Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Differential response of the photosynthetic machinery to dehydration in older and younger resurrection plants.

A group of vascular plants called homoiochlorophyllous resurrection plants evolved unique capabilities to protect their photosynthetic machinery against desiccation-induced damage. This study examined whether the ontogenetic status of the resurrection plant Craterostigma pumilum has an impact on how the plant responds to dehydration at the thylakoid membrane level to prepare cells for the desiccated state. Thus, younger plants (<4 months) were compared with their older (>6 months) counterparts. Ultrastructural analysis provided evidence that younger plants suppressed senescence-like programs that are realized in older plants. During dehydration, older plants degrade specific subunits of the photosynthetic apparatus such as the D1 subunit of PSII and subunits of the cytochrome b6f complex. The latter leads to a controlled down-regulation of linear electron transport. In contrast, younger plants increased photoprotective high-energy quenching mechanisms and maintained a high capability to replace damaged D1 subunits. It follows that depending on the ontogenetic state, either more degradation-based or more photoprotective mechanisms are employed during dehydration of Craterostigma pumilum.

Fundamental helical geometry consolidates the plant photosynthetic membrane.

Plant photosynthetic (thylakoid) membranes are organized into complex networks that are differentiated into 2 distinct morphological and functional domains called grana and stroma lamellae. How the 2 domains join to form a continuous lamellar system has been the subject of numerous studies since the mid-1950s. Using different electron tomography techniques, we found that the grana and stroma lamellae are connected by an array of pitch-balanced right- and left-handed helical membrane surfaces of different radii and pitch. Consistent with theoretical predictions, this arrangement is shown to minimize the surface and bending energies of the membranes. Related configurations were proposed to be present in the rough endoplasmic reticulum and in dense nuclear matter phases theorized to exist in neutron star crusts, where the right- and left-handed helical elements differ only in their handedness. Pitch-balanced helical elements of alternating handedness may thus constitute a fundamental geometry for the efficient packing of connected layers or sheets.

Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves.

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.

Changes in aggregation states of light-harvesting complexes as a mechanism for modulating energy transfer in desert crust cyanobacteria.

In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.

Differential impacts of FtsZ proteins on plastid division in the shoot apex of Arabidopsis.

FtsZ proteins of the FtsZ1 and FtsZ2 families play important roles in the initiation and progression of plastid division in plants and green algae. Arabidopsis possesses a single FTSZ1 member and two FTSZ2 members, FTSZ2-1 and FTSZ2-2. The contribution of these to chloroplast division and partitioning has been mostly investigated in leaf mesophyll tissues. Here, we assessed the involvement of the three FtsZs in plastid division at earlier stages of chloroplast differentiation. To this end, we studied the effect of the absence of specific FtsZ proteins on plastids in the vegetative shoot apex, where the proplastid-to-chloroplast transition takes place. We found that the relative contribution of the two major leaf FtsZ isoforms, FtsZ1 and FtsZ2-1, to the division process varies with cell lineage and position within the shoot apex. While FtsZ2-1 dominates division in the L1 and L3 layers of the shoot apical meristem (SAM), in the L2 layer, FtsZ1 and FtsZ2-1 contribute equally toward the process. Depletion of the third isoform, FtsZ2-2, generally resulted in stronger effects in the shoot apex than those observed in mature leaves. The implications of these findings, along with additional observations made in this work, to our understanding of the mechanisms and regulation of plastid proliferation in the shoot apex are discussed.

Differential Roles of the Thylakoid Lumenal Deg Protease Homologs in Chloroplast Proteostasis.

Deg proteases are involved in protein quality control in prokaryotes. Of the three Arabidopsis (Arabidopsis thaliana) homologs, Deg1, Deg5, and Deg8, located in the thylakoid lumen, Deg1 forms a homohexamer, whereas Deg5 and Deg8 form a heterocomplex. Both Deg1 and Deg5-Deg8 were shown separately to degrade photosynthetic proteins during photoinhibition. To investigate whether Deg1 and Deg5-Deg8 are redundant, a full set of Arabidopsis Deg knockout mutants were generated and their phenotypes were compared. Under all conditions tested, deg1 mutants were affected more than the wild type and deg5 and deg8 mutants. Moreover, overexpression of Deg5-Deg8 could only partially compensate for the loss of Deg1. Comparative proteomics of deg1 mutants revealed moderate up-regulation of thylakoid proteins involved in photoprotection, assembly, repair, and housekeeping and down-regulation of those that form photosynthetic complexes. Quantification of protein levels in the wild type revealed that Deg1 was 2-fold more abundant than Deg5-Deg8. Moreover, recombinant Deg1 displayed higher in vitro proteolytic activity. Affinity enrichment assays revealed that Deg1 was precipitated with very few interacting proteins, whereas Deg5-Deg8 was associated with a number of thylakoid proteins, including D1, OECs, LHCBs, Cyt b 6 f, and NDH subunits, thus implying that Deg5-Deg8 is capable of binding substrates but is unable to degrade them efficiently. This work suggests that differences in protein abundance and proteolytic activity underlie the differential importance of Deg1 and Deg5-Deg8 protease complexes observed in vivo.

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens.

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.

Structural and functional characterisation of the cyanobacterial PetC3 Rieske protein family.

The cyanobacterium Synechocystis PCC 6803 possesses three Rieske isoforms: PetC1, PetC2 and PetC3. While PetC1 and PetC2 have been identified as alternative subunits of the cytochrome b6f complex (b6f), PetC3 was localized exclusively within the plasma membrane. The spatial separation of PetC3 from the photosynthetic and respiratory protein complexes raises doubt in its involvement in bioenergetic electron transfer. Here we report a detailed structural and functional characterization of the cyanobacterial PetC3 protein family indicating that PetC3 is not a component of the b6f and the photosynthetic electron transport as implied by gene annotation. Instead PetC3 has a distinct function in cell envelope homeostasis. Especially proteomic analysis shows that deletion of petC3 in Synechocystis PCC 6803 primarily affects cell envelope proteins including many nutrient transport systems. Therefore, the observed downregulation in the photosynthetic electron transport - mainly caused by photosystem 2 inactivation - might constitute a stress adaptation. Comprehensive in silico sequence analyses revealed that PetC3 proteins are periplasmic lipoproteins tethered to the plasma membrane with a subclass consisting of soluble periplasmic proteins, i.e. their N-terminal domain is inconsistent with their integration into the b6f. For the first time, the structure of PetC3 was determined by X-ray crystallography at an atomic resolution revealing significant high similarities to non-b6f Rieske subunits in contrast to PetC1. These results suggest that PetC3 affects processes in the periplasmic compartment that only indirectly influence photosynthetic electron transport. For this reason, we suggest to rename "Photosynthetic electron transport Chain 3" (PetC3) proteins as "periplasmic Rieske proteins" (Prp).

Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum.

The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C. pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b6 f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light-harvesting complexes and interrupted access to plastoquinone. This well-ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production.