Truncation of the calmodulin binding domain in rice glutamate decarboxylase 4 (OsGAD4) leads to accumulation of γ-aminobutyric acid and confers abiotic stress tolerance in rice seedlings.

GABA (Gamma-aminobutyric acid) is a non-protein amino acid widely known as major inhibitory neurotransmitter. It is synthesized from glutamate via the enzyme glutamate decarboxylase (GAD). GAD is ubiquitous in all organisms, but only plant GAD has ability to bind Ca2+/calmodulin (CaM). This kind of binding suppresses the auto-inhibition of Ca2+/calmodulin binding domain (CaMBD) when the active site of GAD is unfolded resulting in stimulated GAD activity. OsGAD4 is one of the five GAD genes in rice genome. It was confirmed that OsGAD4 has ability to bind to Ca2+/CaM. Moreover, it exhibits strongest expression against several stress conditions among the five OsGAD genes. In this study, CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD from the OsGAD4 gene, to remove its autoinhibitory function. DNA sequence analysis of the genome edited rice plants revealed the truncation of CaMBD (216 bp). Genome edited line (#14-1) produced 11.26 mg GABA/100 g grain, which is almost nine-fold in comparison to wild type. Short deletion in the coding region for CaMBD yielded in mutant (#14-6) with lower GABA content than wild type counterpart. Abiotic stresses like salinity, flooding and drought significantly enhanced GABA accumulation in #14-1 at various time points compared to wild-type and #14-6 under the same stress conditions. Moreover, upregulated mRNA expression in vegetative tissues seems correlated with the stress-responsiveness of OsGAD4 when exposed to the above-mentioned stresses. Stress tolerance of OsGAD4 genome edited lines was evidenced by the higher survival rate indicating the gene may induce tolerance against abiotic stresses in rice. This is the first report on abiotic stress tolerance in rice modulated by endogenous GABA. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01460-1.

Oral intake of rice overexpressing ubiquitin ligase inhibitory pentapeptide prevents atrophy in denervated skeletal muscle.

We previously reported that intramuscular injections of ubiquitin ligase CBLB inhibitory pentapeptide (Cblin; Asp-Gly-pTyr-Met-Pro) restored lost muscle mass caused by sciatic denervation. Here, we detected Cblin on the basolateral side of Caco-2 cells after being placed on the apical side, and found that cytochalasin D, a tight junction opener, enhanced Cblin transport. Orally administered Cblin was found in rat plasma, indicating that intact Cblin was absorbed in vitro and in vivo. Furthermore, transgenic Cblin peptide-enriched rice (CbR) prevented the denervation-induced loss of muscle mass and the upregulation of muscle atrophy-related ubiquitin ligases in mice. These findings indicated that CbR could serve as an alternative treatment for muscle atrophy.

Functional rice with tandemly repeated Cbl-b ubiquitin ligase inhibitory pentapeptide prevents denervation-induced muscle atrophy in vivo.

Ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) play a critical role in nonloading-mediated skeletal muscle atrophy: Cbl-b ubiquitinates insulin receptor substrate-1 (IRS-1), leading to its degradation and a resulting loss in muscle mass. We reported that intramuscular injection of a pentapeptide, DGpYMP, which acts as a mimic of the phosphorylation site in IRS-1, significantly inhibited denervation-induced skeletal muscle loss. In order to explore the possibility of the prevention of muscle atrophy by diet therapy, we examined the effects of oral administration of transgenic rice containing Cblin (Cbl-b inhibitor) peptide (DGYMP) on denervation-induced muscle mass loss in frogs. We generated transgenic rice seeds in which 15 repeats of Cblin peptides with a WQ spacer were inserted into the rice storage protein glutelin. A diet of the transgenic rice seeds had significant inhibitory effects on denervation-induced atrophy of the leg skeletal muscles in frogs, compared with those receiving a diet of wild-type rice.

An In Vivo Targeted Deletion of the Calmodulin-Binding Domain from Rice Glutamate Decarboxylase 3 (OsGAD3) Increases γ-Aminobutyric Acid Content in Grains.

BACKGROUND: Gamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca2+/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice. RESULTS: In this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca2+/CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. Out of 24 transformed rice (T1), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T2) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca2+/bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1. CONCLUSIONS: We have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

Pharmaceuticals in treated wastewater induce a stress response in tomato plants.

Pharmaceuticals remain in treated wastewater used to irrigate agricultural crops. Their effect on terrestrial plants is practically unknown. Here we tested whether these compounds can be considered as plant stress inducers. Several features characterize the general stress response in plants: production of reactive oxygen species acting as stress-response signals, MAPKs signaling cascade inducing expression of defense genes, heat shock proteins preventing protein denaturation and degradation, and amino acids playing signaling roles and involved in osmoregulation. Tomato seedlings bathing in a cocktail of pharmaceuticals (Carbamazepine, Valporic acid, Phenytoin, Diazepam, Lamotrigine) or in Carbamazepine alone, at different concentrations and during different time-periods, were used to study the patterns of stress-related markers. The accumulation of the stress-related biomarkers in leaf and root tissues pointed to a cumulative stress response, mobilizing the cell protection machinery to avoid metabolic modifications and to restore homeostasis. The described approach is suitable for the investigation of stress response of different crop plants to various contaminants present in treated wastewater.

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression.

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5'-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5'-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3'-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

Suppression of hepatic dysfunction in tenascin­X­deficient mice fed a high­fat diet.

Extracellular matrix glycoprotein tenascin­X (TNX) is the largest member of the tenascin family. In the present study, the contribution of TNX to liver dysfunction was investigated by administration of high­fat and high­cholesterol diet with high levels of phosphorus and calcium (HFCD) to wild­type (WT) and TNX­knockout (KO) mice. After 16 weeks of HFCD administration, the ratio of liver weight to body weight was approximately 22% higher in the HFCD­fed WT mice compared with the HFCD­fed TNX­KO mice, indicating hepatomegaly in HFCD­fed WT mice. Histological analyses with hematoxylin and eosin staining at 21 weeks revealed that hepatocyte hypertrophy in HFCD­fed TNX­KO mice was suppressed to 85% of that in HFCD­fed WT mice. By contrast, there was a 1.2­fold increase in lipid deposition in hepatocytes from HFCD­fed TNX­KO mice compared with HFCD­fed WT mice at 18 weeks, as demonstrated by Oil Red O staining. In addition, TNX­KO mice at 21 weeks and 27 weeks post­HFCD administration exhibited significant suppression of inflammatory cell infiltrate to 51 and 24% of that in WT mice, respectively. Immunofluorescence analysis for type I collagen and Elastica van Gieson staining demonstrated a clear hepatic fibrosis progression in HFCD­fed WT mice at 27 weeks, whereas hepatic fibrosis was undetected in HFCD­fed TNX­KO mice. The present findings indicated that TNX deficiency suppressed hepatic dysfunction induced by HFCD administration.

Ancient Plant Glyoxylate/Succinic Semialdehyde Reductases: GLYR1s Are Cytosolic, Whereas GLYR2s Are Localized to Both Mitochondria and Plastids.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2) are considered to be involved in detoxifying harmful aldehydes, thereby preserving plant health during exposure to various abiotic stresses. Phylogenetic analysis revealed that the two GLYR isoforms appeared in the plant lineage prior to the divergence of the Chlorophyta and Streptophyta, which occurred approximately 750 million years ago. Green fluorescent protein fusions of apple (Malus x domestica Borkh.), rice (Oryza sativa L.) and Arabidopsis thaliana [L.] Heynh GLYRs were transiently expressed in tobacco (Nicotiana tabaccum L.) suspension cells or Arabidopsis protoplasts, as well in methoxyfenozide-induced, stably transformed Arabidopsis seedlings. The localization of apple GLYR1 confirmed that this isoform is cytosolic, whereas apple, rice and Arabidopsis GLYR2s were localized to both mitochondria and plastids. These findings highlight the potential involvement of GLYRs within distinct compartments of the plant cell.

Field trial of GABA-fortified rice plants and oral administration of milled rice in spontaneously hypertensive rats.

Hypertension is one of the most critical risk factors accompanying cardiovascular diseases. γ-Aminobutyric acid (GABA) is a non-protein amino acid that functions as a major neurotransmitter in mammals and also as a blood-pressure lowering agent. We previously produced GABA-fortified rice lines of a popular Japonica rice cultivar 'Koshihikari' by genetic manipulation of GABA shunt-related genes. In the study reported here, we grew these same novel rice lines in a field trial and administered the milled rice orally to rats. The yield parameters of the transgenic rice plants were almost unchanged compared to those of untransformed cv. 'Koshihikari' plants, while the rice grains of the transgenic plants contained a high GABA content (3.5 g GABA/kg brown rice; 0.75-0.85 GABA g/kg milled rice) in a greenhouse trial. Oral administration of a diet containing 2.5% GABA-fortified rice, with a daily intake for 8 weeks, had an approximately 20 mmHg anti-hypertensive effect in spontaneous hypertensive rats but not in normotensive Wistar-Kyoto rats. These results suggest that GABA-fortified rice may be applicable as a staple food to control or prevent hypertension.

Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ-aminobutyric acid transaminase (GABA-T) lead to sustained and high levels of GABA accumulation in rice kernels.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T1 and T2 generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T3 generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.

The context of transcription start site regions is crucial for transcription of a plant tRNA(Lys)(UUU) gene group both in vitro and in vivo.

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5' and 3' flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5' flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a ß-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes.

Differential subcellular localization, enzymatic properties and expression patterns of γ-aminobutyric acid transaminases (GABA-Ts) in rice (Oryza sativa).

γ-Aminobutyric acid transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. The rice (Oryza sativa) genome possesses four putative GABA-T genes, which exhibit high amino acid identity (73-82%) but differ in length of the N-terminal region. Transient expression of GABA-T-green fluorescent fusion proteins in onion epidermal cells demonstrated that two of the four enzymes were targeted to mitochondria, a third to chloroplasts, and the fourth to cytosol. Enzymatic analysis of three organelle-targeted GABA-Ts revealed that they used pyruvate and glyoxylate as amino acceptors and that two of the enzymes functioned in mitochondria and chloroplasts at similar levels of activity, whereas the second mitochondrial enzyme displayed very low activity. Transcriptional analysis demonstrated that two of the four genes were more highly expressed in the vegetative organs tested, but exhibited a different pattern during seed maturation. Together, these results suggest that members of the rice GABA-T gene family vary in many respects, such as intracellular targeting, enzymatic activity and regulation of gene expression.

Accumulation mechanism of γ-aminobutyric acid in tomatoes (Solanum lycopersicum L.) under low O2 with and without CO2.

The storage of ripe tomatoes in low-O(2) conditions with and without CO(2) promotes γ-aminobutyric acid (GABA) accumulation. The activities of glutamate decarboxylase (GAD) and α-ketoglutarate-dependent GABA transaminase (GABA-TK) were higher and lower, respectively, following storage under hypoxic (2.4 or 3.5% O(2)) or adjusted aerobic (11% O(2)) conditions compared to the activities in air for 7 days at 25 °C. GAD activity was consistent with the expression level of mRNA for GAD. The GABA concentration in tomatoes stored under hypoxic conditions and adjusted aerobic conditions was 60-90% higher than that when they are stored in air on the same day. These results demonstrate that upregulation of GAD activity and downregulation of GABA-TK activity cause GABA accumulation in tomatoes stored under low-O(2) conditions. Meanwhile, the effect of CO(2) on GABA accumulation is probably minimal.

Seed-specific expression of truncated OsGAD2 produces GABA-enriched rice grains that influence a decrease in blood pressure in spontaneously hypertensive rats.

Gamma-aminobutyric acid (GABA) is a four-carbon amino acid that is commonly present in living organisms and functions as a major inhibitory neurotransmitter in mammals. It is understood to have a potentially anti-hypertensive effect in mammals. GABA is synthesized from glutamate by glutamate decarboxylase (GAD). In plants, GAD is regulated via its calmodulin-binding domain (CaMBD) by Ca(2+)/CaM. We have previously reported that a C-terminal truncated version of one of the five rice GAD isoforms, GAD2DeltaC, revealed higher enzymatic activity in vitro and that its over-expression resulted in exceptionally high GABA accumulation (Akama and Takaiwa, J Exp Bot 58:2699-2607, 2007). In this study, GAD2DeltaC, under the control of the rice glutelin promoter (GluB-1), was introduced into rice cells via Agrobacterium-mediated transformation to produce transgenic rice lines. Analysis of the free amino acid content of rice grains revealed up to about a 30-fold higher level of GABA than in non-transformed rice grains. There were also very high levels of various free protein amino acids in the seeds. GABA-enriched rice grains were milled to a fine powder for oral administration to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). Six weeks of administration showed that transgenic rice brings about a 20 mmHg decrease in blood pressure in two different kinds of SHRs, while there was no significant hypotensive effect in WKYs. These results suggest an alternative way to control and/or cure hypertension in humans with GABA-enriched rice as part of a common daily diet.

Biochemical mechanism on GABA accumulation during fruit development in tomato.

A large amount of gamma-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar 'DG03-9' which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of 'Micro-Tom' and 'DG03-9' fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of alpha-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In 'DG03-9' fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.

Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments.

Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting.

C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells.

Glutamate decarboxylase (GAD) converts L-glutamate to gamma-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca(2+)/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca(2+)/CaM. However, a truncated version of GAD2 (OsGAD2DeltaC) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2DeltaC had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2DeltaC rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo.

A survey of expressed tRNA genes in the chromosome I of Arabidopsis using an RNA polymerase III-dependent in vitro transcription system.

Eukaryotic tRNA genes are transcribed by RNA polymerase III. These tRNA genes are generally predicted using computer programs, and 620 tRNA genes in the Arabidopsis thaliana genome are currently annotated. However, no effort has been made to assay whether these predicted tRNA genes are all expressed, because it has been difficult to assay by routine in vivo methods. We report here a large-scale tRNA expression assay of predicted Arabidopsis tRNA genes using an RNA polymerase III-dependent in vitro transcription system developed by our group. DNA fragments including an annotated tRNA gene each were amplified by PCR and the resulting linear DNA was subjected to in vitro transcription. The addition of poly(dA-dT).poly(dA-dT) enhanced activity significantly and reduced background. The 124 predicted tRNA genes present in the Arabidopsis chromosome I were examined, and transcription activity and transcript stability from individual genes were determined. These results indicated that eight annotated genes are not expressed. Based on previous reports on pseudo-tRNA genes (e.g., Beier and Beier, Mol. Gen. Genet. 1992; 233: 201-208) and the present results, we estimated that 16% or more of the annotated tRNA genes in the chromosome I are not functional.

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.