Insecticidal action of mammalian galectin-1-transfected Arabidopsis thaliana.

BACKGROUND: Galectins (GALs) are a family of mammalian sugar-binding proteins specific for ß-galactosides. Our previous studies have shown that the larval development of the diamondback moth (Plutella xylostella) is significantly disturbed when fed with recombinant mammalian galectin 1 (GAL1) derived from Escherichia coli. To further explore its applicability, two GAL1-overexpressed Arabidopsis [GAL1-Arabidopsis (whole plant) and GAL1-Arabidopsis-vas (vascular bundle-specific)] lines were established for insecticidal activity and mechanism studies. RESULTS: The expression level of GAL1 in transgenic Arabidopsis is 1-0.5% (GAL1-Arabidopsis) and 0.08-0.01% (GAL1-Arabidopsis-vas) of total leaf soluble protein. Survival, body weight, and food consumption significantly decreased in a time-dependent manner in P. xylostella larvae (with chewing mouthparts) fed on GAL1-Arabidopsis. The mortality of Kolla paulula (with piercing-sucking mouthparts and xylem feeder) fed on GAL1-Arabidopsis-vas was also significantly higher than that fed on wild-type Arabidopsis (WT-Arabidopsis), but was lower than that fed on GAL1-Arabidopsis. The histochemical structure and results of immunostaining suggested that the binding of GAL1 to the midgut epithelium of P. xylostella fed on GAL1-Arabidopsis was dose- and time-dependent. Ultrastructural studies further showed the disruption of microvilli, abnormalities in epithelial cells, and fragments of the peritrophic membrane (PM) in P. xylostella larvae fed on GAL1-Arabidopsis. CONCLUSION: The insecticidal mechanism of GAL1 involves interference with PM integrity and suggests that GAL1 is a potential candidate for bioinsecticide development. © 2024 Society of Chemical Industry.

A plant protein farnesylation system in prokaryotic cells reveals Arabidopsis AtJ3 produced and farnesylated in E. coli maintains its function of protecting proteins from heat inactivation.

BACKGROUND: Protein farnesylation involves the addition of a 15-carbon polyunsaturated farnesyl group to proteins whose C-terminus ends with a CaaX motif. This post-translational protein modification is catalyzed by a heterodimeric protein, i.e., farnesyltransferase (PFT), which is composed of an α and a ß subunit. Protein farnesylation in plants is of great interest because of its important roles in the regulation of plant development, responses to environmental stresses, and defense against pathogens. The methods traditionally used to verify whether a protein is farnesylated often require a specific antibody and involve isotope labeling, a tedious and time-consuming process that poses hazardous risks. RESULTS: Since protein farnesylation does not occur in prokaryotic cells, we co-expressed a known PFT substrate (i.e., AtJ3) and both the α and ß subunits of Arabidopsis PFT in E. coli in this study. Farnesylation of AtJ3 was detected using electrophoretic mobility using SDS-PAGE and confirmed using mass spectrometry. AtJ3 is a member of the heat shock protein 40 family and interacts with Arabidopsis HSP70 to protect plant proteins from heat-stress-induced denaturation. A luciferase-based protein denaturation assay demonstrated that farnesylated AtJ3 isolated from E. coli maintained this ability. Interestingly, farnesylated AtJ3 interacted with E. coli HSP70 as well and enhanced the thermotolerance of E. coli. Meanwhile, AtFP3, another known PFT substrate, was farnesylated when co-expressed with AtPFTα and AtPFTß in E. coli. Moreover, using the same strategy to co-express rice PFT α and ß subunit and a potential PFT target, it was confirmed that OsDjA4, a homolog of AtJ3, was farnesylated. CONCLUSION: We developed a protein farnesylation system for E. coli and demonstrated its applicability and practicality in producing functional farnesylated proteins from both mono- and dicotyledonous plants.

Overexpression of OsHsp 18.0 in rice enhanced tolerance to heavy metal stress.

KEY MESSAGE: OsHsp18.0 plays a key role in cross-protection of rice seedlings from damages to photochemical systems and cellular membranes, caused by Cd and Cu stresses.

Environmental Enrichment Components Required to Reduce Methamphetamine-Induced Behavioral Sensitization in Mice: Examination of Behaviors and Neural Substrates.

Environmental enrichment (EE) involves the presentation of various sensory, physical, social, and cognitive stimuli in order to alter neural activity in specific brain areas, which can ameliorate methamphetamine (MAMPH)-induced behavioral sensitization and comorbid anxiety symptoms. No previous studies have comprehensively examined which EE components are critical for effectively reducing MAMPH-induced behavioral sensitization and anxiety. This study examined different housing conditions, including standard housing (SH, No EE), standard EE (STEE), physical EE (PEE), cognitive EE (CEE), and social EE (SEE). In the beginning, mice were randomly assigned to the different combinations of housing conditions and injections, consisting of No EE/Saline, No EE/MAMPH, STEE/MAMPH, PEE/MAMPH, CEE/MAMPH, and SEE/MAMPH groups. Then, the mice received intraperitoneal injections of 1 mg/kg MAMPH or normal saline daily for 7 days, followed by a final injection of 0.5 mg/kg MAMPH or normal saline. After behavioral tests, all mice were examined for c-Fos immunohistochemical staining. The results showed that MAMPH induced behavioral sensitization as measured by distance traveled. MAMPH appeared to induce lowered anxiety responses and severe hyperactivity. All EE conditions did not affect MAMPH-induced lowered anxiety behaviors. STEE was likely more effective for reducing MAMPH-induced behavioral sensitization than PEE, CEE, and SEE. The c-Fos expression analysis showed that the medial prefrontal cortex (i.e., cingulate cortex 1 (Cg1), prelimbic cortex (PrL), and infralimbic cortex (IL)), nucleus accumbens (NAc), basolateral amygdala (BLA), ventral tegmental area (VTA), caudate-putamen (CPu), and hippocampus (i.e., CA1, CA3, and dentate gyrus (DG)) contributed to MAMPH-induced behavioral sensitization. The Cg1, IL, NAc, BLA, VTA, CPu, CA3, and DG also mediated STEE reductions in MAMPH-induced behavioral sensitization. This study indicates that all components of EE are crucial for ameliorating MAMPH-induced behavioral sensitization, as no individual EE component was able to effectively reduce MAMPH-induced behavioral sensitization. The present findings provide insight into the development of non-pharmacological interventions for reducing MAMPH-induced behavioral sensitization.

HSP70-4 and farnesylated AtJ3 constitute a specific HSP70/HSP40-based chaperone machinery essential for prolonged heat stress tolerance in Arabidopsis.

AtJ3 (J3)-a member of the Arabidopsis cytosolic HSP40 family-harbors a C-terminal CaaX motif for farnesylation, which is exclusively catalyzed by protein farnesyltransferase (PFT). Previously, prolonged incubation at 37 °C for 4 d was found to be lethal to the heat-intolerant 5 (hit5) mutant lacking PFT and transgenic j3 plants expressing a CaaX-abolishing J3C417S construct, indicating that farnesylated J3 is essential for heat tolerance in plants. Given the role of HSP40s as cochaperones of HSP70s, the thermal sensitivity of five individual cytosolic HSP70 (HSP70-1 to HSP70-5) knockout mutants was tested in this study. Only hsp70-4 was sensitive to the prolonged heat treatment like hit5 and j3. The bimolecular fluorescence complementation (BiFC) assay revealed that HSP70-4 interacted with J3 and J3C417Sin vivo at normal (23 °C) and high (37 °C) temperatures. At 23 °C, both HSP70-4-J3 and HSP70-4-J3C417S BiFC signals were uniformly distributed across the cell. However, following treatment at 37 °C, HSP70-4-J3, but not HSP70-4-J3C417S, BiFC signals were detected as discernable foci. These heat-induced HSP70-4-J3 BiFC foci were localized in heat stress granules (HSGs). In addition, hsp70-4 and J3C417S accumulated more insoluble proteins than the wild type. Thus, farnesylated J3 dictates the chaperone function of HSP70-4 in HSGs. Collectively, this study identified the first HSP70/HSP40-type chaperone machinery playing a crucial role in protecting plants against prolonged heat stress, and demonstrated the significance of protein farnesylation in its protective function.

Dimerization of the ETO1 family proteins plays a crucial role in regulating ethylene biosynthesis in Arabidopsis thaliana.

ETHYLENE OVERPRODUCER1 (ETO1), ETO1-LIKE1 (EOL1), and EOL2 are members of the Broad complex, Tramtrack, Bric-a-brac (BTB) protein family that collectively regulate type-2 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity in Arabidopsis thaliana. Although ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do not play an equivalent role in regulating ethylene biosynthesis. The mechanistic details underlying the genetic analysis remain elusive. In this study, we reveal that ETO1 collaborates with EOL1/2 to play a key role in the regulation of type-2 ACS activity via protein-protein interactions. ETO1, EOL1, and EOL2 exhibit overlapping but distinct tissue-specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement the eto1 phenotype under control of the ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers with itself and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and is required for homodimerization. However, domain-swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but not sufficient for a full spectrum of ETO1 function. The missense mutation in eto1-5 generates a substitution of phenylalanine with an isoleucine in ETO1F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1F466I in Arabidopsis results in a constitutive triple response phenotype in dark-grown seedlings. Our findings reveal the mechanistic role of protein-protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis.

A CCR4-associated factor 1, OsCAF1B, confers tolerance of low-temperature stress to rice seedlings.

KEY MESSAGE: Rice is an important crop in the world. However, little is known about rice mRNA deadenylation, which is an important regulation step of gene expression at the post-transcriptional level. The CCR4-NOT1 complex contains two key components, CCR4 and CAF1, which are the main cytoplasmic deadenylases in eukaryotic cells. Expression of OsCAF1B was tightly coupled with low-temperature exposure. In the present study, we investigated the function of OsCAF1B in rice by characterizing the molecular and physiological responses to cold stress in OsCAF1B overexpression lines and dominant-negative mutant lines. Our results demonstrate that OsCAF1B plays an important role in growth and development of rice seedlings at low temperatures. Rice is a tropical and subtropical crop that is sensitive to low temperature, and activates a complex gene regulatory network in response to cold stress. Poly(A) tail shortening, also termed deadenylation, is the rate-limiting step of mRNA degradation in eukaryotic cells. CCR4-associated factor 1 (CAF1) proteins are important enzymes for catalysis of mRNA deadenylation in eukaryotes. In the present study, the role of a rice cold-induced CAF1, OsCAF1B, in adaptation of rice plants to low-temperature stress was investigated. Expression of OsCAF1B was closely linked with low-temperature exposure. The increased survival percentage and reduced electrolyte leakage exhibited by OsCAF1B overexpression transgenic lines subjected to cold stress indicate that OsCAF1B plays a positive role in rice growth under low ambient temperature. The enhancement of cold tolerance by OsCAF1B in transgenic rice seedlings involved OsCAF1B deadenylase gene expression, and was associated with elevated expression of late-response cold-related transcription factor genes. In addition, the expression level of OsCAF1B was higher in a cold-tolerant japonica rice cultivar than in a cold-sensitive indica rice cultivar. The results reveal a hitherto undiscovered function of OsCAF1B deadenylase gene expression, which is required for adaptation to cold stress in rice.

Roles of nucleus accumbens shell and core in footshock-induced stress altering behavioral sensitization by methamphetamine in acquisition and testing: Running head: stress, nucleus accumbens, and behavioral sensitization.

How the subregions of the nucleus accumbens (NAc) shell and core and stress are involved in behavioral sensitization induced by psychostimulants remains unclear. The present study manipulated methamphetamine (MAMPH) injections, lesions of the NAc shell or core, and footshock-treatment-induced stress to address this issue. The present data showed that during the acquisition phase, MAMPH injections, lesions of the NAc shell, and footshock treatments induced hyperactivity for the NAc shell. For the NAc core, MAMPH injections induced hyperactivity; however, lesions of the NAc core did not affect locomotor activity. Footshock treatments disrupted hyperactivity of behavioral sensitization. During the testing phase, MAMPH injections, lesions of the NAc shell, and footshock-treatment-induced stress facilitated hyperactivity for the NAc shell. For the NAc core, MAMPH injections and footshock-treatment-induced stress increased hyperactivity. However, the lesion of the NAc core did not affect locomotor activity. In conclusion, MAMPH injections and footshock-treatment-induced stress play an excitatory role for the NAc shell in acquisition and testing. For the NAc core, footshock-treatment-induced stress plays an inhibitory role in acquisition but an excitatory role in testing. The NAc core was not involved in MAMPH-induced behavioral sensitization in acquisition and testing. The NAc shell plays an inhibitory role in acquisition and testing phases. The present data might provide some insights for drug addiction. The results should be discussed further.

AtJ3, a specific HSP40 protein, mediates protein farnesylation-dependent response to heat stress in Arabidopsis.

MAIN CONCLUSION: Despite AtJ3 and AtJ2 sharing a high protein-sequence identity and both being substrates of protein farnesyltransferase (PFT), AtJ3 but not AtJ2 mediates in Arabidopsis the heat-dependent phenotypes derived from farnesylation modification. Arabidopsis HEAT-INTOERANT 5 (HIT5)/ENHANCED RESPONSE TO ABA 1 (ERA1) encodes the ß-subunit of the protein farnesyltransferase (PFT), and the hit5/era1 mutant is better able to tolerate heat-shock stress than the wild type. Given that Arabidopsis AtJ2 (J2) and AtJ3 (J3) are heat-shock protein 40 (HSP40) homologs, sharing 90% protein-sequence identity, and each contains a CaaX box for farnesylation; atj2 (j2) and atj3 (j3) mutants were subjected to heat-shock treatment. Results showed that j3 but not j2 manifested the heat-shock tolerant phenotype. In addition, transgenic j3 plants that expressed a CaaX- abolishing J3C417S construct maintained the same capacity to tolerate heat shock as j3. The basal transcript levels of HEAT-SHOCK PROTEIN 101 (HSP101) in hit5/era1 and j3 were higher than those in the wild type. Although the capacities of j3/hsp101 and hit5/hsp101 double mutants to tolerate heat-shock stress declined compared to those of j3 and hit5/era1, they were still greater than that of the wild type. These results show that a lack of farnesylated J3 contributes to the heat-dependent phenotypes of hit5/era1, in part by the modulation of HSP101 activity, and also indicates that (a) mediator(s) other than J3 is (are) involved in the PFT-regulated heat-stress response. In addition, because HSP40s are known to function in dimer formation, bimolecular fluorescence complementation experiments were performed, and results show that J3 could dimerize regardless of farnesylation. In sum, in this study, a specific PFT substrate was identified, and its roles in the farnesylation-regulated heat-stress responses were clarified, which could be of use in future agricultural applications.

The roles of Arabidopsis HSFA2, HSFA4a, and HSFA7a in the heat shock response and cytosolic protein response.

Previously, we found that Arabidopsis plants transformed with a construct containing the promoter of Oshsp17.3 from rice fused to the ß-glucuronidase gene (GUS), Oshsp17.3Pro::GUS (Oshsp17.3p), showed a GUS signal after heat shock (HS) or azetidine-2-carboxylic acid (AZC) treatment. HS and AZC trigger the heat shock response (HSR) and cytosolic protein response (CPR), respectively, in the cytosol by modulating specific heat shock factor (HSF) activity. Here we further identified that AtHSFA2 (At2g26150), AtHSFA7a (At3g51910), AtHSFB2a (At5g62020), and AtHSFB2b (At4g11660) are HS- and AZC-inducible; AtHSFA4a (At4g18880) is AZC-inducible; and AtHSFA5 (At4g13980) is less AZC- and HS-inducible. To investigate the roles of these 6 AtHSFs in the HSR or CPR, we crossed two independent Oshsp17.3p transgenic Arabidopsis plants with the AtHSF-knockout mutants athsfa2 (SALK_008978), athsfa4a (GABI_181H12), athsfa5 (SALK_004385), athsfa7a (SALK_080138), athsfb2a (SALK_137766), and athsfb2b (SALK_047291), respectively. As compared with the wild type, loss-of-function mutation of AtHSFA2, AtHSFA4a, and AtHSFA7a decreased HS and AZC responsiveness, so these 3 AtHSFs are essential for the HSR and CPR. In addition, loss-of-function results indicated that AthsfB2b is involved in regulating the HSR in Arabidopsis. Furthermore, analysis of the relative GUS activity of two double knockout mutants, athsfA2/athsfA4a and athsfA2/athsfA7a, revealed that AtHSFA2, AtHSFA4a, and AtHSFA7a function differentially in the HSR and CPR. Transcription profiling in athsf mutants revealed positive or negative transcriptional regulation among the 6 AtHSFs in Arabidopsis plants under HS and AZC conditions. Tunicamycin treatment demonstrated that these 6 AtHSFs are not involved in the unfolded protein response.

The Arabidopsis heat-intolerant 5 (hit5)/enhanced response to aba 1 (era1) mutant reveals the crucial role of protein farnesylation in plant responses to heat stress.

Protein farnesylation is a post-translational modification known to regulate abscisic acid (ABA)-mediated drought tolerance in plants. However, it is unclear whether and to what extent protein farnesylation affects plant tolerance to high-temperature conditions. The Arabidopsis heat-intolerant 5 (hit5) mutant was isolated because it was thermosensitive to prolonged heat incubation at 37°C for 4 d but thermotolerant to sudden heat shock at 44°C for 40 min. Map-based cloning revealed that HIT5 encodes the ß-subunit of the protein farnesyltransferase. hit5 was crossed with the aba-insensitive 3 (abi3) mutant, the aba-deficient 3 (aba3) mutant, and the heat shock protein 101 (hsp101) mutant, to characterize the HIT5-mediated heat stress response. hit5/abi3 and hit5/aba3 double mutants had the same temperature-dependent phenotypes as hit5. Additionally, exogenous supplementation of neither ABA nor the ABA synthesis inhibitor fluridone altered the temperature-dependent phenotypes of hit5. The hit5/hsp101 double mutant was still sensitive to prolonged heat incubation, yet its ability to tolerate sudden heat shock was lost. The results suggest that protein farnesylation either positively or negatively affects the ability of plants to survive heat stress, depending on the intensity and duration of high-temperature exposure, in an ABA-independent manner. HSP101 is involved in the hit5-derived heat shock tolerance phenotype.

The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis.

DEAD-box RNA helicases belong to an RNA helicase family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation and RNA decay. This study investigated a DEAD-box RNA helicase, AtRH7/PRH75, in Arabidopsis. Expression of AtRH7/PRH75 was ubiquitous; however, the levels of mRNA accumulation were increased in cell division regions and were induced by cold stress. The phenotypes of two allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled auxin-related developmental defects that were exhibited in several ribosomal protein mutants, and were more severe under cold stress. Northern blot and circular reverse transcription-PCR (RT-PCR) analyses indicated that unprocessed 18S pre-rRNAs accumulated in the atrh7 mutants. The atrh7 mutants were hyposensitive to the antibiotic streptomycin, which targets ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. In addition, the atrh7-2 and atrh7-3 mutants displayed cold hypersensitivity and decreased expression of CBF1, CBF2 and CBF3, which might be responsible for the cold intolerance. The present study indicated that AtRH7 participates in rRNA biogenesis and is also involved in plant development and cold tolerance in Arabidopsis.

A single-repeat MYB transcription repressor, MYBH, participates in regulation of leaf senescence in Arabidopsis.

Leaf senescence, the final stage of leaf development, is regulated tightly by endogenous and environmental signals. MYBS3, a MYB transcription factor with a single DNA-binding domain, mediates sugar signaling in rice. Here we report that an Arabidopsis MYBS3 homolog, MYBH, plays a critical role in developmentally regulated and dark-induced leaf senescence by repressing transcription. Expression of MYBH was enhanced in older and dark-treated leaves. Gain- and loss-of-function analysis indicated that MYBH was involved in the onset of leaf senescence. Plants constitutively overexpressing MYBH underwent premature leaf senescence and showed enhanced expression of leaf senescence marker genes. In contrast, the MYBH mutant line, mybh-1, exhibited a delayed-senescence phenotype. The EAR repression domain was required for MYBH-regulated leaf senescence. Overexpression and knockout of MYBH repressed and enhanced auxin-responsive gene expression, respectively. MYBH repressed the auxin-amido synthase genes DFL1/GH3.6 and DFL2/GH3.10, which regulate auxin homoeostasis, by binding directly to the TA box in each of their regulatory regions. An auxin-responsive phenotype was enhanced in MYBH overexpression lines and reduced in mybh knockout lines. Overexpression of MYBH enhanced gene expression of SAUR36, an auxin-promoted leaf senescence key regulator, and accelerated ABA- and ethylene-induced leaf senescence in transgenic Arabidopsis plants. Our results suggest that the role of MYBH in controlling auxin homeostasis accounts for its capacity to participate in regulation of age- and darkness-induced leaf senescence in Arabidopsis.

Arabidopsis HIT4, a regulator involved in heat-triggered reorganization of chromatin and release of transcriptional gene silencing, relocates from chromocenters to the nucleolus in response to heat stress.

Arabidopsis HIT4 is known to mediate heat-induced decondensation of chromocenters and release from transcriptional gene silencing (TGS) with no change in the level of DNA methylation. It is unclear whether HIT4 and MOM1, a well-known DNA methylation-independent transcriptional silencer, have overlapping regulatory functions. A hit4-1/mom1 double mutant strain was generated. Its nuclear morphology and TGS state were compared with those of wild-type, hit4-1, and mom1 plants. Fluorescent protein tagging was employed to track the fates of HIT4, hit4-1 and MOM1 in vivo under heat stress. HIT4- and MOM1-mediated TGS were distinguishable. Both HIT4 and MOM1 were localized normally to chromocenters. Under heat stress, HIT4 relocated to the nucleolus, whereas MOM1 dispersed with the chromocenters. hit4-1 was able to relocate to the nucleolus under heat stress, but its relocation was insufficient to trigger the decompaction of chromocenters. The hypersensitivity to heat associated with the impaired reactivation of TGS in hit4-1 was not alleviated by mom1-induced release from TGS. HIT4 delineates a novel and MOM1-independent TGS regulation pathway. The involvement of a currently unidentified component that links HIT4 relocation and the large-scale reorganization of chromatin, and which is essential for heat tolerance in plants is hypothesized.

Expression of a gene encoding a rice RING zinc-finger protein, OsRZFP34, enhances stomata opening.

By oligo microarray expression profiling, we identified a rice RING zinc-finger protein (RZFP), OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. As compared with the wild type, rice and Arabidopsis with OsRZFP34 overexpression showed increased relative stomata opening even with ABA treatment. Furthermore, loss-of-function mutation of OsRZFP34 and AtRZFP34 (At5g22920), an OsRZFP34 homolog in Arabidopsis, decreased relative stomata aperture under nonstress control conditions. Expressing OsRZFP34 in atrzfp34 reverted the mutant phenotype to normal, which indicates a conserved molecular function between OsRZFP34 and AtRZFP34. Analysis of water loss and leaf temperature under stress conditions revealed a higher evaporation rate and cooling effect in OsRZFP34-overexpressing Arabidopsis and rice than the wild type, atrzfp34 and osrzfp34. Thus, stomata opening, enhanced leaf cooling, and ABA insensitivity was conserved with OsRZFP34 expression. Transcription profiling of transgenic rice overexpressing OsRZFP34 revealed many genes involved in OsRZFP34-mediated stomatal movement. Several genes upregulated or downregulated in OsRZFP34-overexpressing plants were previously implicated in Ca(2+) sensing, K(+) regulator, and ABA response. We suggest that OsRZFP34 may modulate these genes to control stomata opening.

Divergence of the expression and subcellular localization of CCR4-associated factor 1 (CAF1) deadenylase proteins in Oryza sativa.

Deadenylation, also called poly(A) tail shortening, is the first, rate-limiting step in the general cytoplasmic mRNA degradation in eukaryotic cells. The CCR4-NOT complex, containing the two key components carbon catabolite repressor 4 (CCR4) and CCR4-associated factor 1 (CAF1), is a major player in deadenylation. CAF1 belongs to the RNase D group in the DEDD superfamily, and is a protein conserved through evolution from yeast to humans and plants. Every higher plant, including Arabidopsis and rice, contains a CAF1 multigene family. In this study, we identified and cloned four OsCAF1 genes (OsCAF1A, OsCAF1B, OsCAF1G, and OsCAF1H) from rice. Four recombinant OsCAF1 proteins, rOsCAF1A, rOsCAF1B, rOsCAF1G, and rOsCAF1H, all exhibited 3'-5' exonuclease activity in vitro. Point mutations in the catalytic residues of each analyzed recombinant OsCAF1 proteins were shown to disrupt deadenylase activity. OsCAF1A and OsCAF1G mRNA were found to be abundant in the leaves of mature plants. Two types of OsCAF1B mRNA transcript were detected in an inverse expression pattern in various tissues. OsCAF1B was transient, induced by drought, cold, abscisic acid, and wounding treatments. OsCAF1H mRNA was not detected either under normal conditions or during most stress treatments, but only accumulated during heat stress. Four OsCAF1-reporter fusion proteins were localized in both the cytoplasm and nucleus. In addition, when green fluorescent protein fused with OsCAF1B, OsCAF1G, and OsCAF1H, respectively, fluorescent spots were observed in the nucleolus. OsCAF1B fluorescent fusion proteins were located in discrete cytoplasmic foci and fibers. We present evidences that OsCAF1B colocalizes with AtXRN4, a processing body marker, and AtKSS12, a microtubules maker, indicating that OsCAF1B is a component of the plant P-body and associate with microtubules. Our findings provide biochemical evidence that OsCAF1 proteins may be involved in the deadenylation in rice. The unique expression patterns of each OsCAF1 were observed in various tissues when undergoing abiotic stress treatments, implying that each CAF1 gene in rice plays a specific role in the development and stress response of a plant.

A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings.

The development of juvenile seedlings after germination is critical for the initial establishment of reproductive plants. Ethylene plays a pivotal role in the growth of seedlings under light or dark during early development. Previously, we identified small molecules sharing a quinazolinone backbone that suppressed the constitutive triple response phenotype in dark-grown eto1-4 seedlings. We designated these small molecules as ACSinhibitor quinazolinones (acsinones), which were uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase. To explore the additional roles of acsinones in plants, we screened and identified 19 Arabidopsis mutants with reduced sensitivity to acsinone7303, which were collectively named revert to eto1 (ret) because of their recovery of the eto1 phenotype. A map-based cloning approach revealed that CELLULOSE SYNTHASE6 (CESA6) and DE-ETIOLATED2 (DET2) were mutated in ret8 (cesa6(ret8);eto1-4) and ret41 (det2(ret41);eto1-5), respectively. Etiolated seedlings of both ret8 and ret41 exhibit short hypocotyls and roots. Ethylene levels were similar in etiolated cesa6(ret8) and det2-1 and in eto1 mutants treated with acsinone7303. Chemical inhibitors of ethylene biosynthesis and perception did not significantly suppress the etiolated phenotype of cesa6(ret8) and det2(ret41). However, together with eto1, cesa6(ret8) and det2(ret41) exhibited an enhanced phenotype in the hypocotyls and apical hooks of etiolated seedlings. These results confirm that, in addition to ethylene, cellulose synthesis and brassinolides can independently contribute to modulate hypocotyl development in young seedlings.

Arabidopsis HIT4 encodes a novel chromocentre-localized protein involved in the heat reactivation of transcriptionally silent loci and is essential for heat tolerance in plants.

The Arabidopsis mutant heat-intolerant 4-1 (hit4-1) was isolated from an ethyl methanesulphonate-mutagenized M2 population on the basis of its inability to withstand prolonged heat stress (4 days at 37°C). Further characterization indicated that hit4-1 was impaired specifically in terms of basal but not acquired thermotolerance. Map-based cloning revealed that the HIT4 gene encoded a plant-specific protein for which the molecular function has yet to be studied. To investigate the cellular role of HIT4 and hence elucidate better its protective function in heat tolerance in plants, a GFP-HIT4 reporter construct was created for a protoplast transient expression assay. Results showed that fluorescently tagged HIT4 was localized to the chromocentre, a condensed heterochromatin domain that harbours repetitive elements for which transcription is normally suppressed by transcriptional gene silencing (TGS). DAPI-staining analysis and FISH with a probe that targeted centromeric repeats showed that heat-induced chromocentre decondensation was inhibited in nuclei of hit4-1 subjected to direct heat treatment, but not in those that were allowed to acquire thermotolerance. Moreover, heat reactivation of various TGS loci, regardless of whether they were endogenous or transgenic, or existed as a single copy or as repeats, was found to be attenuated in hit4-1. Meanwhile, the levels of transcripts of heat shock protein genes in response to heat stress were similar in both hit4-1 and wild-type plants. Collectively, these results demonstrated that HIT4 defines a new TGS regulator that acts at the level of heterochromatin organization and is essential for basal thermotolerance in plants.

The Arabidopsis hit1-1 mutant has a plasma membrane profile distinct from that of wild-type plants at optimal growing temperature.

High temperatures alter the physical properties of the plasma membrane and cause loss of function in the embedded proteins. Effective membrane and protein recycling through intracellular vesicular traffic is vital to maintain the structural and functional integrity of the plasma membrane under heat stress. However, in this regard, little experimental data is available. Our characterization of the Arabidopsis hit1-1 mutant, linking a subunit of a vesicle tethering complex to plasma membrane thermostability, provided valuable information to this end. We further dissected the effect of the hit1-1 mutation on plasma membrane properties and found that even at optimal growth temperature (23 °C), the hit1-1 mutant exhibited a plasma membrane protein profile distinct from that of wild-type plants. This result implies that the hit1-1 mutation essentially alters vesicle trafficking and results in changes in the plasma membrane components under non-stress conditions. Such changes do not affect normal plant growth and development, but is significant for plant survival under heat stress.

Involvement of the Arabidopsis HIT1/AtVPS53 tethering protein homologue in the acclimation of the plasma membrane to heat stress.

Arabidopsis thaliana hit1-1 is a heat-intolerant mutant. The HIT1 gene encodes a protein that is homologous to yeast Vps53p, which is a subunit of the Golgi-associated retrograde protein (GARP) complex that is involved in retrograde membrane trafficking to the Golgi. To investigate the correlation between the cellular role of HIT1 and its protective function in heat tolerance in plants, it was verified that HIT1 was co-localized with AtVPS52 and AtVPS54, the other putative subunits of GARP, in the Golgi and post-Golgi compartments in Arabidopsis protoplasts. A bimolecular fluorescence complementation assay showed that HIT1 interacted with AtVPS52 and AtVPS54, which indicated their assembly into a protein complex in vivo. Under heat stress conditions, the plasma membrane of hit1-1 was less stable than that of the wild type, as determined by an electrolyte leakage assay, and enhanced leakage occurred before peroxidation injury to the membrane. In addition, the ability of hit1-1 to survive heat stress was not influenced by exposure to light, which suggested that the heat intolerance of hit-1 was a direct outcome of reduced membrane thermostability rather than heat-induced oxidative stress. Furthermore, hit1-1 was sensitive to the duration (sustained high temperature stress at 37 °C for 3 d) but not the intensity (heat shock at 44 °C for 30 min) of exposure to heat. Collectively, these results imply that HIT1 functions in the membrane trafficking that is involved in the thermal adaptation of the plasma membrane for tolerance to long-term heat stress in plants.