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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2019)

Plants experience wide fluctuations in light intensity and must regulate light harvesting accordingly to prevent damage from excess energy. Several non-photochemical quenching (NPQ) mechanisms exist to harmlessly dissipate the excess energy and protect the photosynthetic apparatus under saturating light conditions. Each NPQ component occurs independently of each other and has different induction and relaxation kinetics involving unique molecular players. These components are outlined in Chapter 1. Under rapid light fluctuations, energy-dependent (qE) and zeaxanthin-dependent (qZ) quenching are critical and continue to be extensively studied. However, I focus here on a sustained form of antenna quenching, known as qH, that occurs under prolonged high light and abiotic stress. qH was recently identified as a distinct NPQ component independent of PsbS, ΔpH, zeaxanthin, D1 inactivation, and other qI processes. Induction of qH occurs under cold and high light stress and takes hours to days to turn off once induced. This slow relaxation rate can compete with light harvesting and limit photosynthetic efficiency. Thus, increasing the rate of qH relaxation may improve photosynthetic efficiency and crop yield. Within the last six years, three molecular players have been identified to be involved in qH. Under non-stress conditions, qH is prevented by the SUPPRESSOR OF QUENCHING1 (SOQ1) protein, which relies on a thioredoxin domain located in the thylakoid lumen. Under cold and high light, qH induction requires the plastid lipocalin protein, LCNP, which is also located in the lumen. In this dissertation, I present molecular insight into qH relaxation, which involves a previously uncharacterized protein, ROQH1.

RELAXATION OF QH1 (ROQH1) is an atypical short chain dehydrogenase/reductase that is conserved throughout the green lineage. In Chapter 2, I present the function of ROQH1 as a qH relaxation factor located in the chloroplast stroma, peripherally bound to the stroma lamellae membrane. Using various mutants and overexpressors, I show that qH does not relax in roqh1 mutants, whereas qH does not occur in ROQH1 overexpressors. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed, and soq1 roqh1 displays constitutive qH and light-limited growth. The antagonistic functions of LCNP and ROQH1 are both dosage-dependent in order to protect the photosynthetic apparatus and maintain light harvesting efficiency in plants.

The site of qH quenching is the peripheral antenna of PSII. In Chapter 3, I focus on which specific antenna component within PSII is responsible. The numerous chlorophyll fluorescence techniques utilized in this chapter collectively support the hypothesis that at least one site of qH quenching is the LHCII trimer. In Chapter 4, I investigate the biochemical mechanism of qH relaxation through interactions between ROQH1 and LHCII. Blue-native PAGE experiments indicate that ROQH1 forms a complex with LHCII under cold and high light conditions. Since the majority of LHCII is located in the grana core and ROQH1 is located in the stroma lamella, our current working hypothesis is that strong qH quenching sites are induced by LCNP in the LHCII trimers located in the grana margins. Through connectivity to these trimers, excitation energy received by the PSII antenna within the grana core are additionally quenched. ROQH1 access to stroma-exposed LHCII is then sufficient to turn off all of qH.

Insight into qH relaxation is important as improving NPQ relaxation has been shown to be a promising way to improve photosynthetic efficiency and crop yield. To this aim, we utilized the antagonistic functions of LCNP and ROQH1 to mitigate or abolish qH in tobacco. We used CRISPR/Cas9 to disrupt both LCNP genes simultaneously, and a leaf specific promoter to overexpress ROQH1. Stable transgenic N. tabacum lines are currently in progress, and we plan to monitor crop performance under greenhouse and field conditions to determine whether qH modification improves photosynthetic efficiency and crop yield. Food production needs to double by 2050 to meet the growing population demand in the face of rapidly changing climates and limitations in available arable land. Therefore, continued research on photosynthetic energy conversion is vital to our future food security.

    Cover page: RELAXATION OF QH1 (ROQH1) Functions in Sustained Photoprotective Energy Dissipation in Plants
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2018)
    Light utilization is finely tuned in photosynthetic organisms to prevent cellular damage. The dissipation of excess absorbed light energy, a process termed nonphotochemical quenching (NPQ), plays an important role in photoprotection. Little is known about the sustained or slowly reversible form(s) of NPQ and whether they are photoprotective, in part due to the lack of mutants. The Arabidopsis thaliana suppressor of quenching1 (soq1) mutant exhibits enhanced sustained NPQ, which we term qH. To identify molecular players involved in qH, we screened for suppressors of soq1 and isolated mutants affecting either chlorophyllide a oxygenase or the chloroplastic lipocalin, now renamed plastid lipocalin (LCNP). Analysis of the mutants confirmed that qH is localized to the peripheral antenna (LHCII) of photosystem II and demonstrated that LCNP is required for qH, either directly (by forming NPQ sites) or indirectly (by modifying the LHCII membrane environment). qH operates under stress conditions such as cold and high light and is photoprotective, as it reduces lipid peroxidation levels. We propose that, under stress conditions, LCNP protects the thylakoid membrane by enabling sustained NPQ in LHCII, thereby preventing singlet oxygen stress.
      Cover page: The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2020)
      Photosynthetic organisms experience wide fluctuations in light intensity and regulate light harvesting accordingly to prevent damage from excess energy. The antenna quenching component qH is a sustained form of energy dissipation that protects the photosynthetic apparatus under stress conditions. This photoprotective mechanism requires the plastid lipocalin LCNP and is prevented by SUPPRESSOR OF QUENCHING1 (SOQ1) under non-stress conditions. However, the molecular mechanism of qH relaxation has yet to be resolved. Here, we isolated and characterized RELAXATION OF QH1 (ROQH1), an atypical short-chain dehydrogenase-reductase that functions as a qH-relaxation factor in Arabidopsis. The ROQH1 gene belongs to the GreenCut2 inventory specific to photosynthetic organisms, and the ROQH1 protein localizes to the chloroplast stroma lamellae membrane. After a cold and high-light treatment, qH does not relax in roqh1 mutants and qH does not occur in leaves overexpressing ROQH1. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed and soq1 roqh1 displays constitutive qH and light-limited growth. We propose that LCNP and ROQH1 perform dosage-dependent, antagonistic functions to protect the photosynthetic apparatus and maintain light-harvesting efficiency in plants.
        Cover page: An atypical short-chain dehydrogenase–reductase functions in the relaxation of photoprotective qH in Arabidopsis
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2014)
        The photosystem II (PSII) protein PsbS and the enzyme violaxanthin deepoxidase (VDE) are known to influence the dynamics of energy-dependent quenching (qE), the component of nonphotochemical quenching (NPQ) that allows plants to respond to fast fluctuations in light intensity. Although the absence of PsbS and VDE has been shown to change the amount of quenching, there have not been any measurements that can detect whether the presence of these proteins alters the type of quenching that occurs. The chlorophyll fluorescence lifetime probes the excited-state chlorophyll relaxation dynamics and can be used to determine the amount of quenching as well as whether two different genotypes with the same amount of NPQ have similar dynamics of excited-state chlorophyll relaxation. We measured the fluorescence lifetimes on whole leaves of Arabidopsis thaliana throughout the induction and relaxation of NPQ for wild type and the qE mutants, npq4, which lacks PsbS; npq1, which lacks VDE and cannot convert violaxanthin to zeaxanthin; and npq1 npq4, which lacks both VDE and PsbS. These measurements show that although PsbS changes the amount of quenching and the rate at which quenching turns on, it does not affect the relaxation dynamics of excited chlorophyll during quenching. In addition, the data suggest that PsbS responds not only to ΔpH but also to the Δψ across the thylakoid membrane. In contrast, the presence of VDE, which is necessary for the accumulation of zeaxanthin, affects the excited-state chlorophyll relaxation dynamics.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2022)
          Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants' survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state. Interestingly, we found that chlorophyll (Chl) fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, indicating that they serve as a site for qH-energy dissipation and providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein, and lipid contents of antenna with active or inactive qH but did not detect any evident differences. Finally, we investigated whether specific subunits of the major antenna complexes were required for qH but found that qH was insensitive to trimer composition. Because we previously observed that qH can occur in the absence of specific xanthophylls, and no evident changes in pigments, proteins, or lipids were detected, we tentatively propose that the energy-dissipative state reported here may stem from Chl-Chl excitonic interaction.
            Cover page: The major trimeric antenna complexes serve as a site for qH-energy dissipation in plants.
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