Arabidopsis thaliana plastidial methionine sulfoxide reductases B,MSRBs, account for most leaf peptide MSR activity and are essential for growth under environmental constraints through a role in the preservation of photosystem antennae

Methionine oxidation to methionine sulfoxide (MetSO) is reversed by two types of methionine sulfoxide reductases (MSRs), A and B, specific to MetSO S‐ and R‐diastereomers, respectively. Two MSRB isoforms, MSRB1 and MSRB2, are present in chloroplasts of Arabidopsis thaliana. To assess their physiological role, we characterized Arabidopsis mutants knockout for the expression of MSRB1, MSRB2 or both genes. Measurements of MSR activity in leaf extracts revealed that the two plastidial MSRB enzymes account for the major part of leaf peptide MSR capacity. Under standard conditions of light and temperature, plants lacking one or both plastidial MSRBs do not exhibit any phenotype, regarding growth and development. In contrast, we observed that the concomitant absence of both proteins results in a reduced growth for plants cultivated under high light or low temperature. In contrast, double mutant lines restored for MSRB2 expression display no phenotype. Under environmental constraints, the MetSO level in leaf proteins is higher in plants lacking both plastidial MSRBs than in Wt plants. The absence of plastidial MSRBs is associated with an increased chlorophyll a/b ratio, a reduced content of Lhca1 and Lhcb1 proteins and an impaired photosynthetic performance. Finally, we show that MSRBs are able to use as substrates, oxidized cpSRP43 and cpSRP54, the two main components involved in the targeting of Lhc proteins to the thylakoids. We propose that plastidial MSRBs fulfil an essential function in maintaining vegetative growth of plants during environmental constraints, through a role in the preservation of photosynthetic antennae.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

Protein phosphatase 2A alleviates cadmium toxicity by modulating ethylene production in Arabidopsis thaliana

Cadmium (Cd) is phytotoxic and detoxified primarily via phytochelatin (PC) complexation in Arabidopsis. Here, we explore Cd toxicity responses and defence mechanisms beyond the PC pathway using forward genetics approach. We isolated an Arabidopsis thaliana Cd-hypersensitive mutant, Cd-induced short root 1 (cdsr1) in the PC synthase mutant (cad1-3) background. Using genomic resequencing and complementation, we identified PP2A-4C as the causal gene for the mutant phenotype, which encodes a catalytic subunit of protein phosphatase 2A (PP2A). Root and shoot growth of cdsr1 cad1-3 and cdsr1 were more sensitive to Cd than their respective wild-type cad1-3 and Col-0. A mutant of the PP2A scaffolding subunit 1A was also more sensitive to Cd. PP2A-4C was localized in the cytoplasm and nucleus and PP2A-4C expression was downregulated by Cd in cad1-3. PP2A enzyme activity was decreased in cdsr1 and cdsr1 cad1-3 under Cd stress. The expression of 1-aminocyclopropane-1-carboxylic acid synthase genes ACS2 and ACS6 was upregulated by Cd more in cad1-3 and cdsr1 cad1-3 than in Col-0 and the double mutant had a higher ACS activity. cdsr1 cad1-3 and cdsr1 overproduced ethylene under Cd stress. The results suggest that PP2A containing 1A and 4C subunits alleviates Cd-induced growth inhibition by modulating ethylene production.     

Identification of a metabolic bottleneck for cold acclimation in Arabidopsis thaliana

Central carbohydrate metabolism of Arabidopsis thaliana is known to play a crucial role during cold acclimation and the acquisition of freezing tolerance. During cold exposure, many carbohydrates accumulate and a new metabolic homeostasis evolves. In the present study, we analyse the diurnal dynamics of carbohydrate homeostasis before and after cold exposure in three natural accessions showing distinct cold acclimation capacity. Diurnal dynamics of soluble carbohydrates were found to be significantly different in cold-sensitive and cold-tolerant accessions. Although experimentally determined maximum turnover rates for sucrose phosphate synthase in cold-acclimated leaves were higher for cold-tolerant accessions, model simulations of diurnal carbohydrate dynamics revealed similar fluxes. This implied a significantly higher capacity for sucrose synthesis in cold-tolerant than cold-sensitive accessions. Based on this implication resulting from mathematical model simulation, a critical temperature for sucrose synthesis was calculated using the Arrhenius equation and experimentally validated in the cold-sensitive accession C24. At the critical temperature suggested by model simulation, an imbalance in photosynthetic carbon fixation ultimately resulting in oxidative stress was observed. It is therefore concluded that metabolic capacities at least in part determine the ability of accessions of Arabidopsis thaliana to cope with changes in environmental conditions.     

Reduced activity of Arabidopsis thaliana HMT2, a methionine biosynthetic enzyme, increases seed methionine content

In the S- methylmethionine cycle of plants, homocysteine methyltransferase (HMT) catalyzes the formation of two molecules of methionine from homocysteine and S- methylmethionine, and methionine methyltransferase (MMT) catalyzes the formation of methionine from S- methylmethionine using S- adenosylmethionine as a methyl group donor. Somewhat surprisingly, two independently isolated knockdown mutations of HMT2 (At3g63250), one of three Arabidopsis thaliana genes encoding homocysteine methyltransferase, increased free methionine abundance in seeds. Crosses and flower stalk grafting experiments demonstrate that the maternal genotype at the top of the flower stalk determines the seed S- methylmethionine and methionine phenotype of hmt2 mutants. Uptake, transport and inter-conversion of [¹³C] S- methylmethionine and [¹³C]methionine in hmt2 , mmt and wild-type plants show that S- methylmethionine is a non-essential intermediate in the movement of methionine from vegetative tissue to the seeds. Together, these results support a model whereby elevated S- methylmethionine in hmt2 vegetative tissue is transported to seeds and either directly or indirectly results in the biosynthesis of additional methionine. Manipulation of the S- methylmethionine cycle may provide a new approach for improving the nutritional value of major grain crops such as rice, as methionine is a limiting essential amino acid for mammalian diets.     

A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis

Disproportionating enzyme (D-enzyme) is a plastidial alpha-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While D-enzyme activity is eliminated in the homozygous dpe1-1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1-1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1-1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1-1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1-1 suggests that malto-oligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1-1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto-oligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.     

Dimerization of the ETO1 family proteins plays a crucial role in regulating ethylene biosynthesis in Arabidopsis thaliana

ETHYLENE OVERPRODUCER1 (ETO1), ETO1-LIKE1 (EOL1), and EOL2 are members of the Broad complex, Tramtrack, Bric-a-brac (BTB) protein family that collectively regulate type-2 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity in Arabidopsis thaliana. Although ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do not play an equivalent role in regulating ethylene biosynthesis. The mechanistic details underlying the genetic analysis remain elusive. In this study, we reveal that ETO1 collaborates with EOL1/2 to play a key role in the regulation of type-2 ACS activity via protein–protein interactions. ETO1, EOL1, and EOL2 exhibit overlapping but distinct tissue-specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement the eto1 phenotype under control of the ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers with itself and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and is required for homodimerization. However, domain-swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but not sufficient for a full spectrum of ETO1 function. The missense mutation in eto1-5 generates a substitution of phenylalanine with an isoleucine in ETO1^F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1^F466I in Arabidopsis results in a constitutive triple response phenotype in dark-grown seedlings. Our findings reveal the mechanistic role of protein–protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis.     

拟南芥乙烯突变体在干旱胁迫下的形态学差异

为了揭示乙烯在植物与环境相互作用过程中的生物学功能,以拟南芥（Arabidopsis thaliana）的ein2-5、ein3-1、EIN3ox、EIL1ox 4种乙烯突变体与Col-0野生型为材料,对比研究它们在干旱胁迫条件下的生长和形态学变化。研究发现,干旱胁迫导致莲座叶直径、叶片面积、花序、水势等指标发生显著变化,同时不同突变体的形态适应特点呈现显著差异。这些结果表明,乙烯积极参与了植物形态塑造过程,与植物的抗旱性具有紧密相关性。     

Role of growth regulators in the senescence of Arabidopsis thaliana leaves

A homozygous, dominant, C₂H₄-resistant line of Arabidopsis thaliana (L.) Heynh (cv. Columbia; er ) was selected from ethylmethylsulfonate-mutagenized seed, and used to test the role of C₂H₄ and other growth regulators in senescence of mature leaves. Chlorophyll (Chl) loss from disks excised from leaves of er was much slower than that from wild-type (WT) disks, whether they were held in the light or in the dark. C₂H₄ accelerated Che loss from WT disks but had no effect on the yellowing of mutant disks. C₂H₄ biosynthesis was higher in disks from the mutant plants, particularly in the light. In the dark, treatment with the cytokinin, 6-benzyladenine (BA), reduced Chl loss from wild-type disks, but had no effect on mutant disks. In the light, BA treatment stimulated chlorophyll breakdown in both wild type and mutant disks. Treatment with abscisic acid (ABA) stimulated chlorophyll loss in wild-type and mutant disks, whether they were held in the light or the dark. C₂H₄ production was stimulated in ABA-treated disks, but they still yellowed even when C₂H₄ production was inhibited by application of aminooxyacetic acid (AOA). These data indicate that C₂H₄ is only one of the factors involved in leaf senescence, and that the promotion of senescence by ABA is not mediated through its stimulation of C₂H₄ production.     

Interaction between ethylene and ABA in the regulation of polyamine level in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> during UV-B stress

The effects of treatment with ethylene (0.01–100 μl/l) on ABA and polyamine contents and treatment with ABA on ethylene synthesis, polyamines content, and the resistance to UV-B radiation of two-week-old Arabidopsis thaliana (L.) Heynh, Columbia ecotype plants grown u?er sterile conditions were studied. Ethylene stimulated the accumulation of polyamines only at concentrations of 0.1–10 μl/l, which could activate ABA synthesis. Treatment with ABA (50–5000 μM, 1 μl per plant) decreased the UV-B-induced ethylene synthesis and a spermine and spermidine loss, increasing the content of putrescine, the precursor of these polyamines. ABA inhibited fresh weight accumulation in irradiated and nonirradiated plants but prevented them from severe damage and death at the high (18 kJ/m²) and lethal (27 kJ/m²) UV-B dose, respectively. The data obtained demonstrated a mutual regulation of ethylene and ABA syntheses and the participation of these hormones in the control of the polyamine level during adaptation of A. thaliana to UV-B stress.     

Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.     

拟南芥乙烯信号转导机理的遗传学和化学生物学研究

乙烯信号途径的建立得益于一系列的突变体研究,EIN3是乙烯信号转导通路的核心转录因子,EIN3的蛋白质含量严格受F-BOX蛋白EBF1/EBF2的降解调控。为了进一步挖掘乙烯信号途径的新组分和深入研究EIN3及其下游的信号组分,作者筛选了四个不同来源的T-DNA库,并利用转基因植物EIN3ox作为遗传背景,进行了EIN3下游的抑制子筛选工作,还利用化学遗传学的方法筛选了四个小分子库。     

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana

Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana , and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants ( ole1 , ole2 , ole3 and ole4 ) and generated three double mutants ( ole1 ole2 , ole1 ole3 and ole2 ole3 ). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2 , which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4°C abolished seed germination of single mutants ( ole1 , ole2 and ole3 ), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22°C instead of 4°C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.     

拟南芥室内培养技术

本文报道了室内培养拟南芥的一些简便易行的改进技术.采用我们改进的营养土、蛭石、素沙混合培养介质和直播方式培养拟南芥,并根据其生物学特性在温度、空气湿度、土壤水分和光照等方面给予适当管理,能培养出生长更健壮、更好地满足实验要求的拟南芥植株.此外还介绍了播种、浇水、生育期调节、种子保存、病虫害防治和防混杂等环节的一些技巧措施.与其他培养方法相比,此法不仅简便、效果好,而且适合较简易的培养条件.     

Evidence for altered methionine methyl-group utilization in the diabetic rat's brain

The methionine (MET) derivative, S-adenosylmethionine (SAM), provides methyl-groups for methylation reactions in many neural processes. In rats made diabetic with streptozotocin (SZ), brain SAM levels were generally lower (10–20%) than in controls, with a constant decrease being observed five weeks after onset of diabetes. This decrease in SAM levels may be due to reduced precursor (MET) availability because greatly elevating plasma MET concentrations in SZ diabetic rats by dietary manipulation increased their neural SAM concentrations to be approximately or even greater than (5–20%) those of controls. In contrast, neural levels of SAM's demethylated product, S-adenosylhomocysteine (SAH), were reduced to a greater extent (17–44%) than SAM levels in all groups of SZ diabetic rats independent of their plasma MET concentrations or brain SAM levels. This indicates that the decrease in SAH levels is not simply due to substrate (SAM) restriction. These changes in MET metabolites appear to be a general effect of diabetes rather than a non-pancreatic side-effect of SZ, because genetically diabetic BB Wistar rats also exhibited reduced brain SAM (25%) and brain SAH (46%) levels. These results indicate that methyl-groups from MET are handled differently in the brain of the diabetic rat, which considering the variety and importance of neural methylation reactions, could have important consequences for the diabetic.Abbreviations MET methionine - SAM S-adenosylmethionine - SAH S-adenosylhomocysteine - SZ streptozotocin - BBW BB Wistar - LNAA large neutral amino acids - BCAA branchedchain amino acids - MET:BCAA methionine to branched-chain amino acid ratio - MET:LNAA methionine to large neutral amino acid ratio     

Effect of ABA on the UV-B-Induced Ethylene Evolution by the etr and ctr Mutants of Arabidopsis thaliana

The responses of 14-day-old Arabidopsis thaliana (L.) Heynh. plants to UV-B irradiation (280–320 nm) and ABA treatment were investigated. Wild-type plants as well as ethylene-insensitive etr1-1 and ctr1-1 mutants were used. Theetr1-1 mutant considerably differed from the ctr1-1 one in the fresh weight production after UV-B treatment (29.5 kJ/m²). The irradiated etr1-1 plants fell well behind the nonirradiated ones during the first two days after stress, but by the 8th day, their weight attained 70% of control plant weight. In contrast, Ctr1-1 mutant weight comprised 70% of control level after two days of stress but, by the 8th day, it was only 56% of the weight of control plants. In wild-type and ctr1-1 plants, ABA, in the 8 × 10^–6 to 2 × 10^–4 M concentration range, increased the difference between the weights of nonirradiated and irradiated plants, but in etr1-1 plants, ABA decreased this difference. The etr1-1, ctr1-1, and wild-type plants were very similar in the dynamics of ethylene evolution after UV-B treatment (7.4 kJ/m²). In wild-type, etr1-1, and ctr1-1 plants, ABA, in a concentration-dependent manner, inhibited UV-B-induced ethylene evolution to the same extent. The results obtained show that ABA exerted an opposite effect on UV-B-dependent growth in the plants with active (wild type and ctr1-1) and blocked (etr1-1) ethylene signal pathway, whereas the inhibition of ethylene synthesis by ABA was not related to ethylene signal transmission.     

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.