Comparative Analysis of Structural Variations Due to Genome Shuffling of Bacillus Subtilis VS15 for Improved Cellulase Production.

Cellulose is one of the most abundant and renewable biomass products used for the production of bioethanol. Cellulose can be efficiently hydrolyzed by Bacillus subtilis VS15, a strain isolate obtained from decomposing logs. A genome shuffling approach was implemented to improve the cellulase activity of Bacillus subtilis VS15. Mutant strains were created using ethyl methyl sulfonate (EMS), N-Methyl-N' nitro-N-nitrosoguanidine (NTG), and ultraviolet light (UV) followed by recursive protoplast fusion. After two rounds of shuffling, the mutants Gb2, Gc8, and Gd7 were produced that had an increase in cellulase activity of 128%, 148%, and 167%, respectively, in comparison to the wild type VS15. The genetic diversity of the shuffled strain Gd7 and wild type VS15 was compared at whole genome level. Genomic-level comparisons identified a set of eight genes, consisting of cellulase and regulatory genes, of interest for further analyses. Various genes were identified with insertions and deletions that may be involved in improved celluase production in Gd7.. Strain Gd7 maintained the capability of hydrolyzing wheatbran to glucose and converting glucose to ethanol by fermentation with Saccharomyces cerevisiae of the wild type VS17. This ability was further confirmed by the acidified potassium dichromate (K2Cr2O7) method.

Effect of oxygen on the non-photochemical quenching of vascular plants and potential oxygen deficiency in the stroma of PsbS-knock-out rice.

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.

Post-transcriptional regulation of Ghd7 protein stability by phytochrome and OsGI in photoperiodic control of flowering in rice.

Rice (Oryza sativa) is a facultative short-day (SD) plant, flowering early under SD and late under long-day (LD) conditions. Ghd7 is a major regulator of flowering time in rice, which strongly delays flowering under LD. Induction of Ghd7 expression by phytochromes has been shown to contribute to photoperiodic regulation of flowering in rice. Here, we show that Ghd7 also is regulated by phytochromes at a post-transcriptional level. We found that constitutive expression of Ghd7 delays flowering in the wild-type (WT) background, but not in the se5 mutant background (deficient in functional phytochromes) under LD and that Ghd7 protein fails to accumulate in the se5 mutant. We also found that co-expressing OsGIGANTEA (OsGI) with Ghd7 causes reduced accumulation of Ghd7 protein and partially suppresses the delayed flowering phenotype in the WT background, suggesting that phytochromes and OsGI play antagonist roles in regulating Ghd7 protein stability and flowering time. We show that OsPHYA, OsPHYB and OsGI could directly interact with Ghd7. Interestingly, OsPHYA and OsPHYB could inhibit the interaction between OsGI and Ghd7, thus helping to stabilize Ghd7 protein. Our results revealed a new level of Ghd7 regulation by phytochromes and OsGI in photoperiodic control of flowering in rice.

TGACG-BINDING FACTORs (TGAs) and TGA-interacting CC-type glutaredoxins modulate hyponastic growth in Arabidopsis thaliana.

TGACG-BINDING FACTORs (TGAs) control the developmental or defense-related processes. In Arabidopsis thaliana, the functions of at least TGA2 and PERIANTHIA (PAN) can be repressed by interacting with CC-type glutaredoxins, which have the potential to control the redox state of target proteins. As TGA1 can be redox modulated in planta, we analyzed whether some of the 21 CC-type glutaredoxins (ROXYs) encoded in the Arabidopsis genome can influence TGA1 activity in planta and whether the redox active cysteines of TGA1 are functionally important. We show that the tga1 tga4 mutant and plants ectopically expressing ROXY8 or ROXY9 are impaired in hyponastic growth. As expression of ROXY8 and ROXY9 is activated upon transfer of plants from hyponasty-inducing low light to normal light, they might interfere with the growth-promoting function of TGA1/TGA4 to facilitate reversal of hyponastic growth. The redox-sensitive cysteines of TGA1 are not required for induction or reversal of hyponastic growth. TGA1 and TGA4 interact with ROXYs 8, 9, 18, and 19/GRX480, but ectopically expressed ROXY18 and ROXY19/GRX480 do not interfere with hyponastic growth. Our results therefore demonstrate functional specificities of individual ROXYs for distinct TGAs despite promiscuous protein-protein interactions and point to different repression mechanisms, depending on the TGA/ROXY combination.

Genome shuffling and high-throughput screening of Brevibacterium flavum MDV1 for enhanced L-valine production.

L-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for L-valine grows rapidly, there is an increasing interest to develop an efficient L-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield L-valine strains to replace the traditional L-valine assay. L-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six L-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an L-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of L-valine (0.598 mol L-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m3 fermentor, this strain produced > 66.8 g/L L-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of L-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.

mRNA Interactome Capture from Plant Protoplasts.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called "mRNA interactome capture," which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Optogenetics in Plants: Red/Far-Red Light Control of Gene Expression.

Optogenetic tools to control gene expression have many advantages over the classical chemically inducible systems, overcoming intrinsic limitations of chemical inducers such as solubility, diffusion, and cell toxicity. They offer an unmatched spatiotemporal resolution and permit quantitative and noninvasive control of the gene expression. Here we describe a protocol of a synthetic light-inducible system for the targeted control of gene expression in plants based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). The synthetic toggle switch system is in the ON state when plant protoplasts are illuminated with red light (660 nm) and can be returned to the OFF state by subsequent illumination with far-red light (760 nm). In this protocol, the implementation of a red light-inducible expression system in plants using Light-Emitting Diode (LED) illumination boxes is described, including the isolation and transient transformation of plant protoplasts from Arabidopsis thaliana and Nicotiana tabacum.

Photoprotection by foliar anthocyanins mitigates effects of boron toxicity in sweet basil (Ocimum basilicum).

Boron (B) toxicity is an important agricultural problem in arid environments. Excess edaphic B compromises photosynthetic efficiency, limits growth and reduces crop yield. However, some purple-leafed cultivars of sweet basil (Ocimum basilicum) exhibit greater tolerance to high B concentrations than do green-leafed cultivars. We hypothesised that foliar anthocyanins protect basil leaf mesophyll from photo-oxidative stress when chloroplast function is compromised by B toxicity. Purple-leafed 'Red Rubin' and green-leafed 'Tigullio' cultivars, grown with high or negligible edaphic B, were given a photoinhibitory light treatment. Possible effects of photoabatement by anthocyanins were simulated by superimposing a purple polycarbonate filter on the green leaves. An ameliorative effect of light filtering on photosynthetic quantum yield and on photo-oxidative load was observed in B-stressed plants. In addition, when green protoplasts from both cultivars were treated with B and illuminated through a screen of anthocyanic protoplasts or a polycarbonate film which approximated cyanidin-3-O-glucoside optical properties, the degree of photoinhibition, hydrogen peroxide production, and malondialdehyde content were reduced. The data provide evidence that anthocyanins exert a photoprotective role in purple-leafed basil mesophyll cells, thereby contributing to improved tolerance to high B concentrations.

Difference in abscisic acid perception mechanisms between closure induction and opening inhibition of stomata.

Abscisic acid (ABA) induces stomatal closure and inhibits light-induced stomatal opening. The mechanisms in these two processes are not necessarily the same. It has been postulated that the ABA receptors involved in opening inhibition are different from those involved in closure induction. Here, we provide evidence that four recently identified ABA receptors (PYRABACTIN RESISTANCE1 [PYR1], PYRABACTIN RESISTANCE-LIKE1 [PYL1], PYL2, and PYL4) are not sufficient for opening inhibition in Arabidopsis (Arabidopsis thaliana). ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant. ABA inhibition of the opening of the mutant's stomata remained intact. ABA did not induce either the production of reactive oxygen species and nitric oxide or the alkalization of the cytosol in the quadruple mutant, in accordance with the closure phenotype. Whole cell patch-clamp analysis of inward-rectifying K(+) current in guard cells showed a partial inhibition by ABA, indicating that the ABA sensitivity of the mutant was not fully impaired. ABA substantially inhibited blue light-induced phosphorylation of H(+)-ATPase in guard cells in both the mutant and the wild type. On the other hand, in a knockout mutant of the SNF1-related protein kinase, srk2e, stomatal opening and closure, reactive oxygen species and nitric oxide production, cytosolic alkalization, inward-rectifying K(+) current inactivation, and H(+)-ATPase phosphorylation were not sensitive to ABA.

Calcium-dependent protein kinase CPK6 positively functions in induction by yeast elicitor of stomatal closure and inhibition by yeast elicitor of light-induced stomatal opening in Arabidopsis.

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca(2+)-dependent signaling pathway. A Ca(2+)-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca(2+)-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca(2+) concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K(+) channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.

Analysis of the P1 promoter in response to UV-B radiation in allelic variants of high-altitude maize.

BACKGROUND: Plants living at high altitudes are typically exposed to elevated UV-B radiation, and harbor mechanisms to prevent the induced damage, such as the accumulation of UV-absorbing compounds. The maize R2R3-MYB transcription factor P1 controls the accumulation of several UV-B absorbing phenolics by activating a subset of flavonoid biosynthetic genes in leaves of maize landraces adapted to high altitudes. RESULTS: Here, we studied the UV-B regulation of P1 in maize leaves of high altitude landraces, and we investigated how UV-B regulates P1 binding to the CHS promoter in both low and high altitude lines. In addition, we analyzed whether the expansion in the P1 expression domain between these maize landraces and inbred lines is associated to changes in the molecular structure of the proximal promoter, distal enhancer and first intron of P1. Finally, using transient expression experiments in protoplasts from various maize genotypes, we investigated whether the different expression patterns of P1 in the high altitude landraces could be attributed to trans- or cis-acting elements. CONCLUSIONS: Together, our results demonstrate that, although differences in cis-acting elements exist between the different lines under study, the different patterns of P1 expression are largely a consequence of effects in trans.

Inhibitory effects of methylglyoxal on light-induced stomatal opening and inward K+ channel activity in Arabidopsis.

Methylglyoxal (MG) is a reactive aldehyde derived by glycolysis. In Arabidopsis, MG inhibited light-induced stomatal opening in a dose-dependent manner. It significantly inhibited both inward-rectifying potassium (K(in)) channels in guard-cell protoplasts and an Arabidopsis K(in) channel, KAT1, heterologously expressed in Xenopus oocytes. Thus it appears that MG inhibition of stomatal opening involves MG inhibition of K(+) influx into guard cells.

Nitric oxide inhibits blue light-induced stomatal opening by regulating the K+ influx in guard cells.

Blue light (BL)-induced stomatal opening and nitric oxide (NO)-promoted stomatal closure comprise two main aspects of stomatal regulation. Stomatal movement depends on ion fluxion in guard cells, whereas the physiological roles of BL or NO in regulating ion channel activities remain largely unknown. For gaining further insights into NO function in mediating BL-induced stomatal opening, guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that twice BL pulses (100 µmol m⁻² s⁻¹ for 30s) effectively activated inward rectifying K⁺ channels by 67% and 20% in Vicia GCPs, respectively. In contrast, Red light (RL) showed little effect on inward rectifying K⁺ channels. In accord with this, BL also increased inward K⁺ currents by 54% in Arabidopsis thaliana wild type gl1, but not in phot1-5 phot2-1 (BL receptor phototropin deletion mutant). Sodium nitroprusside (SNP, a NO donor), at 100 µM, inhibited BL-dependent K⁺ influx and stomatal opening, which were abolished by c-PTIO (a specific NO scavenger). These results indicated that NO inhibits BL-induced stomatal opening maybe through restricting the K⁺ influx across plasma membrane in guard cells.

Hybrid inflorescences derived from gamma-fusion of Arabidopsis thaliana with Bupleurum scorzonerifolium.

In our early experiments, a variety of Bupleurum scorzonerifolium-like somatic hybrid plants were obtained from protoplast fusion between Arabidopsis thaliana and UV-treated/untreated B. scorzonerifolium. To compare the effects of UV and γ-ray irradiation on the B. scorzonerifolium partner and obtain Arabidopsis-like hybrids, we designed a novel combination of somatic hybridization between A. thaliana and B. scorzonerifolium. Before protoplast isolation and fusion, the suspension cells of B. scorzonerifolium were irradiated by gamma ray ((60)Co, 50 Gy with 1.3 Gy min(-1)). Both parental protoplasts lost regeneration capacity, but over 100 somatic hybrids restored the capacity and developed to Arabidopsis-like inflorescences and flowers with some characteristics of B. scorzonerifolium. Some hybrid flowers showed yellow sepal, petal, or carpel, whose color was similar to the petal of B. scorzonerifolium; the others had silique of Arabidopsis with angularity of B. scorzonerifolium, and their parts possessed five stamens, the same as B. scorzonerifolium. Cytological analysis showed that three hybrids had Arabidopsis-like karyotypes. Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR) profiles revealed that both parental fragments were amplified from these hybrids. These results indicated chromatin introgression from B. scorzonerifolium to A. thaliana, which may be related to the complementation of hybrid inflorescence and flower generation.

Cell type-specific regulation of ion channels within the maize stomatal complex.

The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K(+)-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear. In order to bridge this gap, we performed patch-clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca(2+) and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2',7'-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined. Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.

Role of salicylic acid in alleviating photochemical damage and autophagic cell death induction of cadmium stress in Arabidopsis thaliana.

As a widespread pollutant in the environment, cadmium (Cd) would be accumulated in leaves and cause phytotoxic effect on plants. Salicylic acid (SA), a natural signal molecule, plays an important role in eliciting specific responses to biotic and abiotic stresses. In our case, the effect of SA on Cd-induced photochemical damage and cell death in Arabidopsis was studied. The results illustrated that Cd could cause a series of physiological events such as chloroplast structure change (e.g. irregular mesophyll cell as well as ultrastructure change), reactive oxygen species (ROS) production and cell death. Furthermore, chlorophyll fluorescence parameters (F(v)/F(m), qN and ETR) showed a rapid decrease in wild-type (WT) Arabidopsis after treatment with 50 µM CdCl(2), identical with the change in chlorophyll delayed fluorescence (DF) intensity. The changes of these parameters showed the damage of Cd toxicity to photosynthetic apparatus. We found that cell death might be autophagic cell death, which might be caused by Cd toxicity induced oxidative stress just like photosynthetic damage. The NahG plants with lower SA accumulation level showed more sensitivity to Cd toxicity, although they exhibited a decrease both in chlorophyll fluorescence parameters and DF intensity. Exogenously SA prevented the Cd-induced photochemical efficiency decrease and mitigated Cd toxicity. Additionally, SA pretreatment could alleviate Cd-induced ROS overproduction. In conclusion, our results suggested that SA could prevent Cd-induced photosynthetic damage and cell death, which might be due to the inhibition of ROS overproduction.

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis.

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation.

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO2. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components--ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate-glutathione (Asc-GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.

Real-time detection of caspase-3-like protease activation in vivo using fluorescence resonance energy transfer during plant programmed cell death induced by ultraviolet C overexposure.

Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the time scale of caspase-3-like protease activation was investigated in single living plant cells undergoing PCD induced by ultraviolet C (UV-C) overexposure. The real-time detection of caspase-3-like protease activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis (Arabidopsis thaliana) protoplasts. At 30 min after exposure to UV-C, caspase-3-like protease activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in the DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like protease activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western-blot analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with a mutant DEVD tag. In summary, these results represent direct evidence for the activation of caspase-3-like protease in UV-C-induced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time.