Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants

To increase the specific free amino acid content in the japonica rice (Oryza sativa L.) cultivar Donganbyeo, mutant cell lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from embryo-cultured callus irradiated with 50 Gy gamma-rays. Four 5MT-resistant homozygous M₄ lines, MRI-40, MRI-116, MRII-8, and MRII-12, were obtained. The mean content of nine free essential amino acids were 70.1, 72.5, 31.7, and 35.4% greater than the original variety in these four mutant lines, respectively. For AFLP analysis, 8 EcoRI (+2) and 8 MseI (+3) primers used in 45 primer combinations generated a total of 3,684 bands with a mean of 82 bands, of which 361 (9.8%) were clearly polymorphic with the control cultivar, the four 5MT-resistant mutants, and five sensitive lines. The lines were grouped into three clusters through cluster analysis using unweighted pair grouping method of averages. The 36 polymorphic PCR products present only in the four homozygous 5MT-resistant lines were cloned and sequenced, and 10 of these sequenced products were converted into sequence tagged site (STS) markers. These STS primer sets were designated OSMR1–OSMR10. Six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4, OSMR5, and OSMR6) generated a single monomorphic PCR product identical in size to the original AFLP fragments. The broad applicability of these STS markers for the screening of 5MT resistance was evaluated with seven putative 5MT-resistant M₂ plants (PM-1 to PM-7). Four STS markers (OSMR1, OSMR2, OSMR4, and OSMR5) out of six STS primer sets were revealed as polymorphic products between the control cultivar and the seven M₂ plants. These markers can be utilized for the fine selection of 5MT resistance in rice, and this PCR-screening technique is less time-consuming, less labor-intensive, and more accurate and reliable than selection based solely on phenotypic evaluation involving soaking in 5MT solutions.Abbreviations AFLP Amplified fragment length polymorphism - MAS Marker-assisted selection - STS Sequence tagged site - UPGMA Unweighted pair grouping method of averages - 5MT 5-Methyltryptophan     

Evaluation of amino acid profile in contrasting arsenic accumulating rice genotypes under arsenic stress

Amino acids (AAs) play significant roles in metal binding, antioxidant defense, and signaling in plants during heavy metal stress. In the present study, the essential amino acids (EAAs), non-essential amino acids (NEAAs), as well as the enzymes of proline and cysteine biosynthetic pathways were studied in contrasting arsenic accumulating rice genotypes grown in hydroponic solutions with addition of arsenate (As^V) or arsenite (As^III). Under a mild As stress, the total AAs content significantly increased in both the rice genotypes with a greater increase in a low As accumulating rice genotype (LAARG; IET-19226) than in a high As accumulating rice genotype (HAARG; BRG-12). At the equimolar concentration (10 μM), As^III had a greater effect on EAAs than As^V. Conversely, As^V was more effective in inducing a proline accumulation than As^III. Among NEAAs, As significantly induced the accumulation of histidine, aspartic acid, and serine. In contrast, a higher As concentration (50 μM) reduced the content of most AAs, the effect being more prominent during As^III exposure. The inhibition of glutamate kinase activity was noticed in HAARG, conversely, serine acetyltransferase and cysteine synthase activities were increased which was positively correlated with the cysteine synthesis.     

Modification of amino acid composition of endosperm proteins from in-vitro-selected high lysine mutants in rice

Summary Endosperm protein mutants in rice may be recovered by biochemical selections with inhibitory levels of lysine and threonine. Among the phenotypes recovered from in vitro selections are lines with increased protein and percent lysine in the protein. This work was designed to identify changes in proteins of rice mutants and to further our understanding of the mechanisms of lysine plus threonine selections in rice. Among the most obvious amino acid changes in mutants was a higher lysine level in all protein solubility fractions and a decrease in tyrosine. Methionine and glutamate are reduced in some protein fractions. However, methionine is significantly higher in the mutant than the control in the glutelin fraction. Several other aspartate pathway amino acids are higher in the mutant than the unselected controls. Separation of proteins in SDS-PAGE gels showed shifts in the protein profiles in the mutants, including a decrease in the major 30 kDa low lysine globulin component, and an increase in several high-molecular-weight components, approximately 60–100 kDa. Increases in the lysine content of proteins of different solubility classes and different proteins within classes are detailed.     

Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds

Aspartate aminotransferase (AAT) is a key enzyme in the synthesis of amino acids. It plays an important role in regulating carbon and nitrogen metabolism in almost all organisms. In this study, we over-expressed in rice separately all three AAT genes from rice (OsAAT1~3) and one AAT gene from Escherichia coli (EcAAT). Over-expression was driven by the CaMV 35S promoter and constructs were introduced into rice by Agrobacterium tumefaciens-mediated transformation. Compared with control plants, the transformants showed significantly increased leaf AAT activity and greater seed amino acid and protein contents. No other phenotypic changes were observed. The total leaf AAT activities in plants over-expressing OsAAT1, OsAAT2, and EcAAT were 26.6, 23.6, and 19.6 A min⁻¹ mg⁻¹ FW (A: units of activity, defined as increase of absorbency per min per mg; FW: fresh weight), which were significantly higher than that in the wild-type control (17.7 A min⁻¹ mg⁻¹ FW). The amino acid content in seeds of transgenic plants over-expressing OsAAT1, OsAAT2, and EcAAT was 119.36, 115.36, and 113.72 mg g⁻¹, respectively, which were 16.1, 12.0, and 5.4% higher, respectively, than that in the control plants. The transgenic plants over-expressing OsAAT1, OsAAT2, and EcAAT had significantly higher protein contents (increased 22.2, 21.1, and 11.1%, respectively) than wild-type plants. No significant changes were found in leaf AAT activity, seed amino acid content or protein content in OsAAT3 over-expressed plants. The expression patterns of the three OsAAT genes and their different functions are also discussed.     

Defence response produced during photodynamic damage in transgenic rice overexpressing 5-aminolevulinic acid synthase

Photodynamic and photoprotective responses at different irradiances were investigated in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum 5-aminolevulinic acid synthase (ALA-S). With high irradiance (HI) of 350 µmol m^?2 s^?1, transgenic lines P5 and P14 showed a decrease in contents of chlorophyll (Chl) and the chloroplast-encoded gene psbA mRNA, whereas a decrease in light-harvesting Chl-binding proteins was observed only in P14. These effects were not observed in the wild-type (WT) line treated with HI or all of the lines treated with low irradiance (LI) of 150 µmol m^?2 s^?1. HI resulted in a greater decrease in the quantum yield of photosystem 2 and a greater increase in non-photochemical quenching (NPQ) in the transgenic lines, particularly in P14, compared to WT. Photoprotective zeaxanthin contents increased at HI, even though carotenoid contents were lower in the transgenic lines compared to WT. When exposed to HI, superoxide dismutase greatly increased in transgenic lines P5 and P14, but peroxidase and glutathione reductase increased only in P14, in which more photodynamic damage occurred. Thus the greater expression of ALA-S in the transgenic plants developed the stronger protective functions, i.e. the increased values of NPQ and zeaxanthin, as well as more photodynamic reactions, i.e. decreased photosynthetic component and efficiency, in the photosynthetic complexes. However, the photodynamic reactions indicate that the antioxidant capacity was insufficient to cope with the severe stress triggered by photoactive porphyrins in the transgenic rice expressing ALA-S.     

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.     

Endosomal microautophagy is an integrated part of the autophagic response to amino acid starvation

Starvation is a fundamental type of stress naturally occurring in biological systems. All organisms have therefore evolved different safeguard mechanisms to cope with deficiencies in various types of nutrients. Cells, from yeast to humans, typically respond to amino acid starvation by initiating degradation of cellular components by inducing autophagy. This degradation releases metabolic building blocks to sustain essential core cellular processes. Increasing evidence indicates that starvation-induced autophagy also acts to prepare cells for prolonged starvation by degrading key regulators of different cellular processes. In a recent study, we found that within the first hours of amino acid starvation cells elicit an autophagic response causing rapid degradation of specific proteins. The response is executed independently of both MTOR and canonical macroautophagy. Based on RNAi-mediated knockdown of essential components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery and electron microscopy we conclude that the response relies on some sort of endosomal microautophagy, hence vesicle budding into endosomes. Substantiated by the different substrates that are selectively degraded by this novel pathway we propose that the response predominantly acts to prepare cells for prolonged starvation. Intriguingly, this includes shutting down selective macroautophagy in preparation for a massive induction of bulk macroautophagy.     

The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar-insensitive (sis) mutants of Arabidopsis that, unlike wild-type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA-biosynthesis mutant aba2 and the ABA-insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose- and mannose-insensitive phenotypes. Wild-type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.     

Incorporation of glycosylated amino acid into protein by an in vitro translation system

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.     

Alteration of ribosomal protein S5 from two spectinomycin-resistant mutants of Bacillus subtilis: deletion of one amino acid residue

Summary The ribosomal protein S5 was isolated from Bacillus subtilis ATCC 6633 (wild-type) and two spectinomycin-resistant mutants (BSPC 31 and BSPC 61). Analyses of the protein revealed that in each of these mutants one of the two consecutive arginine residues (position 29 or 30 in the wild-type) is missing.     

A novel pyridoxal 5'-phosphate-dependent amino acid racemase in the Aplysia californica central nervous system

D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.     

Excitatory amino acid receptors may mediate the effects of light on the reproductive system of the golden hamster.

Seasonal regulation of reproduction occurs in golden hamsters (Mesocricetus auratus) in response to changes in day length (photoperiod). We report here the results of experiments designed to evaluate whether excitatory amino acid neurotransmission is involved in mediating the effects of light on reproduction. We found that MK-801, a specific antagonist of the N-methyl-D-aspartate subclass of excitatory amino acid receptors, blocked the stimulatory action of short pulses of light on gonadal function in hamsters maintained in short days. Furthermore, MK-801 also inhibited the light-induced decline in pineal melatonin content. These findings suggest that excitatory amino acids may mediate the effects of light on the reproductive system.