LIGHT STRESS — TOO MUCH OF A GOOD THING

BY CAROLINE WOOD

Light is the fundamental energy that drives plant growth and productivity, but outside the controlled conditions of our research laboratories, its intensity can fluctuate between extremes in a matter of seconds. This can present plants with the constant challenge of striking a delicate balance between maximising gains whilst keeping the delicate photosynthetic machinery safe from damage. Here, we take a look at fascinating new insights into the exquisite photoprotective mechanisms that help plants to stay safe from light stress.

Getting The Gradient Right

Intense spikes in luminescence—a sudden break in cloud cover, for instance—expose plants to potentially damaging amounts of excess light energy, capable of causing irreparable damage. Photoprotective mechanisms counteract this by dissipating excess light energy and downregulating photosynthesis during these events. Often, this involves adjusting the proton gradient across the thylakoid membrane that transiently stores the energy that powers photosynthesis. “Ongoing energy dissipation in shade periods, however, would decrease the efficiency of photosynthesis under natural, dynamically changing light conditions,” says Ute Armbruster, from the Max Planck Institute of Molecular Plant Physiology. “Consequently, plants rapidly turn off photoprotective mechanisms as soon as light intensity drops again. Our research has demonstrated that these switches in response to changes in light intensity are heavily accelerated by ion transporters in the thylakoid membrane.”

An intriguing question was whether the activity of these photoprotective mechanisms varied depending on the environment of the plant during its early development. To investigate this, Ute and her colleagues grew Arabidopsis thaliana plants under a variety of different light regimes, with some that fluctuated according to regular or random patterns and non-fluctuating control regimes.1 Plants from these different light environments were then exposed to a sudden, ten-fold increase in light intensity. By measuring changes in chlorophyll fluorescence and absorptive processes related to photosynthesis, the team assessed the activity of two thylakoid ion transport proteins, VCCN1 and KEA3. As a Cl− channel, VCCN1 lowered the membrane potential and promotes the induction of nonphotochemical quenching to dissipate excess light energy. Conversely, the K+/H+ exchanger KEA3 activated once light levels dropped again, to accelerate the relaxation of quenching mechanisms.

For both transporters, activity during high light stress strongly depended on the growth environment, with VCCN1 activity during high light stress correlating negatively and KEA3 activity positively with the average growth light intensity. “Together, this results in increased and faster activation of photoprotection in plants coming from growth conditions with low light intensities, and decreased activation in plants from growth conditions with higher average light intensities,” says Thekla von Bismarck, lead author of the study.

Their investigations also revealed that KEA3 suppresses the accumulation of zeaxanthin, a pigment which delays the relaxation of energydependent quenching mechanisms. The effect was particularly evident in plants grown under high or strongly fluctuating light intensities. “In fluctuating growth light conditions, this function of KEA3 would contribute to a more rapid response of a major photoprotective mechanism to changes in light intensity,” adds Thekla. “This discovery mandates future crop improvement strategies, which seek to accelerate photosynthetic responses, to consider acclimation effects on target regulator functions.”

According to Ute, another key finding from the study is that the photoprotective activities of thylakoid ion transport proteins only activate when the capacity for photosynthetic assimilation is saturated. This suggests that it may be possible to enhance the light reactions by manipulating carbon assimilation to lift downstream metabolic limitations. “At the same time, it is also plausible that the activities of thylakoid ion transport proteins have a tendency to ‘overprotect’ the system, ultimately restricting photosynthesis,” she adds.

“We are still in the infancy of understanding how thylakoid ion transport proteins sense and respond to metabolic changes, and the next step for our work is to resolve this. Additionally, we are interested to understand how the function and regulation of thylakoid ion transport proteins differ between photosynthetic organisms with variable morphological and physiological characteristics as well as environmental requirements.”

A Rapid Readjustment

The thylakoid membrane may also be implicated in another intriguing photoprotective mechanism through a physical link with plastoglobules. These may be one of the more underappreciated plant organelles, but recent studies indicate that plastoglobules have important roles in mediating stress perception and adaptative responses in plants. Found within chloroplasts, these lipid droplets are continuous with the photosynthetically active thylakoid membrane and, according to Peter Lundquist (Michigan State University), “represent an underexplored component of plant stress tolerance, with clear potential for enhancing crop resilience”. The evidence for this includes the fact that loss-of-function mutations in plastoglobulerelated genes demonstrate greater sensitivity to many abiotic stresses (including light stress), and the observation that virtually any stressful event causes plastoglobules to swell dramatically: often a more than 400-fold increase in volume.

But the exact role that plastoglobules may play in stress responses, besides the mechanisms they use, remain largely unknown. “My theory was that one key process may be a rapid adjustment of the lipid and protein composition in thylakoid membranes,” says Peter. “Despite the physical connectivity between plastoglobules and thylakoids, the plastoglobules maintain a distinct population of proteins and lipids, suggesting the existence of a proactive selection mechanism. Furthermore, the dramatic changes in plastoglobule size during

Chlorophyll fluorescence is captured from Arabidopsis thaliana to determine the activity of photoprotective mechanisms that protect against the damaging effects of excess light energy Photo credit: Ute Armbruster Below Peter with his highperformance liquid chromatography instruments used for measuring carotenoids and other prenyl-lipid compounds Photo credit: Peter Lundquist stresses imply a substantial influx of additional lipids, whether shuttled from the thylakoid membrane or synthesized de novo on the plastoglobule surface.”

To investigate this, Peter and his team compared unstressed plastoglobules from Arabidopsis with those that had been subjected to 5 days of light stress.2 Their results demonstrated that light stress induced highly specific, tailored changes to the lipid and protein composition of plastoglobules. In particular, specific carotenoid lipids used in light harvesting, such as zeaxanthin, over-accumulated within the light-stressed plastoglobules. “This indicates that turnover of light-harvesting complexes is occurring and the released cofactor lipids are subsequently shifted to the plastoglobule for storage,” Peter says.

Consistent with this, the plastoglobules also accumulated proteins involved in leaf senescence processes, including a lipase and protease, and an enzyme of the jasmonic acid biosynthetic pathway. “Although the light stress treatment that was applied does not induce visible senescence, our results suggest that senescent processes related to turnover of light harvesting machinery are employed by the plant as an adaptive response to the light stress treatment,” adds Peter. “Early senescent processes, including turnover of thylakoid lipids and protein, do not represent a commitment to cell death and are in fact important to protect the plant from overproduction of harmful reactive oxygen species.”

Potentially, these responses may be driven by changes in kinase activity. The group found that isolated plastoglobules demonstrated kinase activity towards multiple target proteins, and that this was more pronounced in plastoglobules of unstressed than light-stressed leaf tissue. “Our current theory is that kinase activity is the master regulator of the adaptive molecular changes that take place in plastoglobules during stress events,” says Peter. “Thus, the transition from low to high light intensities tailors the activity of plastoglobule proteins through phosphorylation to accumulate compounds derived from thylakoid membrane remodelling, such as carotenes. The next stage of our experiments will be to elucidate the effect that phosphorylation may have on plastoglobule-localised proteins. Possibilities include regulating sub-plastid localisation, activating or inactivating enzymes, or altering the oligomeric state of the proteins.”

A Versatile Hormone

Many of the cellular adaptations that plants use to combat abiotic stresses are coordinated by hormones, which can act as chemical messengers at the whole-plant level. Strigolactones (SLs) are phytohormones derived from carotenoids and are important for root/shoot growth and for promoting the symbiotic interaction with arbuscular mycorrhizal fungi. Recent evidence indicates that they may also have a role in adaptations to high light stress, as Sibu Simon (formerly Mar Athanasios College for Advanced Studies, India, now Mahatma Gandhi University) explains. “We carried out a SL-based transcriptome study in Arabidopsis, and conducted a meta-analysis comparing existing transcriptome data available with a wide range of different plant hormones. We identified that SLs uniquely regulated photosynthesis-related genes, in comparison to other plant hormones. Specifically, they upregulated genes in the light reaction pathway related to photosystem I and photosystem II. This made us ask whether SLs have any direct role in regulating photosynthesis and resulting high light stress tolerance in plants.”3

To investigate this, Sibu and his colleagues measured the photosynthesis rate of Arabidopsis plants after exogenous application of SL and compared their tolerance to high light stress (a jump from 60 to 1000 µmol/m2/s). The SL signalling mutant d14 exhibited a lower photosynthesis rate and was hypersensitive to high light stress, showing a more severe phenotype and a poorer recovery. Wild-type seedlings treated with SL, however, showed less damage and a stronger recovery in high light treatment compared with nontreated plants. Furthermore, SL treatment was ineffective on d14, adding further evidence that the hormonal pathway was responsible for the phenotype.

Additional investigations revealed that the SL treatment significantly reduced both chlorophyll and carotenoid content in the wild-type seedlings.

“Because chlorophyll and carotenoids act to protect light harvesting complexes from photoinhibition, this reduction could serve to increase the rate of the light-dependent photosynthesis reactions,” says Sibu. In support of this, chlorophyll fluorescence experiments revealed that SL treatment increased the quantum yield of photochemistry (the fraction of absorbed photons used for photochemical reactions) and decreased nonphotochemical quenching. “This confirms that SL treatment led to better utilisation of light via the photochemical electron transport pathway, and consequently a higher light reaction activity of photosynthesis,” says Sibu.

Metabolite profiling indicated that this had a downstream effect on the carbon fixation reactions. In Arabidopsis seedlings exposed to high light stress and treated with SL, there was a significant upregulation of intermediate compounds in both glycolysis (e.g. glucose-6-phosphate, fructose-6phosphate and pyruvate) and the tricarboxylic acid cycle (e.g. citric acid, succinic acid and-malic acid).

“Our results suggest there is a dynamic change in the metabolite levels as the plants respond to high light stress following SL treatment, suggesting that light acclimation involves modulation in the rate of carbon fixation,” says Sibu.

“This is the first study to have demonstrated a role of SLs in high light stress tolerance in plants. Furthermore, this appears to be through a direct effect of SLs in regulating photosynthesis rate,” he adds. “Upregulating photosynthesis would be an effective means to deal with the excess light energy. Whilst this study was limited to the initial phase of SL treatment during stress conditions, further time series could provide more insights into the dynamic changes throughout the recovery of the seedlings.”