Priming avocado with sodium hydrosulfide prior to frost conditions induces the expression of genes involved in protection and stress responses.

Priming plants with chemical agents has been extensively investigated as a means for improving their tolerance to many biotic and abiotic stresses. Earlier, we showed that priming young avocado (Persea americana Mill cv. 'Hass') trees with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide, improves the response of photosynthesis to simulated frost (cold followed by high light) conditions. In the current study, we performed a transcriptome analysis to gain insight into the molecular response of avocado 'Hass' leaves to frost, with or without NaHS priming. The analysis revealed 2144 (down-regulated) and 2064 (up-regulated) differentially expressed genes (DEGs) common to both non-primed and primed trees. Non-primed trees had 697 (down) and 559 (up) unique DEGs, while primed trees exhibited 1395 (down) and 1385 (up) unique DEGs. We focus on changes in the expression patterns of genes encoding proteins involved in photosynthesis, carbon cycle, protective functions, biosynthesis of isoprenoids and abscisic acid (ABA), as well as ABA-regulated genes. Notably, the differential expression results depict the enhanced response of primed trees to the frost and highlight gene expression changes unique to primed trees. Amongst these are up-regulated genes encoding pathogenesis-related proteins, heat shock proteins, enzymes for ABA metabolism, and ABA-induced transcription factors. Extending the priming experiments to field conditions, which showed a benefit to the physiology of trees following chilling, suggests that it can be a possible means to improve trees' response to cold stress under natural winter conditions.

Expression Profiling of Four Mango FT/TFL1-Encoding Genes under Different Fruit Load Conditions, and Their Involvement in Flowering Regulation.

Plant flowering is antagonistically modulated by similar FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) proteins. In mango (Mangifera indica L.), flowering is induced by cold temperatures, unless the tree is juvenile or the adult tree had a high fruit load (HFL) in the summer. Here, we studied the effects of juvenility and fruit load on the expression of four MiFT/TFL1 genes cloned from the mango 'Shelly' cultivar. Ectopic expression of MiFT1 in Arabidopsis resulted in early flowering, whereas over-expression of MiFT2 and the two cloned MiTFL1 genes repressed flowering. Moreover, juvenility was positively correlated with higher transcript levels of MiFT2 and both MiTFL1s. In trees with a low fruit load, leaf MiFT1 expression increased in winter, whereas HFL delayed its upregulation. MiFT2 expression was upregulated in both leaves and buds under both fruit load conditions. Downregulation of both MITFL1s in buds was associated with a decrease in regional temperatures under both conditions; nevertheless, HFL delayed the decrease in their accumulation. Our results suggest that cold temperature has opposite effects on the expression of MiFT1 and the MiTFL1s, thereby inducing flowering, whereas HFL represses flowering by both suppressing MiFT1 upregulation and delaying MiTFL1s downregulation. The apparent flowering-inhibitory functions of MiFT2 are discussed.

Bumblebees (Bombus terrestris) Improve 'Hass' Avocado (Persea americana) Pollination.

Pollination is limiting for avocado production. We examined whether adding bumblebees (BBs; ca. 10 hives/ha) to conventional honeybees (HB; 5 hives/ha) would improve 'Hass' avocado pollination and yields. A preliminary trial (2017/18) in an avocado orchard with four consecutive rows of 'Hass' followed by one row of 'Ettinger' serving as a pollenizer (20% 'Ettinger') showed a considerable increase in 'Hass' yield in rows adjacent to (up to 80 m from) the BB hives vs. distant rows (=controls). In 2018/19, the trials were extended to three additional orchards. A significant yield increase was obtained in the BB hive-adjacent trees compared to BB hive-distant ones. Similar results were obtained in 2019/20, in experiments conducted throughout the country. The SNP analysis, to determine the parents of 'Hass' fruit at varying distances from the BB hives, showed no differences in the cross-pollination rate ('Hass' × 'Ettinger'). However, pollination rates and the number of germinating pollen grains per stigma decreased with distance from the hives, and correlated to the negative gradient in yield. Taken together, our data suggest that adding BB hives to 'Hass' avocado orchards, at ca. 10 hives/ha resulting in 0.5-1.0 BB visits/tree per min, increases pollination and, accordingly, total yield.

Characterization of Two Ethephon-Induced IDA-Like Genes from Mango, and Elucidation of Their Involvement in Regulating Organ Abscission.

In mango (Mangifera indica L.), fruitlet abscission limits productivity. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide acts as a key component controlling abscission events in Arabidopsis. IDA-like peptides may assume similar roles in fruit trees. In this study, we isolated two mango IDA-like encoding-genes, MiIDA1 and MiIDA2. We used mango fruitlet-bearing explants and fruitlet-bearing trees, in which fruitlets abscission was induced using ethephon. We monitored the expression profiles of the two MiIDA-like genes in control and treated fruitlet abscission zones (AZs). In both systems, qRT-PCR showed that, within 24 h, both MiIDA-like genes were induced by ethephon, and that changes in their expression profiles were associated with upregulation of different ethylene signaling-related and cell-wall modifying genes. Furthermore, ectopic expression of both genes in Arabidopsis promoted floral-organ abscission, and was accompanied by an early increase in the cytosolic pH of floral AZ cells-a phenomenon known to be linked with abscission, and by activation of cell separation in vestigial AZs. Finally, overexpression of both genes in an Atida mutant restored its abscission ability. Our results suggest roles for MiIDA1 and MiIDA2 in affecting mango fruitlet abscission. Based on our results, we propose new possible modes of action for IDA-like proteins in regulating organ abscission.

Short De-Etiolation Increases the Rooting of VC801 Avocado Rootstock.

Dark-grown (etiolated) branches of many recalcitrant plant species root better than their green counterparts. Here it was hypothesized that changes in cell-wall properties and hormones occurring during etiolation contribute to rooting efficiency. Measurements of chlorophyll, carbohydrate and auxin contents, as well as tissue compression, histological analysis and gene-expression profiles were determined in etiolated and de-etiolated branches of the avocado rootstock VC801. Differences in chlorophyll content and tissue rigidity, and changes in xyloglucan and pectin in cambium and parenchyma cells were found. Interestingly, lignin and sugar contents were similar, suggesting that de-etiolated branches resemble the etiolated ones in this respect. Surprisingly, the branches that underwent short de-etiolation rooted better than the etiolated ones, and only a slight difference in IAA content between the two was observed. Gene-expression profiles revealed an increase in ethylene-responsive transcripts in the etiolated branches, which correlated with enrichment in xyloglucan hydrolases. In contrast, transcripts encoding pectin methylesterase and pectolyases were enriched in the de-etiolated branches. Taken together, it seems that the short de-etiolation period led to fine tuning of the conditions favoring adventitious root formation in terms of auxin-ethylene balance and cell-wall properties.

Sodium hydrosulfide priming improves the response of photosynthesis to overnight frost and day high light in avocado (Persea americana Mill, cv. 'Hass').

Radiation frost events, which have become more common in the Mediterranean Basin in recent years, inflict extensive damage to tropical/subtropical fruit crops. During radiation frost, sub-zero temperatures are encountered in the dark, followed by high light during the subsequent clear-sky day. One of the key processes affected by these conditions is photosynthesis, which, when significantly inhibited, leads to the enhanced accumulation of reactive oxygen species (ROS) and damage. The use of 'chemical priming' treatments that induce plants' endogenous stress responses is a possible strategy to improve their coping with stress conditions. Herein, we studied the effects of priming with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H2 S), on the response of photosynthesis to overnight frost and day high-light conditions in 'Hass' avocado (Persea americana Mill). We found that priming with a single foliar application of NaHS had positive effects on the response of grafted 'Hass' plants. Primed plants exhibited significantly reduced inhibition of CO2 assimilation, a lower accumulation of hydrogen peroxide as well as lower photoinhibition, as compared to untreated plants. The ability to maintain a high CO2 assimilation capacity after the frost was attained on the background of considerable inhibition in stomatal conductance. Thus, it was likely related to the lower accumulation of ROS and photodamage observed in primed 'Hass' plants. This work contributes toward the understanding of the response of photosynthesis in a subtropical crop species to frost conditions and provides a prospect for chemical priming as a potential practice in orchards during cold winters.

Juvenility and Vegetative Phase Transition in Tropical/Subtropical Tree Crops.

In plants, juvenile to adult phase transition is regulated by the sequential activity of two microRNAs: miR156 and miR172. A decline in miR156 and increase in miR172 abundance is associated with phase transition. There is very limited information on phase transition in economically important horticultural tree crops, which have a significantly long vegetative phase affecting fruit bearing. Here, we profiled various molecular cues known to be involved in phase transition and flowering, including the microRNAs miR156 and miR172, in three horticultural tree crops: avocado (Persea americana), mango (Mangifera indica), and macadamia (Macadamia integrifolia). We observed that miR156 expression decreases as these trees age and can potentially be used as a juvenility marker. Consistent with findings in annual plants, we also observed conserved regulation of the miR156-SPL3/4/5 regulatory module in these genetically distant tree crops, suggesting that this pathway may play a highly conserved role in vegetative identity. Meanwhile, the abundance of miR172 and its target AP2-like genes as well as the accumulation level of SPL9 transcripts were not related with plant age in these crops except in avocado where miR172 expression increased steadily. Finally, we demonstrate that various floral genes, including AP1 and SOC1 were upregulated in the reproductive phase and can be used as potential markers for the reproductive phase transition. Overall, this study provides an insight into the molecular associations of juvenility and phase transition in horticultural trees where crop breeding and improvement are encumbered by long juvenile phases.

Genetic diversity of avocado (Persea americana Mill.) germplasm using pooled sequencing.

BACKGROUND: Discovering a genome-wide set of avocado (Persea americana Mill.) single nucleotide polymorphisms and characterizing the diversity of germplasm collection is a powerful tool for breeding. However, discovery is a costly process, due to loss of loci that are proven to be non-informative when genotyping the germplasm. RESULTS: Our study on a collection of 100 accessions comprised the three race types, Guatemalan, Mexican, and West Indian. To increase the chances of discovering polymorphic loci, three pools of genomic DNA, one from each race, were sequenced and the reads were aligned to a reference transcriptome. In total, 507,917 polymorphic loci were identified in the entire collection. Of these, 345,617 were observed in all three pools, 117,692 in two pools, 44,552 in one of the pools, and only 56 (0.0001%) were homozygous in the three pools but for different alleles. The polymorphic loci were validated using 192 randomly selected SNPs by genotyping the accessions within each pool. The sensitivity of polymorphic locus prediction ranged from 0.77 to 0.94. The correlation between the allele frequency estimated from the pooled sequences and actual allele frequency from genotype calling of individual accessions was r = 0.8. A subset of 109 SNPs were then used to evaluate the genetic relationships among avocado accessions and the genetic diversity of the collection. The three races were distinctly clustered by projecting the genetic variation on a PCA plot. As expected, by estimating the kinship coefficient for all the accessions, many of the cultivars from the California breeding program were closely related to each other, especially, the Hass-like ones. The green-skin avocados, e.g., 'Bacon', 'Zutano', 'Ettinger' and 'Fuerte' were also closely related to each other. CONCLUSIONS: A framework for SNP discovery and genetically characterizing of a breeder's accessions was described. Sequencing pools of gDNA is a cost-effective approach to create a genome-wide stock of polymorphic loci for a breeding program. Reassessing the botanical and the genetic knowledge about the germplasm accessions is valuable for future breeding. Kinship analysis may be used as a first step in finding a parental candidates in a parentage analyses.

Distinct organ-specific and temporal expression profiles of auxin-related genes during mango fruitlet drop.

In mango, fruitlet abscission initiates with a decrease in polar auxin transport through the abscission zone (AZ), triggered by ethylene. To explore the molecular components affecting this process, we initially conducted experiments with developing fruitlet explants in which fruitlet drop was induced by ethephon, and monitored the expression patterns of distinct indole-3-acetic acid (IAA)-related genes, comparing control vs. ethephon-treated pericarp and AZ profiles. Over the examined time period (48 h), the accumulation of MiPIN1 and MiLAX2 IAA-efflux and influx genes decreased in both control and treated tissues. Nevertheless, ethephon-treated tissues displayed significantly lower levels of these transcripts within 18-24 h. An opposite pattern was observed for MiLAX3, which overall exhibited up-regulation in treated fruitlet tissues. Ethephon treatment also induced an early and pronounced down-regulation of five out of six IAA-responsive genes, and a substantial reduction in the accumulation of two IAA-synthesis related transcripts, contrasting with significant up-regulation of Gretchen Hagen3 transcript (MiGH3.1) encoding an IAA-amino synthetase. Furthermore, for both control and treated AZ, the decrease in IAA-carrier transcripts was associated with a decrease in IAA content and an increase in IAA-Asp:IAA ratio, suggesting that fruitlet drop is accompanied by formation of this non-hydrolyzed IAA-amino acid conjugate. Despite these similarities, ethephon-treated AZ displayed a sharper decrease in IAA content and higher IAA-Asp:IAA ratio within 18 h. Lastly, the response of IAA-related genes to exogenous IAA treatment was also examined. Our results are discussed, highlighting the roles that distinct IAA-related genes might assume during mango fruitlet drop.

Expression profiling of FLOWERING LOCUS T-like gene in alternate bearing 'Hass' avocado trees suggests a role for PaFT in avocado flower induction.

In many perennials, heavy fruit load on a shoot decreases the ability of the plant to undergo floral induction in the following spring, resulting in a pattern of crop production known as alternate bearing. Here, we studied the effects of fruit load on floral determination in 'Hass' avocado (Persea americana). De-fruiting experiments initially confirmed the negative effects of fruit load on return to flowering. Next, we isolated a FLOWERING LOCUS T-like gene, PaFT, hypothesized to act as a phloem-mobile florigen signal and examined its expression profile in shoot tissues of on (fully loaded) and off (fruit-lacking) trees. Expression analyses revealed a strong peak in PaFT transcript levels in leaves of off trees from the end of October through November, followed by a return to starting levels. Moreover and concomitant with inflorescence development, only off buds displayed up-regulation of the floral identity transcripts PaAP1 and PaLFY, with significant variation being detected from October and November, respectively. Furthermore, a parallel microscopic study of off apical buds revealed the presence of secondary inflorescence axis structures that only appeared towards the end of November. Finally, ectopic expression of PaFT in Arabidopsis resulted in early flowering transition. Together, our data suggests a link between increased PaFT expression observed during late autumn and avocado flower induction. Furthermore, our results also imply that, as in the case of other crop trees, fruit-load might affect flowering by repressing the expression of PaFT in the leaves. Possible mechanism(s) by which fruit crop might repress PaFT expression, are discussed.

PaKRP, a cyclin-dependent kinase inhibitor from avocado, may facilitate exit from the cell cycle during fruit growth.

Previous studies using 'Hass' avocado cultivar showed that its small-fruit (SF) phenotype is limited by cell number. To explore the molecular components affecting avocado cell production, we isolated four cDNAs encoding: an ICK/KRP protein, known to play cell cycle-regulating roles through modulation of CDK function; two CDK proteins and a D-type cyclin, and monitored their expression patterns, comparing NF (normal fruit) versus SF profiles. The accumulation of PaKRP gradually deceased during growth in both fruit populations. Despite these similarities, SF exhibited higher levels of PaKRP accumulation at early stages of growth. Moreover, in NF, augmented PaKRP expression coincided with a decrease in CDK and PaCYCD1 levels, whereas in SF, enhanced PaKPR expression was coupled with an earlier decline of CDK and PaCYCD1 levels. For both NF and SF, enhanced mesocarp PaKRP transcript accumulation, was associated with elevated abscisic acid (ABA) and ABA catabolites content. Nevertheless, the collective ABA levels, including catabolites, were substantially higher in SF tissues, as compared with NF tissues. Finally, additional expression analysis revealed that in cultured cells, PaKRP could be induced by ABA. Together, our data links PaKRP with exit from the fruit cell cycle and suggest a role for ABA in controlling its expression.

Cloning and molecular characterization of an ethylene receptor gene, MiERS1, expressed during mango fruitlet abscission and fruit ripening.

We isolated and characterized a mango (Mangifera indica L.) cDNA homolog of the ethylene receptor gene ERS1, designated MiERS1. Genomic Southern blot analysis suggested the existence of a second gene with homology to MiERS1. Spatial and temporal expression patterns of MiERS1 were first studied during fruitlet drop and compared with those of a previously identified MiETR1 gene that encodes an ETR1-type ethylene receptor. Experiments were conducted on developing fruitlet explants in which fruitlet abscission was induced by ethephon treatment. Northern analysis revealed a notable increase in MiERS1 mRNA levels in the fruitlet's activated abscission zone within 24 h of ethephon application, followed by a decreasing pattern 48 h post-treatment. A transient, albeit lesser, increase in MiERS1 mRNA levels was also observed in treated fruitlet seed and mesocarp tissues. In contrast, in the abscission zone, accumulation of MiETR1 transcript remained unchanged; a temporal increase in MiETR1 transcript level was observed in the fruitlet mesocarp, whereas in the seed, MiETR1 expression had already dropped by 24 h. Expression profiles of MiERS1 and MiETR1 were then studied during fruit ripening. In agreement with a previous study and coinciding with the climacteric rise in ethylene production, RNA blot analysis revealed that during fruit ripening, MiETR1 mRNA level increases in both mesocarp and seed tissues. Unexpectedly, however, in those same tissues, MiERS1 transcript accumulation was barely detected. Collectively, our data highlight MiERS1's possible specific function in regulating fruitlet abscission rather than fruit ripening.

A proposed conserved role for an avocado FW2.2-like gene as a negative regulator of fruit cell division.

Previous studies using 'Hass' avocado and its small fruit (SF) phenotype as a model showed that SF is limited by cell number, not by cell size. In an attempt to explore the molecular mechanisms regulating avocado fruit cell division, we isolated four distinct avocado cell proliferation-related genes and investigated their expression characteristics, comparing normal fruit (NF) and SF developmental patterns. Three cDNAs termed PaCYCA1, PaCYCB1 and PaPCNA, encoding two mitotic cyclins and a proliferating cell nuclear antigen (PCNA), were first isolated from young NF tissues. The accumulation of their transcripts was predominant in mitotically active organs, including young fruitlets, leaves and roots. Furthermore, a fourth full-length cDNA, designated Pafw2.2-like, encoding a FW2.2 (fruit-weight)-like protein, was isolated from SF tissues. FW2.2 is postulated to function as a negative regulator of cell division in tomato fruit. Remarkably, northern analysis revealed that the accumulation of the mitotic cyclins and of PCNA transcripts gradually decreased in NF tissues during growth, whereas in SF, their levels had already decreased at earlier stages of fruit development, concomitant with an earlier arrest of fruit cell division activity. In contrast, parallel sq-RT-PCR analysis showed that Pafw2.2-like mRNA accumulation was considerably higher in SF tissues than in the same NF tissues essentially at all examined stages of fruit growth. Together, our data suggest essential roles for the two mitotic cyclins genes and the PCNA gene in regulating avocado fruit development. Furthermore, the possibility that Pafw2.2-like acts as does fw2.2 in tomato, is discussed.

Phosphate and sulfur limitation responses in the chloroplast of Chlamydomonas reinhardtii.

Phosphorus (P) and sulfur (S) are two macronutrients that photosynthetic organisms require in relatively large amounts despite their levels in the environment often being limited. Accordingly, to adapt to random changes in macronutrient concentrations, plants and algae must sense and respond in a coordinated fashion. The unicellular green alga Chlamydomonas reinhardti is a widely used model organism for the study of P and S stress responses. Herein, we review the current knowledge of P and S nutrient stress responses, highlighting the roles of P and S key global-regulator proteins in mediating signals that link P and S detection to different chloroplast nutrient stress responses.

The sulfur acclimation SAC3 kinase is required for chloroplast transcriptional repression under sulfur limitation in Chlamydomonas reinhardtii.

Sulfur (S) deprivation responses have been studied extensively in algae and land plants; however, little is known of the signals that link perception of S status to chloroplast gene expression. Here, we have compared the chloroplast S limitation response in WT vs. sac1 and sac3 sulfur acclimation mutants of the green alga Chlamydomonas reinhardtii. We provide evidence that in the WT, chloroplast transcriptional activity rapidly decreases after removal of S from the medium, leading to reduced transcript accumulation. This decrease correlates with reduced abundance of a sigma70-like factor, Sig1, which is most likely the unique chloroplast transcription specificity factor. We further show that reduced transcription activity and diminished Sig1 accumulation are mediated by the SAC3 gene product, a putative Snf1-type Ser/Thr kinase previously shown to have both positive and negative effects on nuclear gene expression. Inclusion of the protein kinase inhibitor 6-dimethylaminopurine during S limitation yielded a pattern of expression that was largely similar to that seen in the sac3 mutant, lending support to the hypothesis that Sac3 kinase activation leads to transcriptional repression and Sig1 proteolysis. The finding that Sac3 regulates chloroplast gene expression suggests that it has a previously unknown role in integrating the S limitation response in multiple subcellular compartments.

Cloning, expression, and purification of functional Sec11a and Sec11b, type I signal peptidases of the archaeon Haloferax volcanii.

Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features with traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile, and oligomeric status. In this study, genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii, were cloned. Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. By employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea such as Haloferax volcanii, the signal peptide-cleaving activities of both isolated membranes and purified Sec11a and Sec11b were addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions.

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription.

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.

A proposed mechanism for the inhibitory effects of oxidative stress on Rubisco assembly and its subunit expression.

In Chlamydomonas reinhardtii, a light-induced oxidative stress shifts the glutathione pool toward its oxidized form, resulting in a translational arrest of the large subunit (LSU) of Rubisco. We show here that the translational arrest of LSU is tightly coordinated with cessation of Rubisco assembly, and both processes take place after a threshold level of reactive oxygen species is reached. As a result, the small subunit is also eliminated by rapid degradation. We previously showed that the amino terminus of the LSU could bind RNA in a sequence-independent manner, as it shares a structural similarity with the RNA recognition motif. This domain becomes exposed only under oxidizing conditions, thus restricting the RNA-binding activity. Here we show that in vitro, thiol groups of both subunits become oxidized in the presence of oxidized glutathione. The structural changes are mediated by oxidized glutathione, whereas only very high concentrations of H2O2 confer similar results in vitro. Changes in the redox state of the LSU thiol groups are also observed in vivo, in response to a physiological light shock caused by transfer of cells from low light to high light. We propose that during a photooxidative stress, oxidation of thiol groups occurs already in nascent LSU chains, perhaps hindering their association with chaperones. As a result, their RNA recognition motif domain becomes exposed and will bind any RNA in its vicinity, including its own transcript. Due to this binding the ribosome stalls, preventing the assembly of additional ribosomes on the transcript. Polysome analysis using Suc gradients indeed shows that the rbcL RNA is associated with the polysomal fraction at all times but shifts toward fractions that contain smaller polysomes and monosomes during oxidative stress. Thus, translational arrest of the LSU most likely occurs at a postinitiation stage.

RNA binding activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit from Chlamydomonas reinhardtii.

Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289-16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5'-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG.

Move it on over: getting proteins across biological membranes.

The translocation of proteins across membranes is a central problem in biology. Regardless of the system in question, delivering proteins across a given membrane relies on many of the same basic themes. At the same time, however, each membrane translocation system, be it signal-gated or signal-assembled, makes use of components unique to that system. The latest findings on protein translocation across a variety of biological membranes have been presented in a recent review article.