Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages

Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3′,5′-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway.     

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 ( tdd1 ) , which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase β-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1 , indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1 , which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR , which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.     

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate

Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.     

Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra)

The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.     

L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants

L-Ascorbic acid (AsA) was found to be loaded into phloem of source leaves and transported to sink tissues. When L-[(14)C]AsA was applied to leaves of intact plants of three different species, autoradiographs and HPLC analysis demonstrated that AsA was accumulated into phloem and transported to root tips, shoots, and floral organs, but not to mature leaves. AsA was also directly detected in Arabidopsis sieve tube sap collected from an English green aphid (Sitobion avenae) stylet. Feeding a single leaf of intact Arabidopsis or Medicago sativa with 10 or 20 mM L-galactono-1,4-lactone (GAL-L), the immediate precursor of AsA, lead to a 7- to 8-fold increase in AsA in the treated leaf and a 2- to 3-fold increase of AsA in untreated sink tissues of the same plant. The amount of AsA produced in treated leaves and accumulated in sink tissues was proportional to the amount of GAL-L applied. Studies of the ability of organs to produce AsA from GAL-L showed mature leaves have a 3- to 10-fold higher biosynthetic capacity and much lower AsA turnover rate than sink tissues. The results indicate AsA transporters reside in the phloem, and that AsA translocation is likely required to meet AsA demands of rapidly growing non-photosynthetic tissues. This study also demonstrates that source leaf AsA biosynthesis is limited by substrate availability rather than biosynthetic capacity, and sink AsA levels may be limited to some extent by source production. Phloem translocation of AsA may be one factor regulating sink development because AsA is critical to cell division/growth.     

Activity of 1-aminocyclopropane carboxylic acid synthase and growth of mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) following defoliation

To examine the possible relationship between the activity of 1-aminocyclopropane carboxylic acid synthase (ACS; EC 4.4.1.14) and growth of mustard (Brassica juncea L.), ACS activity, ethylene and plant growth were studied in the presence of ACS activity modulators in no-defoliation and defoliated plants. Growth of plants was greatest when subjected to defoliation of 50% lower leaves in the plant axis compared to defoliation of 25% lower leaves or no-defoliation. The activity of ACS in no-defoliation and defoliated plants was correlative with growth of plants. ACS activity and ethylene evolution in no-defoliation plants treated with 10 μM indole-3-acetic acid (IAA) and defoliated plants treated with water were equal and resulted in maximum plant growth. On the contrary, the application of 10 μM IAA on defoliated plants resulted in the increase in ACS activity and ethylene evolution to an extent that inhibited the growth. The application of 100 μM IAA on no-defoliation and defoliated plants increased ACS activity and ethylene evolution maximally and proved inhibitory for the plant growth. The association of ACS activity, ethylene evolution and growth of plants was further substantiated with the use of 50 μM aminoethoxyvinyl glycine (AVG) applied alone or in combination with 10 or 100 μM IAA. The application of AVG resulted in the inhibition of ACS activity and the growth of no-defoliation or defoliated plants. The results indicate that there exists a correlation between ACS activity, ethylene and the growth of mustard plants.     

Synthesis of <Emphasis Type="SmallCaps">L</Emphasis>-ascorbic acid in the phloem

Background  

Although plants are the main source of vitamin C in the human diet, we still have a limited understanding of how plants synthesise L-ascorbic acid (AsA) and what regulates its concentration in different plant tissues. In particular, the enormous variability in the vitamin C content of storage organs from different plants remains unexplained. Possible sources of AsA in plant storage organs include in situ synthesis and long-distance transport of AsA synthesised in other tissues via the phloem. In this paper we examine a third possibility, that of synthesis within the phloem.     

Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique

Water-soluble chlorophyll protein (WSCP) has been found in many Brassicaceae, most often in leaves. In many cases, its expression is stress-induced, therefore, it is thought to be involved in some stress response. In this work, recombinant WSCP from Arabidopsis thaliana (AtWSCP) is found to form chlorophyll-protein complexes in vitro that share many properties with recombinant or native WSCP from Brassica oleracea, BoWSCP, including an unusual heat resistance up to 100°C in aqueous solution. A polyclonal antibody raised against the recombinant apoprotein is used to identify plant tissues expressing AtWSCP. The only plant organs containing significant amounts of AtWSCP are the gynoecium in open flowers and the septum of developing siliques, specifically the transmission tract. In fully grown but still green siliques, the protein has almost disappeared. Possible implications for AtWSCP functions are discussed.     

Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat

Although ascorbic acid (AsA) is one of the most important and abundantly occurring water soluble antioxidants in plants, relatively little is known about its role in counteracting the adverse effects of salt stress on plant growth. To address this issue that whether exogenous application of ascorbic acid (AsA) through rooting medium could alleviate the adverse effects of salt stress on wheat plants, a hydroponic experiment was conducted under glasshouse conditions using two wheat cultivars, S-24 (salt tolerant) and MH-97 (moderately salt sensitive). Plants of both cultivars were subjected to 0 or 150 mM NaCl solution supplemented with 0, 50, or 150 mg L⁻¹ AsA for 58 days. Imposition of salt stress reduced the growth of both wheat cultivars by causing reduction in photosynthesis, and endogenous AsA level, and enhancing accumulation of Na⁺ and Cl⁻ coupled with a decrease in K⁺ and Ca²⁺ in the leaves and roots of both cultivars thereby decreasing tissue K⁺/Na⁺ ratio. However, root applied AsA counteracted the adverse effects of salt stress on the growth of cv. S-24 only, particularly at 100 mg L⁻¹ AsA level. AsA-induced enhancement in growth of salt-stressed plants of S-24 was associated with enhanced endogenous AsA level and CAT activity, and higher photosynthetic capacity, and accumulation of K⁺ and Ca²⁺ in the leaves. Although root applied AsA did not improve the growth of salt-stressed plants of MH-97, it enhanced endogenous level of AsA, CAT activity, photosynthetic capacity, and leaf K⁺ and Ca²⁺. These findings led us to conclude that root applied AsA counteracts the adverse effects of salt stress on growth of wheat by improving photosynthetic capacity of wheat plants against salt-induced oxidative stress and maintaining ion homeostasis, however, these effects were cultivar specific.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

The Changes of The Level of Triterpenoids in Calendula officinalis during Vegetation

Using thin-layer chromatography for separation and colorimetric test with CoClj for determination, qualitative and quantitative composition of triterpenoids was estimated in Calendula officinalis dining vegetation. It was found that sterols and oleanolic acid as well as trace amounts of triterpenic monols occur in all organs of the plant during the whole vegetative period. Triterpenic diols and greater quantities of triterpenic monols appear in flowers. The biosynthesis rate of triterpcnoids is highest in young organs of the plant that is in seedlings, young leaves and flower buds. During flowering the content of sterols and oleanolic acid increases in all plant organs except of old leaves. A high level of oleanolic acid in the root during the flowering period implies that oleanolic acid glycosides can be transported from older leaves to underground parts of the plant. Biosynthesis of triterpenic monols, Ψ-taraxasterol and taraxasterol, precedes the formation of the diols faradiol and arnidiol. It can be inferred from the course of accumulation of these compounds that monols are the precursors of diols and that hydroxylation occurs in flowers.     

Photoperiodic control of growth, development and phytohormone balance in Solanum tuberosum

Some lines of Solanum tuberosum ssp. andigena are strictly photoperiodic, forming tubers only in short days and flowers only in long days. We used this advantageous phenomenon to study phytohormone involvement in the development of the plants, mainly that of tuber formation. Plants grown for 2 months under short days (SD) of 14 h darkness, night break (1 h white light in the middle of the 14‐h dark period) and continuous light (LD) were compared. Short day‐grown plants formed tubers, while plants in LD flowered. Night break prevented tuber formation, but caused flowering, although it was weaker than in LD. Plants grown under night break displayed many growth characteristics intermediate between SD and LD. Under LD and night break regimes ABA levels in all organs were about one‐fourth of those under SD. An opposite trend was found for gibberellin content: it was very low in SD‐grown plants and 4‐10 times higher under both other conditions with the exception of roots and stolons in night break. Thus, the ratio of ABA/GA, known to be important for tuber formation, was high in SD and low in night break and LD. The level of free indoleacetic acid (IAA) was high in LD‐ and night break‐grown plants and it was much lower in SD‐grown plants, including tubers. Cytokinin (CK) levels were also high in LD‐ and night break‐grown plants. In SD, leaves had slightly decreased and stems and roots + stolons had more pronouncedly decreased cytokinin levels. The CK/IAA ratio was thus higher in SD in stems and roots + stolons; it was also high in tubers and no difference was found in leaves. These results indicate that the effect of photoperiod on tuber formation and development may be mediated by photoperiod‐induced changes in hormone levels.     

Melatonin stimulates the expansion of etiolated lupin cotyledons

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine which is structurally related to tryptophan, serotonin and indole-3-acetic acid (IAA), among other important substances. Many studies have clearly demonstrated its presence in different plant organs, including roots, stems, leaves, flowers, fruits and seeds. Since it discovery in plants in 1995, authors have postulated many physiological roles for melatonin, although research into this molecule in plants is still in its infancy. The data presented in this study demonstrate that melatonin stimulates the expansion of etiolated cotyledons of lupin (Lupinus albus L.) to a similar extent to that observed for IAA but less than in the case of kinetin. Endogenous melatonin in imbibed cotyledons has been quantified using a liquid chromatography method with fluorescence detection and capacity of cotyledons to absorb melatonin has been determined. The observed effect of melatonin on lupin cotyledon expansion can be added to the other effects demonstrated by our group such as its role as growth promoter and rooting promotor in adventitious and lateral roots.     

Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between D-fructose 6-phosphate and D-mannose 6-phosphate (Man-6P). The apparent K(m) and V(max) values for Man-6P of purified recombinant PMI1 were 41.3+/-4.2 microm and 1.89 micromol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372+/-13 microm and 22.5 micromol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn(2+), Cd(2+), and L-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.     

Quantitative profiling of polar glycerolipid species from organs of wild-type Arabidopsis and a phospholipase Dalpha1 knockout mutant

Lipid profiling is a targeted metabolomics platform that provides a comprehensive analysis of lipid species with high sensitivity. Profiling based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides quantitative data and is adaptable to high throughput analyses. Here we report the profiling of 140 apparent molecular species of polar glycerolipids in Arabidopsis leaves, flower stalks, flowers, siliques, roots, and seeds. Considerable differences in lipid species occur among these organs, providing insights into the different lipid metabolic activities in a specific organ. In addition, comparative profiling between wild-type and a knockout mutant pldalpha1 (locus ID: AT3G15730) provides insight into the metabolic function of phospholipase D (PLD) in different organs. PLDalpha1 contributes significantly to phosphatidic acid (PA) levels in roots, seeds, flowers, and flower stalks, but little to basal PA levels in siliques and leaves. In seeds of the pldalpha1 mutant plants, levels of PA, lysophosphatidylcholine, and lysophosphatidylethanolamine were significantly lower than those of wild-type seeds, suggesting a role for PLDalpha1 in membrane lipid degradation in seeds.