Wilt-induced ABA biosynthesis, gene expression and down-regulation of rbcS mRNA levels in Arabidopsis thaliana

The kinetics of wilt-induced abscisic acid (ABA) biosynthesis were investigated in shoots of Arabidopsis thaliana (L.) Heynh Landsberg erecta. ABA concentrations were measured using a radioimmunoassay (RIA) based on the monoclonal antibody MAC 252, and the RIA validated by comparison with combined gas chromatography-mass spectrometry using a [²H₃] labelled internal standard. The basal ABA content of Arabidopsis shoots was ca 10 ng g^?1 fresh weight; the concentrations had increased ca 4-fold within 30 min of the initiation of wilting, increased ca 8-fold after 4 h and 11-fold after 8 h. This stress-induced ABA production was dependent on de novo gene expression; pre-treatment of leaves and shoots with the metabolic inhibitors cordycepin and cycloheximide reduced the rate of subsequent stress-induced ABA biosynthesis from 12.5 ng g^?1 h^?1 to 1 ng g^?1 h^?1 and 0 ng g^?1 h^?1, respectively. In vitro translation of mRNA isolated from shoots subjected to wilting or ABA treatment followed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed only minor changes. The effects of wilting and ABA on the content of total ribulose 1,5-bisphosphate carboxylase/oxygenase small sub-unit (rbcS) mRNA were also determined. Both wilting and exogenous ABA resulted in a substantial reduction in the amount of rbcS mRNA, an effect readily reversed by rehydration of wilted shoots. However, the effects of wilting were not mediated solely by newly-synthesised endogenous ABA, as wilting also reduced rbcS mRNA levels in the ABA-deficient aba-1 mutant, which did not produce ABA in response to loss of turgor. The amount of rbcS mRNA was higher in aba-1 shoots, suggesting that cellular rbcS mRNA levels are normally down-regulated by ABA. Cold treatment induced ABA production in wild type shoots only, but resulted in an increased rbcS mRNA content of both wild type and aba-1 shoots.     

Thi1, a thiamine biosynthetic gene inArabidopsis thaliana, complements bacterial defects in DNA repair

AnArabidopsis thaliana cDNA was isolated by complementation of theEscherichia coli mutant strain BW535 (xth, nfo, nth), which is defective in DNA base excision repair pathways. This cDNA partially complements the methyl methane sulfonate (MMS) sensitive phenotype of BW535. It also partially corrects the UV-sensitive phenothpe ofE. coli AB1886 (uvrA) and restores its ability to reactivate UV-irradiated phage. It has an insert of ca. 1.3 kb with an open reading frame of 1047 bp (predicting a protein with a molecular mass of 36 kDa). This cDNA presents a high homology to a stress related gene from two species ofFusarium (sti35) and to genes whose products participate in the thiamine biosynthesis pathway,THI4, fromSaccharomyces cerevisiae andnmt2 fromSchizosaccharomyces pombe. TheArabidopsis predicted polypeptide has homology to several protein motifs: amino-terminal chloroplast transit peptide, dinucleotide binding site, DNA binding and bacterial DNA polymerases. The auxotrophy for thiamine in the yeastthi4::URA3 disruption strain is complemented by theArabidopsis gene. Thus, the cloned gene, namedthi1, is likely to function in the biosynthesis of thiamine in plants. The data presented in this work indicate thatthi1 may also be involved in DNA damage tolerance in plant cells.Depto. de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo     

The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development,photoprotection and retrograde signalling

Carotenoids, a class of natural pigments found in all photosynthetic organisms, are involved in a variety of physiological processes, including coloration, photoprotection, biosynthesis of abscisic acid （ABA） and chloroplast biogenesis. Although carotenoid biosynthesis has been well studied biochemically, the genetic basis of the pathway is not well understood. Here, we report the characterization of two allelic Arabidopsis mutants, spontaneous cell death1-1 （spcl-1） and spc1-2. The weak allele spc1-1 mutant showed characteristics of bleached leaves, accumulation of superoxide and mosaic cell death. The strong mutant allele spc1-2 caused a complete arrest of plant growth and development shortly after germination, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that SPC1 encodes a putative ζ-carotene desaturase （ZDS） in the carotenoid biosynthesis pathway. Analysis of carotenoids revealed that several major carotenoid compounds downstream of SPC 1/ZDS were substantially reduced in spc1-1, suggesting that SPC 1 is a functional ZDS. Consistent with the downregulated expression of CAO and PORB, the chlorophyll content was decreased in spc1-1 plants. In addition, expression of Lhcb1. 1, Lhcbl. 4 and RbcS was absent in spc1-2, suggesting the possible involvement of carotenoids in the plastid-to-nucleus retrograde signaling. The spc1-1 mutant also displays an ABA-deficient phenotype that can be partially rescued by the externally supplied phytohormone. These results suggest that SPC1/ZDS is essential for biosynthesis of carotenoids and plays a crucial role in plant growth and development.     

Integrated metabolite and gene expression profiling revealing phytochrome A regulation of polyamine biosynthesis of Arabidopsis thaliana

In this study, metabolite profiling was demonstrated as a usefultool to plot a specific metabolic pathway, which is regulatedby phytochrome A (phyA). Etiolated Arabidopsis wild-type (WT)and phyA mutant seedlings were irradiated with either far-redlight (FR) or white light (W). Primary metabolites of the irradiatedseedlings were profiled by gas chromatography time-of-flightmass spectrometry (GC/TOF-MS) to obtain new insights on phyA-regulatedmetabolic pathways. Comparison of metabolite profiles in phyAand WT seedlings grown under FR revealed a number of metabolitesthat contribute to the differences between phyA and the WT.Several metabolites, including some amino acids, organic acids,and major sugars, as well as putrescine, were found in smalleramounts in WT compared with the content in phyA seedlings grownunder FR. There were also significant differences between metaboliteprofiles of WT and phyA seedlings during de-etiolation underW. The polyamine biosynthetic pathway was investigated further,because putrescine, one of the polyamines existing in a widevariety of living organisms, was found to be present in loweramounts in WT than in phyA under both light conditions. Theexpression levels of polyamine biosynthesis-related genes wereinvestigated by quantitative real-time RT-PCR. The gene expressionprofiles revealed that the arginine decarboxylase 2 (ADC2) genewas transcribed less in the WT than in phyA seedlings underboth light conditions. This finding suggests that ADC2 is negativelyregulated by phyA during photomorphogenesis. In addition, S-adenosylmethioninedecarboxylase 2 and 4 (SAMDC2 and SAMDC4) were found to be regulatedby phyA but in a different manner from the regulation of ADC2. Key words: Arabidopsis thaliana, gene expression profiling, metabolite profiling, phytochrome A, polyamine biosynthesis Received 19 October 2007; Revised 17 January 2008 Accepted 18 January 2008     

拟南芥和作物中维生素C生物合成与代谢研究进展

维生素C（vitaminC,Vc）是动植物体内含量较为丰富且发挥着重要功能的小分子物质。该文综述了近年来以模式植物拟南芥为实验材料研究Vc生物合成和代谢取得的进展,并对作物中类似的研究进行了概述。总结的信息对于在作物中进一步开展Vc合成与代谢研究并通过分子育种提高作物的抗逆性和营养价值具有参考意义。     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Jasmonate biosynthesis in Arabidopsis thaliana--enzymes, products, regulation

Among the plant hormones jasmonic acid and related derivatives are known to mediate stress responses and several developmental processes. Biosynthesis, regulation, and metabolism of jasmonic acid in Arabidopsis thaliana are reviewed, including properties of mutants of jasmonate biosynthesis. The individual signalling properties of several jasmonates are described.     

Abscisic-acid-deficient mutants at the aba gene locus of Arabidopsis thaliana are impaired in the epoxidation of zeaxanthin

Abstract. The xanthophyll content of wild type and abscisic acid (ABA) - deficient mutants of pea and Arabidopsis thaliana was determined. The wilty mutant of pea was indistinguishable from the non-mutant control. In contrast, plants homozygous for mutant alleles at the aba locus of Arabidopsis were very different from wild type. In these mutants, zeaxanthin accumulated to abnormally high levels. The major carotenoids, violaxanthin and 9'- cis -neoxanthin were virually absent from the mutant chromatograms. It was concluded that the aba genetic lesion impairs the epoxidation of zeaxanthin to violaxanthin and that this results in an inability to accumulate ABA. This provides clear evidence that zeaxanthin is a precursor of ABA.     

Cloning, sequencing and function ofsanA, a gene involved in nikkomycin biosynthesis ofStreptomyces ansochromogenes

Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores of Streptomyces ansochromogenes, a nikkomycin producer, were treated with ultra violet radiation. One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19. A DMA library was constructed using plasmid plJ702 as cloning vector, NBB19 as cloning recipient. A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity. Sequence analysis showed that the 3 kb Bgl II-Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2). ORF1 is designated as sanA. sanA is 1 365 bp, encoding a protein consisting of 454 amino acid residues. Database searching indicated that sanA is homologous to the hypothetical methyltransferase in Pyrococcus horikoshli with 25% identities and 41% positives. Disruptant of sanA lost the ability to synthesize nikkomycin. It indicated that sa     

Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis

In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6-X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g(-1) protein or 1-2% of the amount found in wild-type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.     

An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense

Emission of methylsalicylate (MeSA), and occasionally of methylbenzoate (MeBA), from Arabidopsis thaliana leaves was detected following the application of some forms of both biotic and abiotic stresses to the plant. Maximal emission of MeSA was observed following alamethicin treatment of leaves. A gene (AtBSMT1) encoding a protein with both benzoic acid (BA) and salicylic acid (SA) carboxyl methyltransferase activities was identified using a biochemical genomics approach. Its ortholog (AlBSMT1) in A. lyrata, a close relative of A. thaliana, was also isolated. The AtBSMT1 protein utilizes SA more efficiently than BA, whereas AlBSMT1 catalyzes the methylation of SA less effectively than that of BA. The AtBSMT1 and AlBSMT1 genes showed expression in leaves under normal growth conditions and were more highly expressed in the flowers. In A. thaliana leaves, the expression of AtBSMT1 was induced by alamethicin, Plutella xylostella herbivory, uprooting, physical wounding, and methyl jasmonate. SA was not an effective inducer. Using a beta-glucuronidase (GUS) reporter approach, the promoter activity of AtBSMT1 was localized to the sepals of flowers, and also to leaf trichomes and hydathodes. Upon thrip damage to leaves, AtBSMT1 promoter activity was induced specifically around the lesions.     

HAL1 mediate salt adaptation in Arabidopsis thaliana

INTRODUCTIONSalinity is a major environmental stress that isa substantial constraint to crop production both fordry land and irrigated agriculture. The detrimental impact of this stress is perpetuated and exacerbated by management practices used to facilitatehigh-output crop production. To overcome theselimitations and improve production efficiency in theface of a burgeoning world population, more salt tolerant crops must be developed. In contrast with traditional breeding, the direct ill…     

Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana

Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. We have used ozone (O₃) as a tool to elicit ROS-activated stress responses, and to activate cell death in plant leaves. Here, we have investigated the roles and interactions of ROS and NO in the induction and regulation of O₃-induced cell death. Treatment with O₃ induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. During the later time points, NO production coincided with the formation of hypersensitive response (HR)-like lesions. The NO donor sodium nitroprusside (SNP) and O₃ individually induced a large set of defence-related genes; however, in a combined treatment SNP attenuated the O₃ induction of salicylic acid (SA) biosynthesis and other defence-related genes. Consistent with this, SNP treatment also decreased O₃-induced SA accumulation. The O₃-sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 ( Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1 ), a mutant with decreased production of NO, was also O₃ sensitive. This, together with experiments combining O₃ and the NO donor SNP suggested that NO can modify signalling, hormone biosynthesis and gene expression in plants during O₃ exposure, and that a functional NO production is needed for a proper O₃ response. In summary, NO is an important signalling molecule in the response to O₃.     

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.     

l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.