Thylakoid ultrastructural variations in chlorophyll-deficient wheat: aberrations or structural acclimation?

MAIN CONCLUSION: A structural re-modeling of the thylakoid system, including granum size and regularity, occurs in chlorophyll-deficient wheat mutants affected by photosynthetic membrane over-reduction. In the chloroplast of land plants, the thylakoid system is defined by appressed grana stacks and unstacked stroma lamellae. This study focuses on the variations of the grana organization occurring in outdoor-grown wheat mutants characterized by low chlorophyll content and a tendency for photosynthetic membrane over-reduction. Triticum aestivum ANK-32A and Triticum durum ANDW-7B were compared to their corresponding WT lines, NS67 and LD222, respectively. Electron micrographs of chloroplasts were used to calculate grana ultrastructural parameters. Photosynthetic parameters were obtained by modulated chlorophyll fluorescence and applying Light Curves (LC) and Rapid Light Curves (RLC) protocols. For each photosynthetic parameter, the difference Δ(RLC-LC) was calculated to evaluate the flexible response to light in the examined lines. In the mutants, fewer and smaller disks formed grana stacks characterized by a marked increase in lateral and cross-sectional irregularity, both negatively correlated with the number of layers per granum. A relationship was found between membrane over-reduction and granum structural irregularity. The possible acclimative significance of a greater proportion of stroma-exposed grana domains in relieving the excess electron pressure on PSI is discussed.

Ultrastructural organization of the thylakoid system during the afternoon relocation of the giant chloroplast in Selaginella martensii Spring (Lycopodiophyta).

Within the ancient vascular plant lineage known as lycophytes, many Selaginella species contain only one giant chloroplast in the upper epidermal cells of the leaf. In deep-shade species, such as S. martensii, the chloroplast is cup-shaped and the thylakoid system differentiates into an upper lamellar region and a lower granal region (bizonoplast). In this report, we describe the ultrastructural changes occurring in the giant chloroplast hosted in the epidermal cells of S. martensii during the daily relocation of the organelle. The process occurs in up to ca. 40% of the microphylls without the plants being exposed to high-light flecks. The relocated chloroplast loses its cup shape: first, it flattens laterally toward the radial cell wall and then assumes a more globular shape. The loss of the conical cell shape, the side-by-side lateral positioning of vacuole and chloroplast, and the extensive rearrangement of the thylakoid system to only granal cooperate in limiting light absorption. While the cup-shaped chloroplast emphasizes the light-harvesting capacity in the morning, the relocated chloroplast is suggested to support the renewal of the thylakoid system during the afternoon, including the recovery of photosystem II (PSII) from photoinhibition. The giant chloroplast repositioning is part of a complex reversible reshaping of the whole epidermal cell.

Thylakoid membrane appression in the giant chloroplast of Selaginella martensii Spring: A lycophyte challenges grana paradigms in shade-adapted species.

In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid system among plant species is mainly determined by the adaptation to the growth light regime, according to a paradigm stating that shade-tolerant species are featured by a high membrane extension with an enhanced number of thylakoid layers per granum. In this study, the thylakoid system was analysed in Selaginella martensii Spring, a shade-adapted rainforest species belonging to lycophytes, a diminutive plant lineage, sister clade of all other vascular plants (euphyllophytes, including ferns and seed plants). The species is characterized by giant cup-shaped chloroplasts in the upper epidermis and, quantitatively less important, disk-shaped chloroplasts in the mesophyll and lower epidermis. The study aimed at the quantitative assessment of the thylakoid appression exploiting a combination of complementary methods, including electron microscopy, selective thylakoid solubilisation, electron paramagnetic resonance, and simultaneous analysis of fast chlorophyll a fluorescence and P700 redox state. With a chlorophyll a/b ratio of 2.6 and PSI/PSII ratio of 0.31, the plant confirmed two typical hallmarks of shade-adaptation. The morphometric analysis of electron micrographs revealed a 33% fraction of non-appressed thylakoid domains. However, contrasting with the structural paradigm of thylakoid shade-adaptation in angiosperms, S. martensii privileges the increase in the granum diameter in place of the increase in the number of layers building the granum. The very wide grana diameter, 727 nm on average, largely overcame the threshold of 500 nm currently hypothesized to allow an effective diffusion of long-range electron carriers. The fraction of non-appressed membranes based on the selective solubilisation of thylakoids with digitonin was 26%, lower than the morphometric determination, indicating the presence of non-appressed domains inaccessible to the detergent, most probably because of the high three-dimensional complexity of the thylakoid system in S. martensii. Particularly, strong irregularity of grana stacks is determined by assembling thylakoid layers of variable width that tend to slide apart from each other as the number of stacked layers increases.

Long-Term Alleviation of the Functional Phenotype in Chlorophyll-Deficient Wheat and Impact on Productivity: A Semi-Field Phenotyping Experiment.

Wheat mutants with a reduced chlorophyll synthesis are affected by a defective control of the photosynthetic electron flow, but tend to recover a wild-type phenotype. The sensitivity of some mutants to light fluctuations suggested that cultivation outdoors could significantly impact productivity. Six mutant lines of Triticum durum or Triticum aestivum with their respective wild-type cultivars were cultivated with a regular seasonal cycle (October-May) in a semi-field experiment. Leaf chlorophyll content and fluorescence parameters were analysed at the early (November) and late (May) developmental stages, and checked for correlation with morphometric and grain-production parameters. The alleviation of the phenotype severity concerned primarily the recovery of the photosynthetic-membrane functionality, but not the leaf chlorophyll content. Photosystem II (PSII) was less photoprotected in the mutants, but a moderate PSII photoinhibition could help control the electron flow into the chain. The accumulation of interchain electron carriers was a primary acclimative response towards the naturally fluctuating environment, maximally exploited by the mature durum-wheat mutants. The mutation itself and/or the energy-consuming compensatory mechanisms markedly influenced the plant morphogenesis, leading especially to reduced tillering, which in turn resulted in lower grain production per plant. Consistently with the interrelation between early photosynthetic phenotype and grain-yield per plant, chlorophyll-fluorescence indexes related to the level of photoprotective thermal dissipation (pNPQ), photosystem II antenna size (ABS/RC), and pool of electron carriers (Sm) are proposed as good candidates for the in-field phenotyping of chlorophyll-deficient wheat.

Fast chlorophyll a fluorescence induction (OJIP) phenotyping of chlorophyll-deficient wheat suggests that an enlarged acceptor pool size of Photosystem I helps compensate for a deregulated photosynthetic electron flow.

The wheat lines affected by a decrease in the leaf chlorophyll content typically experience a biomass loss. A known major problem of the chlorophyll-deficient wheat mutants is their limited prevention of Photosystem I (PSI) over-reduction brought about by an insufficient cyclic electron flow, potentially exposing them to a higher sensitivity to light fluctuations. However, the resistance of some mutant lines against fluctuating light suggests the occurrence of regulatory processes compensating for the defect in cyclic electron flow. In this study, a phenotyping approach based on fast chlorophyll a fluorescence induction (OJIP transient), corroborated by P700 redox kinetics, was applied to a collection of chlorophyll-deficient wheat lines, grown under continuous or fluctuating light. Quantitative parameters calculated from the OJIP transient are considered informative about Photosystem II (PSII) functional antenna size and photochemistry, as well as the functioning of the entire photosynthetic electron transport chain. The mutants tended to recover a wild-type-like chlorophyll content, and mature plants could hardly be distinguished based on their effective PSII antenna size. Nevertheless, specific OJIP-derived parameters were strongly correlated with the phenotype severity, in particular the amplitude of the I-P phase and the I-P/J-P amplitude ratio, which are indicative of a more capacitive pool of PSI final electron acceptors (ferredoxin and ferredoxin-NADP+ oxidoreductase, FNR). We propose that the enlargement of such pool of electron carriers is a compensatory response operating at the acceptor side of PSI to alleviate potentially harmful over-reduced states of PSI. Our results also suggest that, in chlorophyll-deficient mutants, higher FV /FM cannot prove a superior PSII photochemistry and wider I-P phase is not indicative of a higher relative content of PSI.

Photosystem II photoinhibition and photoprotection in a lycophyte, Selaginella martensii.

The Lycophyte Selaginella martensii efficiently acclimates to diverse light environments, from deep shade to full sunlight. The plant does not modulate the abundance of the Light Harvesting Complex II, mostly found as a free trimer, and does not alter the maximum capacity of thermal dissipation (NPQ). Nevertheless, the photoprotection is expected to be modulatable upon long-term light acclimation to preserve the photosystems (PSII, PSI). The effects of long-term light acclimation on PSII photoprotection were investigated using the chlorophyll fluorometric method known as "photochemical quenching measured in the dark" (qPd ). Singularly high-qPd values at relatively low irradiance suggest a heterogeneous antenna system (PSII antenna uncoupling). The extent of antenna uncoupling largely depends on the light regime, reaching the highest value in sun-acclimated plants. In parallel, the photoprotective NPQ (pNPQ) increased from deep-shade to high-light grown plants. It is proposed that the differences in the long-term modulation in the photoprotective capacity are proportional to the amount of uncoupled LHCII. In deep-shade plants, the inconsistency between invariable maximum NPQ and lower pNPQ is attributed to the thermal dissipation occurring in the PSII core.

In an ancient vascular plant the intermediate relaxing component of NPQ depends on a reduced stroma: Evidence from dithiothreitol treatment.

In plants, the non-photochemical quenching of chlorophyll fluorescence (NPQ) induced by high light reveals the occurrence of a multiplicity of regulatory processes of photosynthesis, primarily devoted to photoprotection of photosystem I and II (PSI and PSII). The study of NPQ relaxation in darkness allows the separation of three kinetically distinct phases: the fast relaxing high-energy quenching qE, the intermediate relaxing phase and the nearly non-relaxatable photoinhibitory quenching. Several processes can underlie the intermediate phase. In the ancient vascular plant Selaginella martensii (Lycopodiophyta) this component, here termed qX, was previously proposed to reflect mainly a photoprotective energy-spillover from PSII to PSI. It is hypothesized that qX is induced by an over-reduced photosynthetic electron transport chain from PSII to final acceptors. To test this hypothesis the leaves were treated with the reductant dithiothreitol (DTT) and the chlorophyll fluorescence changes were analysed during the induction with high irradiance and the subsequent relaxation in darkness. DTT treatment caused the well-known decrease in NPQ induction and expectedly resulted in a disturbed photosynthetic electron flow. The relaxation curves of Y(NPQ), formally representing the quantum yield of the regulatory thermal dissipation, revealed a DTT dose-dependent decrease in amplitude not only of qE, but also of qX, up to the complete disappearance of the latter. Modelling of the relaxation curves under alternative scenarios led to the conclusion that DTT is permissive with respect to qX induction but suppresses its dark relaxation. The strong dependence of qX on the chloroplast redox state is discussed with respect to its proposed energy-spillover photoprotective significance in a lycophyte.