低氮下拟南芥叶酰聚谷氨酸合成酶突变体atdfb根发育研究

利用叶酰聚谷氨酸合成酶功能缺失突变体atdfb解析叶酸在拟南芥根发育过程中的生物学功能。纯合T-DNA插入功能缺失突变体atdfb 在土壤培养条件下生长3周,与野生型表型无明显差异。在氮源充足的1/2MS培养基上,atdfb的主根显著短于野生型,互补植株的主根长度恢复到野生型水平,说明主根缩短的表型是由AtDFB基因功能缺失造成的。在1/2MS培养基生长11 d的突变体主根长度只有野生型的23%。在低氮条件下,突变体的生长发育几乎停滞,培养11 d的突变体主根长度只有野生型的4%;5-甲酰四氢叶酸(5-F-THF)可以恢复低氮条件下atdfb-3的表型,其主根长度、根毛长度及静止中心的细胞排列均得到恢复。进一步分析发现,低氮条件下培养少于3 d的atdfb-3补充充足的5-F-THF,3 d后能像野生型一样适应低氮环境。由此说明叶酸对拟南芥根部发育及对低氮环境的适应是必需的。     

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.     

Apical meristem exhaustion during determinate primary root growth in the moots koom 1 mutant of Arabidopsis thaliana

An indeterminate developmental program allows plant organs to grow continuously by maintaining functional meristems over time. The molecular mechanisms involved in the maintenance of the root apical meristem are not completely understood. We have identified a new Arabidopsis thaliana mutant named moots koom 1 (mko1) that showed complete root apical meristem exhaustion of the primary root by 9?days post-germination. MKO1 is essential for maintenance of root cell proliferation. In the mutant, cell division is uncoupled from cell growth in the region corresponding to the root apical meristem. We established the sequence of cellular events that lead to meristem exhaustion in this mutant. Interestingly, the SCR and WOX5 promoters were active in the mko1 quiescent center at all developmental stages. However, during meristem exhaustion, the mutant root tip showed defects in starch accumulation in the columella and changes in auxin response pattern. Therefore, contrary to many described mutants, the determinate growth in mko1 seedlings does not appear to be a consequence of incorrect establishment or affected maintenance of the quiescent center but rather of cell proliferation defects both in stem cell niche and in the rest of the apical meristem. Our results support a model whereby the MKO1 gene plays an important role in the maintenance of the root apical meristem proliferative capacity and indeterminate root growth, which apparently acts independently of the SCR/SHR and WOX5 regulatory pathways.     

RCD1 and SRO1 are necessary to maintain meristematic fate in Arabidopsis thaliana

The radical-induced cell death1 and similar to RCD ONE1 genes of Arabidopsis thaliana encode members of the poly(ADP-ribose) polymerase (PARP) superfamily and have pleiotropic functions in development and abiotic stress response. In order to begin to understand the developmental and molecular bases of the defects seen in rcd1-3; sro1-1 plants, this study used the root as a model. Double mutant roots are short and display abnormally organized root apical meristems. However, acquisition of most cell fates within the root is not significantly disrupted. The identity of the quiescent centre is compromised, the zone of cell division is smaller than in wild-type roots and abnormal divisions are common, suggesting that RCD1 and SRO1 are necessary to maintain cells in a division-competent state and to regulate division plane placement. In addition, differentiation of several cell types is disrupted in rcd1-3; sro1-1 roots and shoots, demonstrating that RCD1 and SRO1 are also necessary for proper cell differentiation. Based on the data shown in this article and previous work, we hypothesize that RCD1 and SRO1 are involved in redox control and, in their absence, an altered redox balance leads to abnormal development.     

OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis gene AtCyt-inv1. This gene was designated as OsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in the Oscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of the Oscyt-inv1 mutant. These results indicated that OsCyt-inv1 played important roles in root cell development and reproductivity in rice.     

Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles

gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.     

A turanose-insensitive mutant suggests a role for WOX5 in auxin homeostasis in Arabidopsis thaliana

Sugars acting as signalling molecules regulate many developmental processes in plants, including lateral and adventitious root production. Turanose, a non-metabolizable sucrose analogue, profoundly affects the growth pattern of Arabidopsis seedlings. Turanose-treated seedlings are characterized by a very short primary root and a short hypocotyl showing the production of adventitious roots. A turanose-insensitive (tin) mutant was identified and characterized. Because of a T-DNA insertion and a chromosomal translocation, tin expresses a chimeric form of WOX5, a gene known to be expressed in the root quiescent centre. The tin mutation can be complemented by overexpression of WOX5, suggesting it is a loss-of-function mutant. We found that WOX5 is both turanose- and auxin-inducible. Moreover, turanose insensitivity is associated with altered auxin homeostasis, as demonstrated by the constitutive activation of indole acetic acid (IAA) conjugation and SUPERROOT2 expression in tin. On the basis of turanose effects on wild-type seedlings and the tin molecular and hormonal phenotype, we propose a role for WOX5 in the root apical meristem as a negative trigger of IAA homeostatic mechanisms allowing the maintenance of a restricted area of auxin maximum, which is required for a correct root-formation pattern.     

Activation of cell division in the quiescent center of excised maize root tip

The phenomenon of activation of cell proliferation in the quiescent center of excised maize roots is described. The root tips were grown on wet filter paper in Petri dishes. This phenomenon was observed in 8 to 14 maize cultivars and was absent in excised Arabidopsis root tips. The distribution of mitoses in meristems greatly varied in roots of individual seedlings from the same seed lot and seedlings of different cultivars. Meristem opening was observed after the removal of small root tips not longer than 3 mm and intact seminal roots. Sucrose (2%) and 10(-6)-10(-8) M indole-3-acetic acid did not prevent meristem opening. These findings indicate that the state of quiescent center is maintained by a system of intercellular and interorgan relations, which are to be clarified.     

Binding of ATP as well as tetrahydrofolate induces conformational changes in Lactobacillus casei folylpolyglutamate synthetase in solution

Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate to folate derivatives to form folate polyglutamates. FPGS is essential for folate biosynthesis in bacteria and retention of folate pools in eukaryotes. X-ray crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS suggest that binding of folate triggers a conformational change that activates FPGS. We used EPR and CD spectroscopy to further characterize the conformational change in the FPGS reaction. For EPR spectroscopy, two cysteine residues were introduced into FPGS by site-directed mutagenesis, K172C in the N-terminal domain and D345C in the C-terminal domain. The mutant protein was expressed, purified, and labeled with methanethiosulfonate. Addition of ATP, tetrahydrofolate, or 5,10-methylenetetrahydrofolate but not glutamate to FPGS showed broadening of EPR spectra, which is due to stronger spin-spin interactions, suggesting that both ATP and tetrahydrofolates cause a conformational change. ATP binding had an EPR spectrum distinct from that of tetrahydrofolate binding, indicating that it caused a different conformational change. When both ATP and THF were bound, the spectrum was identical to that seen when THF alone bound to the enzyme, showing that the THF-induced conformation was dominant. The spectral broadening suggests that the conformation change involves the two domains moving closer together, which is consistent with the rigid-body rotation of the C-terminal domain observed in the FPGS crystal structure with AMPPCP and 5,10-methylenetetrahydrofolate bound. No changes in the CD spectra were observed with the addition of FPGS substrates, suggesting that the conformational changes did not affect the secondary structure elements of the enzyme. These studies confirm the conformational change seen in the crystal structure by an independent method but also show that ATP binds to the free enzyme and affects its conformation.     

The BIG gene is required for auxin-mediated organ growth in Arabidopsis

Control of organ size by cell expansion and cell proliferation is a fundamental process during development, but the importance of BIG in this process is still poorly understood. Here, we report the isolation and characterization of a new allele mutant of BIG in Arabidopsis: big-j588. The mutant displayed small aerial organs that were characterized by reduced cell size in the epidermis and short roots with decreased cell numbers. The big-j588 axr1 double and big-j588 arf7 arf19 triple mutants displayed more severe defects in leaf expansion and root elongation than their parents, implying BIG is involved in auxin-dependent organ growth. Genetic analysis suggests that BIG may act synergistically with PIN1 to affect leaf growth. The PIN1 protein level decreased in both the root cells and the tips of leaf pavement cell lobes of big-j588. Further analysis showed that the auxin maxima in the roots and the leaves of big-j588 decreased. Therefore, we concluded that the small leaves and the short roots of big-j588 were associated with reduction of auxin maxima. Overall, our study suggested that BIG is required for Arabidopsis organ growth via auxin action.     

Folate-binding triggers the activation of folylpolyglutamate synthetase

Folic acid is an essential vitamin for normal cell growth, primarily through its central role in one-carbon metabolism. Folate analogs (antifolates) are targeted at the same reactions and are widely used as therapeutic drugs for cancer and bacterial infections. Effective retention of folates in cells and the efficacy of antifolate drugs both depend upon the addition of a polyglutamate tail to the folate or antifolate molecule by the enzyme folylpolyglutamate synthetase (FPGS). The reaction mechanism involves the ATP-dependent activation of the free carboxylate group on the folate molecule to give an acyl phosphate intermediate, followed by attack by the incoming L-glutamate substrate. FPGS shares a number of structural and mechanistic details with the bacterial cell wall ligases MurD, MurE and MurF, and these enzymes, along with FPGS, form a subfamily of the ADP-forming amide bond ligase family. High-resolution crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS reveal that binding of the first substrate (ATP) is not sufficient to generate an active enzyme. However, binding of folate as the second substrate triggers a large conformational change that activates FPGS and allows the enzyme to adopt a form that is then able to bind the third substrate, L-glutamate, and effect the addition of a polyglutamate tail to the folate.     

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zea mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development. Received: 22 September 1999 / Accepted: 9 November 1999     

In situ autoradiographic detection of folylpolyglutamate synthetase activity

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.     

Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis.

Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.     

hoc: An Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity

A novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokinin-overproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth-regulator mutants. In darkness the hoc mutant is de-etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two- and sevenfold the cytokinin level of wild-type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the first cytokinin-overproducing Arabidopsis mutant capable of auto-regenerating shoots without exogenous growth regulators.