The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated amino acid metabolism

Amino acids and amino acid analogs have been used in numerous genetic screens to isolate mutants deficient in amino acid biosynthetic pathways or in the regulation of amino acid metabolism. Several of these mutants exhibit relaxed feedback control of branched amino acid biosynthetic pathways and are thus resistant to accumulation of pathway end products. For example, feedback-regulated enzymes of the shikimate pathway are anthranilate synthase on the branch leading to Trp and chorismate mutase on the branch leading to Phe and Tyr. A feedback-insensitive mutant of anthranilate synthase alpha, trp5-1, is resistant to toxic Trp analogs. Mutants resistant to Phe have not previously been reported, and this article describes the isolation of the recessive Arabidopsis Phe insensitive growth mutant pig1-1 by a forward genetic screen. pig1-1 was not only tolerant to Phe, Tyr, and Trp, but also to other, not biosynthetically related amino acids. Amino acid contents in pig1-1 were significantly elevated with respect to wild-type controls but, in contrast to the wild type, dramatically decreased when plants were supplemented with 2 mm Phe. Protein contents were similar in the mutant and the wild type at all tested conditions. Phe catabolism was similar to the wild type in pig1-1 roots but was significantly increased in pig1-1 shoots. Phenylalanine uptake into the root, its root-to-shoot translocation, and Phe and phenylpropanoid contents were unaltered in pig1-1, indicating that pig1-1 is not affected in amino acid translocation or the shikimate pathway. Instead, the response of pig1-1 toward amino acid feeding indicates that amino acid metabolism is generally deregulated in pig1-1.     

Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway

Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome p450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants. It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR. When [(3)H]Phe was fed, the majority of metabolized [(3)H]Phe was incorporated into p-[(3)H]coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity. PAL alone expressers metabolized very little phenylalanine into cinnamic acid. To test for intermediate channeling between PAL and C4H, we fed [(3)H]Phe and [(14)C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [(3)H]cinnamate into p-coumarate. Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway. This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants.     

高梁肉桂酸羟化酶基因SbC4H1降低拟南芥的木质素合成

肉桂酸羟化酶（C4H）是苯丙烷代谢通路的关键酶,其活性和含量直接影响木质素合成的效率。本文研究通过高粱bmr突变体的抑制差减杂交筛选、克隆到了一个C4H基因sbC4H。半定量RT-PCR发现,SbC4H1在多个bmr突变体中上调表达。将SbC4H1-GFP融合基因转化拟南芥原生质体进行瞬时表达,发现SbC4H1表达产物蛋白定位于细胞质。SbC4H1在拟南芥中的异源表达明显降低其茎的木质素含量,并且下调了拟南芥4CL1、FSH和HCT质素合成基因的表达。这些结果表明,高粱SbC4H1抑制了拟南芥木质素的合成。     

Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis

The end products of the phenylpropanoid pathway play important roles in plant structure and development, as well as in plant defense mechanisms against biotic and abiotic stresses. From a human perspective, phenylpropanoid pathway-derived metabolites influence both human health and the potential utility of plants in agricultural contexts. The last known enzyme of the phenylpropanoid pathway that has not been characterized is p-coumarate 3-hydroxylase (C3H). By screening for plants that fail to accumulate soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. We have now shown that the ref8 mutant is defective in the gene encoding C3H. Phenotypic characterization of the ref8 mutant has revealed that the lack of C3H activity in the mutant leads to diverse changes in phenylpropanoid metabolism. The ref8 mutant accumulates p-coumarate esters in place of the sinapoylmalate found in wild-type plants. The mutant also deposits a lignin formed primarily from p-coumaryl alcohol, a monomer that is at best a minor component in the lignin of other plants. Finally, the mutant displays developmental defects and is subject to fungal attack, suggesting that phenylpropanoid pathway products downstream of REF8 may be required for normal plant development and disease resistance.     

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non‐oxidative deamination of Phe to trans‐cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81–94% led to an 18‐fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate‐derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL‐RNAi transgenic plants resulted in 1.6‐fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies.     

An Arabidopsis mutant defective in the general phenylpropanoid pathway.

Mutants of Arabidopsis deficient in a major leaf phenylpropanoid ester, 2-O-sinapoyl-L-malate, were identified by thin-layer chromatographic screening of methanolic leaf extracts from several thousand mutagenized plants. Mutations at a locus designated SIN1 also eliminate accumulation of the sinapic acid esters characteristic of seed tissues. Because of increased transparency to UV light, the sin1 mutants exhibit a characteristic red fluorescence under UV light, whereas wild-type plants have a blue-green appearance due to the fluorescence of sinapoyl malate in the upper epidermis. As determined by in vivo radiotracer feeding experiments, precursor supplementation studies, and enzymatic assays, the defect in the sin1 mutants appears to block the conversion of ferulate to 5-hydroxyferulate in the general phenylpropanoid pathway. As a result, the lignin of the mutant lacks the sinapic acid-derived components typical of wild-type lignin.     

Redirection of the phenylpropanoid pathway to feruloyl malate in <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants deficient for cinnamoyl-CoA reductase 1

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25–35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.     

Root uptake of cationic amino acids by Arabidopsis depends on functional expression of amino acid permease 5

* Specific transporters mediate uptake of amino acids by plant roots. Earlier studies have indicated that the lysine histidine transporter 1 and amino acid permease 1 participate in this process, but although plant roots have been shown to absorb cationic amino acids with high affinity, neither of these transporters seems to mediate transport of L-arginine (L-Arg) or L-lysine (L-Lys). * Here, a collection of T-DNA knockout mutants were screened for alterations in Arabidopsis root uptake rates of L-Arg and it was found that only the AAP5 mutant displayed clear phenotypic divergence on high concentrations of L-Arg. A second screen using low concentrations of (15)N-labelled L-Arg in the growth media also identified AAP5 as being involved in L-Arg acquisition. * Momentaneous root uptake of basic amino acids was strongly affected in AAP5 mutant lines, but their uptake of other types of amino acids was only marginally affected. Comparisons of the root uptake characteristics of AAP5 and LHT1 mutants corroborated the hypothesis that the two transporters have distinct affinity spectra in planta. * Root uptake of all tested amino acids, except L-aspartic acid (L-Asp), was significantly affected in double AAP5*LHT1 mutants, suggesting that these two transporters account for a major proportion of roots' uptake of amino acids at low concentrations.     

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.     

Specific accumulation of polysaccharide-linked hydroxycinnamoyl esters in the cell walls of irregularly shaped and collapsed internode parenchyma cells of the dwarf rice mutant Fukei 71

We examined a novel rice mutant, Fukei 71 (Oryza sativa L.), for alterations in the levels of hydroxycinnamoyl esters that are linked to cell wall polysaccharides and lignin units. In this mutant, a recessive mutation at a single locus caused the collapse of parenchyma cells in the internodes. Light microscopy revealed that the abnormal walls of internode parenchyma cells of Fukei 71 were stained by the M?ule reaction, which is specific for syringyl units in phenolic compounds. These walls were not stained by Wiesner's reagent (phloroglucinol-HCl), which reacts cinnamaldehyde in lignin. Levels of p-coumaric acid (PCA) and ferulic acid (FA) were apparently elevated in the abnormal tissue of the mutant. Western blotting analysis with antibodies specific for phenylalanine ammonia-lyase (PAL) revealed higher levels of PAL in the abnormal parenchyma tissue of Fukei 71 than in the parenchyma tissue of the parent cultivar Fujiminori. These results and the observation that PAL was produced at a greatly elevated level indicated that the phenylpropanoid pathway that leads to the biosynthesis of polysaccharide-linked FA and PCA was abnormally activated in the irregularly shaped and collapsed internode parenchyma cells, in which the biosynthesis of lignin is normally repressed.     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.     

The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes

The Arabidopsis ref2 mutant was identified in a screen for plants having altered fluorescence under UV light. Characterization of the ref2 mutants showed that they contained reduced levels of a number of phenylpropanoid pathway-derived products: sinapoylmalate in leaves, sinapoylcholine in seeds, and syringyl lignin in stems. Surprisingly, positional cloning of the REF2 locus revealed that it encodes CYP83A1, a cytochrome P450 sharing a high degree of similarity to CYP83B1, an enzyme involved in glucosinolate biosynthesis. Upon further investigation, ref2 mutants were found to have reduced levels of all aliphatic glucosinolates and increased levels of indole-derived glucosinolates in their leaves. These results show that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates and suggest a novel metabolic link between glucosinolate biosynthesis, a secondary biosynthetic pathway found only in plants in the order Capparales, and phenylpropanoid metabolism, a pathway found in all plants and considered essential to the survival of terrestrial plant species.     

Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis

Systemic acquired resistance (SAR) is a plant defense state that is induced, for example, after previous pathogen infection or by chemicals that mimic natural signaling compounds. SAR is associated with the ability to induce cellular defense responses more rapidly and to a greater degree than in noninduced plants, a process called "priming." Arabidopsis plants were treated with the synthetic SAR inducer benzothiadiazole (BTH) before stimulating two prominent cellular defense responses, namely Phe AMMONIA-LYASE (PAL) gene activation and callose deposition. Although BTH itself was essentially inactive at the immediate induction of these two responses, the pretreatment with BTH greatly augmented the subsequent PAL gene expression induced by Pseudomonas syringae pv. tomato infection, wounding, or infiltrating the leaves with water. The BTH pretreatment also enhanced the production of callose, which was induced by wounding or infiltrating the leaves with water. It is interesting that the potentiation by BTH pretreatment of PAL gene activation and callose deposition was not seen in the Arabidopsis nonexpresser of PR genes 1/noninducible immunity 1 mutant, which is compromised in SAR. In a converse manner, augmented PAL gene activation and enhanced callose biosynthesis were found, without BTH pretreatment, in the Arabidopsis constitutive expresser of pathogenesis-related genes (cpr)1 and constitutive expresser of pathogenesis-related genes 5 mutants, in which SAR is constitutive. Moreover, priming for potentiated defense gene activation was also found in pathogen-induced SAR. In sum, the results suggest that priming is an important cellular mechanism in acquired disease resistance of plants that requires the nonexpresser of PR genes 1/noninducible immunity 1 gene.