Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis.

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

MHP1 and MHL generate odd-chain fatty acids from 2-hydroxy fatty acids in sphingolipids and are related to immunity in Arabidopsis thaliana.

In plants, the 2-hydroxy fatty acids (HFAs) of sphingolipids are important for plant growth and stress responses. Although the synthetic pathway of HFAs is well understood, their degradation has not yet been elucidated. In Saccharomyces cerevisiae, Mpo1 has been identified as a dioxygenase that degrades HFAs. This study examined the functions of two homologs of yeast Mpo1, MHP1 and MHL, in Arabidopsis thaliana. The mhp1 and mhp1mhl mutants showed a dwarf phenotype compared to that of the wild type. Lipid analysis of the mutants revealed the involvement of MHP1 and MHL in synthesizing odd-chain fatty acids (OCFAs), possibly by the degradation of HFAs. OCFAs are present in trace amounts in plants; however, their physiological significance is largely unknown. RNA sequence analysis of the mhp1mhl mutant revealed that growth-related genes decreased, whereas genes involved in stress response increased. Additionally, the mhp1mhl mutant had increased expression of defense-related genes and increased resistance to infection by Pseudomonas syringae pv. tomato DC3000 (Pto), and Pto carrying the effector AvrRpt2. Phytohormone analysis demonstrated that jasmonic acid in mhp1mhl was higher than that in the wild type. These results indicate that MHP1 and MHL are involved in synthesizing OCFAs and immunity in Arabidopsis.

The effect of exogenous dihydroxyacetone and methylglyoxal on growth, anthocyanin accumulation, and the glyoxalase system in Arabidopsis.

Dihydroxyacetone (DHA) occurs in wide-ranging organisms, including plants, and can undergo spontaneous conversion to methylglyoxal (MG). While the toxicity of MG to plants is well-known, the toxicity of DHA to plants remains to be elucidated. We investigated the effects of DHA and MG on Arabidopsis. Exogenous DHA at up to 10 mm did not affect the radicle emergence, the expansion of green cotyledons, the seedling growth, or the activity of glyoxalase II, while DHA at 10 mm inhibited the root elongation and increased the activity of glyoxalase I. Exogenous MG at 1.0 mm inhibited these physiological responses and increased both activities. Dihydroxyacetone at 10 mm increased the MG content in the roots. These results indicate that DHA is not so toxic as MG in Arabidopsis seeds and seedlings and suggest that the toxic effect of DHA at high concentrations is attributed to MG accumulation by the conversion to MG.

Zinc deficiency-induced defensin-like proteins are involved in the inhibition of root growth in Arabidopsis.

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number.

Application of Trehalose Mitigates Short-Styled Flowers in Solanaceous Crops.

Trehalose is a disaccharide and is often foliar applied by farmers aiming at increasing stress resistance or crop production. However, the physiological effect of exogenously applied trehalose on crops remains obscure. Here, we explored the effect of foliar trehalose application on style length of solanaceous crops, Solanum melongena and S. lycopersicum. Trehalose application promotes pistil to stamen ratio by gaining style length. Another disaccharide consisting of two glucose molecules, maltose, showed the same effect on style length of S. lycopersicum, while monosaccharide glucose did not. Trehalose is found to affect style length through uptake via roots or interaction with rhizosphere but not through absorption by shoots in S. lycopersicum. Our study suggests that yield improvement of solanaceous crops by trehalose application under stressed conditions is brought about by suppression of the occurrence of short-styled flowers. This study suggests that trehalose holds potential to act as a plant biostimulant in preventing short-styled flowers in solanaceous crops.

Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators.

The ubiquitin-proteasome system is vital to hormone-mediated developmental and stress responses in plants. Ubiquitin ligases target hormone-specific transcriptional activators (TAs) for degradation, but how TAs are processed by proteasomes remains unknown. We report that in Arabidopsis, the salicylic acid- and ethylene-responsive TAs, NPR1 and EIN3, are relayed from pathway-specific ubiquitin ligases to proteasome-associated HECT-type UPL3/4 ligases. Activity and stability of NPR1 were regulated by sequential action of three ubiquitin ligases, including UPL3/4, while proteasome processing of EIN3 required physical handover between ethylene-responsive SCFEBF2 and UPL3/4 ligases. Consequently, UPL3/4 controlled extensive hormone-induced developmental and stress-responsive transcriptional programs. Thus, our findings identify unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases, which may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates.

Functional roles of ALMT-type anion channels in malate-induced stomatal closure in tomato and Arabidopsis.

Guard-cell-type aluminium-activated malate transporters (ALMTs) are involved in stomatal closure by exporting anions from guard cells. However, their physiological and electrophysiological functions are yet to be explored. Here, we analysed the physiological and electrophysiological properties of the ALMT channels in Arabidopsis and tomato (Solanum lycopersicum). SlALMT11 was specifically expressed in tomato guard cells. External malate-induced stomatal closure was impaired in ALMT-suppressed lines of tomato and Arabidopsis, although abscisic acid did not influence the stomatal response in SlALMT11-knock-down tomato lines. Electrophysiological analyses in Xenopus oocytes showed that SlALMT11 and AtALMT12/QUAC1 exhibited characteristic bell-shaped current-voltage patterns dependent on extracellular malate, fumarate, and citrate. Both ALMTs could transport malate, fumarate, and succinate, but not citrate, suggesting that the guard-cell-type ALMTs are dicarboxylic anion channels activated by extracellular organic acids. The truncation of acidic amino acids, Asp or Glu, from the C-terminal end of SlALMT11 or AtALMT12/QUAC1 led to the disappearance of the bell-shaped current-voltage patterns. Our findings establish that malate-activated stomatal closure is mediated by guard-cell-type ALMT channels that require an acidic amino acid in the C-terminus as a candidate voltage sensor in both tomato and Arabidopsis.

Cytokinin increases vegetative growth period by suppressing florigen expression in rice and maize.

Increasing the vegetative growth period of crops can increase biomass and grain yield. In rice (Oryza sativa), the concentration of trans -zeatin, an active cytokinin, was high in the leaves during vegetative growth and decreased rapidly upon induction of florigen expression, suggesting that this hormone is involved in the regulation of the vegetative phase. To elucidate whether exogenous cytokinin application influences the length of the vegetative phase, we applied 6-benzylaminopurine (BAP) to rice plants at various developmental stages. Our treatment delayed flowering time by 8-9 days when compared with mock-treated rice plants, but only at the transition stage when the flowering signals were produced. Our observations also showed that flowering in the paddy field is delayed by thidiazuron, a stable chemical that mimics the effects of cytokinin. The transcript levels of florigen genes Heading date 3a (Hd3a) and Rice Flowering locus T1 (RFT1) were significantly reduced by the treatment, but the expression of Early heading date 1 (Ehd1), a gene found directly upstream of the florigen genes, was not altered. In maize (Zea mays), similarly, BAP treatment increased the vegetative phage by inhibiting the expression of ZCN8, an ortholog of Hd3a. We showed that cytokinin treatment induced the expression of two type-A response regulators (OsRR1 and OsRR2) which interacted with Ehd1, a type-B response regulator. We also observed that cytokinin did not affect flowering time in ehd1 knockout mutants. Our study indicates that cytokinin application increases the duration of the vegetative phase by delaying the expression of florigen genes in rice and maize by inhibiting Ehd1.

Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in Arabidopsis thaliana.

Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces, including raindrops, to anticipate pathogen infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) and mitogen-activated protein kinases. We propose an early layer of plant immunity in which trichomes function as mechanosensory cells that detect potential risks.

Abscisic acid is required for exodermal suberization to form a barrier to radial oxygen loss in the adventitious roots of rice (Oryza sativa).

To acclimate to waterlogged conditions, wetland plants form a barrier to radial oxygen loss (ROL) that can enhance oxygen transport to the root apex. We hypothesized that one or more hormones are involved in the induction of the barrier and searched for such hormones in rice. We previously identified 98 genes that were tissue-specifically upregulated during ROL barrier formation in rice. The RiceXPro database showed that most of these genes were highly enhanced by exogenous abscisic acid (ABA). We then examined the effect of ABA on ROL barrier formation by using an ABA biosynthesis inhibitor (fluridone, FLU), by applying exogenous ABA and by examining a mutant with a defective ABA biosynthesis gene (osaba1). FLU suppressed barrier formation in a stagnant solution that mimics waterlogged soil. Under aerobic conditions, rice does not naturally form a barrier, but 24 h of ABA treatment induced barrier formation. osaba1 did not form a barrier under stagnant conditions, but the application of ABA rescued the barrier. In parallel with ROL barrier formation, suberin lamellae formed in the exodermis. These findings strongly suggest that ABA is an inducer of suberin lamellae formation in the exodermis, resulting in an ROL barrier formation in rice.

Salicylic Acid Acts Antagonistically to Plastid Retrograde Signaling by Promoting the Accumulation of Photosynthesis-associated Proteins in Arabidopsis.

Plastids are involved in phytohormone metabolism as well as photosynthesis. However, the mechanism by which plastid retrograde signals and phytohormones cooperatively regulate plastid biogenesis remains elusive. Here, we investigated the effects of an inhibitor and a mutation that generate biogenic plastid signals on phytohormones and vice versa. Inhibition of plastid biogenesis by norflurazon (NF) treatment and the plastid protein import2 (ppi2) mutation caused a decrease in salicylic acid (SA) and jasmonic acid (JA). This effect can be attributed in part to the altered expression of genes involved in the biosynthesis and the metabolism of SA and JA. However, SA-dependent induction of the PATHOGENESIS-RELATED1 gene was virtually unaffected in NF-treated plants and the ppi2 mutant. Instead, the level of chlorophyll in these plants was partially restored by the exogenous application of SA. Consistent with this observation, the levels of some photosynthesis-associated proteins increased in the ppi2 and NF-treated plants in response to SA treatment. This regulation in true leaves seems to occur at the posttranscriptional level since SA treatment did not induce the expression of photosynthesis-associated genes. In salicylic acid induction deficient 2 and lesions simulating disease resistance 1 mutants, endogenous SA regulates the accumulation of photosynthesis-associated proteins through transcriptional and posttranscriptional mechanisms. These data indicate that SA acts antagonistically to the inhibition of plastid biogenesis by promoting the accumulation of photosynthesis-associated proteins in Arabidopsis, suggesting a possible link between SA and biogenic plastid signaling.

Cadmium uptake via apoplastic bypass flow in Oryza sativa.

It is known that rice roots take up cadmium (Cd) via the symplastic route mediated by membrane-bound mineral transporters. Here we provide evidence that apoplastic bypass flow is another Cd uptake route in rice. High concentrations of Cd rendered apoplastic bypass flow rate increased in rice seedlings. These concentrations of Cd compromised membrane integrity in the root meristem and transition zone. Polyethleneglycol and proline inhibited the Cd-induced apoplastic bypass flow and Cd transfer to the shoots. Loss-of-function mutant of the Cd uptake transporter, nramp5, showed Cd transport to the shoot comparable to the wild type. At a low Cd concentration, increased apoplastic bypass flow rate by NaCl stress resulted in an elevation of Cd transport to shoots both in the wildtype and nramp5. These observations indicate that apoplastic bypass flow in roots carries Cd transport leading to xylem loading of Cd in addition to the symplastic pathway mediated by mineral transporters under stressed conditions.

Possible roles for phytohormones in controlling the stomatal behavior of Mesembryanthemum crystallinum during the salt-induced transition from C3 to crassulacean acid metabolism.

The halophyte ice plant (Mesembryanthemum crystallinum) converts its mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) during severe water stress. During the transition to CAM, the plant induces CAM-related genes and changes its diurnal stomatal behavior to take up CO2 efficiently at night. However, limited information concerning this signaling exists. Here, we investigated the changes in the diurnal stomatal behavior of M. crystallinum during its shift in photosynthesis using a detached epidermis. M. crystallinum plants grown under C3 conditions opened their stomata during the day and closed them at night. However, CAM-induced plants closed their stomata during the day and opened them at night. Quantitative analysis of endogenous phytohormones revealed that trans-zeatin levels were high in CAM-induced plants. In contrast, the levels of jasmonic acid (JA) and JA-isoleucine were severely reduced in CAM-induced plants, specifically at night. CAM induction did not alter the levels of abscisic acid; however, inhibitors of abscisic acid synthesis suppressed CAM-induced stomatal closure. These results indicate that M. crystallinum regulates the diurnal balance of cytokinin and JA during CAM transition to alter stomatal behavior.

Large magnitude of force leads to NO-mediated cell shrinkage in single osteocytes implying an initial apoptotic response.

Damage accumulation in the bone under continuous daily loading causes local mechanical overloading known to induce osteocyte apoptosis, which promotes bone resorption to repair bone damage. However, only a few studies have investigated the mechanism of apoptosis in mechanically overloaded osteocytes. As mechanically stimulated osteocytes produce nitric oxide (NO), which triggers apoptosis in various cell types, we aimed to elucidate the mechanism underlying apoptosis in mechanically overloaded osteocytes, focusing on intracellular NO. To investigate the effects of force magnitude on apoptosis and intracellular NO production, we isolated osteocytes from DMP1-EGFP mice and subjected them to quantitative local forces via fibronectin-coated micro beads targeting integrin on the cell surface using a magnetic tweezer. Cell shrinkage was microscopically examined, and intracellular NO production was visualized using DAR-4 M. Mechanical stimulation revealed relationships between force magnitude, apoptosis, and intracellular NO production. The application of a smaller force resulted in no significant cell shrinkage or intracellular NO production; however, a larger force caused a rapid increase in intracellular NO production followed by cell shrinkage. Besides, intracellular NOS (NO synthase) inhibition and NO donation revealed the pro-apoptotic roles of NO in osteocytes. L-NAME (NOS inhibitor)-treated cells displayed no significant shrinkage under a larger force, whereas SNP (NO donor)-treated cells showed cell shrinkage and Annexin V fluorescence, indicating apoptosis. Collectively, our study demonstrates that larger force leads to NO production-mediated osteocyte shrinkage, implying an initial apoptotic response and highlighting the importance of NO production in bone damage.

[Diffuse cerebral atrophy and reversible polyneuropathy in a patient with chronic Bromvalerylurea intoxication: a case report].

A 37-year-old man who had been on bromvalerylurea (BU) medication for 11 years at a maximum dose of 2,400 mg per day for headache therapy was admitted to our hospital due to gait disturbance. He had weight loss and exanthema all over his body. Cognitive dysfunction, intellectual deterioration, attention disturbance, decreased muscle strength, and decreased vibratory sense in the lower limbs were observed. Brain MRI showed diffuse brain atrophy, and a peripheral nerve conduction examination revealed decreased nerve conduction velocity and action potential amplitude in the extremities. We diagnosed him with chronic BU intoxication based on pseudohyperchloremia, BU detected in the blood, and bromide elevation. By discontinuing BU and performing intravenous infusion, neurological symptoms and exanthema were improved, and peripheral nerve conduction examination findings also improved. There are few reports of peripheral neuropathy cases of chronic BU intoxication; herein we report one such case along with previously reported cases.

BdWRKY38 is required for the incompatible interaction of Brachypodium distachyon with the necrotrophic fungus Rhizoctonia solani.

Rhizoctonia solani is a soil-borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3-1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession-specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3-1 and Tek-3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY-dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling.

Manganese Treatment Alleviates Zinc Deficiency Symptoms in Arabidopsis Seedlings.

Plant phenotypes caused by mineral deficiencies differ depending on growth conditions. We recently reported that the growth of Arabidopsis thaliana was severely inhibited on MGRL-based zinc (Zn)-deficient medium but not on Murashige-Skoog-based Zn-deficient medium. Here, we explored the underlying reason for the phenotypic differences in Arabidopsis grown on the different media. The root growth and chlorophyll contents reduced by Zn deficiency were rescued by the addition of extra manganese (Mn) during short-term growth (10 or 14 d). However, this treatment did not affect the growth recovery after long-term growth (38 d). To investigate the reason for plant recovery from Zn deficiency, we performed the RNA-seq analysis of the roots grown on the Zn-basal medium and the Zn-depleted medium with/without additional Mn. Principal component analysis of the RNA-seq data showed that the gene expression patterns of plants on the Zn-basal medium were similar to those on the Zn-depleted medium with Mn, whereas those on the Zn-depleted medium without Mn were different from the others. The expression of several transcription factors and reactive oxygen species (ROS)-related genes was upregulated in only plants on the Zn-depleted medium without Mn. Consistent with the gene expression data, ROS accumulation in the roots grown on this medium was higher than those grown in other conditions. These results suggest that plants accumulate ROS and reduce their biomass under undesirable growth conditions, such as Zn depletion. Taken together, this study shows that the addition of extra Mn to the Zn-depleted medium induces transcriptional changes in ROS-related genes, thereby alleviating short-term growth inhibition due to Zn deficiency.

Low temperature modulates natural peel degreening in lemon fruit independently of endogenous ethylene.

Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit (Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 ºC, moderately low storage temperatures of 5-20 °C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit.

Interaction of intracellular hydrogen peroxide accumulation with nitric oxide production in abscisic acid signaling in guard cells.

Reactive oxygen species and nitric oxide (NO•) concomitantly play essential roles in guard cell signaling. Studies using catalase mutants have revealed that the inducible and constitutive elevations of intracellular hydrogen peroxide (H2O2) have different roles: only the inducible H2O2 production transduces the abscisic acid (ABA) signal leading stomatal closure. However, the involvement of inducible or constitutive NO• productions, if exists, in this process remains unknown. We studied H2O2 and NO• mobilization in guard cells of catalase mutants. Constitutive H2O2 level was higher in the mutants than that in wild type, but constitutive NO• level was not different among lines. Induced NO• and H2O2 levels elicited by ABA showed a high correlation with each other in all lines. Furthermore, NO• levels increased by exogenous H2O2 also showed a high correlation with stomatal aperture size. Our results demonstrate that ABA-induced intracellular H2O2 accumulation triggers NO• production leading stomatal closure. ABBREVIATIONS: ABA: abscisic acid; CAT: catalase; cGMP: cyclic guanosine monophosphate; DAF-2DA: 4,5-diaminofluorescein-2 diacetate; H2DCF-DA: 2',7'-dichlorodihydrofluorescein diacetate; MeJA: methyljasmonate; NOS: nitric oxide synthetase; NR: nitrate reductase; POX: peroxidase; ROS: reactive oxygen species; SNAP: S-nitroso-N-acetyl-DL-penicillamine; SNP: sodium nitroprusside; NOX: NADP(H) oxidase.

Application of the cellular oxidation biosensor to Toxicity Identification Evaluations for high-throughput toxicity assessment of river water.

Toxicity Identification Evaluation (TIE) is a useful method for the classification and identification of toxicants in a composite environment water sample. However, its extension to a larger sample size has been restrained owing to the limited throughput of toxicity bioassays. Here we reported the development of a high-throughput method of TIE Phase I. This newly developed method was assisted by the fluorescence-based cellular oxidation (CO) biosensor fabricated with roGFP2-expressing bacterial cells in 96-well microplate format. The assessment of four river water samples from Langat river basin by this new method demonstrated that the contaminant composition of the four samples can be classified into two distinct groups. The entire toxicity assay consisted of 2338 tests was completed within 12 h with a fluorescence microplate reader. Concurrently, the sample volume for each assay was reduced to 50 µL, which is 600 to 4700 times lesser to compare with conventional bioassays. These imply that the throughput of the CO biosensor-assisted TIE Phase I is now feasible for constructing a large-scale toxicity monitoring system, which would cover a whole watershed scale.