Previous work showed a transient but dramatic arrest in the synthesis of Rubisco large subunit (LSU) upon transfer of Chlamydomonas reinhardtii cells from low light (LL) to high light (HL). Using dichlorofluorescin, a short-term increase in reactive oxygen species (ROS) was demonstrated, suggesting that their excessive formation could signal LSU down-regulation. A decrease in LSU synthesis occurred at LL in the presence of methyl viologen and was prevented at HL by ascorbate. Interfering with D1 function by mutations or by incubation with DCMU prevented the increase in ROS formation at HL and the concomitant down-regulation of LSU synthesis. If the electron transport was blocked further downstream, by mutation in the cytochrome b 6 /f or by incubation with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, ROS formation increased, and LSU synthesis ceased. The elevation of ROS occurred concurrently with a change in the redox state of the glutathione pool, which shifted toward its oxidized form immediately after the transfer to HL and returned to its original value after 6 h. The decrease in the reduced/oxidized glutathione ratio at HL was prevented by ascorbate and could be induced at LL by methyl viologen. We suggest that excess ROS mediate a decrease in the reduced/oxidized glutathione ratio that in turn signals the translational arrest of the rbcL transcript.Exposure of photosynthetic organisms to light intensities that exceed the limits of photosynthesis saturation can cause severe damage to the photosynthetic machinery, referred to as photoinhibition (1-3). Most plants and algae have the capacity to recover from light stress through photoacclimation, which normally involves a reduction in either the number or size of the light harvesting complexes and increased synthesis of the photodamaged D1, the core protein of photosystem II (PSII) (4, 5). Excess light energy generates reactive oxygen species (ROS), 1 which in turn lead to the induction of antioxidant photoprotective mechanisms enabling the plant to combat the danger posed by the presence of ROS.Ribulose biphosphate carboxylase-oxygenase (Rubisco) is the key enzyme in carbon assimilation during photosynthesis. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family (6, 7). Assembly of the Rubisco holoenzyme is driven by the chloroplast Cpn60 and Cpn10, encoded by groEL and groES, respectively. Previously we observed unique and opposite patterns for translational regulation of the chloroplast LSU and D1 polypeptides in response to changes in light intensity. Within minutes of shifting cells of C. reinhardtii from low light to higher light intensities, LSU synthesis was down-regulated dramatically for a period that did not exceed 4 -6 h, whereas that of D1 was gradually up-regulated. Translation of other genes was hardly affected, including photosynthesis-related genes such as the chloroplast encoded ATPase ␤-sub...

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 H 2 O 2 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.Rubisco is responsible for CO 2 fixation during photosynthesis. In vascular plants and green algae, it exists as a holoenzyme composed of eight large subunits (LSUs; 55 kD) encoded by the chloroplast rbcL gene and eight small subunits (SSUs; 15 kD) produced by a nuclear family of rbcS genes (Spreitzer, 1993). SSU precursors are processed during entry into the chloroplast and are then assembled with the LSUs to yield the holoenzyme. Assembly of the oligomeric protein (approximately 500 kD) is mediated by cpn60 and cpn10 (Gatenby and Ellis, 1990), and a complex of the LSU and cpn60 serves as an intermediate of the assembly process. The chloroplast cpn60 is a homolog of bacterial groEL, and prokaryotic subunits of Rubisco expressed in Escherichia coli can be successfully assembled into a holoenzyme (Goloubinoff et al., 1989). A role for additional chaperone molecules, in accumulation of mature Rubisco complexes, was recently described (Brutnell et al., 1999).Many protein complexes in the chloroplast are composed of multiple polypeptides, which are expressed in a tightly coordinated manner, as removal of one subunit can have an effect on expression of the other ...

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.

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.

Although protein secretion occurs post-translationally in bacteria and is mainly a cotranslational event in Eukarya, the relationship between the translation and translocation of secreted proteins in Archaea is not known. To address this question, the signal peptideencoding region of the surface layer glycoprotein gene from the Haloarchaea Haloferax volcanii was fused either to the cellulose-binding domain of the Clostridium thermocellum cellulosome or to the cytoplasmic enzyme dihydrofolate reductase from H. volcanii. Signal peptide-cleaved mature versions of both the cellulose-binding domain and dihydrofolate reductase could be detected in the growth medium of transformed H. volcanii cells. Immunoblot analysis revealed, however, the presence of full-length signal peptide-bearing forms of both proteins inside the cytoplasm of the transformed cells. Proteinase accessibility assays confirmed that the presence of cell-associated signal peptide-bearing proteins was not due to medium contamination. Moreover, the pulse-radiolabeled signal peptide cellulose-binding domain chimera could be chased from the cytoplasm into the growth medium even following treatment with anisomycin, an antibiotic inhibitor of haloarchaeal protein translation. Thus, these results provide evidence that, in Archaea, at least some secreted proteins are first synthesized inside the cell and only then translocated across the plasma membrane into the medium.