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Blood S-adenosylmethionine concentrations and lymphocyte methylenetetrahydrofolate reductase activity in diabetes mellitus and diabetic nephropathy.

The erythrocyte concentrations of the body's chief physiologic methyl donor S-adenosylmethionine (SAM) and of its metabolite and inhibitor S-adenosylhomocysteine (SAH), the plasma concentrations of total homocysteine (tHcy), and the activity of N(5,10) methylenetetrahydrofolate reductase (MTHFR) in lymphocytes were determined in healthy subjects and patients with diabetes mellitus without complications and at various stages of diabetic nephropathy, categorized according to the degree of progression of the disease. These groups were as follows: 1, control; 2, diabetics with no complications; 3, patients with albuminuria; 4, patients with an elevated plasma creatinine; and 5, patients on dialysis. No parameter studied exhibited significant differences between the type 1 and the type 2 diabetics. In control subjects, the blood concentrations of SAM were proportional to the activity of MTHFR; in diabetics, it was not. Consistent with previous observations, progression of nephropathy was accompanied by increased concentrations of tHcy. Increased erythrocyte concentrations of SAH, decreased erythrocyte concentrations of SAM, SAM/SAH ratios, and lymphocyte MTHFR activity also accompanied disease progression. The blood concentrations of SAH paralleled those of tHcy, while the concentrations of SAM showed a bimodal relationship with those of tHcy. These results provide further evidence that alterations in the blood concentrations of SAM and related compounds are abnormal in patients with diabetes, particularly in those with nephropathy. The deficiency of SAM may lead to methyl deficiencies, which may contribute to the high morbidity and mortality in patients with diabetic nephropathy. We have also demonstrated a decrease in lymphocyte MTHFR activity in patients with advanced nephropathy, suggesting that hyperhomocysteinemia in these patients may be due to a generalized metabolic abnormality. Further studies are needed to determine the pathogenesis of these abnormalities and whether they are present in renal failure due to causes other than diabetes or whether they are specific to diabetic nephropathy.

Adult↗

Influence of buthionine sulfoximine, S-adenosylmethionine and glutathione on liver regeneration following partial hepatectomy.

This study was designed to evaluate the effect of derangement in methionine metabolism, and the effects of S-adenosylmethionine (SAM) and glutathione (GSH) on liver regeneration. Rats were treated immediately and 6 h after 70% hepatectomy (PH) as follows: (1) SAM, (2) GSH, (3) buthionine sulfoximine (BSO; inhibitor of SAM and GSH synthesis), (4) BSO + SAM, (5) BSO + GSH, and (6) buffer solution (B). The control group consisted of laparotomized animals treated with B. The increase in hepatic DNA synthesis, hepatocyte mitotic activity, and protein synthesis were observed in PH rats while in BSO rats, a delayed onset of liver regeneration was observed. SAM induced an earlier onset of hepatocyte mitotic activity, and reduced the inhibitory effect of BSO. GSH treatment exerted an inhibitory effect on liver regeneration and worsened the negative effect of BSO. It is concluded that derangement in methionine metabolism can impair the development of liver regeneration. The data also demonstrate the favourable effect of SAM and negative effect of GSH.

Amino Acids↗

Metal ion recognition and molecular templating in self-assembled monolayers of cyclic and acyclic polyethers.

Our recent work with cyclic and acyclic polyether self-assembled monolayers (SAMs) on gold is presented. A series of dithia-crown-tetrathiafulvalene derivatives with one or two disulfide groups has been prepared, and their SAMs on gold have been characterized by electrochemistry and by reflection-absorption infrared spectroscopy. These SAMs are extremely stable on repeated electrochemical potential scans and can selectively recognize alkali metal ions. Acyclic polyether derivatives can also self-assemble on gold to yield selective metal ion recognition domains. Impedance spectroscopy data for these SAMs fit the Langmuir isotherm and allow the determination of ion association constants. Some of the SAMs prepared with new acyclic polyether derivatives are able to detect potassium cations selectively when templated in their presence. These structures are believed to be a consequence of ion "imprinting" during the process of SAM formation.

Cations↗

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

4-Butyrolactone↗

Expression of a novel murine phospholipase D homolog coincides with late neuronal development in the forebrain.

Members of the phospholipase D (PLD) superfamily are defined by the conserved HXKXXXXD motif, which is essential for the catalytic function of mammalian PLD. PLD enzymes are thought to play roles in signal transduction and membrane vesicular trafficking in mammalian cells. Here we describe a 54-kDa novel murine polypeptide (designated SAM-9) that is predicted to be a membrane-associated member of the PLD superfamily. SAM-9 shares 40, 30, and 29% amino acid identity with potential orthologs, in vaccinia virus, Caenorhabditis elegans, and Dictyostelium discoideum, respectively, and belongs to a subclass of PLD homologs in which the second HXKXXXXD motif is imperfect and harbors a conserved Asp to Glu substitution. The sam-9 gene has more than eight exons, and the two HXKXXXXD motifs are encoded by two highly conserved exons. The expression of the sam-9 gene is greater in the brain than in non-nervous tissue and appears to be predominantly of neuronal origin. sam-9 expression is pronounced in mature neurons of the forebrain and appears to be turned on at late stages of neurogenesis as revealed by in situ hybridization analysis of sam-9 expression during postnatal development of the hippocampal formation and the primary somatosensory cortex.

Amino Acid Sequence↗

Low ratio of S-adenosylmethionine to S-adenosylhomocysteine is associated with vitamin deficiency in Brazilian pregnant women and newborns.

BACKGROUND: Pregnant women with low cobalamin concentrations are unable to provide the necessary amount of cobalamin to their fetuses. The effect of low maternal cobalamin concentrations on transmethylation metabolism in pregnant women and their newborns is unknown. OBJECTIVE: We investigated the relation between maternal and neonatal cobalamin concentrations and changes in total homocysteine (tHcy), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH). DESIGN: Hematologic data and concentrations of cobalamin, red blood cell folate, serum folate, tHcy, methylmalonic acid, SAM, SAH, and other metabolites were measured in 119 serum specimens from pregnant Brazilian women (gestational age: 37-42 wk) and their newborns' placental veins at the time of delivery. RESULTS: The tHcy concentrations were higher in placental vein serum from newborns whose mothers had low cobalamin. Serum SAH concentrations were elevated and serum SAM and methionine concentrations were decreased in pregnant women with lower cobalamin concentrations. SAM:SAH was significantly decreased in both cobalamin-deficient pregnant women and their newborns. CONCLUSIONS: Lower maternal cobalamin concentrations are associated with higher tHcy and lower SAM:SAH in newborns. Because SAM:SAH is closely linked with the activity of numerous enzymatic methylation reactions, these results suggest that methylation could be impaired in cobalamin-deficient pregnant women and their newborns.

Adult↗

Improving population attributable fraction methods: examining smoking-attributable mortality for 87 geographic regions in Canada.

Smoking-attributable mortality (SAM) is the number of deaths in a population caused by smoking. In this study, the authors examined and empirically quantified the effects of methodological problems in the estimation of SAM through population attributable fraction methods. In addition to exploring common concerns regarding generalizability and residual confounding in relative risks, the authors considered errors in measuring estimates of risk exposure prevalence and mortality in target populations and estimates of relative risks from etiologic studies. They also considered errors resulting from combining these three sources of data. By modifying SAM estimates calculated using smoking prevalence obtained from the 2000-2001 Canadian Community Health Survey, a population-based survey of 131,535 Canadian households, the authors observed the following effects of potential errors on estimated national SAM (and the range of effects on 87 regional SAMs): 1) using a slightly biased, mismatched definition of former smoking: 5.3% (range, 1.8% to 11.6%); 2) using age-collapsed prevalence and relative risks: 6.9% (range, 1.1% to 15.5%) and -15.4% (range, -7.9% to -21.0%), respectively; 3) using relative risks derived from the same cohort but with a shorter follow-up period: 8.7% (range, 4.5% to 11.8%); 4) using relative risks for all diseases with age-collapsed prevalence: 49.7% (range, 24.1% to 82.2%); and 5) using prevalence estimates unadjusted for exposure-outcome lag: -14.5% (range, -20.8% to 42.6%) to -1.4% (range, -0.8% to -2.7%), depending on the method of adjustment. Applications of the SAM estimation method should consider these sources of potential error.

Canada↗

Stem fasciated, a recessive mutation in sunflower (Helianthus annuus), alters plant morphology and auxin level.

BACKGROUND AND AIMS: Plant lateral organs such as leaves arise from a group of initial cells within the flanks of the shoot apical meristem (SAM). Alterations in the initiation of lateral organs are often associated with changes in the dimension and arrangement of the SAM as well as with abnormal hormonal homeostasis. A mutation named stem fasciated (stf) that affects various aspects of plant development, including SAM shape and auxin level, was characterized in sunflower (Helianthus annuus). METHODS: F1, F2 and F3 generations were obtained through reciprocal crosses between stf and normal plants. For the genetic analysis, a chi2 test was used. Phenotypic observations were made in field-grown and potted plants. A histological analysis of SAM, hypocotyl, epicotyl, stem and root apical meristem was also conducted. To evaluate the level of endogenous indole-3-acetic acid (IAA), a capillary gas chromatography-mass spectrometry-selected ion monitoring analysis was performed. KEY RESULTS: stf is controlled by a single nuclear recessive gene. stf plants are characterized by a dramatically increased number of leaves and vascular bundles in the stem, as well as by a shortened plastochron and an altered phyllotaxis pattern. By histological analysis, it was demonstrated that the stf phenotype is related to an enlarged vegetative SAM. Microscopy analysis of the mutant's apex also revealed an abnormal enlargement of nuclei in both central and peripheral zones and a disorganized distribution of cells in the L2 layer of the central zone. The stf mutant showed a high endogenous free IAA level, whereas auxin perception appeared normal. CONCLUSIONS: The observed phenotype and the high level of auxin detected in stf plants suggest that the STF gene is necessary for the proper initiation of primordia and for the establishment of a phyllotactic pattern through control of both SAM arrangement and hormonal homeostasis.

Flowers↗

Hidden Markov models for detecting remote protein homologies.

MOTIVATION: A new hidden Markov model method (SAM-T98) for finding remote homologs of protein sequences is described and evaluated. The method begins with a single target sequence and iteratively builds a hidden Markov model (HMM) from the sequence and homologs found using the HMM for database search. SAM-T98 is also used to construct model libraries automatically from sequences in structural databases. METHODS: We evaluate the SAM-T98 method with four datasets. Three of the test sets are fold-recognition tests, where the correct answers are determined by structural similarity. The fourth uses a curated database. The method is compared against WU-BLASTP and against DOUBLE-BLAST, a two-step method similar to ISS, but using BLAST instead of FASTA. RESULTS: SAM-T98 had the fewest errors in all tests-dramatically so for the fold-recognition tests. At the minimum-error point on the SCOP (Structural Classification of Proteins)-domains test, SAM-T98 got 880 true positives and 68 false positives, DOUBLE-BLAST got 533 true positives with 71 false positives, and WU-BLASTP got 353 true positives with 24 false positives. The method is optimized to recognize superfamilies, and would require parameter adjustment to be used to find family or fold relationships. One key to the performance of the HMM method is a new score-normalization technique that compares the score to the score with a reversed model rather than to a uniform null model. AVAILABILITY: A World Wide Web server, as well as information on obtaining the Sequence Alignment and Modeling (SAM) software suite, can be found at http://www.cse.ucsc.edu/research/compbi o/ CONTACT: karplus@cse.ucsc.edu; http://www.cse.ucsc.edu/karplus

Algorithms↗

Ornithine decarboxylase activity in chemically induced mouse skin papillomas.

The activity levels of L-ornithine carboxy-lyase (ODC) (E.C. 4.1.1.17) and S-adenosyl-L-methionine carboxy-lyase (SAM-D) (E.C.4.1.1.50) were determined in individual papillomas induced in mouse skin by a two-stage technique, and in normal mouse epidermis. Cycloheximide treatment abolished both enzyme activities. In normal epidermis the ODC activity was barely detectable, whereas the tumors exhibited high levels of ODC. Levels of SAM-D activity above those of normal epidermis were detected in some papillomas, but in contrast to ODC the SAM-D activity levels were not consistently increased in skin tumors. By pooling a great number of papillomas, the variations in ODC and SAM-D activities between different papillomas could be minimized so that reliable measurements of the biological half-lives of ODC and SAM-D in the tumors were obtained using cycloheximide treatment. The half-life of SAM-D in squamous papillomas was 45 min, almost identical to the 41 min half-life of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced level of this enzyme in normal mouse epidermis. In contrast, the ODC activity of the mouse skin papillomas declined at a rate similar to that in TPA-treated epidermis for only the first 15-20 min after cycloheximide injection. Thereafter, at time points when protein synthesis was approximately 90% inhibited, the ODC activity reverted to high levels. These results show that the high level of ODC activity in squamous papillomas is stabilized. This observation is compatible with the hypothesis that the control mechanism of the ODC activity level in these tumors is severely deranged. This change in polyamine turnover pattern may be related to altered differentiation of the epidermal cells, which constitute the main bulk of cells in these tumors.

Adenosylmethionine Decarboxylase↗

Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

Arabidopsis↗

PGC-1beta down-regulation is associated with reduced ERRalpha activity and MCAD expression in skeletal muscle of senescence-accelerated mice.

Mitochondrial dysfunction is involved in the development of aging. Here, we examined the effect of aging on the skeletal muscle expression of two isoforms of the transcriptional peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1 (PGC-1) in an experimental murine model of accelerated aging, the senescence-accelerated mouse (SAM). The senescence-accelerated prone mice (SAM-P8) showed no changes in PGC-1alpha, but a decrease in PGC-1beta expression (52% reduction, p <.001) was observed compared to the senescence-accelerated resistant mice (SAM-R1). In agreement with the proposed role of PGC-1beta as an estrogen-related receptor (ERR) protein ligand, the expression of the ERRalpha target gene medium-chain acyl-coenzyme A dehydrogenase was strongly suppressed (85%, p <.001) in SAM-P8. The decrease in the expression of medium-chain acyl-coenzyme A dehydrogenase was consistent with the reduction in ERRalpha DNA-binding activity of SAM-P8. These findings indicate that the age-mediated decrease in PGC-1beta expression in SAM-P8 skeletal muscle affects the expression of genes involved in mitochondrial fatty acid oxidation.

Acyl-CoA Dehydrogenase↗

Hepatic content of S-adenosylmethionine, S-adenosylhomocysteine and glutathione in rats receiving treatments modulating methyl donor availability.

Because of evidence linking methyl group deficiency and increased tumor formation in experimental animals, we explored other possible methods of producing a methyl group deficiency. Rats fed a low methionine diet lacking choline (MCD) were injected intraperitoneally daily for 3 wk with large doses of nicotinamide. Hepatic levels of lipids were elevated, S-adenosylmethionine (SAM) levels and the SAM:S-adenosylhomocysteine (SAH) ratio were decreased, and SAH level was not consistently changed. In livers of rats fed the MCD diet without folate (MCFD), lipids were also elevated and SAM reduced as compared to MCD-fed rats. In rats fed the MCD diet plus a methionine (Met) supplement (MCD + Met), hepatic SAM levels and the SAM:SAH ratio were higher and lipid levels lower than in MCD-fed rats, indicating that the MCD diet is marginally deficient in methyl donor groups. The injection of nicotinamide or the removal of folate from the MCD diet increased the severity of methyl donor deficiency, as shown by lower hepatic SAM levels and higher hepatic lipid levels. Hepatic glutathione levels were similar in MCD- and MCFD-fed rats and were lower than in rats fed the methionine-supplemented MCD diet or injected with nicotinamide.

Animals↗

A comparison of the effects of betaine and S-adenosylmethionine on ethanol-induced changes in methionine metabolism and steatosis in rat hepatocytes.

Previous studies showed that chronic ethanol administration alters methionine metabolism in the liver, resulting in increased intracellular S-adenosylhomocysteine (SAH) levels and increased homocysteine release into the plasma. We showed further that these changes appear to be reversed by betaine administration. This study compared the effects of betaine and S-adenosylmethionine (SAM), another methylating agent, on ethanol-induced changes of methionine metabolism and hepatic steatosis. Wistar rats were fed ethanol or control Lieber-Decarli liquid diet for 4 wk and metabolites of the methionine cycle were measured in isolated hepatocytes. Hepatocytes from ethanol-fed rats had a 50% lower intracellular SAM:SAH ratio and almost 2-fold greater homocysteine release into the media compared with controls. Supplementation of betaine or SAM in the incubation media increased this ratio in hepatocytes from both control and ethanol-fed rats and attenuated the ethanol-induced increased hepatocellular triglyceride levels by approximately 20%. On the other hand, only betaine prevented the increase in generation of homocysteine in the incubation media under basal and methionine-loaded conditions. SAM can correct only the ratio and the methylation defects and may in fact be detrimental after prolonged use because of its propensity to increase homocysteine release. Both SAM and betaine are effective in increasing the SAM:SAH ratio in hepatocytes and in attenuating hepatic steatosis; however, only betaine can effectively methylate homocysteine and prevent increased homocysteine release by the liver.

Animals↗

Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice.

Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects.

Aging, Premature↗

Alteration of soluble adhesion molecules during aging and their modulation by calorie restriction.

To investigate the status of soluble adhesion molecules (sAMs) during aging, the present study determined protein levels of several major sAMs in serum samples obtained from rats at different ages. These sAMs include E-selectin, P-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). Fischer 344 rats, ages 6, 12, 18, and 24 months, fed ad libitum (AL) and calorie restricted (CR) diets were used in this study. Analysis by Western blotting showed that the levels of all sAMs studied increased during aging in AL rats, but were effectively blunted in the CR rats. Total reactive oxygen species/reactive nitrogen species (ROS/RNS) levels were measured by fluorescent probe 2',7'-dichlorofluorescin diacetate. Increased ROS/RNS levels were found to coincide with increased levels of superoxide-generating xanthine oxidase in serum during aging, but were found suppressed by CR. Increases in sAMs levels were duplicated in another experiment in which young (13-month-old) and old (31-month-old) rats were injected with proinflammatory lipopolysaccharide. These findings suggest that the altered expressions of sAMs may be due to increased oxidative stress with advanced age and that these increases were prevented by CR through its antioxidative action.

Aging↗

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli.

Regulation of methionine biosynthesis in Escherichia coli involves a complex of the MetJ aporepressor protein and S-adenosylmethionine (SAM) repressing expression of most genes in the met regulon. To test the role of SAM in the regulation of met genes directly, SAM pools were depleted by the in vivo expression of the cloned plasmid vector-based coliphage T3 SAM hydrolase (SAMase) gene. Cultures with in vivo SAMase activity were assayed for expression of the metA, B, C, E, F, H, J, K and R genes in cells grown in methionine-rich complete media as well as in defined media with and without l-methionine. In vivo SAMase activity dramatically induced expression between 11- and nearly 1000-fold depending on the gene assayed for all but metJ and metH, and these genes were induced over twofold. metJ : : Tn5 (aporepressor defective) and metK : : Tn5 (SAM synthetase impaired; produces <5 % of wild-type SAM) strains containing in vivo SAMase activity produced even higher met gene activity than that seen in comparably prepared cells with wild-type genes for all but metJ in a MetJ-deficient background. The SAMase-mediated hyperinduction of metH in wild-type cells and of the met genes assayed in metJ : : Tn5 and metK : : Tn5 cells provokes questions about how other elements such as the MetR activator protein or factors beyond the met regulon itself might be involved in the regulation of genes responsible for methionine biosynthesis.

Artificial Gene Fusion↗

The polar auxin transport inhibitor N-1-naphthylphthalamic acid disrupts leaf initiation, KNOX protein regulation, and formation of leaf margins in maize.

Maize (Zea mays) leaves develop basipetally (tip to base); the upper blade emerges from the shoot apical meristem (SAM) before the expansion of the lower sheath. Founder cells, leaf initials located in the periphery of the SAM, are distinguished from the SAM proper by the differential accumulation of KNOX proteins. KNOX proteins accumulate in the SAM, but are excluded from maize leaf primordia and leaf founder cells. As in Arabidopsis and tomato (Lycopersicon esculentum), maize shoots failed to initiate new leaves when cultured in the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). We demonstrate that NPA-induced arrest of leaf initiation in maize is correlated with the failure to down-regulate KNOX accumulation in the SAM. In addition, NPA-cultured shoots formed abnormal tubular leaf bases in which the margins failed to separate in the lower leaf zone. The tubular leaf bases always formed in the fourth leaf from the arrested meristem. Moreover, the unseparated margin domains of these tubular leaf bases accumulated ectopic KNOX protein(s). Transfer of NPA-cultured apices to NPA-free media resulted in the resumption of leaf initiation from the SAM and the restoration of normal patterns of KNOX down-regulation, accordingly. These data suggest that the lower sheath margins emerge from the leaf base late in maize leaf development and that the separation of these leaf margin domains is correlated with auxin transport and down-regulation of KNOX proteins. In addition, these results suggest that the down-regulation of KNOX accumulation in maize apices is not upstream of polar auxin transport, although a more complicated feedback network may exist. A model for L1-derived margin development in maize leaves is presented.

Biological Transport↗