Seawater fish use an electrogenic boric acid transporter, Slc4a11A, for boric acid excretion by the kidney.

Boric acid is a vital micronutrient in animals; however, excess amounts are toxic to them. Little is known about whole-body boric acid homeostasis in animals. Seawater (SW) contains 0.4 mM boric acid, and since marine fish drink SW, their urinary system was used here as a model of the boric acid excretion system. We determined that the bladder urine of a euryhaline pufferfish (river pufferfish, Takifugu obscurus) acclimated to fresh water and SW contained 0.020 and 19 mM of boric acid, respectively (a 950-fold difference), indicating the presence of a powerful excretory renal system for boric acid. Slc4a11 is a potential animal homolog of the plant boron transporter BOR1; however, mammalian Slc4a11 mediates H+ (OH-) conductance but does not transport boric acid. We found that renal expression of the pufferfish paralog of Slc4a11, Slc4a11A, was markedly induced after transfer from fresh water to SW, and Slc4a11A was localized to the apical membrane of kidney tubules. When pufferfish Slc4a11A was expressed in Xenopus oocytes, exposure to media containing boric acid and a voltage clamp elicited whole-cell outward currents, a marked increase in pHi, and increased boron content. In addition, the activity of Slc4a11A was independent of extracellular Na+. These results indicate that pufferfish Slc4a11A is an electrogenic boric acid transporter that functions as a B(OH)4- uniporter, B(OH)3-OH- cotransporter, or B(OH)3/H+ exchanger. These observations suggest that Slc4a11A is involved in the kidney tubular secretion of boric acid in SW fish, probably induced by the negative membrane potential and low pH of urine.

Boron-dependent regulation of translation through AUGUAA sequence in yeast.

Under high boron (B) conditions, nodulin 26-like intrinsic protein 5;1 (NIP5;1) mRNA, a boric acid channel, is destabilized to avoid excess B entry into roots of Arabidopsis thaliana. In this regulation, the minimum upstream open reading frame (uORF), AUGUAA, in its 5'-untranslated region (5'-UTR) is essential, and high B enhances ribosome stalling at AUGUAA and leads to suppression of translation and mRNA degradation. This B-dependent AUGUAA-mediated regulation occurs also in animal transient expression and reticulocyte lysate translation systems. Thus, uncovering the ubiquitousness of B-dependent unique regulation is important to reveal the evolution of translational regulation. In the present study, we examined the regulation in Saccharomyces cerevisiae. Reporter assay showed that in yeast, carrying ATGTAA in 5'-UTR of NIP5;1 upstream of the reporter gene, the relative reporter activities were reduced significantly under high B conditions compared with control, whereas deletion of ATGTAA abolished such responses. This suggests that AUGUAA mediates B-dependent regulation of translation in Saccharomyces cerevisiae. Moreover, the deletion of ATGTAA resulted in up to 10-fold increase in general reporter activities indicating the suppression effect of AUGUAA on translation of the main ORF. Interestingly, mRNA level of the reporter gene was not affected by B in both yeast cells with and without AUGUAA. This finding reveals that in yeast, unlike the case in plants, mRNA degradation is not associated with AUGUAA regulation. Together, results suggest that B-dependent AUGUAA-mediated translational regulation is common among eukaryotes.

Boron-Dependent Translational Suppression of the Borate Exporter BOR1 Contributes to the Avoidance of Boron Toxicity.

Boron (B) is an essential element for plants; however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (Arabidopsis thaliana) that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is down-regulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism. In vivo and in vitro reporter assays demonstrated that BOR1 translation was reduced in a B-dependent manner and that the 5'-untranslated region was both necessary and sufficient for this process. Mutational analysis revealed that multiple upstream open reading frames in the 5'-untranslated region were required for BOR1 translational suppression, and this process depended on the efficiency of translational reinitiation at the BOR1 open reading frame after translation of the upstream open reading frames. To understand the physiological significance of BOR1 regulation, we characterized transgenic plants defective in either one or both of the BOR1 regulation mechanisms. BOR1 translational suppression was induced at higher B concentrations than those triggering BOR1 degradation. Plants lacking both regulation mechanisms exhibited more severe shoot growth reduction under high-B conditions than did plants lacking BOR1 degradation alone, thus demonstrating the importance of BOR1 translational suppression. This study demonstrates that two mechanisms of posttranscriptional BOR1 regulation, each induced under different B concentrations, contribute to the avoidance of B toxicity in plants.

Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose.

Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two ß-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.

The effects of administering lactic acid bacteria sealed in a capsule on the intestinal bacterial flora of cattle.

We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 X 10¹¹ colony forming unit (cfu) of freeze-dried Lactobacillus coryniformis subsp. torquens (JCM1099) encapsulated in an enteric capsule capable of bypassing the rumen was administered for seven days. DNA was extracted from feces 0 and 24 hr after daily administration. Metagenomic analysis showed an increasing trend of the alpha diversity, an index of the species diversity. Furthermore, principal component analysis of intestinal flora revealed that cattle could be differentiated by JCM1099 capsule and suspension administration via principal components 1, 2, and 3. We conclude that administration of encapsulated JCM1099 can alter the intestinal bacterial flora of cattle.

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012.

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.

Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains.

Accumulation of cadmium (Cd) in rice (Oryza sativa L.) grains poses a potential health problem, especially in Asia. Most Cd in rice grains accumulates through phloem transport, but the molecular mechanism of this transport has not been revealed. In this study, we identified a rice Cd transporter, OsLCT1, involved in Cd transport to the grains. OsLCT1-GFP was localized at the plasma membrane in plant cells, and OsLCT1 showed Cd efflux activity in yeast. In rice plants, strong OsLCT1 expression was observed in leaf blades and nodes during the reproductive stage. In the uppermost node, OsLCT1 transcripts were detected around large vascular bundles and in diffuse vascular bundles. RNAi-mediated knockdown of OsLCT1 did not affect xylem-mediated Cd transport but reduced phloem-mediated Cd transport. The knockdown plants of OsLCT1 accumulated approximately half as much Cd in the grains as did the control plants. The content of other metals in rice grains and plant growth were not negatively affected by OsLCT1 suppression. These results suggest that OsLCT1 functions at the nodes in Cd transport into grains and that in a standard japonica cultivar, the regulation of OsLCT1 enables the generation of "low-Cd rice" without negative effects on agronomical traits. These findings identify a transporter gene for phloem Cd transport in plants.

Roles of pollen-specific boron efflux transporter, OsBOR4, in the rice fertilization process.

Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.

Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways.

Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.

Long-Term Dietary Fish Meal Substitution with the Black Soldier Fly Larval Meal Modifies the Caecal Microbiota and Microbial Pathway in Laying Hens.

Feeding laying hens with black soldier fly larval (BSFL) meal improves their performance. However, the beneficial mechanism of BSFL meals in improving the performance of laying hens remains unclear. This study investigated the effects of the BSFL diet on liver metabolism, gut physiology, and gut microbiota in laying hens. Eighty-seven Julia hens were randomly assigned to three groups based on their diets and fed maize grain-and soybean meal-based diets mixed with either 3% fish meal (control diet), 1.5% fish and 1.5% BSFL meals, or 3% BSFL meal for 52 weeks. No significant differences were observed in biochemical parameters, hepatic amino acid and saturated fatty acid contents, intestinal mucosal disaccharidase activity, and intestinal morphology between BSFL diet-fed and control diet-fed laying hens. However, the BSFL diet significantly increased the abundance of acetic and propionic acid-producing bacteria, caecal short-chain fatty acids, and modified the caecal microbial pathways that are associated with bile acid metabolism. These findings indicate that consuming a diet containing BSFL meal has minimal effects on plasma and liver nutritional metabolism in laying hens; however, it can alter the gut microbiota associated with short-chain fatty acid production as well as the microbial pathways involved in intestinal fat metabolism. In conclusion, this study provides evidence that BSFL can enhance enterocyte metabolism and gut homeostasis in laying hens.

Boric acid transport activity of marine teleost aquaporins expressed in Xenopus oocytes.

Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin-like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana.

Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.

VvBOR1, the grapevine ortholog of AtBOR1, encodes an efflux boron transporter that is differentially expressed throughout reproductive development of Vitis vinifera L.

Boron (B) is an essential micronutrient for normal development of roots, shoots and reproductive tissues in plants. Due to its role in the structure of rhamnogalacturonan II, a polysaccharide required for pollen tube growth, B deficiency has been associated with the occurrence of parthenocarpic seedless grapes in some varieties of Vitis vinifera L. Despite that, it is unclear how B is mobilized and accumulated in reproductive tissues. Here we describe the characterization of an efflux B transporter, VvBOR1, homolog to AtBOR1, which is involved in B xylem loading in Arabidopsis thaliana roots. VvBOR1-green fluorescent protein (GFP) fusion protein expressed in A. thaliana localizes in the proximal plasma membrane domain in root pericycle cells, and VvBOR1 overexpression restores the wild-type phenotype in A. thaliana bor1-3 mutant plants exposed to B deficiency. Complementation of a mutant yeast strain indicates that VvBOR1 corresponds to a B efflux transporter. Transcriptional analyses during grapevine reproductive development show that the VvBOR1 gene is preferentially expressed in flowers at anthesis and a direct correlation between the expression pattern and B content in grapes was established, suggesting the involvement of this transporter in B accumulation in grapevine berries.

Egg quality and laying performance of Julia laying hens fed with black soldier fly (Hermetia illucens) larvae meal as a long-term substitute for fish meal.

The use of insects in animal feed appears to be an efficient approach that contributes to solving the environmental issues related to leftover disposal; however, it has not been approved in some countries due to concerns about pathogenic infections. This study aimed to evaluate the feasibility of long-term substitution of fish meal in poultry feed with organic defatted black soldier fly larvae (BSFL) meal prepared from BSFL raised on leftovers. The 87 Julia laying hens (178-day-old) were allotted in a completely randomized design with three treatments (29 layers in each treatment). The laying hens were fed maize grain and soybean meal-based diet containing either 3% fish meal, 1.5% fish meal and 1.5% BSFL meal, or 3% BSFL meal supplements for 52 wk (541-day-old). Results showed that substituting fish meal with BSFL meal had no effect on the laying rate, feed intake, and feed conversion ratio of laying hens, and only the complete replacement (3% BSFL meal) significantly increased the body weight of laying hens. In terms of egg quality, there was no significant effect on eggshell parameters (weight, thickness, and strength), albumen weight, yolk height, yolk color, and Haugh unit. However, both half (1.5% fish meal and 1.5% BSFL meal) and complete substitution of fish meal increased yolk weight (P < 0.01) and egg weight (P < 0.05). In conclusion, even if BSFL were fed leftovers and the meal was defatted with organic solvents, it can be used as a poultry feed ingredient without any adverse effect. Moreover, the complete substitution of fish meal with BSFL meal may be a feasible way to effectively contribute to the laying hens' performances and poultry farming costs. In addition to fish meal, the replacement of soybean meal with BSFL meal may also needs to be further studied for the extensive BSFL meal application in poultry feed.

Lactate released from human fibroblasts enhances Ni elution from Ni plate.

Elution of Ni ions from medical devices induces inflammation and toxicity. We previously reported that elution of Ni ions from Ni wires induced COX-2 expression and increased lactate production, but whether lactate is involved in the further elution of Ni ions remains unclear. In this study, using KMST-6, a human fibroblast cell line, we examined the molecular mechanisms by which Ni ions increase lactate release and the role of lactate in enhancing the elution of Ni ions. When KMST-6 cells were incubated on a Ni plate or stimulated with NiCl2 (1 mM), the expression of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), and the release of lactate were enhanced. The NiCl2 (1 mM)-induced expression of these genes was inhibited by a hypoxia-inducible factor-1α (HIF-1α) inhibitor, PX-478 (10-25 µM). Stimulation of cells with a prolyl hydroxylase domain (PHD) inhibitor, roxadustat, increased the expression of these genes, lactate release, and elution of Ni ions at 10 µM. A monocarboxylate transporter-4 (MCT4) inhibitor, syrosingopine, inhibited lactate release from roxadustat-treated cells and reduced the elution of Ni ions by the cells at 10 µM. Finally, syrosingopine (10 µM) reduced the elution of Ni ions by the cells from the Ni plate. These results suggest that elution of Ni ions from metals promotes the production of lactate via HIF-1α-mediated gene expression and causes further Ni elution. Thus, Ni ions show a positive feedback mechanism of Ni elution, and this step may be potentially targeted to protect against metal elution from metal devices.

Evaluation of Black Soldier Fly (Hermetia illucens) Larvae and Pre-Pupae Raised on Household Organic Waste, as Potential Ingredients for Poultry Feed.

Black soldier fly (BSF) larvae and pre-pupae could be satisfactorily raised on household organic waste and used as poultry feed, offering a potential sustainable way to recycle untapped resources of waste. The present study was conducted to determine if whole (non-defatted) BSF larvae and pre-pupae raised on experimental household waste could substitute soybean meal and oil as ingredients for laying hen diets. While no significant differences in feed intake and the egg-laying rate of hens were observed throughout the experiment, egg weight and eggshell thickness were greater in the pre-pupae-fed group than in the other groups. Moreover, although diversity of the cecal microbiota was significantly higher in the pre-pupae-fed than in the control group, no significant differences in bacterial genera known to cause food poisoning were observed when comparing the treatment groups. Nonetheless, Lactobacillus and Bifidobacterium populations were significantly lower in the treatment than in the control group. Fat content in BSF was possibly related with the changes in the cecal microbiota. Hence, since BSF fat was deficient in essential fatty acids, special attention should be paid to the fat content and its fatty acid composition in the case of regular inclusion of BSF larvae and pre-pupae oil as an ingredient in poultry diets.

Nickel ions bind to HSP90ß and enhance HIF-1α-mediated IL-8 expression.

Nickel ions (Ni2+) eluted from biomedical devices cause inflammation and Ni allergy. Although Ni2+ and Co2+ elicit common effects, Ni2+ induces a generally stronger inflammatory reaction. However, the molecular mechanism by which Ni2+ and Co2+ induce such different responses remains to be elucidated. In the present study, we compared the effects of Ni2+ and Co2+ on the expression of interleukin (IL)-8 in human monocyte THP-1 cells. We report that NiCl2 but not CoCl2 induced the expression of IL-8; in contrast, CoCl2 elicited a higher expression of hypoxia-inducible factor-1α (HIF-1α). The NiCl2-induced expression of IL-8 in late phase was blocked by a HIF-1α inhibitor, PX-478, indicating that NiCl2 targets additional factors responsible for activating HIF-1α. To identify such targets, proteins that bound preferentially to Ni-NTA beads were analyzed by LC/MS/MS. The analysis yielded heat shock protein 90ß (HSP90ß) as a possible candidate. Furthermore, Ni2+ reduced the interaction of HSP90ß with HIF-1α, and instead promoted the interaction between HIF-1α and HIF-1ß, as well as the nuclear localization of HIF-1α. Using various deletion variants, we showed that Ni2+ could bind to the linker domain on HSP90ß. These results suggest that HSP90ß plays important roles in Ni2+-induced production of IL-8 and could be a potential target for the regulation of Ni2+-induced inflammation.

Excretion rates of indigestible plastic balls of different specific gravities and diameters in dairy cattle.

We used plastic balls to investigate how their specific gravity and diameter affect excretion rate and rumination in dairy cattle, to develop a capsule that can be used for reaching the lower gastrointestinal tract without physical breakdown and/or degradation in the rumen. Twelve types of indigestible plastic balls composed of a combination of four specific gravities (0.95, 1.19, 1.41, or 2.20) and three diameters (3.97, 6.35, or 7.94 mm) were orally administered to lactating dairy cows, and the balls were collected from feces, after 120 h post-administration, to evaluate the recovery rate. Recovery rate of the balls with specific gravity 1.19 or 1.41 and diameter 6.35 or 7.94 mm was higher than those with specific gravity 0.95 or 2.20 and diameter 3.97 mm. The cumulative recovery rate at 24 and 48 h post-administration was higher for balls with specific gravity 1.19 than that for balls with other specific gravities. These results suggest that specific gravity 1.19 or 1.41 and diameters 6.35-7.94 mm are optimal for use in bypass capsules for administration to cattle. In addition, the passage time of capsules differed between specific gravities 1.19 and 1.41.

Development of three-layered rumen escapable capsules for cattle.

A new rumen escapable tool is presented for cattle in prospect of developing medical treatment or supplementing trace elements for disease prevention. This tool consists of a three-layered capsule that dissolves in the lower digestive tract, but not in the rumen. The capsule was manufactured by capsule-forming techniques through the use of liquid surface tension. This method does not involve high-temperature treatment, so the capsule can contain not only lipophilic substances but also hydrophilic or heat-sensitive substances. Furthermore, the capsule has a specific gravity of 1.3 and diameter of 6.0 mm, which were previously shown to be appropriate to avoid rumination. The objective of this study was to confirm the effectiveness of the capsule pertinent to rumen escaping. In order to validate rumen escape, capsules containing 30 g of water-soluble vitamin (thiamine hydrochloride) per head were administered to four lactating cows assigned in a crossover trial. In the group administered encapsulated thiamine hydrochloride, blood thiamine levels increased from 12.4 ± 1.03 ng/ml before administration to 54.8 ± 2.21 ng/ml at 6 hr following administration, whereas the level remained at 13.3 ± 2.05 ng/ml in the control group administered via aqueous solution. This indicates that the three-layered capsules passed through the rumen and dissolved in the lower digestive tract, thus functioning as a rumen escapable tool.

Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline.

Chloroplasts possess bacterial-type systems for transcription and translation. On the basis of the identification of a Chlamydomonas reinhardtii gene encoding a RelA-SpoT homolog (RSH) that catalyzes the synthesis of guanosine tetra- or pentaphosphate [(p)ppGpp], we have previously suggested the operation of stringent control in the chloroplast genetic system. Although RSH genes have also been identified in several higher plants, the activities of the encoded enzymes and their mode of action in chloroplasts have remained uncharacterized. We have now characterized the intrinsic (p)ppGpp synthase activity of chloroplast extracts prepared from pea (Pisum sativum). Fractionation by ultracentrifugation suggested that the (p)ppGpp synthase activity of a translationally active chloroplast stromal extract was associated with 70S ribosomes. Furthermore, this enzymatic activity was inhibited by tetracycline, as was the peptide elongation activity of the extract. Structural comparisons between rRNA molecules of Escherichia coli and pea chloroplasts revealed the conservation of putative tetracycline-binding sites. These observations demonstrate the presence of a ribosome-associated (p)ppGpp synthase activity in the chloroplasts of a higher plant, further implicating (p)ppGpp in a genetic system of chloroplasts similar to that operative in bacteria.