Unraveling chloroplast
State of the art - Senescence is the last stage of leaf development that is indispensable for the life on earth. Through senescence plants disassemble macromolecules in a programmed manner for relocating nutrients from source leaves to sink tissues (e.g. roots and seeds). Because plants are sessile, efficient use and salvage of assimilated nutrients are particularly important for plants' fitness and yield under ever-changing environments. Decisions concerning 'when to die' and 'how to die' must be tightly regulated by an intricate genetic program that integrates information about leaf age and various environmental factors. However, the nature of these signals and molecular mechanisms underlying senescence remains largely unknown.
- As the defining organelles of green plants, chloroplasts are the first organelles to be dismantled during senescence, representing a rich source of recycled nitrogen as a result of protein and chlorophyll breakdown. Amounting evidence indicate that chloroplasts also contribute signals that determine the initiation and proceeding of senescence. These signals could be linked to redox status of photosynthetic electron transfer chain and diverse metabolism in chloroplasts. However, the proposed senescence regulatory network mediated by chloroplasts remain yet to be established.
Objective - My overarching research goal is to establish an innovative chloroplast-mediated post-translational regulatory network that drive programmed leaf senescence. My strategy involves using large-scale genetic screening in combination with whole-genome resequencing analysis and omics-based technologies to understand and model the nature of photosynthetic pigment metabolism, reactive oxygen species signaling (ROS), and chloroplast degradation during senescence, and to characterize committed key regulators. Enabled by cutting-edge genome-editing tools, the applicant will integrate system data in a predictable model that will guide me in redesigning and engineering chloroplast events, that improving crop yield and grain quality.
Preliminary work - My early work (PhD and Postdoc) was focused on the investigation of the novel post-translational regulatory mechanisms that control various aspects of chloroplast development and chlorophyll metabolism in plants. Following my interests in chloroplast biology, the applicant joined the group of Prof. Dr. Bernhard Grimm in the Department of Biology/Plant Physiology, Humboldt University of Berlin. The applicant has revealed several novel post-translational regulatory mechanisms in chloroplasts, including chloroplast thioredoxin-mediated thiol-based redox regulation of the photosystem biogenesis, plastid gene expression, plant high-light acclimation, and chlorophyll biosynthesis (Wang et al. Plant Physiol, 2013, Liu1, Wang1 et al., Plant J, 2013; Wang et al., Mol Plant, 2014; Richter1, Wang1 et al., Plant Cell Physiol, 2016; Da1, Wang1 et al., Plant Physiol, 2017; 1equal contribution), chloroplast signal recognition particle 43-mediated chaperoning of key chlorophyll-synthesizing enzymes (Wang and Grimm, Photosynth Res, 2015; Wang and Grimm, Plant Physiol, 2016; Wang et al., PNAS, 2018), and BALANCE OF CHLOROPHYLL METABOLISM (BCM)-mediated coordination between chlorophyll synthesis and breakdown (Wang* et al., Nat Commun, 2020, in press, *corresponding author).
Work programme - A particular achievement of this work was the establishment of a powerful chlorophyll metabolism-based genetic screening platform, by which the novel players involved in the regulation of chlorophyll/carotenoid metabolism, ROS signaling, and chloroplast degradation during senescence could be identified in the prime model plant Arabidopsis thaliana.
·Post-translational regulation of chlorophyll breakdown. Chlorophyll breakdown is responsible for the detoxification of the potentially phototoxic pigments and is highly associated with degradation of thylakoid proteins and dismantling of thylakoid membranes during senescence. While many post-translational mechanisms have been documented in chlorophyll biosynthesis, post-translational control of chlorophyll catabolism awaits further study. The applicant employed the forward-genetic strategy and identified two paralogous BCMs, which simultaneously stimulate chlorophyll synthesis and attenuate chlorophyll degradation, respectively through interaction with the Mg-chelatase and Mg-dechelatase (Wang* et al., Nat Commun, 2020, in press, *corresponding author). It is intended to ask whether other auxiliary factors, such as thioredoxins, proteases, protein kinase, phosphatase, and molecular chaperone are involved in the regulation of chlorophyll breakdown. The reverse-genetic strategy and in vivo immunoprecipitation in combination with high sensitivity mass spectrometry will be applied to search for the novel regulators, which may interact physically or genetically with the BCMs or key chlorophyll catalytic enzymes.
·Post-translational of carotenoid metabolism. Carotenoids play crucial roles in both plant development and human nutrition and health. In comparison with intensive chlorophyll degradation during fruit ripening, carotenoid levels increase concomitantly with enlarged size of plastoglobules and collapse of thylakoids. However, the intrinsic mechanisms that coordinate chlorophyll breakdown, de novo carotenoid synthesis, and assembly of plastoglobules during senescence are poorly understood. It has been recently suggested that the BCM paralogs in soybean, tomato, and rice play a conserved role in the regulation of seed dormancy by the interaction with the rate-limiting enzymes in carotenoid metabolism and in turn modulating the synthesis of abscisic acid (Wang et al., Nat Genetics, 2018). The question that stem from these findings is that whether the BCMs act as a linker between chlorophyll metabolism and carotenoid metabolism, and in turn affect the organization of plastoglobules. The answer for this question is expected to improve understanding of post-translational regulatory network in carotenoid metabolism. In addition, since the proposed function of the H subunit of Mg-chelatase as the receptor of abscisic acid (Shen et al., Nature, 2006) are still under debate, this study may re-examine the function of Mg-chelatase and BCMs in the synthesis and signaling of abscisic acid.
·Identification of new players in the ROS signaling. The ROS species are essential signal compounds that determines a vast set of developmental and physiological processes in plants. However, little is known about the complete plant ROS signaling pathways, including the sensors, second messengers, and executers of ROS signals. Photosynthetic active chloroplasts are the major site of the ROS production in plant cells particularly under various stress conditions. Among diverse ROS species, the singlet oxygen (1O2) is exclusive germinated in chloroplasts with short half-life (~4 μs), when electronically excited porphyrins transfer energy to molecular oxygens. Deficiency of key regulators or enzymes of tetrapyrrole biosynthesis, such as FLUORESCENT and ferrochelatase, caused light-dependent seedling lethality in a controlled and noninvasive manner, due to the 1O2 induced cell death or chloroplast degradation. The crucial players of 1O2 signaling have emerged by genetically screening for the second-site mutations that abrogate the fatal effect of 1O2 boost on plants (Wanger et al., Science, 2004; Woodson et al., Science, 2015). Intriguingly, the Arabidopsis bcm1 knock-out mutant showed seedling lethal phenotype when the mutant seedlings were re-illuminated after 5 days of dark incubation. It is intended to identify novel ROS signaling players through searching for the second-site suppressors of the lethal bcm1 mutation, that surviving prolonged darkness.
·Screen for novel factors of chloroplast degradation. Degradation of chloroplasts is a hallmark of leaf senescence that is achieved via three cellular pathways relying respectively on autophagy, senescence-associated vacuoles, and chloroplast vesiculation protein. Despite their importance, only little is known about molecular control of chloroplast degradation in plants. Chlorophyll breakdown is the most visible symptom of leaf senescence and fruit ripening. The interrupted chloroplast degradation or chlorophyll breakdown usually lead to the retention of chlorophylls in the thylakoids, a syndrome called stay-green trait. Thus, it is feasible to use the stay-green phenotype as a marker to screen for the new factors of leaf senescence by phenotyping the Arabidopsis or tomato random gene mutation pools achieved by T-DNA insertion or CRISPR-Cas9-mediated genome editing under senescing conditions, which are triggered by elongated dark incubation, exogenous treatment of abscisic acid and jasmonic acid, or nutrient depletion.
Final Remark - It is intended to expand the existing strengthens of the Department of Plant Sciences at the University of Oxford with expertise in various chloroplast biology, pigment metabolism, and senescence related methodologies. The applicant will contribute to the Plant Physiology lecture courses and the lab courses, including but not limited to photosynthesis, chloroplast biology, leaf senescence, plant metabolisms, and plant cellular signaling. The funded research projects will also be benefit for the training undergraduate and graduate students.
