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Plant organ development to a specific size and shape is controlled by cell proliferation and cell expansion. Here, we identify a novel Myb-like Arabidopsis gene, Development Related Myb-like1 (DRMY1), which controls cell expansion in both vegetative and reproductive organs. DRMY1 is strongly expressed in developing organs and its expression is reduced by ethylene while it is induced by ABA. DRMY1 has a Myb-like DNA-binding domain, which is predominantly localized in the nucleus and does not exhibit transcriptional activation activity. The loss-of-function T-DNA insertion mutant drmy1 shows reduced organ growth and cell expansion, which is associated with changes in the cell wall matrix polysaccharides. Interestingly, overexpression of DRMY1 in Arabidopsis does not lead to enhanced organ growth. Expression of genes involved in cell wall biosynthesis/remodeling, ribosome biogenesis and in ethylene and ABA signaling pathways is changed with the deficiency of DRMY1. Our results suggest that DRMY1 plays an essential role in organ development by regulating cell expansion either directly by affecting cell wall architecture and/or cytoplasmic growth or indirectly through the ethylene and/or ABA signaling pathways.

The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two different subcellular sites (the plasma membrane and the nucleus), and contains four distinct domains: (i) a RING domain that mediates degradation of phosphate transporters at the plasma membrane; (ii) an SPX domain that facilitates NLA’s interaction with the phosphate transporters, and also exists in other proteins that regulate the nuclear transcription factors that control the phosphate starvation response pathway; (iii) a linker domain that lies between the RING and SPX domains; and (iv) a C-terminal domain, which, like the linker region, is of unknown function. Here we carried out a mutational analysis of NLA, which indicated that all the domains are not only essential for proper functioning of the protein, but also mediate its localization to the plasma membrane and (or) nucleus, as well as to different subdomains within the nucleus. Overall, the results provide new insights to the distinct protein motifs within NLA and the role(s) that this protein serves at different subcellular sites with respect to the regulation of nitrogen-dependent phosphate homeostasis as well as other possible physiological functions.

A one-pot base-mediated reaction of azides and β- or α-vinyl bromides has been reported. The effects of bases and solvents have been investigated in the process. A variety of 1,5-disubstituted triazoles were prepared in low to good yields. Further studies reveal that the corresponding alkynes were produced as intermediates via elimination reaction. Under the same reaction conditions, the reactions of alkyl alkynes with phenyl azide would give 1,5-disubstituted 1,2,3-triazoles.

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation.

The sorption of aqueous lead on carbonate-hydroxyapatite (CHAp) is a complicated non-homogeneous solid/water reaction, which from the kinetic point of view has two stages. In the first stage, the reaction rate is so fast and the kinetic pathway so intricate that further research is required. In the second stage, the reaction rate slows down and the reaction process follows that of a first-order kinetic equation. Experimental results show that the relationship between the reaction rate constant k 1 and temperature T agrees with the Arrhenius equation, and that the activation energy of sorption ( E a ) is 11.93 kJ/mol and the frequency factor ( A ) is 2.51/s. The reaction rate constant k 1 increases with the Pb 2+ initial concentration and decreasing pH, but with increasing CHAp dosage. X-ray diffraction (XRD), scanning electron microscopy with energy dispersion spectrum (SEM-EDS) and toxicity characteristic leaching procedure (TCLP) tests indicate that the main sorption mechanism is dissolution-precipitation, in conjunction with surface sorption.

Nitrogen is an essential mineral nutrient and is required in great abundance for plant growth and development. Insufficient nitrogen triggers extensive physiological and biochemical changes in plants which constitute a set of adaptive responses to nitrogen limitation. In this study, to determine the genome-wide transcriptome response to nitrogen limitation, Arabidopsis plants were grown with limiting (3 mM) and sufficient (10 mM) nitrate, respectively, and their gene expression profiles were analyzed using Affymetrix GeneChip arrays. In addition to inducing the adaptive responses in Arabidopsis, nitrogen limitation altered the expression levels of 629 genes with 340 up-regulated and 289 down-regulated. The up-regulated group included the genes involved in protein degradation and the biosynthesis of anthocyanin and phenylpropanoids. The down-regulated group contained the genes functioning in photosynthesis and in the synthesis of nitrogenous macromolecules such as chlorophyll, proteins, amino acids and nucleotides. Numerous nitrogen limitation responsive genes encode transcription factors, signal transduction components, and proteins required for hormone synthesis and response. The Arabidopsis nitrogen limitation adaptation mutant (nla) is defective in developing the nitrogen limitation adaptive responses. The microarray analysis revealed that the absence of the functional NLA in the nla mutant extensively altered its responsive transcriptome to nitrogen limitation. In this mutant 1122 genes were up-regulated and 622 repressed. It was also found that the nla mutant phenotype was associated with the early induction of senescence-associated genes. This study presents a genome-wide view of Arabidopsis transcriptome response to nitrogen limitation and its regulation by NLA, and provides information to probe the molecular mechanism controlling plant adaptability to nitrogen limitation.

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of ‘proteomics-grade’ intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione–bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.

Summary Abundant nitrogen is required for the optimal growth and development of plants, while numerous biotic and abiotic factors that consume soil nitrogen frequently create a nitrogen limitation growth condition. To cope with this, plants have evolved a suite of adaptive responses to nitrogen limitation. However, the molecular mechanism governing the adaptability of plants to nitrogen limitation is totally unknown because no reported mutant defines this trait. Here we isolated an Arabidopsis mutant, nla (nitrogen limitation adaptation), and identified the NLA gene as an essential component in this molecular mechanism. Supplied with insufficient inorganic nitrogen (nitrate or ammonium), the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild type. Under other stress conditions including low phosphorus nutrient, drought and high temperature, the nla mutant did not show this early senescence phenotype, but closely resembled the wild type in growth and development. Map-based cloning of NLA revealed that this gene encodes a RING-type ubiquitin ligase, and nla is a deletion mutation which does not code for the RING domain in the NLA protein. The NLA protein is localized to the nuclear speckles, where this protein interacts with the Arabidopsis ubiquitin conjugase 8 (AtUBC8). In the nla mutant, the deletion of the RING domain from NLA altered its subcellular localization, disrupted the interaction between NLA and AtUBC8 and caused the early senescence phenotype induced by low inorganic nitrogen. All the results indicate that NLA is a positive regulator for the development of the adaptability of Arabidopsis to nitrogen limitation.

A one-pot base-mediated reaction of azides and β- or α-vinyl bromides has been reported. The effects of bases and solvents have been investigated in the process. A variety of 1,5-disubstituted triazoles were prepared in low to good yields. Further studies reveal that the corresponding alkynes were produced as intermediates via elimination reaction. Under the same reaction conditions, the reactions of alkyl alkynes with phenyl azide would give 1,5-disubstituted 1,2,3-triazoles.

γ-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 Km values (0.5–2 μm) 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 ∼50 and ∼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.

Two full-length cDNA, wSsI-I and wSsI-II, encoding wheat starch synthase I (wSSI; E.C. 2.4.1.21) were isolated from a wheat endosperm cDNA library. Both cDNA clones were able to complement a glycogen synthase mutation present in the E. coli strain, RH98, thus demonstrating that the cDNA clones encoded functional starch synthases. RNA gel-blot analysis revealed that wSsI transcripts were produced in leaf, stem, root, pre-anthesis floret, ovary, pollen and endosperm. In the endosperm, the amount of wSsI transcripts accumulated was higher at 5–10 days-post-anthesis (DPA) than at 15–25 DPA. During the endosperm development, the relative abundance of wSSI did not vary in starch granules, whereas, wSSI concentration in the endosperm soluble fractions was highest from ten to 15 DPA, and below detection levels at five DPA. On a per kernel basis, wSSI exhibited similar concentration from 15 to 25 DPA in endosperm soluble fractions, but at considerably higher concentrations in starch granules as compared to endosperm soluble fractions.

Mature wheat (Triticum aestivum L.) endosperm contains two types of starch granules: large A-type and small B-type. Two methods, microsieving or centrifugal sedimentation through aqueous solutions of sucrose, maltose, or Percoll were used to separate A- and B-type starch granules. Microsieving could not completely separate the two types of starch granules, while centrifuging through maltose and sucrose solutions gave a homogenous population for B-type starch granules only. Centrifuging through two Percoll solutions (70 and 100%, v/v) produced purified populations of both the A- and B-type starch granules. Analysis of starch granule size distribution in the purified A- and B-type granule populations and in the whole-starch granule population obtained directly from wheat endosperm confirmed that the purified A- and B-type starch granule populations represented their counterparts in mature wheat endosperm. Centrifugations through two Percoll solutions were used to purify A- and B-type starch granule...

Development of asparagus microspores in cold-treated buds of varying sizes and shed microspores from these buds in in vitro culture were observed cytologically for the G459 genotype. Before cold pretreatment, more than 75% of the microspores in flower buds of the 1.4–1.6, 1.7–1.9, 2.0–2.2, 2.3–2.5, and 2.6–2.8 mm size classes were at the early-, mid-, late-uninucleate, early-, and late-binucleate stages, respectively. After 7 d in cold treatment, percentages of microspores at different stages changed in all flower buds. Most notable was the appearance of binucleate microspores resulting from symmetric rather than asymmetric division. For flower buds of 1.7–1.9, 2.0–2.2, and 2.3–2.5 mm size classes, 4.9%, 27.2%, and 11.4% of the microspores had divided symmetrically, respectively. When microspores from buds of each size category were cultured in androgenesis induction medium, only microspores completing symmetric pollen mitosis I during cold treatment were observed to divide further, and calluses were only obtained from microspores of flower bud size classes where symmetric divisions were observed after several days of cold treatment. Significant correlations existed among microspore callus yield, the percentage of late-uninucleate microspores in vivo before cold treatment, and the frequency of symmetric pollen mitosis I after 7 d of cold treatment. Consequently, asparagus microspore androgenesis may occur through one developmental pathway, where a symmetric first mitotic division is a prerequisite for continued development.

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Over the last several decades, increased agricultural production has been driven by improved agronomic practices and a dramatic increase in the use of nitrogen-containing fertilizers to maximize the yield potential of crops. To reduce input costs and to minimize the potential environmental impacts of nitrogen fertilizer that has been used to optimize yield, an increased understanding of the molecular responses to nitrogen under field conditions is critical for our ability to further improve agricultural sustainability. Using maize (Zea mays) as a model, we have characterized the transcriptional response of plants grown under limiting and sufficient nitrogen conditions and during the recovery of nitrogen-starved plants. We show that a large percentage (approximately 7%) of the maize transcriptome is nitrogen responsive, similar to previous observations in other plant species. Furthermore, we have used statistical approaches to identify a small set of genes whose expression profiles can quantitatively assess the response of plants to varying nitrogen conditions. Using a composite gene expression scoring system, this single set of biomarker genes can accurately assess nitrogen responses independently of genotype, developmental stage, tissue type, or environment, including in plants grown under controlled environments or in the field. Importantly, the biomarker composite expression response is much more rapid and quantitative than phenotypic observations. Consequently, we have successfully used these biomarkers to monitor nitrogen status in real-time assays of field-grown maize plants under typical production conditions. Our results suggest that biomarkers have the potential to be used as agronomic tools to monitor and optimize nitrogen fertilizer usage to help achieve maximal crop yields.

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion., Author Summary Higher crop yields require increased use of fertilizers, especially for the prime macronutrients nitrogen and phosphorus. Increasing nitrogen and phosphorus use efficiency in plants would decrease crop production cost and reduce environmental pollution. In an attempt to isolate the regulatory genes for nitrogen and phosphorus homeostasis in plants, we identified the NLA gene as having a role in plant adaptation under low-nitrogen conditions. In the current work, detailed genetic and molecular analysis for the functionality of this gene revealed that NLA has a key role in the maintenance of phosphate (Pi) homeostasis in plants in a nitrate-dependent fashion. Further, Pi has an antagonistic crosstalk with nitrate, not only with regards to its accumulation, but also in its differential effects on flowering time. Interestingly, the antagonistic genetic interaction of Pi is with nitrate, but not with ammonium.

Mutations within the Arabidopsis METHYL-CpG BINDING DOMAIN 9 gene (AtMBD9) cause pleotropic phenotypes including early flowering and multiple lateral branches. Early flowering was previously attributed to the repression of flowering locus C (FLC) due to a reduction in histone acetylation. However, the reasons for other phenotypic variations remained obscure. Recent studies suggest an important functional correlation between DNA methylation and histone modifications. By investigating this relationship, we found that the global genomic DNA of atmbd9 was over-methylated, including the FLC gene region. Recombinant AtMBD9 does not have detectable DNA demethylation activity in vitro, but instead has histone acetylation activity. Ectopic over-expression of AtMBD9 and transient DNA demethylation promotes flowering and causes partial recovery of the normal branching phenotype. Co-immunoprecipitation assays suggest that AtMBD9 interacts in vivo with some regions of the FLC gene and binds to histone 4 (H4). Gene expression profile analysis revealed earlier up-regulation of some flower-specific transcriptional factors and alteration of potential hormonal and signal transducer axillary branching regulatory genes. In accordance with this result, AtMBD9 itself was found to be localized in the nucleus and expressed in the flower and axillary buds. Together, these results suggest that AtMBD9 controls flowering time and axillary branching by modulating gene expression through DNA methylation and histone acetylation, and reveal another component of the epigenetic mechanism controlling gene expression.

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.

A quasi rail-to-rail (VDD -Vdsa, -Vss) operational amplifier with a single differential pair is presented. A new common-mode adapter circuit based on a differential pair structure is proposed. With the common-mode adapter circuit, the common-mode input range is extended; the differential-mode signal is not affected. A two-stage operational amplifier with the proposed common-mode adapter is designed with TSMC 0.18 mum CMOS technology. With a 1 V supply voltage and a 15 pF capacitance load, the simulation shows the DC gain is about 82 dB, unity-gain frequency is about 6 MHz, and the phase margin is about 60deg.

Thermal effects on current mirror performance are significant. It is especially important to consider the thermal effects for current mirrors designed with dielectrically isolated bipolar devices. Thermal effects in integrated circuits can be categorized as thermal coupling, self-heating and the package thermal effect. This paper describes the effect of the self-heating on the output characteristics of the current mirrors. In general all the frequently used current mirrors in analog IC design are simulated and measured with the dielectrically isolated self-heating vertical bipolar inter-company (VBIC) devices. In a step by step procedure it is shown that this thermal effect is reduced or can be made negligible for the advanced mirrors; i.e. for the cascode connected and Wilson current mirrors.

Abundant nitrogen is required for the optimal growth and development of plants, while numerous biotic and abiotic factors that consume soil nitrogen frequently create a nitrogen limitation growth condition. To cope with this, plants have evolved a suite of adaptive responses to nitrogen limitation. However, the molecular mechanism governing the adaptability of plants to nitrogen limitation is totally unknown because no reported mutant defines this trait. Here we isolated an Arabidopsis mutant, nla (nitrogen limitation adaptation), and identified the NLA gene as an essential component in this molecular mechanism. Supplied with insufficient inorganic nitrogen (nitrate or ammonium), the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild type. Under other stress conditions including low phosphorus nutrient, drought and high temperature, the nla mutant did not show this early senescence phenotype, but closely resembled the wild type in growth and development. Map-based cloning of NLA revealed that this gene encodes a RING-type ubiquitin ligase, and nla is a deletion mutation which does not code for the RING domain in the NLA protein. The NLA protein is localized to the nuclear speckles, where this protein interacts with the Arabidopsis ubiquitin conjugase 8 (AtUBC8). In the nla mutant, the deletion of the RING domain from NLA altered its subcellular localization, disrupted the interaction between NLA and AtUBC8 and caused the early senescence phenotype induced by low inorganic nitrogen. All the results indicate that NLA is a positive regulator for the development of the adaptability of Arabidopsis to nitrogen limitation.

*Summary The functional characterization of mammalian proteins containing a methyl-CpG-binding domain (MBD) has revealed that MBD proteins can decipher the epigenetic information encoded by DNA methylation, and integrate DNA methylation, modification of chromatin structure and repression of gene expression. The Arabidopsis genome has 13 putative genes encoding MBD proteins, and no specific biological function has been defined for any AtMBD genes. In this study, we identified three T-DNA insertion mutant alleles at the AtMBD9 locus, and found that all of them exhibited obvious developmental abnormalities. First, the atmbd9 mutants flowered significantly earlier than wild-type plants. The expression of FLOWERING LOCUS C (FLC), a major repressor of Arabidopsis flowering, was markedly attenuated by the AtMBD9 mutations. This FLC transcription reduction was associated with a significant decrease in the acetylation level in histone H3 and H4 of FLC chromatin in the atmbd9 mutants. Secondly, the atmbd9 mutants produced more shoot branches by increasing the outgrowth of axillary buds when compared with wild-type plants. The two known major factors controlling the outgrowth of axillary buds in Arabidopsis, auxin and the more axillary growth (MAX) pathway, were found not to be involved in producing this enhanced shoot branching phenotype in atmbd9 mutants, indicating that AtMBD9 may regulate a novel pathway to control shoot branching. This pathway is not related to FLCexpression as over-expression of FLCin atmbd9-2 restored itsflowering time to one similar to that of the wild type, but did not alter the shoot branching phenotype.

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.

This paper proposes an explicit rate-based end-to-end congestion control mechanism to alleviate congestion of multimedia traffic in packet switched networks such as the Internet. The congestion is controlled by adjusting the transmission rates of the sources in response to the feedback information from destination such as the buffer occupancy, packet arrival rate and service rate at the outgoing link, so that a desired quality of service (QoS) can be met. The QoS is defined in terms of packet loss ratio, transmission delay, power, and network utilization. Comparison studies demonstrate the effectiveness of the proposed scheme over New-Reno TCP (a variant of AIMD: additive increase multiplicative decrease) technique during simulated congestion. Since it is end-to-end, no router support is necessary, the proposed methodology can be readily applied to today's Internet, as well as for real-time video and voice data transfer in unicast networks.

Plants are a particularly rich source of ABC proteins. The genome of the model plant Arabidopsis thaliana is capable of encoding a total of 129 ABC proteins, which fall into 12 or more subfamilies. This gene count outstrips those for the human genome and any other animal genome sequenced to date. The human genome is estimated to encode a mere 48 ABC proteins while those of the nematode worm, Caenorhabditis elegans , and the fruit fly, Drosophila melanogaster , encode only 58 and 51, respectively. Of the total ABC protein open reading frames (ORFs) identified in Arabidopsis , at least 103 probably encode membrane proteins–proteins possessing membrane spans contiguous with one or two nucleotide-binding domains. Arabidopsis allocates nearly twice as many of its transporter genes to ABC transporters as C. elegans and Drosophila. This chapter uses the Arabidopsis ABC protein superfamily as a model for plants in general. Arabidopsis is the first plant whose ORFs encoding ABC proteins have been systematically inventoried in their entirety. The nomenclature adopted is one based on the acronyms of the prototypes, when available, of a particular ABC protein subfamily defined from another source. Searches of the earlier literature and comparisons with the ABC proteins found in organisms other than plants are thereby simplified and expedited. A nomenclature similar to that applied to the ABC protein-encoding ORFs of yeast is favored.

This paper proposes a fair, rate-based end-to-end congestion control mechanism for multimedia traffic in packet switched networks such as the Internet. The scheme is modeled after a non-linear system. The congestion is controlled by adjusting the transmission rates of the sources in response to the feedback information from destination such as the buffer occupancy, packet arrival rate and service rate, so that a desired quality of service (QoS) can be met. The QoS is defined in terms of packet loss ratio, transmission delay, power and fairness. The performance and stability of the system is analyzed mathematically. The proposed scheme has been implemented in the NS-2 simulator. Simulation results demonstrate the performance of the scheme to be in agreement with mathematical analysis. The proposed scheme is shown to provide considerable improvements in terms of the QoS metrics over New-Reno TCP technique.

Two starch granule-bound proteins (SGP), SGP-140 and SGP-145, were preferentially associated with A-type starch granules (>10 μm) in developing and mature wheat (Triticum aestivum) kernels. Immunoblotting and N-terminal sequencing suggested that the two proteins were different variants of SBEIc, a 152-kD isoform of wheat starch-branching enzyme. Both SGP-140 and SGP-145 were localized to the endosperm starch granules but were not found in the endosperm soluble fraction or pericarp starch granules younger than 15 d post anthesis (DPA). Small-size starch granules (10 μm). In contrast, small-size starch granules harvested after 15 DPA contained only low amounts of SGP-140 and SGP-145 and developed mainly into B-type starch granules (

Clear visualization of asparagus (Asparagus officinalis L.) microspore nuclei with common stains such as acetocarmine or DAPI is difficult, hindering cytological analyses. The addition of saturated aqueous ferric chloride solution to Carnoy's I fixative (30 μL·mL-1) resulted in clear visualization of nuclei. A distinct nucleus was observed in uninucleate cells and the vegetative and generative nuclei were clearly visible in binucleate microspores. This method can be used reliably for determination of asparagus microspore developmental stage. Chemical name used: 4′,6-diamidino-2-phenylindole-2HCL (DAPI).