UC Berkeley

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.