in 1,4-ß-glucan chains remodeling, which is required for cell wall loosening. Moreover, the overexpression of PtrGH9A7 significantly increases lateral root number, which might result from improved lateral root primordium development due to enhanced cell expansion. Taken together, these results demonstrate that this ß-type EGase induced by auxin signaling has a novel role in promoting lateral root formation as well as in enhancing plant growth.

Sinorhizobium meliloti Glutathione Reductase Is Required for both Redox Homeostasis and Symbiosis.

Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. melilotigor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2 and sodium nitroprusside. Interestingly, the expression of gshA, gshB1, katA, and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCE The antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacterium S. meliloti, the de novo synthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection in S. meliloti cells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

Enzyme-PISA: An Efficient Method for Preparing Well-Defined Polymer Nano-Objects under Mild Conditions.

Enzyme catalysis is a mild, efficient, and selective technique that has many applications in organic synthesis as well as polymer synthesis. Here, a novel enzyme-catalysis-induced reversible addition-fragmentation chain transfer (RAFT)-mediated dispersion polymerization for preparing AB diblock copolymer nano-objects with complex morphologies at room temperature is described. Taking advantage of the room-temperature feature, it is shown that pure, worm-like polymer nano-objects can be readily prepared by just monitoring the viscosity. Moreover, it is demonstrated that inorganic nanoparticles and proteins can be loaded in situ into vesicles by this method. Finally, a novel oxygen-tolerant RAFT-mediated dispersion polymerization initiated by enzyme cascade reaction that can be carried out in open vessels is developed. The enzyme-initiated RAFT dispersion polymerization provides a facile platform for the synthesis of various functional polymer nano-objects under mild conditions.

[Protective effect of Ligustri Lucidi Ait Polysaccharide against lipopolysaccharide-induced inflammatory injury of Sertoli cells in rats].

OBJECTIVE: To explore the effect of Ligustri Lucidi Ait Polysaccharide (LLP) on lipopolysaccharide (LPS)-induced inflammatory injury of Sertoli cells. METHODS: Rat Sertoli cells were isolated and cultured in vitro and then divided into five groups, blank control, LPS, LPS + low-dose LLP, LPS + medium-dose LLP, and LPS + high-dose LLP. After 48 hours of treatment, the proliferation of the cells was detected by CCK-8, their apoptosis determined by FMC, and the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) in the supernatant of the cell culture medium measured by ultraviolet spectrophotometry. The contents of IL-1α, IL-6 and TGF-ß in the culture medium were detected by ELISA before and after removal of LPS. RESULTS: The proliferation of the cells showed statistically significant differences among different groups (F = 153.93, P < 0.01), markedly reduced in the LPS group as compared with the blank control (P < 0.01), but remarkably increased in the high- and medium-dose LLP groups in comparison with the LPS group (both P < 0.01), and so did the apoptosis of the cells (F = 64.06, P < 0.01), significantly increased in the LPS group as compared with the blank control (P < 0.05), but markedly decreased in the high- and medium-dose LLP groups in comparison with the LPS group (both P < 0.01). Statistically significant differences were also observed among different groups in the levels of SOD (F = 56.07, P < 0.01), CAT (F = 41.57, P < 0.01), GSH-Px activity (F = 238.46, P < 0.01), and MDA (F = 285.31, P < 0.01), with decreased SOD, CAT and GSH-Px activity (P < 0.01) and increased MDA (P < 0.01) in the LPS group as compared with the control, but elevated SOD and CAT in the high- and medium-dose LLP groups and increased GSH-Px activity and decreased MDA concentration in all the three LLP groups in comparison with the LPS group (P < 0.01). Before the removal of LPS, the contents of IL-1α, IL-6 and TGF-ß in the culture medium were markedly higher in the LPS than in the control group (all P < 0.01), that of IL-1α was increased significantly in the high- and medium-dose LLP groups (P < 0.01 and P < 0.05) while those of IL-6 and TGF-ß showed no statistically significant differences in the three LPS groups as compared with the LLP group (P > 0.05). After the removal of LPS, the contents of IL-1α and IL-6 were remarkably reduced (t = 25.26 and 61.43, P < 0.01) and that of TGF-ß increased (t = －18.16, P < 0.01), even more significantly in the LLP+LPS groups (P < 0.01). CONCLUSIONS: Ligustri Lucidi Ait Polysaccharide plays a protective role in LPS-induced inflammatory injury of Sertoli cells by reducing cell apoptosis and regulating the contents of IL-1α, IL-6 and TGF-ß from Sertoli cells in inflammation.

Regulation of cysteine residues in LsrB proteins from Sinorhizobium meliloti under free-living and symbiotic oxidative stress.

The development of legume nitrogen-fixing nodules is regulated by reactive oxygen species (ROS) produced by symbionts. Several regulators from Rhizobium are involved in ROS sensing. In a previous study, we found that Sinorhizobium meliloti LsrB regulates lipopolysaccharide production and is associated with H2 O2 accumulation in alfalfa (Medicago sativa) nodules. However, its underlying regulatory mechanism remains unclear. Here, we report that the cysteine residues in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa nodulation and nitrogen fixation. Moreover, LsrB directly activated the transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase, responsible for glutathione synthesis) and this regulation required the cysteine (Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense oxidative stress via the formation of intermolecular disulfide bonds, generating LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive regulatory site for the induction of downstream genes, whereas C146 and C275 play negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions displayed altered phenotypes in respect to their adaptation to oxidative stress, nodulation and nitrogen fixation-related plant growth. Our findings demonstrate that S. meliloti LsrB modulates alfalfa nodule development by directly regulating downstream gene expression via a post-translational strategy.

Overexpression of phytosulfokine-α induces male sterility and cell growth by regulating cell wall development in Arabidopsis.

KEY MESSAGE: Over-production of functional PSK-α in Arabidopsis caused increases in both plant cell growth and biomass and induced male sterility by regulating cell wall development. Phytosulfokine-α (PSK-α) is a novel disulfated pentapeptide hormone that is involved in promoting plant cell growth. Although a role for PSK-α in stimulating protoplast expansion has been suggested, how PSK-α regulates cell growth in planta remains poorly understood. In this study, we found that overexpression of the normal PSK-α precursor gene AtPSK4, which resulted in high levels of PSK-α, caused longer roots and larger leaves with enlarged cells. As expected, these changes were not observed in transgenic plants overexpressing mutated AtPSK4, which generated unsulfated PSK-α. These findings confirmed the role of PSK-α in promoting plant cell growth. Furthermore, we found that overexpressing AtPSK4, but not mutated AtPSK4, induced a phenotype of male sterility that resulted from the failure of fibrous cell wall development in the endothecium. In addition, overexpressing AtPSK4 enhanced expression of a number of genes encoding expansins, which are involved in cell wall loosening. Accordingly, in addition to its role in cell growth, we propose a novel function for PSK-α signaling in the modulation of plant male sterility via regulation of cell wall development.

Phytosulfokine Is Involved in Positive Regulation of Lotus japonicus Nodulation.

Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation, and innate immunity. The potential role of PSK in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1 to LjPSK5. Three of them (LjPSK1, LjPSK4, and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-ß-glucuronidase analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 1M synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules but not the events of rhizobial infection and nodule initiation was increased by overexpressing LjPSK1 in transgenic hairy roots, in which the expression of jasmonate-responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.

Same duodenal neuroendocrine tumors, different endoscopic resection methods: a case report and literature review.

Duodenal neuroendocrine tumors (NETs), comprising 2-3% of all gastrointestinal NETs and 1-3% of all duodenal tumors, are remarkably uncommon. In this report, we described a patient diagnosed with two submucosal tumors in the duodenal bulb. We used two distinct endoscopic resection methods, including endoscopic submucosal dissection (ESD) and submucosal tunneling endoscopic resection (STER), to achieve en bloc resection of the lesions without complications. Pathological evaluation, involving hematoxylin-eosin staining and immunohistochemistry, confirmed the diagnosis of NET. Given the limited operative field and space in the duodenal bulb, STER proved to be a viable endoscopic resection technique.