Heta Mattila

Fast cytoplasmic streaming enables extensive chloroplast movements in the giant cells of unicellular, siphonous macroalgae. We studied chloroplast movements by light microscopy, macro-photography and pulse modulated chlorophyll a fluorescence in two such algae: Dasycladalean Acetabularia acetabulum and Bryopsidales Bryopsis sp.. Chemical inhibitors of actin polymerization and microtubule assembly were used to confirm that the observed effects were controlled by the cytoskeleton. Macro-imaging suggest that A. acetabulum responded to high light by reversible chloroplast aggregation, and inhibition of the movements led to increased Photosystem II photoinhibition, probed by the fluorescence parameter FV/FM. In A. acetabulum, wounding caused by cutting or feeding by the sap-sucking sea slug Elysia timida triggered appearance of darker and lighter cell regions, presumably chloroplast aggregation, within minutes of incurring damage. Addition of media collected from vicinity of cut algae was sufficient to induce these aggregations in intact algae, suggesting that water-borne cues or signals triggered the response. On the other hand, only minimal cytoplasmic changes were observed in Bryopsis sp. in response to high light or cutting. The relevance of chloroplast movements in protection against abiotic and biotic stressors in A. acetabulum, and the potential reasons behind the different defence strategies of the algae, are discussed.

In Metazoa, long-term sequestration of photosynthetically active algal chloroplasts – kleptoplasts – is a distinctive physiological trait of sacoglossan sea slugs. In this study, we examined the effects of the light spectrum on the body weight and photosynthetic activity of the sea slug Elysia crispata. Laboratory-reared specimens were exposed to equivalent irradiances of white, red, or blue light during feeding and starvation experiments. Sea slugs fed under red light increased their weight at a faster pace than specimens fed under white or blue light. At the end of the feeding trial, chlorophyll a concentration – a proxy for kleptoplast abundance – was significantly higher under blue light. Progressive weight loss was observed in starved sea slugs under all light spectra, but an initial transient weight increase was observed under red light. Photosystem II activity and photosynthetic electron transport rate declined in starvation, with the decrease being fastest under red light and slowest under blue light. Results suggest a trade-off between increased photosynthetic rates and kleptoplast degradation, leading to increased growth (under red light) and enhanced kleptoplast longevity, possibly due to reduced photooxidative damage (under blue light). We hypothesise that the higher degree of parapodia closure observed under blue light shields kleptoplasts from light-induced damage, effectively extending their lifespan.

Bryopsidales green macroalgae can induce non-photochemical quenching (NPQ) only slowly, presumably due to the lack of the proton gradient-induced component (qE) of NPQ and xanthophyll cycle. Here, two morphologically rather similar siphonous macroalgae, a Bryopsidales alga Bryopsis sp. and a Dasycladales alga Acetabularia acetabulum , latter of which is capable of qE, were given high light treatments with both constant and fluctuating intensity. No differences in the rate of photoinhibition of Photosystem II (PSII), estimated with the chlorophyll a fluorescence parameter F _V /F _M , in the absence or presence of lincomycin, were observed between constant and fluctuating light, nor between the two algae. Bryopsis sp. showed slower PSII recovery than A. acetabulum , possibly reflecting a regulatory response rather than increased oxidative stress, as the recovery rates increased with increasing amounts of PSII photoinhibition in both algae. In Bryopsis sp., however, high light treatments led to decreased electron transfer rates, estimated by both chlorophyll a fluorescence and net oxygen production, whereas a stimulation was observed in A. acetabulum . Nigericin, which prevents the formation of qE, increased photoinhibition in A. acetabulum but not in Bryopsis sp. Microoxic conditions as well as inhibitors of plastid terminal oxidase and mitochondrial respiration, on the other hand, enhanced photoinhibition only in Bryopsis sp., suggesting that, in the absence of qE, oxygen-dependent pathways (including flavodiiron proteins) are important for photoprotection. Near-infra-red absorption measurements suggest decreased Photosystem I (PSI) donor side limitation in Bryopsis sp., compared to A. acetabulum , and a lower capacity to keep P700 oxidised.

Fast cytoplasmic streaming enables extensive chloroplast movements in the giant cells of unicellular, siphonous macroalgae. Here, we studied chloroplast movements in two such algae: the Dasycladalean Acetabularia acetabulum and the Bryopsidales Bryopsis sp.. We hypothesised that chloroplast movements function as a protective avoidance mechanism under excess light, particularly in Bryopsis sp., which lacks capacity for fast induction of photoprotective non-photochemical quenching (NPQ) and state transitions. In addition, we also investigated whether chloroplast movements are involved in responses to wounding and herbivory. The movements were studied by light microscopy, photography and pulse modulated chlorophyll a fluorescence quenching analysis. Chemical inhibitors of actin polymerization and microtubules assembly were used to confirm that the observed effects were active responses controlled by the cytoskeleton. A. acetabulum responded to high light by reversible chloroplast aggregation, probed by macro-imaging; and chemical inhibition of chloroplast movements led to an enhancement of Photosystem II photoinhibition, as probed by the fluorescence parameter F _V /F _M . No chloroplast movements were observed in Bryopsis sp. in response to high light. In A. acetabulum , wounding caused either by cutting or due to feeding by the sap-sucking sea slug Elysia timida triggered aggregation of chloroplasts within minutes of incurring the damage. Interestingly, the aggregation also occurred in intact cells away from the cutting site. Furthermore, the addition of media collected from the vicinity of cut algae was sufficient to induce chloroplast aggregation in intact algae, suggesting that water-borne cues or signals triggered the aggregation response in A. acetabulum . Bryopsis sp., however, responded to cutting by only local chloroplast aggregation. The relevance of chloroplast movements in protection against both abiotic and biotic stressors in A. acetabulum, and the potential reasons behind the different defence strategies of the algae, are discussed.

Phosphorus and nitrogen are two fundamental macronutrients needed for photosynthesis, and their balance in photosymbiotic relationships, like those between corals and their zooxanthellae, is known to be highly important for maintaining a healthy partnership between the host and the symbiont. Kleptoplasty is a special case of algae-herbivore interaction, where the host animal or protist sequesters the chloroplasts from their prey algae as kleptoplasts inside their own cells. In some kleptoplastic sea slugs, the kleptoplasts can remain functional for months without the help from the algal nucleus, and the fundamental mechanisms in place to achieve this are still largely unsolved. Unlike in purely symbiotic systems like corals, the importance of nutrient balance to kleptoplast longevity has not been addressed in kleptoplastic sea slugs. We studied the phosphate and nitrogen exchange between the sea slug Elysia timida and its Acetabularia acetabulum derived kleptoplasts in various nutrient depletion and replenishment experiments. Our results show that sea slugs consumed phosphate from their surrounding medium and were able to recover electron transfer rates of kleptoplasts originating from phosphate depleted algae, suggesting that the sea slug provided the acquired organelles with phosphate to the extent that they increased the photosynthetic capacity remarkably well. Additional available phosphate in the water also increased the overall longevity of the kleptoplasts. The sea slugs were not able to recover the photosynthesis of nitrogen-depleted kleptoplasts, and additional dissolved nitrate and ammonium were not beneficial for kleptoplast longevity. Our results suggest that the phosphate-nitrogen balance inside the sea slug cells is mainly limited by phosphate, the addition of which can help the kleptoplasts maintain photosynthesis, perhaps by allowing the use of the glutamine synthetase (GS) and glutamate synthase (GOGAT) nitrogen assimilation pathway to function as an efficient alternative electron sink.

To combat light-induced damage, photosynthetic organisms have evolved several photoprotective mechanisms, including non-photochemical quenching (NPQ) of excess light energy. Bryopsidales green macroalgae lack the energy-dependent quenching (qE) and can therefore induce NPQ only slowly. We illuminated a Bryopsidales macroalga ( Bryopsis sp.) and a morphologically similar Dasycladales alga ( Acetabularia acetabulum ), capable of qE, with high light. No differences in the rate of Photosystem II (PSII) damage, probed by the chlorophyll a fluorescence parameter F _V /F _M in the absence and presence of lincomycin, were observed between constant and fluctuating light in Bryopsis sp., nor between the two algae. In Bryopsis sp., however, photoinhibition lead to decreased rates of electron transfer (estimated by fluorescence and oxygen evolution), while a stimulation was observed in A. acetabulum . Compared to A. acetabulum , Bryopsis sp. showed slow PSII repair, but as the rate of repair increased with increasing rates of the damage, the slow repair may not indicate increased oxidative stress but be a regulatory response. We observed, in A. acetabulum , a post-illumination dip in PSII activity and a concurring bump in NPQ, which were removed by an inhibitor of the NDA2-dependent cyclic electron transfer route and enhanced in the presence of inhibitor of mitochondrial respiration, suggesting that these pathways reduce and oxidise, respectively, the plastoquinone pool in the dark in this alga. Nigericin, which prevents the formation of proton gradient and thus qE, increased photoinhibition in A. acetabulum but not in Bryopsis sp.. Anoxia and inhibitors of the plastid terminal oxidase and mitochondrial respiration, on the other hand, enhanced photoinhibition only in Bryopsis sp., suggesting that oxygen-dependent pathways (including flavodiiron proteins) are important for photoprotection in the qE-deficient Bryopsidales algae.

In order to reduce the expenses of plant tissue culture for plant industry, cheaper materials and/or equipment would be welcome especially in developing countries. In the present study, we tested mucilage extracted from seeds of quince (Cydonia oblonga) as alternative for agar in tissue culture. The seeds are currently disposed of as waste products of fruit industry. Tomato and tobacco lateral buds and stems were used as explants, and callus formation and the number of shoots, roots, and leaves were measured after thirty days of in vitro culture. The performance of quince mucilage based medium (QM) in comparison to agar was studied, varying the amounts of hormones, salts and sucrose in the medium. Statistical analyses, based on negative binomial regression, show that QM improved the production of callus, shoots, roots and leaves from tobacco explants. In tomato, results from QM and agar were similar to each other except with root production in which QM excelled. The results suggest that QM has potential for use as a gelling agent in plant tissue culture.

Green macroalgae within the order Bryopsidales lack the fundamental photoprotective mechanisms of green algae, the xanthophyll cycle and energy-dependent dissipation of excess light. Here, by measuring chlorophyll fluorescence at 77K after specific light treatments, we show that Bryopsidales algae also lack state transitions, another ubiquitous photoprotection mechanism present in other green algae. Certain Sacoglossa sea slugs can feed on Ulvophyceae algae, including some Bryopsidales, and steal chloroplasts –kleptoplasts– that remain functional inside the animal cells for months without the support of the algal nucleus. Our data reveal that the state transition capacity is not retained in the kleptoplasts of the sea slugs, and we provide evidence that the loss is caused by structural changes during their incorporation by the animals. This enforced chloroplast sphericity was observed in all studied kleptoplastic associations, and we propose that it is a fundamental property supporting long-term retention of kleptoplasts in photosynthetic sea slugs.

Instead of red anthocyanins, birches synthesise colourless (to human eye), UV-absorbing flavonols during autumn senescence. To test if flavonols protect against insects, and if leaves with high or low amounts of flavonols differ in their photosynthetic functions, aphid-free and aphid-infested green and senescing birch leaves were collected from outdoor-grown trees and analysed. Photosynthetic parameters were greatly affected by the leaf chlorophyll content (i.e. the phase of senescence). Photochemical quenching and the amount of functional Photosystem I decreased linearly with chlorophyll content, while FV/FM (Photosystem II functionality) decreased strongly only at the end of senescence. Non-photochemical quenching of excitation energy (NPQ) increased towards the end of senescence. However, no significant differences in the total flavonol amounts, nor in individual flavonol species, were found between aphid-free and aphid-infested leaves, suggesting that flavonols play no role in defence against aphid herbivory. Interestingly, both green and senescing leaves with a high flavonol content showed low FV/FM values. High flavonol content slowed down PSII photoinhibition and improved recovery, but only in green leaves. Previously, we proposed that anthocyanins provide an additional sink for photosynthates at the nitrogen resorption phase during autumn senescence, and the present data may suggest that flavonol synthesis plays a similar role.

Autumn senescence is characterised by spatial and temporal heterogeneity. We show that senescing birch (Betula spp.) leaves had lower PSII activity (probed by the FV/FM chlorophyll a fluorescence parameter) in late autumn than in early autumn. We confirmed that PSII repair slows down with decreasing temperature, while rates of photodamage and recovery, measured under laboratory conditions at 20°C, were similar in these leaves. We propose that low temperatures during late autumn hinder repair and lead to accumulation of non-functional PSII units in senescing leaves. Fluorescence imaging of birch revealed that chlorophyll preferentially disappeared from inter-veinal leaf areas. These areas showed no recovery capacity and low non-photochemical quenching while green veinal areas of senescing leaves resembled green leaves. However, green and yellow leaf areas showed similar values of photochemical quenching. Analyses of thylakoids isolated from maple (Acer platanoides) leaves showed that red, senescing leaves contained high amounts of carotenoids and α-tocopherol, and our calculations suggest that α-tocopherol was synthesised during autumn. Thylakoids isolated from red maple leaves produced little singlet oxygen, probably due to the high antioxidant content. However, the rate of PSII photodamage did not decrease. The data show that the heterogeneity of senescing leaves must be taken into account to fully understand autumn senescence.