Identification of Key Ubiquitination Sites Involved in the Proteasomal Degradation of AtACS7 in Arabidopsis.

The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis.

miR-494-5p mediates the antioxidant activity of EPA by targeting the mitochondrial elongation factor 1 gene MIEF1 in HepG2 cells.

Eicosapentaenoic acid (EPA) shows antioxidant activity, which may be attributed to its regulatory effect on microRNA expression. Our preliminary study indicated that EPA upregulated miR-494-5p, which was possibly involved in the regulation of cellular stress responses. The current study aimed to address whether miR-494-5p was targeted by EPA to regulate cellular oxidative stress and its possible functional mechanism. The results showed that miR-494-5p mediated the antioxidant effect of EPA and miR-494-5p reduction deteriorated EPA-induced increase in the cellular antioxidant capacity of HepG2 cells. Moreover, the mitochondrial elongation factor 1 (MIEF1) gene was a target gene of miR-494-5p. Both miR-494-5p overexpression and MIEF1 knockdown significantly enhanced cellular antioxidant capacity, as indicated by a reduction in the reactive oxygen species level and an increase in the total cellular antioxidant capacity, along with enhancing antioxidant enzymes. Thus, miR-494-5p and MIEF1 had opposite effects on cellular antioxidant capacity. Furthermore, their regulatory effects on oxidative stress may have been attributed to modulation of mitochondrial function, biogenesis and homeostasis. Taken together, the findings indicated that miR-494-5p mediated EPA activity and promoted cellular antioxidant capacity by inhibiting the expression of MIEF1, which further modulated mitochondrial structure and activity. This study may provide novel insights into the post-translational regulation of antioxidation reactions, which involves the coordinated control of mitochondria.

Domain Swapping between AtACS7 and PpACL1 Results in Chimeric ACS-like Proteins with ACS or Cß-S Lyase Single Enzymatic Activity.

The gaseous hormone ethylene plays a pivotal role in plant growth and development. In seed plants, the key rate-limiting enzyme that controls ethylene biosynthesis is ACC synthase (ACS). ACS has, for a long time, been believed to be a single-activity enzyme until we recently discovered that it also possesses Cß-S lyase (CSL) activity. This discovery raises fundamental questions regarding the biological significance of the dual enzymatic activities of ACS. To address these issues, it is highly necessary to obtain ACS mutants with either ACS or CSL single activity. Here, domain swapping between Arabidopsis AtACS7 and moss CSL PpACL1 were performed. Enzymatic activity assays of the constructed chimeras revealed that, R10, which was produced by replacing AtACS7 box 6 with that of PpACL1, lost ACS but retained CSL activity, whereas R12 generated by box 4 substitution lost CSL and only had ACS activity. The activities of both chimeric proteins were compared with previously obtained single-activity mutants including R6, AtACS7Q98A, and AtACS7D245N. All the results provided new insights into the key residues required for ACS and CSL activities of AtACS7 and laid an important foundation for further in-depth study of the biological functions of its dual enzymatic activities.

Identification and Functional Analysis of Key Autophosphorylation Residues of Arabidopsis Senescence Associated Receptor-like Kinase.

Reversible protein phosphorylation mediated by protein kinases and phosphatases plays important roles in the regulation of leaf senescence. We previously reported that the senescence-associated leucine-rich repeat receptor-like kinase AtSARK autophosphorylates on both serine/threonine and tyrosine residues and functions as a positive regulator of Arabidopsis leaf senescence; the senescence-suppressed protein phosphatase SSPP interacts with and dephosphorylates the cytoplasmic domain of AtSARK, thereby negatively regulating leaf senescence. Here, 27 autophosphorylation residues of AtSARK were revealed by mass spectrometry analysis, and six of them, including two Ser, two Thr, and two Tyr residues, were further found to be important for the biological functions of AtSARK. All site-directed mutations of these six residues that resulted in decreased autophosphorylation level of AtSARK could significantly inhibit AtSARK-induced leaf senescence. In addition, mutations mimicking the dephosphorylation form of Ser384 (S384A) or the phosphorylation form of Tyr413 (Y413E) substantially reduced the interaction between AtSARK and SSPP. All results suggest that autophosphorylation of AtSARK is essential for its functions in promoting leaf senescence. The possible roles of S384 and Y413 residues in fine-tuning the interaction between AtSARK and SSPP are discussed herein.

N7 -SSPP fusion gene improves salt stress tolerance in transgenic Arabidopsis and soybean through ROS scavenging.

Considerable signal crosstalk exists in the regulatory network of senescence and stress response. Numerous senescence-associated genes are also involved in plant stress tolerance. However, the underlying mechanisms and application potential of these genes in stress-tolerant crop breeding remain poorly explored. We found that overexpression of SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), a negative regulator of leaf senescence, significantly improved plant salt tolerance by increasing reactive oxygen species (ROS) scavenging in both Arabidopsis and soybean. However, overexpression of SSPP severely suppressed normal plant growth, limiting its direct use in agriculture. We previously revealed that the N-terminal 1-14 residues of ACS7 (termed 'N7 ') negatively regulated its protein stability through the ubiquitin/proteasome pathway, and the N7 -mediated protein degradation was suppressed by environmental and senescence signals. To avoid the adverse effects of SSPP, the N7 element was fused to the N-terminus of SSPP. We demonstrated that N7 -SSPP fusion gene effectively rescued SSPP-induced growth suppression but maintained enhanced salt tolerance in Arabidopsis and soybean. Particularly, N7 -SSPP enhanced tolerance to long-term salt stress and increased seed yield in soybean. These results suggest that N7 -SSPP overcomes the disadvantages of SSPP on plant growth inhibition and effectively improves salt tolerance through enhanced ROS scavenging, providing an effective strategy of using posttranslational regulatory element for salt-tolerant crop breeding.

Dual activities of ACC synthase: Novel clues regarding the molecular evolution of ACS genes.

Ethylene plays profound roles in plant development. The rate-limiting enzyme of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which is generally believed to be a single-activity enzyme evolving from aspartate aminotransferases. Here, we demonstrate that, in addition to catalyzing the conversion of S-adenosyl-methionine to the ethylene precursor ACC, genuine ACSs widely have Cß-S lyase activity. Two N-terminal motifs, including a glutamine residue, are essential for conferring ACS activity to ACS-like proteins. Motif and activity analyses of ACS-like proteins from plants at different evolutionary stages suggest that the ACC-dependent pathway is uniquely developed in seed plants. A putative catalytic mechanism for the dual activities of ACSs is proposed on the basis of the crystal structure and biochemical data. These findings not only expand our current understanding of ACS functions but also provide novel insights into the evolutionary origin of ACS genes.

Overexpression of GmAAP6a enhances tolerance to low nitrogen and improves seed nitrogen status by optimizing amino acid partitioning in soybean.

Amino acid transport via phloem is one of the major source-to-sink nitrogen translocation pathways in most plant species. Amino acid permeases (AAPs) play essential roles in amino acid transport between plant cells and subsequent phloem or seed loading. In this study, a soybean AAP gene, annotated as GmAAP6a, was cloned and demonstrated to be significantly induced by nitrogen starvation. Histochemical staining of GmAAP6a:GmAAP6a-GUS transgenic soybean revealed that GmAAP6a is predominantly expressed in phloem and xylem parenchyma cells. Growth and transport studies using toxic amino acid analogs or single amino acids as a sole nitrogen source suggest that GmAAP6a can selectively absorb and transport neutral and acidic amino acids. Overexpression of GmAAP6a in Arabidopsis and soybean resulted in elevated tolerance to nitrogen limitation. Furthermore, the source-to-sink transfer of amino acids in the transgenic soybean was markedly improved under low nitrogen conditions. At the vegetative stage, GmAAP6a-overexpressing soybean showed significantly increased nitrogen export from source cotyledons and simultaneously enhanced nitrogen import into sink primary leaves. At the reproductive stage, nitrogen import into seeds was greatly enhanced under both sufficient and limited nitrogen conditions. Collectively, our results imply that overexpression of GmAAP6a enhances nitrogen stress tolerance and source-to-sink transport and improves seed quality in soybean. Co-expression of GmAAP6a with genes specialized in source nitrogen recycling and seed loading may represent an interesting application potential in breeding.

SAUR49 Can Positively Regulate Leaf Senescence by Suppressing SSPP in Arabidopsis.

The involvement of SMALL AUXIN-UP RNA (SAUR) proteins in leaf senescence has been more and more acknowledged, but the detailed mechanisms remain unclear. In the present study, we performed yeast two-hybrid assays and identified SAUR49 as an interactor of SENESCENCE SUPPRESSED PROTEIN PHOSPHATASE (SSPP), which is a PP2C protein phosphatase that negatively regulates Arabidopsis leaf senescence by suppressing the leucine-rich repeat receptor-like protein kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK), as reported previously by our group. The interaction between SAUR49 and SSPP was further confirmed in planta. Functional characterization revealed that SAUR49 is a positive regulator of leaf senescence. The accumulation level of SAUR49 protein increased during natural leaf senescence in Arabidopsis. The transcript level of SAUR49 was upregulated during SARK-induced premature leaf senescence but downregulated during SSPP-mediated delayed leaf senescence. Overexpression of SAUR49 significantly accelerated both natural and dark-induced leaf senescence in Arabidopsis. More importantly, SAUR49 overexpression completely reversed SSPP-induced delayed leaf senescence. In addition, overexpression of SAUR49 reversed the decreased plasma membrane H+-ATPase activity mediated by SSPP. Taken together, the results showed that SAUR49 functions in accelerating the leaf senescence process via the activation of SARK-mediated leaf senescence signaling by suppressing SSPP. We further identified four other SSPP-interacting SAURs, SAUR30, SAUR39, SAUR41 and SAUR72, that may act redundantly with SAUR49 in regulating leaf senescence. All these observations indicated that certain members of the SAUR family may serve as an important hub that integrates various hormonal and environmental signals with senescence signals in Arabidopsis.

AHK3-Mediated Cytokinin Signaling Is Required for the Delayed Leaf Senescence Induced by SSPP.

Leaf senescence is a highly-programmed developmental process regulated by an array of multiple signaling pathways. Our group previously reported that overexpression of the protein phosphatase-encoding gene SSPP led to delayed leaf senescence and significantly enhanced cytokinin responses. However, it is still unclear how the delayed leaf senescence phenotype is associated with the enhanced cytokinin responses. In this study, we introduced a cytokinin receptor AHK3 knockout into the 35S:SSPP background. The phenotypic analysis of double mutant revealed that AHK3 loss-of-function reversed the delayed leaf senescence induced by SSPP. Moreover, we found the hypersensitivity of 35S:SSPP to exogenous cytokinin treatment disappeared due to the introduction of AHK3 knockout. Collectively, our results demonstrated that AHK3-mediated cytokinin signaling is required for the delayed leaf senescence caused by SSPP overexpression and the detailed mechanism remains to be further elucidated.

The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.

The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.

N-Terminus-Mediated Degradation of ACS7 Is Negatively Regulated by Senescence Signaling to Allow Optimal Ethylene Production during Leaf Development in Arabidopsis.

Senescence is the final phase of leaf development, characterized by key processes by which resources trapped in deteriorating leaves are degraded and recycled to sustain the growth of newly formed organs. As the gaseous hormone ethylene exerts a profound effect on the progression of leaf senescence, both the optimal timing and amount of its biosynthesis are essential for controlled leaf development. The rate-limiting enzyme that controls ethylene synthesis in higher plants is ACC synthase (ACS). In this study, we evaluated the production of ethylene and revealed an up-regulation of ACS7 during leaf senescence in Arabidopsis. We further showed that the promoter activity of ACS7 was maintained at a relatively high level throughout the whole rosette development process. However, the accumulation level of ACS7 protein was extremely low in the light-grown young seedlings, and it was gradually restored as plants aging. We previously demonstrated that degradation of ACS7 is regulated by its first 14 N-terminal residues, here we compared the phenotypes of transgenic Arabidopsis overexpressing a truncated ACS7 lacking the 14 residues with transgenic plants overexpressing the full-length protein. Results showed that seedlings overexpressing the truncated ACS7 exhibited a senescence phenotype much earlier than their counterparts overexpressing the full-length gene. Fusion of the 14 residues to SSPP, a PP2C-type senescence-suppressed protein phosphatase, effectively rescued the SSPP-induced suppression of rosette growth and development but had no effect on the delayed senescence. This observation further supported that N-terminus-mediated degradation of ACS7 is negatively regulated by leaf senescence signaling. All results of this study therefore suggest that ACS7 is one of the major contributors to the synthesis of 'senescence ethylene'. And more importantly, the N-terminal 14 residue-mediated degradation of this protein is highly regulated by senescence signaling to enable plants to produce the appropriate levels of ethylene required.

Functional investigation of two 1-aminocyclopropane-1-carboxylate (ACC) synthase-like genes in the moss Physcomitrella patens.

KEY MESSAGE: Two ACC synthase-like (ACL) proteins in the moss Physcomitrella patens have no ACS activity, and PpACL1 functions as an L -cystine/ L -cysteine C-S lyase. The ethylene biosynthetic pathway has been well characterized in higher plants, and homologs of a key enzyme in this pathway, ACS, have been reported in several algae and mosses, including Physcomitrella patens. However, the function of the ACS homologs in P. patens has not been investigated. In this research, we cloned two putative ACS genes from the P. patens genome, namely PpACS-Like 1 and 2, and investigated whether their encoded proteins had in vitro and in vivo ACS activity. In vitro biochemical assays using purified PpACL1 and PpACL2 showed that neither protein had ACS activity. Subsequently, we generated transgenic Arabidopsis lines expressing 35S:PpACL1 and 35S:PpACL2, and found that the transgenic etiolated seedlings that overexpressed either of these proteins lacked the constitutive triple response phenotype and did not emit excess levels of ethylene, indicating that neither of the PpACS-Like proteins had in vivo ACS activity. Furthermore, we found that PpACL1 functions as a C-S lyase that uses L-cystine and L-cysteine as substrates, rather than as an aminotransferase. Together, these results indicated that PpACL1 and PpACL2 are not true ACS genes as those found in higher plants.

Depositing CdS nanoclusters on carbon-modified NaYF4:Yb,Tm upconversion nanocrystals for NIR-light enhanced photocatalysis.

High-quality hexagonal NaYF4:Yb,Tm upconversion nanocrystals (UCNs) prepared in organic solutions display uniform sizes and strong UC emissions, but they possess a hydrophobic surface which hinders combining them with various semiconductor nanocrystals (NCs) to form a hybrid NIR-activated photocatalyst. Herein we present a facile approach to modify hydrophobic UCNs with a uniform carbon layer and enable them with hydrophilicity and surface functionalization. The carbon shell provides a good substrate for enriching with metal ions and in situ generation of CdS nanoclusters on the particle surface which can utilize both the upconverted UV and visible emissions. The developed NaYF4:Yb,Tm@C@CdS nanoparticles are characterized with TEM, SEM, XRD, PL and UV-Vis spectra and their formation mechanism is elucidated. The products display good photocatalytic activity under visible light and obviously enhanced performance under Vis-NIR light, due to the efficient utilization of UC emissions and the strong adsorption capacity of the carbon shell. The working mechanism of the hybrid photocatalysts is also proposed.

Synthesis of rhombic hierarchical YF3 nanocrystals and their use as upconversion photocatalysts after TiO2 coating.

A facile method has been developed to synthesize uniform nanoscale YF3 architectures. Interestingly, the unique YF3 nanostructure exhibits a flat and rhombic appearance which is formulated through the hierarchical assembly of YF3 nanocrystals along a specific crystalline orientation. Investigations on the formation process suggest that an assembly disassembly process is responsible for the construction of this novel structure. Enabled by doping with different lanthanides ions, the products can exhibit various down- or up-conversion luminescences, showing their potentials in serving as versatile host matrixes. The tunable luminescent properties allow designing effective upconversion photocatalysts when the doped YF3 nanostructures are coated with a TiO2 shell on their surface. In particular, the YF3@TiO2 hybrid structures have the porous nature that is partially inherited from the YF3 architectures, whose high surface-to-volume ratio facilitates their use as photocatalysts. In this article, we have demonstrated that the YF3:Yb,Tm@TiO2 structures exhibit satisfactory photocatalytic activities under the irradiation of both UV and near IR light. As compared with the conventional TiO2 catalysts, the hybrid structures here offer better performance in photocatalysis in the full solar spectrum. It is anticipated that this work provides a new approach to designing photocatalysts with responses to a broader spectral range.

Controlled synthesis of uniform LaF3 polyhedrons, nanorods and nanoplates using NaOH and ligands.

A facile and productive method has been developed to synthesize uniform LaF3 nanocrystals with controllable shapes, including polyhedrons, nanorods and nanoplates. By tuning the amount of NaOH and ligands (oleic acid and octadecylamine), we can finely tailor the shapes and sizes of LaF3 nanocrystals. Three prepared LaF3 nanostructures were well characterized, followed by a series of control experiments to propose a mechanism for the shape control. Based on the success in materials synthesis, controlled patterning of LaF3 nanoplates on substrates has also been achieved. After Yb/Er or Yb/Tm was co-doped in these LaF3 nanostructures, they could serve as nanoparticulate host matrices to give strong upconversion luminescence, showing great potential in biomedical applications considering their small sizes and well-defined shapes.