Ectopic RING zinc finger gene from hot pepper induces totally different genes in lettuce and tobacco.

Advances in molecular biology have improved crops through transferring genes from one organism to new hosts, and these efforts have raised concerns about potential unexpected outcomes. Here, we provide evidence that a gene with a specific function in one organism can yield completely different effects in a new host. CaRZFP1 is a C3HC4-type RING zinc finger protein gene previously isolated from a cDNA library for heat-stressed hot pepper. In our previous work investigating in vivo CaRZFP1 function, we transferred CaRZFP1 into tobacco; transgenic tobacco exhibited enhanced growth and tolerance to abiotic stresses. As further analysis of CaRZFP1 ectopic expression in a heterologous host plant, here we mobilized and constitutively overexpressed CaRZFP1 in lettuce. In contrast to tobacco, transgenic lettuce exhibited poorer growth and delayed flowering compared with vector-only controls. To identify genes that might be involved in this phenotypic effect, transcriptome analyses on transgenic plants of both species were performed, uncovering dozens of genes that reflect the different outcomes between tobacco and lettuce. These included protein kinase, transcriptional factor, transporter protein, hormone and metabolism-related genes, and some unannotated genes. The opposite effects of CaRZFP1 ectopic expression in lettuce and tobacco address concerns of unexpectedly different outcomes in different host species.

Small heat shock proteins can release light dependence of tobacco seed during germination.

Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination.

Tobacco class I cytosolic small heat shock proteins are under transcriptional and translational regulations in expression and heterocomplex prevails under the high-temperature stress condition in vitro.

Seven genomic clones of tobacco (Nicotiana tabacum W38) cytosolic class I small heat shock proteins (sHSPs), probably representing all members in the class, were isolated and found to have 66 to 92% homology between their nucleotide sequences. Even though all seven sHSP genes showed heat shock-responsive accumulation of their transcripts and proteins, each member showed discrepancies in abundance and timing of expression upon high-temperature stress. This was mainly the result of transcriptional regulation during mild stress conditions and transcriptional and translational regulation during strong stress conditions. Open reading frames (ORFs) of these genomic clones were expressed in Escherichia coli and the sHSPs were purified from E. coli. The purified tobacco sHSPs rendered citrate synthase and luciferase soluble under high temperatures. At room temperature, non-denaturing pore exclusion polyacrylamide gel electrophoresis on three sHSPs demonstrated that the sHSPs spontaneously formed homo-oligomeric complexes of 200 ∼ 240 kDa. However, under elevated temperatures, hetero-oligomeric complexes between the sHSPs gradually prevailed. Atomic force microscopy showed that the hetero-oligomer of NtHSP18.2/NtHSP18.3 formed a stable oligomeric particle similar to that of the NtHSP18.2 homo-oligomer. These hetero-oligomers positively influenced the revival of thermally inactivated luciferase. Amino acid residues mainly in the N-terminus are suggested for the exchange of the component sHSPs and the formation of dominant hetero-oligomers under high temperatures.

Tobacco mitochondrial small heat shock protein NtHSP24.6 adopts a dimeric configuration and has a broad range of substrates.

There is a broad range of different small heat shock proteins (sHSPs) that have diverse structural and functional characteristics. To better understand the functional role of mitochondrial sHSP, NtHSP24.6 was expressed in Escherichia coli with a hexahistidine tag and purified. The protein was analyzed by non-denaturing PAGE, chemical cross-linking and size exclusion chromatography and the H6NtHSP24.6 protein was found to form a dimer in solution. The in vitro functional analysis of H6NtHSP24.6 using firefly luciferase and citrate synthase demonstrated that this protein displays typical molecular chaperone activity. When cell lysates of E. coli were heated after the addition of H6NtHSP24.6, a broad range of proteins from 10 to 160 kD in size remained in the soluble state. These results suggest that NtHSP24.6 forms a dimer and can function as a molecular chaperone to protect a diverse range of proteins from thermal aggregation.

Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.).

Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, aMAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47-93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK.

A BAX inhibitor-1 gene in Capsicum annuum is induced under various abiotic stresses and endows multi-tolerance in transgenic tobacco.

Programmed cell death (PCD) is a highly conserved cellular suicide process important in developmental processes and elimination of damaged cells upon environmental stresses. Among the important regulators of PCD, much interest has been centered on BCL2-associated x protein (BAX) as the pro-PCD factor. On the other hand, BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor that balances out the activity of BAX in the developmental processes and responses to environment. A cDNA clone coding a BI-1 gene was isolated from a cDNA library of heat-stressed hot pepper (Capsicum annuum) and named as CaBI-1. This gene contains an open reading frame (ORF) of 248 amino acids encoding a BI-1 protein. Genomic DNA-blot analysis for CaBI-1 suggested one or two loci in the C. annuum genome. Transcription of CaBI-1 was induced in response to high or low temperatures, drought, high salinity, flooding and heavy metal stresses, and ABA. We introduced the ORF of CaBI-1 under the control of the CaMV 35S promoter (P(35S)) into tobacco (Nicotiana tabacum cv. Wisconsin 38) genome by Agrobacterium-mediated transformation. The P(35S):CaBI-1 transgenic plants displayed markedly improved tolerance to high temperature, water deficit, and high salinity in comparison to the control plants. The results indicate that CaBI-1 is a BI-1 gene of which expression induced under various abiotic stresses and endows tolerance to several types of environmental stresses.

Flooding stress-induced glycine-rich RNA-binding protein from Nicotiana tabacum.

A cDNA clone for a transcript preferentially expressed during an early phase of flooding was isolated from Nicotiana tabacum. Nucleotide sequencing of the cDNA clone identified an open reading frame that has high homology to the previously reported glycine-rich RNA-binding proteins. The open reading frame consists of 157 amino acids with an N-terminal RNA-recognition motif and a C-terminal glycine-rich domain, and thus the cDNA clone was designated as Nicotiana tabaccum glycine-rich RNA-binding protein-1 (NtGRP1). Expression of NtGRP1 was upregulated under flooding stress and also increased, but at much lower levels, under conditions of cold, drought, heat, high salt content, and abscisic acid treatment. RNA homopolymer-binding assay showed that NtGRP1 binds to all the RNA homopolymers tested with a higher affinity to poly r(G) and poly r(A) than to poly r(U) and poly r(C). Nucleic acid-binding assays showed that NtGRP1 binds to ssDNA, dsDNA, and mRNA. NtGRP1 suppressed expression of the fire luciferase gene in vitro, and the suppression of luciferase gene expression could be rescued by addition of oligonucleotides. Collectively, the data suggest NtGRP1 as a negative modulator of gene expression by binding to DNA or RNA in bulk that could be advantageous for plants in a stress condition like flooding.

Submergence-inducible and circadian rhythmic basic helix-loop-helix protein gene in Nicotiana tabacum.

Submergence stress leads to diverse changes in transcription and translation of genes involved in developmental and physiological metabolisms of plants. The basic helix-loop-helix (bHLH) protein family is one of the largest transcriptional factor families in plants, and has been shown to play pivotal roles in diverse biological responses. However, there has been no report on bHLH protein related to submergence stress response. In this study, a novel bHLH gene, NtbHLH, was isolated from tobacco (Nicotiana tabacum) by differential screening of a submergence-stress-induced cDNA library. NtbHLH cDNA is 1027bp in length, with an open reading frame (ORF) of 702 nucleotides encoding 233 amino acid residues that contain the bHLH domain. RNA-blot analyses showed that transcription of NtbHLH was induced by submergence stress, while cold, heat shock, and drought decreased its expression. The gene expression was down-regulated by gibberellins, but ABA and ethylene seemed not to affect it. It was also apparent that NtbHLH expression follows circadian rhythmicity. The electrophoretic mobility shift and chemical cross-linking assays showed that NtbHLH specifically binds to G-box and forms homo-dimers.

Heat-inducible C3HC4 type RING zinc finger protein gene from Capsicum annuum enhances growth of transgenic tobacco.

Capsicum annuum RING Zinc Finger Protein 1 (CaRZFP1) gene is a novel C3HC4-type RING zinc finger protein gene which was previously isolated from a cDNA library for hot pepper plants treated of heat-shock. The CaRZFP1 was inducible to diverse environmental stresses in hot pepper plants. We introduced the CaRZFP1 into the Wisconsin 38 cultivar of tobacco (Nicotiana tabacum) by Agrobacterium mediated transformation under the control of the CaMV 35S promoter. Expression of the transgene in the transformed tobacco plants was demonstrated by RNA blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T(1) lines showed that the transgene segregated in a Mendelian fashion. Transgenic tobacco lines that expressed the CaRZFP1 gene were compared with several different empty vector lines and they exhibited enhanced growth; they have larger primary root, more lateral root, larger hypocotyls and bigger leaf size, resulting in heavier fresh weight. Enhanced growth of transgenic lines accompanied with longer vegetative growth that resulted in bigger plants with higher number of leaves. Microarray analysis revealed the up-regulation of some growth related genes in the transgenic plants which were verified by specific oligomer RNA blot analyses. These results indicate that CaRZFP1 activates and up-regulates some growth related proteins and thereby effectively promoting plant growth.

Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants.

HSP70, a heat shock protein, is a molecular chaperone responsive to various environmental stresses. Here, NtHSP70-1 was a drought-/ABA-inducible gene. We monitored the expression of CaERD15 (early responsive to dehydration) with exposing plants to progressive drought stress. Its activity was used as an indicator of water-deficit conditions. To analyze the protective role of HSP70, we obtained transgenic tobacco plants that constitutively expressed elevated levels of the tobacco HSP70, NtHSP70-1, as well as transgenic plants containing either the vector alone or else having NtHSP70-1 in the antisense orientation. Plants with enhanced levels of NtHSP70-1 in their transgenic sense lines exhibited tolerance to water stress. Under progressive drought, the amount of leaf NtHSP70-1 was correlated with maintenance of optimum water content, with contents being higher in the leaves of dehydrated transgenic sense plants than in those of either the control (vector-only) or the transgenic antisense plants. Moreover, the expression of CaERD15 was considerably reduced in tobacco plants that over-expressed NtHSP70-1. These results suggest that elevated levels of NtHSP70-1 can confer drought-stress tolerance.

Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members.

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the beta-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.

A cyclophilin from Griffithsia japonica has thermoprotective activity and is affected by CsA.

Members of the multifunctional Cyp family have been isolated from a wide range of organisms. However, few functional studies have been performed on the role of these proteins as chaperones in red alga. For studying the function of cDNA GjCyp-1 isolated from the red alga (Griffithsia japonica), we expressed and purified a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus in Escherichia coli. An expressed fusion protein, H6GjCyp-1 maintained the stability of E. coli proteins up to 50 degrees C. For a functional bioassay for recombinant H6GjCyp-1, the viability of E. coli cells overexpressing H6GjCyp-1 was compared with that of cells not expressing H6GjCyp-1 at 50 degrees C. After high temperature treatment for 1 h, E. coli overexpressing H6GjCyp-1 survived about three times longer than E. coli lacking H6GjCyp-1. Measurement of the light scattering of luciferase (luc) showed that GjCyp-1 prevents the aggregation of luc during mild heat stress and that the thermoprotective activity of GjCyp-1 is blocked by cyclosporin A (CsA), an inhibitor of Cyps. Furthermore, the Cyp-CsA complex inhibited the growth of E. coli under normal conditions. The results of the GjCyp-1 bioassays as well as in vitro studies strongly suggest that Cyp confers thermotolerance to E. coli.

Biochemical analysis of a cytosolic small heat shock protein, NtHSP18.3, from Nicotiana tabacum.

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70 degrees . Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.

Dimeric configuration of SeqA protein bound to a pair of hemi-methylated GATC sequences.

The binding of SeqA protein to hemi-methylated GATC sequences (hemi-sites) regulates chromosome initiation and the segregation of replicated chromosome in Escherichia coli. We have used atomic force microscopy to examine the architecture of SeqA and the mode of binding of one molecule of SeqA to a pair of hemi-sites in aqueous solution. SeqA has a bipartite structure composed of a large and a small lobe. Upon binding of a SeqA molecule to a pair of hemi-sites, the larger lobe becomes visibly separated into two DNA binding domains, each of which binds to one hemi-site. The two DNA binding domains are held together by association between the two multimerization domains that make up the smaller lobe. The binding of each DNA binding domain to a hemi-site leads to bending of the bound DNA inwards toward the bound protein. In this way, SeqA adopts a dimeric configuration when bound to a pair of hemi-sites. Mutational analysis of the multimerization domain indicates that, in addition to multimerization of SeqA polypeptides, this domain contributes to the ability of SeqA to bind to a pair of hemi-sites and to its cooperative behavior.

A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C.

Posttranscriptional RNA metabolism plays versatile roles in the regulation of gene expression during eukaryotic growth and development. It is mediated by a group of RNA binding proteins with distinct conserved motifs. In this study, an Arabidopsis (Arabidopsis thaliana) gene, designated FLK, was identified and shown to encode a putative RNA binding protein with K homology motifs. A mutant in which FLK was inactivated by T-DNA insertion exhibited a severe late flowering phenotype both in long and short days. The late flowering phenotype was reversed by gibberellin and vernalization treatments. The FLOWERING LOCUS C (FLC) transcription was greatly upregulated, whereas those of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 decreased in the mutant. These observations demonstrate that FLK regulates the autonomous flowering pathway via FLC. It is now evident that a battery of different RNA binding proteins are involved in the posttranscriptional regulation of flowering time in Arabidopsis.

The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis.

The floral transition in Arabidopsis is regulated by at least four flowering pathways: the long-day, autonomous, vernalization, and gibberellin (GA)-dependent pathways. Previously, we reported that the MADS-box transcription factor SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) integrates the long-day and vernalization/autonomous pathways. Here, we present evidences that SOC1 also integrates signaling from the GA-dependent pathway, a major flowering pathway under non-inductive short days. Under short days, the flowering time of GA-biosynthetic and -signaling mutants was well correlated with the level of SOC1 expression; overexpression of SOC1 rescued the non-flowering phenotype of ga1-3, and the soc1 null mutant showed reduced sensitivity to GA for flowering. In addition, we show that vernalization-induced repression of FLOWERING LOCUS C (FLC), an upstream negative regulator of SOC1, is not sufficient to activate SOC1; positive factors are also required. Under short days, the GA pathway provides a positive factor for SOC1 activation. In contrast to SOC1, the GA pathway does not regulate expression of other flowering integrators FLC and FT. Our results explain why the GA pathway has a strong effect on flowering under short days and how vernalization and GA interact at the molecular level.

A cDNA clone for cyclophilin from Griffithsia japonica and phylogenetic analysis of cyclophilins.

A cDNA clone, designated as Griffithsia japonica cyclophilin-1 (GjCyp-1), was isolated by differential screening of a cDNA library for a red alga, G. japonica. The transcript that corresponded to GjCyp-1 was abundant in vegetative, male, and tetrasporangial thalli, but only the basal level of the transcript was detected in female gametophytes. Determination of the nucleotide sequence of GjCyp-1 identified an open reading frame (ORF), which shared high homologies with cyclophilins that were previously reported in other organisms. Currently available amino acid sequences of eukaryotic cyclophilins were compared in order to examine their phylogenetic relationship to GjCyp-1. A phylogenetic analysis, based on the aligned sequences, showed two major clades - cytosolic cyclophilins (CypA) and ER cyclophilins (CypB). The clade of CypA was divided into six groups - plant, nematode, mammal, euglenozoa, fungi, and platyhelminthes CypA. GjCyp-1 appeared to be closely allied with the euglenozoan CypAs, but constituted an independent lineage. GjCyp-1 showed little relationship with other algal Cyps. A green alga, Chlamydomonas (Chl a + b group), was located in a green plant clade, but a brown alga, Fucus (Chl a + c group), formed an independent clade with a fungus Uromyces (Basidiomycota).