A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

Differential inactivation of seed exudate stimulation of Pythium ultimum sporangium germination by Enterobacter cloacae influences biological control efficacy on different plant species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

Priming and accelerated ageing affect L-isoaspartyl methyltransferase activity in tomato (Lycopersicon esculentum Mill.) seed

Damage and degradation of cellular proteins is observed duringage-induced seed deterioration. L-Isoaspartyl protein methyltransferase(EC 2.1.1.77 [EC] ) is an enzyme hypothesized to play a role in limitingand repairing age-induced damage to proteins. Tomato (Lycopersiconesculentum Mill. ‘New Yorker’) seeds were assayedfor changes in L-isoaspartyl methyl-transferase activity duringaccelerated ageing and after osmotic priming. Accelerated ageingof seeds for 1–4 d at 45C and 100% relative humidityreduced germination from 94% to 71%, increased the mean timeof germination (MTG) from 2.4 to 5.8 d, and was accompaniedby a correlative decrease in L-isoaspartyl methyltransferaseactivity (r²=0.90). Aged and untreated seeds were primed for7 d at 20C in darkness using aerated solutions of 3% KNO₃ orpolyethylene glycol 8000 (PEG) with equivalent osmotic potential(–1.25 MPa). Priming with KNO₃ decreased the MTG, butdid not improve germination percentage for untreated seeds.Priming did not affect L-isoaspartyl methyltransferase activityin untreated seeds, but restored activity in aged seeds primedin KNO₃ to levels near that of untreated seeds. Priming withPEG did not effectively improve the MTG or increase L-isoaspartylmethyltransferase activity. During germination, L-isoaspartylmethyltransferase activity remained constant for 48 h post-imbibitionand then declined, suggesting that the enzyme was developmentallyregulated and inactivated or degraded as radicle emergence occurred. Key words: L-Isoaspartyl methyltransferase, protein repair, seed priming, accelerated ageing, Lycopersicon esculentum     

Widespread phylogenetic distribution of a protein methyltransferase that modifies L-isoaspartyl residues.

Protein L-isoaspartyl methyltransferase is implicated in the repair or degradation of age-damaged proteins that contain atypical, L-isoaspartyl residues. The enzyme has previously been demonstrated in a variety of vertebrates and in the bacterium S. typhimurium (O'Connor, C.M. and Clarke, S. (1985) Biochem. Biophys. Res. Commun. 132, 1144-1150). We report here that the enzyme is present in a mollusc (great slug), a crustacean (pill woodlouse), a fungus (mushroom), and a plant (wheat germ). Using mushroom as an example, we show that the enzyme activity may, in some instances, require a partial purification before its presence is clearly detectable. Our findings significantly extend the known phylogenetic distribution of this enzyme and suggest that it may play an indispensable role in protein metabolism.     

Phytoene desaturase is present in a large protein complex in the plastid membrane

Phytoene desaturase (PDS; EC 1.14.99.-) represents one of the key enzymes in the carotenoid biosynthetic pathway and is present in nearly all types of plastids in plants. To further characterize PDS, we isolated the PDS cDNA from cauliflower ( BoPDS ) and confirmed its function by heterologous expression in a strain of Escherichia coli containing a carotenoid-producing plasmid. The BoPDS cDNA encodes a predicted mature protein of approximately 55 kDa. In comparison with PDS from a few other plant species, BoPDS exhibited a high enzyme activity in E. coli , and its expression in plastids was independent of carotenoid levels. Plastids were purified from tissues of different plant species including cauliflower curds, tomato fruits, carrot roots and Arabidopsis leaves. By employing both Blue Native PAGE and SDS-PAGE approaches in conjunction with Western blot analysis, it was found that PDS in these plants existed in two forms. The plastid membrane form was present in a large protein complex of approximately 350 kDa, whereas the stroma version was in an approximately 660 kDa complex.     

New proteomic developments to analyze protein isomerization and their biological significance in plants

Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.     

Differential Inactivation of Seed Exudate Stimulation of Pythium ultimum Sporangium Germination by Enterobacter cloacae Influences Biological Control Efficacy on Different Plant Species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Effect of low temperature stress on the expression of sucrose synthetase in spring and winter wheat plants. Development of a monoclonal antibody against wheat germ sucrose synthetase.

A monoclonal antibody against wheat germ sucrose synthetase is developed and characterized. Its use in studying the effect of cold acclimation on the expression of sucrose synthetase in winter and spring wheat plants is described. The antibody shows cross-reactivity with sucrose synthetase from maize and pea plants, as well as carrot cells. A gradual accumulation of the enzyme as a function of time spent at 2 degrees C is observed in both wheat varieties. In contrast, an initial sharp rise in the mRNA level is observed, which agrees with the previously reported response of maize plants subjected to anaerobic stress.     

云南南亚热带北缘地区稻田亩产吨粮的种植模式及其生态生理学研究

1996～ 1 999年 ,在云南省蒙自县草坝镇 (南亚热带北缘 )进行了亩产吨粮的种植模式研究。通过对春、夏、秋、冬播玉米 ,早、中、晚稻以及冬小麦的系统研究 ,以地上部分总干物重、籽粒产量、总入射辐照量、叶面积持续期 (灌浆期 )、氮肥的利用率和生产力、水生产力为指标 ,对早稻—晚稻、早稻—秋玉米、早稻—冬小麦、春玉米—晚稻、夏玉米—冬小麦、中稻—冬小麦和中稻—冬玉米 7种复种模式作了比较 ,选出中稻—冬玉米为一年两熟亩产吨粮的最佳种植模式。     

Seed aging and survival mechanisms

Aging and death are universal to living systems. In temperate climate latitudes the mature seeds of higher plants are exposed to aging and have developed resistance mechanisms allowing survival and plant propagation. In addition to the physicochemical properties of the seed that confer stress resistance, the protein metabolism contributes importantly to longevity mechanisms. Recently, genetic studies have demonstrated the occurrence of the Protein L-isoaspartyl methyltransferase repair enzyme in controlling age-related protein damages and seed survival. These protective mechanisms by protein repair are widespread in all kingdoms, so that the use of seeds as models to study these controlling processes offers the prospect of understanding longevity mechanisms better.     

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.     

Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli

Mammalian tissues contain protein carboxyl methyltransferases that catalyze the transfer of methyl groups from S-adenosylmethionine to the free carboxyl groups of D-aspartyl or L-isoaspartyl residues (EC 2.1.1.77). These enzymes have been postulated to play a role in the repair and/or degradation of spontaneously damaged proteins. We have now characterized a similar activity from Escherichia coli that recognizes L-isoaspartyl-containing peptides as well as protein substrates such as ovalbumin. The enzyme was purified by DEAE-cellulose, hydroxylapatite, Sephadex G-100, polyaspartate, and reversed-phase chromatography and was shown to consist of a single 24-kDa polypeptide chain. The sequence determined for the N-terminal 39 residues was used to design an oligonucleotide probe that allowed the precise localization of its structural gene (pcm) on the physical map of the E. coli chromosome at 59 min. Transformation of E. coli cells with a plasmid containing DNA from this region results in a 3-4-fold overproduction of enzyme activity. The nucleotide sequence determined for the pcm gene and its flanking regions was used to deduce a mature amino acid sequence of 207 residues with a calculated molecular weight of 23,128. This sequence shows 30.8% sequence identity with the human L-isoaspartyl/D-aspartyl methyltransferase and suggests that this enzyme catalyzes a fundamental reaction in both procaryotic and eucaryotic cells.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

拟南芥γ-生育酚甲基转移酶(γ-TMT)启动子的分离及表达特性分析

维生素E是一类人体所必需的脂溶性的维生素,具有重要的生理功能。γ-生育酚甲基转移酶(γ-TMT)是维生素E生物合成途径中的关键酶之一,催化γ、δ-生育酚甲基化,生成α、β-生育酚。从拟南芥中分离了γ-生育酚甲基转移酶基因1552bp的启动子序列,构建了含有该启动子和GUS报告基因的植物表达载体,通过农杆菌介导转化拟南芥,获得了转基因植株。GUS组织化学染色结果表明,在γ-TMT启动子的驱动下,报告基因GUS在拟南芥的叶、茎以及花均有表达,且在茎尖、雄蕊和幼叶中表达最强,而在根、种子和种荚中则没有检测到GUS基因的表达,表明γ-TMT基因可能仅在拟南芥某些组织中特异性高表达。     

Constitutive Expression of Soybean Ferritin cDNA Intransgenic Wheat and Rice Results in Increased Iron Levels in Vegetative Tissues but not in Seeds

We used particle bombardment to produce transgenic wheat and rice plants expressing recombinant soybean ferritin, a protein that can store large amounts of iron. The cDNA sequence was isolated from soybean by RT-PCR and expressed using the constitutive maize ubiquitin-1 promoter. The presence of ferritin mRNA and protein was confirmed in the vegetative tissues and seeds of transgenic wheat and rice plants by northern and western blot analysis, respectively. The levels of ferritin mRNA were similar in the vegetative tissues of both species, but ferritin protein levels were higher in rice. Both ferritin mRNA and protein levels were lower in wheat and rice seeds. ICAP spectrometry showed that iron levels increased only in vegetative tissues of transgenic plants, and not in the seeds. These data indicate that recombinant ferritin expression under the control of the maize ubiquitin promoter significantly increases iron levels invegetative tissues, but that the levels of recombinant ferritin in seeds are not sufficient to increase iron levels significantly over those in the seeds of non-transgenic plants.     

Inhibitors of animal cell-free protein synthesis from grains

We described earlier the purification and properties of a protein (tritin) from wheat that enzymatically inhibits translation in cell-free systems from animals but not plants. In this report, we have examined 11 additional grains (Family Gramineae) and three other seeds for the presence of tritin-like proteins. In addition to wheat species, barley, oats, rye, triticale and corn were found to be sources of inhibitor; no inhibitor could be detected in rice, millet, sesame, alfalfa, mung bean or common bean seeds. The inhibitors from barley and rye were purified and found to differ from tritin with respect to heat inactivation, although they are similar to tritin with respect to molecular weight, behavior during purification and specific activity. The inhibitor from corn was purified and found to differ from tritin with respect to heat inactivation and molecular weight, although it is similar to tritin in behavior during purification and specific activity. These inhibitors constitute 2–17% of the total extractable protein in these grain s. Thus, wheat, barley, rye and corn can serve as convenient sources of a family of closely related inhibitors of protein synthesis which, when conjugated with lectins, antibodies, or hormones, could prove useful as chimeric toxins.