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.

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.

Enhancing Urban Wastewater Treatment through Isolated Chlorella Strain-Based Phytoremediation in Centrate Stream: An Analysis of Algae Morpho-Physiology and Nutrients Removal Efficiency.

The release of inadequately treated urban wastewater is the main cause of environmental pollution of aquatic ecosystems. Among efficient and environmentally friendly technologies to improve the remediation process, those based on microalgae represent an attractive alternative due to the potential of microalgae to remove nitrogen (N) and phosphorus (P) from wastewaters. In this work, microalgae were isolated from the centrate stream of an urban wastewater treatment plant and a native Chlorella-like species was selected for studies on nutrient removal from centrate streams. Comparative experiments were set up using 100% centrate and BG11 synthetic medium, modified with the same N and P as the effluent. Since microalgal growth in 100% effluent was inhibited, cultivation of microalgae was performed by mixing tap-freshwater with centrate at increasing percentages (50%, 60%, 70%, and 80%). While algal biomass and nutrient removal was little affected by the differently diluted effluent, morpho-physiological parameters (FV/FM ratio, carotenoids, chloroplast ultrastructure) showed that cell stress increased with increasing amounts of centrate. However, the production of an algal biomass enriched in carotenoids and P, together with N and P abatement in the effluent, supports promising microalgae applications that combine centrate remediation with the production of compounds of biotechnological interest; for example, for organic agriculture.

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.

Removal of Nitrogen and Phosphorus from Thickening Effluent of an Urban Wastewater Treatment Plant by an Isolated Green Microalga.

Microalgae are photosynthetic microorganisms and are considered excellent candidates for a wide range of biotechnological applications, including the removal of nutrients from urban wastewaters, which they can recover and convert into biomass. Microalgae-based systems can be integrated into conventional urban wastewater treatment plants (WW-TP) to improve the water depuration process. However, microalgal strain selection represents a crucial step for effective phytoremediation. In this work, a microalga isolated from the effluent derived from the thickening stage of waste sludge of an urban WW-TP was selected and tested to highlight its potential for nutrient removal. Ammonium and phosphate abatements by microalgae were evaluated using both the effluent and a synthetic medium in a comparative approach. Parallelly, the isolate was characterized in terms of growth capability, morphology, photosynthetic pigment content and photosystem II maximum quantum yield. The isolated microalga showed surprisingly high biomass yield and removal efficiency of both ammonium and phosphate ions from the effluent but not from the synthetic medium. This suggests its clear preference to grow in the effluent, linked to the overall characteristics of this matrix. Moreover, biomass from microalgae cultivated in wastewater was enriched in photosynthetic pigments, polyphosphates, proteins and starch, but not lipids, suggesting its possible use as a biofertilizer.