Adina Breiman

Arabidopsis ROF1 (FKBP62) modulates thermotolerance by interacting with HSP90.1 and affecting the accumulation of HsfA2-regulated sHSPs.

Arabidopsis ROF1 (AtFKBP62) is a peptidyl prolyl cis/trans isomerase and a member of the FKBP (FK506 binding protein) family. ROF1 expression is induced by heat stress and developmentally regulated. In this study, we show that ROF1 binds heat shock proteins HSP90.1 via its tetratricopeptide repeat domain, and localizes in the cytoplasm under normal conditions. Exposure to heat stress induces nuclear localization of the ROF1-HSP90.1 complex, which is dependent upon the presence of the transcription factor HsfA2, which interacts with HSP90.1 but not with ROF1. Nuclear localization of ROF1 was not detected in Arabidopsis HSP90.1 and HsfA2 knockout mutants. The rof1 knockout plants exhibited collapse when 24-48 h passed between acclimation at 37 degrees C and exposure to 45 degrees C. Transgenic ROF1 over-expressors showed better survival in response to exposure to 45 degrees C than wild-type plants did. In rof1 knockout mutants, the level of expression of small HSPs regulated by HsfA2 was dramatically reduced after exposure to 37 degrees C and recovery for 24-48 h, and correlates well with the mutant phenotype. We suggest a role for ROF1 in prolongation of thermotolerance by sustaining the levels of small HSPs that are essential for survival at high temperatures.

Citrus cybrids: production by donor-recipient protoplast-fusion and verification by mitochondrial-DNA restriction profiles

SummaryEmbryogenic nucellar-callus from three Citrus types was used for protoplast isolation. The protoplasts were fused by the ‘donor-recipient’ procedure by which the nuclear-division of the donor-protoplasts was arrested by gamma-irradiation and the metabolism of unfused recipient-protoplasts was transiently inhibited by iodoacetate. The following fusion combinations were performed: (1) Poorman x Poncirus trifoliata with Villafranca lemon; (2) Poorman x P. trifoliata with Sour orange; (3) Sour orange with Villafranca. Combinations 1, 2 and 3 resulted in 120 (125), 70 (5) and 19 (89) calli (regenerated plants), respectively. The mitochondrial-DNA (mtDNA) restriction profiles of Poorman x P. trifoliata obtained by fragmentation with Bcl I, Bam H1 or Sal I differed from the respective profiles of Villafranca and Sour orange but no differences in mtDNA restriction profiles were detected between Villafranca and Sour orange. When Southern blots of Villafranca and Sour orange mtDNAs were hybridized with radiolabelled heterologous mtDNA probes, the mtDNAs of these two Citrus types could be differentiated. The fusion-derived plants from all three donor-recipient combinations had the recipients morphological (nuclear-coded) features. MtDNA restriction profiles, with and without hybridization to heterologous probes, indicated that the analysed plants were cybrids.

Heritable somaclonal variation in wild barley (Hordeum spontaneum).

SummaryProgenies of H. spontaneum plants regenerated from immature embryo derived calli were analysed for somaclonal variation on the following traits: (1) organization of the intergenic spacer of the rRNA genes; (2) B and C hordein pattern on SDS-PAGE; (3) genomic organization of the B and C hordein coding sequences; (4) mitochondrial DNA organization assayed by hybridization of Southern blots of total DNA with mitochondrial coding genes; (5) cytology. One out of twelve progeny plants was characterized as variant for two traits: (a) a loss of 1.8 and 2.5 kb Taq I intergenic rDNA spacer fragments and (b) a variant pattern of hordeins on 1-D SDS-PAGE. No numerical or structural chromosome variation was detected among the control plants therefore it is assumed that the variation was caused by the in vitro culture and transmitted, through sexual reproduction, to the analysed progeny.

Involvement of Arabidopsis ROF2 (FKBP65) in thermotolerance

The ROF2 (FKBP65) is a heat stress protein which belongs to the FK506 Binding Protein (FKBP) family. It is homologous to ROF1 (FKBP62) which was recently shown to be involved in long term acquired thermotolerance by its interaction with HSP90.1 and modulation of the heat shock transcription factor HsfA2. In this study, we have demonstrated that ROF2 participates in long term acquired thermolerance, its mode of action being different from ROF1. In the absence of ROF2, the small heat shock proteins were highly expressed and the plants were resistant to heat stress, opposite to the effect observed in the absence of ROF1. It was further demonstrated that ROF2 transcription is modulated by HsfA2 which is also essential for keeping high levels of ROF2 during recovery from heat stress. ROF2 localization to the nucleus was observed several hours after heat stress exposure and its translocation to the nucleus was independent from the presence of HSP90.1 or HsfA2. ROF2 has been shown to interact with ROF1, to form heterodimers and it is suggested that via this interaction it can join the complex ROF1-HSP90.1- HsfA2. Transient expression of ROF2 together with ROF1 repressed transcription of small HSPs. A model describing the mode of action of ROF2 as a heat stress modulator which functions in negative feedback regulation of HsfA2 is proposed.

A novel plant peptidyl-prolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression.

A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat. It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned. It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site. The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin. Northern blot analysis showed that wheat FKBP was found mainly in young tissues. Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots. The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.

Sumoylation of Arabidopsis heat shock factor A2 (HsfA2) modifies its activity during acquired thermotholerance.

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulate many cellular processes. In this work we show SUMOylation of the heat shock transcription factor, AtHsfA2, in connection with the plant’s response to heat stress and acquired thermotolerance. Using the Yeast two hybrid and the bimolecular fluorescence complementation system, we have found that AtSUMO1 physically interacts with AtHsfA2. Further investigation allowed us to determine that Lys 315 of AtHsfA2 is the main SUMOylation site. Overexpression of AtSUMO1 led to a decrease in AtHsfA2 transcriptional activation of heat shock promoters. We have examined the effect of AtSUMO1 on AtHsfA2 during heat shock treatments. The phenotype of seedlings overexpressing AtSUMO1 resembled the phenotype of AtHsfA2 knock out seedlings, which were more sensitive than wild type seedlings to repeated heat treatment. Furthermore, AtSUMO1 overexpressing seedlings exhibited lower expression levels of small heat shock proteins as compared with wild type seedlings after heat treatment. Based on our findings, we suggest that AtSUMO1 is involved in the regulation of AtHsfA2 in acquired thermotolerance.

MULTIPLE CDNAS OF WHEAT VOLTAGE-DEPENDENT ANION CHANNELS (VDAC) : ISOLATION, DIFFERENTIAL EXPRESSION, MAPPING AND EVOLUTION

The mitochondrial outer membrane of eukaryotic cells contains voltage-dependent anion channels (VDAC) also termed porins. Three cDNAs from wheat (Triticum aestivum) were isolated and sequenced (Tavdac 1–3). They share 65% similarity of their amino acid sequences, and therefore they probably represent isoforms. The deduced amino acid sequence of one of the cDNAs was found to be identical to the purified VDAC protein from wheat mitochondria [8]. Secondary structure analysis of the deduced amino acid sequences of the three vdac cDNAs revealed a characteristic α helix at their N-terminal and β-barrel cylinders characteristic of VDAC channels.The Tavdac cDNAs are differentially expressed in meristematic tissues. The transcript levels of Tavdac 1 in all wheat tissues is at least 2.5-fold higher than Tavdac 2 and Tavdac 3. Tavdac 2 has a low level of expression in all floral tissues whereas Tavdac 3 is highly expressed in anthers. This is the first report on differential expression of vdac genes in plants.The Tavdac genes have been mapped on the wheat genome. Tavdac 1 is located on the long arm of chromosome 5, Tavdac 2 on the long arm of chromosome 1 and Tavdac 3 on the long arm of chromosome 3.A phylogenetic reconstruction indicates that vdac genes underwent numerous duplication events throughout their evolution. All duplications occurred after the separation of plants from animals and fungi, and no orthologous genes are shared among phyla. Within plants, some of the vdac gene duplications probably occurred before the monocotydelon-dicotydelon split.

Mitochondrial DNA diversity in the genera of Triticum and Aegilops revealed by southern blot hybridization.

SummarySouthern blot hybridization of total DNA to defined mitochondrial DNA sequences provides a sensitive assay for mtDNA variation in the genera of Triticum and Aegilops. A clear distinction between cytoplasms of tetraploid species sharing the “AG” haploid genome is reported for the first time. The Sitopsis section of the genus Aegilops showed the most extensive intra- and inter-specific variation, whereas no variation could be detected among the cytoplasms of polyploid Triticum species (wheats) sharing the AB haploid genome. Extensive cytoplasmic intraspecific diversity was revealed in Ae. speltoides.

The involvement of mammalian and plant FK506-binding proteins (FKBPs) in development

The FK506-binding proteins (FKBPs) are peptidyl prolyl cis/trans isomerases and the information gathered in the last 10 years reveals their involvement in diverse biological systems affecting the function and structure of target proteins. Members of the FKBP family were shown to be growth-regulated and participate in signal transduction. In this review we have chosen to focus on a few examples of the mammalian and plant systems in which members of the FKBP family have been demonstrated to affect the function of proteins or development. The technologies that enable production of knockout mice, Arabidopsis mutants and overexpression in transgenic organisms have revealed the contribution of FKBP to development in higher eukaryotes. It appears that members of the FKBP family have conserved some of their basic functions in the animal and plant kingdom, whereas other functions became unique. Studies that will take advantage of the full genome sequence available for Arabidopsis and the human genome, DNA chip technologies and the use of transgenic complementation system will contribute to the elucidation of the molecular mechanism and biological function of FKBPs.

Functional Differences among Wheat Voltage-dependent Anion Channel (VDAC) Isoforms Expressed in Yeast INDICATION FOR THE PRESENCE OF A NOVEL VDAC-MODULATING PROTEIN?

VDAC is a voltage-gated anion channel located in the mitochondrial outer membrane, presumably participating in controlling aerobic metabolism. Three distinct wheat vdac cDNAs were expressed in a vdac-minus yeast strain and successfully complemented its defective phenotype. The growth curves of these transformants were different. The wheat channel isoforms were functionally characterized following purification from yeast mitochondria and reconstitution into soybean phospholipid planar membranes. All three isoforms yielded voltage-dependent anion channels with electrophysiological parameters comparable to known VDACs. Isoform-related functional features (specific conductance levels, kinetics, and gating behaviors) are reported for the first time in VDACs. The presence (or absence) of protease inhibitors during the purification procedure, and the use of Pronase on reconstituted channels, strongly suggest that some of the unique wheat VDAC properties are due to co-purification of a yeast channel-modulating protein. Its effects, different from the reported functional interactions of the channel with hexo- or creatine kinases, could not be mimicked by the protein termed VDAC modulator, indicating the presence of a novel VDAC modulator. In addition to strengthening VDAC presumed role in metabolism, the functional diversity of the channels (as shown here in two different systems) implies a highly dynamic outer membrane permeability. Our results are consistent with VDAC functioning as a heteromer including one pore protein and other modulating subunits.