AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

yangzhou university

Yangzhou University

Chlorophyllase (CLH), which catalyzes the release of the phytol chain from chlorophyll (Chl), has been long considered to catalyze the first step of Chl degradation. Arabidopsis contains two isoforms of CLH (CLH1 and CLH2), and CLH1 was previously demonstrated to be localized in tonoplast and endoplasmic reticulum, and not be involved in Chl degradation. In contrast, CLH2 possesses a predicted signal-peptide for chloroplast localization, and phylogenetic analysis of CLHs in Arabidopsis and other species also indicate that CLH2 forms a different clade than CLH1. Therefore, the possibility remains that CLH2 is involved in the breakdown of Chl. In the current study, clh mutants lacking CLH2 or both CLH isoforms were analyzed after the induction of senescence. Results indicated that the clh knockout lines were still able to degrade Chl at the same rate as wild-type plants. Transgenic Arabidopsis plants were generated that constitutively expressed either CLH2 or CLH2 fused to a yellow fluorescent protein (YFP). Observations made using confocal microscopy indicated that CLH2-YFP was located external to chloroplasts. Additionally, in overexpression plants, CLH2 was enriched in tonoplast and endoplasmic reticulum fractions following membrane fractionation. Based on the collective data, we conclude that CLH2 is not involved in Chl breakdown during senescence in Arabidopsis.

Subcellular localization of chlorophyllase2 reveals it is not involved in chlorophyll degradation during senescence in Arabidopsis thaliana.

Edible fleshy fruits are important food sources in the human diet. Their yield and nutritional quality have long been considered as breeding targets for improvement. Various developing fleshy fruits with functional chloroplasts are capable of photosynthesis and contribute to fruit photosynthate, leading to the accumulation of metabolites associated with nutritional quality in ripe fruit. Although tomato high-pigment mutants with dark-green fruits have been isolated for more than 100 years, our understanding of the mechanism of chloroplast development in fleshy fruit remain poor. During the past few years, several transcription factors that regulate chloroplast development in fleshy fruit were identified through map-based cloning. In addition, substantial progress has been made in elucidating the mechanisms that how these transcription factors regulate chloroplast development. This review provides a summary and update on this progress, with a framework for further investigations of the multifaceted and hierarchical regulation of chloroplast development in fleshy fruit.

Progress on Understanding Transcriptional Regulation of Chloroplast Development in Fleshy Fruit.

The chlorophyll (Chl) cycle is the metabolic pathway for Chl  and Chl  inter-conversion. In this pathway, Chl  is synthesized from Chl  by the catalyzing action of chlorophyllide  oxygenase (CAO). In contrast, Chl  is firstly reduced to produce 7-hydroxymethyl Chl (HMChl) , which is catalyzed by two isozymes of Chl  reductase (CBR), non-yellow coloring 1 (NYC1) and NYC1-like (NOL). Subsequently, HMChl  is reduced to Chl  by HMChl  reductase (HCAR). CAO plays a pivotal role in Chl / ratio regulation and plants over-accumulate Chl  in CAO-overexpressing plants. NYC1 is more accumulated in Chl--overproducing plants, while HCAR is not changed. To investigate the role of HCAR in Chl cycle regulation, the Chl metabolites of Chl--overproducing plants were analyzed. The results showed that HMChl  accumulated in these plants, and it decreased and the Chl / ratio increased by overexpressing HCAR, implying HCAR is insufficient for Chl cycle in Chl--overproducing plants. Furthermore, during dark-induced senescence, the non-programmed cell death symptoms (leaves dehydrated with green color retained) of Chl--overproducing plants were obviously alleviated, and the content of HM pheophorbide (HMPheide)  and Pheide  were sharply decreased by overexpressing HCAR. These results imply that HCAR is also insufficient for Chl degradation in Chl--overproducing plants during senescence, thus causing the accumulation of Chl metabolites and non-programmed cell death of leaves. With these results taken together, we conclude that HCAR is not well regulated and it is a limiting factor for Chl cycle and Chl  degradation in Chl--overproducing plants.

HCAR Is a Limitation Factor for Chlorophyll Cycle and Chlorophyll  Degradation in Chlorophyll--Overproducing Plants.

Leaf senescence, the last stage of leaf development, is a well-regulated and complex process for investigation. For simplification, dark-induced leaf senescence has frequently been used to mimic the natural senescence of leaves because many typical senescence symptoms, such as chlorophyll (Chl) and protein degradation, also occur under darkness. In this study, we compared the phenotypes of leaf senescence that occurred when detached leaves or intact plants were incubated in darkness to induce senescence. We found that the symptoms of non-programmed cell death (non-PCD) with remaining green coloration occurred more heavily in the senescent leaves of whole plants than in the detached leaves. The pheophorbide  (Pheide ) content was also shown to be much higher in senescent leaves when whole plants were incubated in darkness by analyses of leaf Chl and its metabolic intermediates. In addition, more serious non-PCD occurred and more Pheide  accumulated in senescent leaves during dark incubation if the soil used for plant growth contained more water. Under similar conditions, the non-PCD phenotype was alleviated and the accumulation of Pheide  was reduced by overexpressing 7-hydroxymethyl Chl  (HMChl ) reductase (HCAR). Taken together, we conclude that a high soil water content induced non-PCD by decreasing HCAR activity when whole plants were incubated in darkness to induce senescence; thus, the investigation of the fundamental aspects of biochemistry and the regulation of leaf senescence are affected by using dark-induced leaf senescence.

Overexpressing 7-Hydroxymethyl Chlorophyll  Reductase Alleviates Non-Programmed Cell Death during Dark-Induced Senescence in Intact  Plants.

Global warming is a serious challenge plant production has to face. Heat stress not only affects plant growth and development but also reduces crop yield and quality. Studying the response mechanisms of plants to heat stress will help humans use these mechanisms to improve the heat tolerance of plants, thereby reducing the harm of global warming to plant production. Research on plant heat tolerance has gradually become a hotspot in plant molecular biology research in recent years. In view of the special role of chloroplasts in the response to heat stress in plants, this review is focusing on three perspectives related to chloroplasts and their function in the response of heat stress in plants: the role of chloroplasts in sensing high temperatures, the transmission of heat signals, and the improvement of heat tolerance in plants. We also present our views on the future direction of research on chloroplast related heat tolerance in plants.

Progress in Research on the Mechanisms Underlying Chloroplast-Involved Heat Tolerance in Plants.

Chlorophylls (Chls, Chl  and Chl ) are tetrapyrrole molecules essential for photosynthetic light harvesting and energy transduction in plants. Once formed, Chls are noncovalently bound to photosynthetic proteins on the thylakoid membrane. In contrast, they are dismantled from photosystems in response to environmental changes or developmental processes; thus, they undergo interconversion, turnover, and degradation. In the last twenty years, fruitful research progress has been achieved on these Chl metabolic processes. The discovery of new metabolic pathways has been accompanied by the identification of enzymes associated with biochemical steps. This article reviews recent progress in the analysis of the Chl cycle, turnover and degradation pathways and the involved enzymes. In addition, open questions regarding these pathways that require further investigation are also suggested.

Research Progress in the Interconversion, Turnover and Degradation of Chlorophyll.

Chlorophyllase (Chlase, CLH) is one of the earliest discovered enzymes present in plants and green algae. It was long considered to be the first enzyme involved in chlorophyll (Chl) degradation, while strong evidence showed that it is not involved in Chl breakdown during leaf senescence. On the other hand, it is possible that CLH is involved in Chl breakdown during fruit ripening. Recently, it was discovered that  CLH1 is located in developing chloroplasts but not in mature chloroplasts, and it plays a role in protecting young leaves from long-term photodamage by catalysing Chl turnover in the photosystem II (PSII) repair cycle. However, there remain other important questions related to CLH. In this article, we briefly reviewed the research progress on CLH and listed the main unanswered questions related to CLH for further study.

Chlorophyllase, a Common Plant Hydrolase Enzyme with a Long History, Is Still a Puzzle.

The J-proteins, also called DNAJ-proteins or heat shock protein 40 (HSP40), are one of the famous molecular chaperones. J-proteins, HSP70s and other chaperones work together as constitute ubiquitous types of molecular chaperone complex, which function in a wide variety of physiological processes. J-proteins are widely distributed in major cellular compartments. In the chloroplast of higher plants, around 18 J-proteins and multiple J-like proteins are present; however, the functions of most of them remain unclear. During the last few years, important progress has been made in the research on their roles in plants. There is increasing evidence that the chloroplast J-proteins play essential roles in chloroplast development, photosynthesis, seed germination and stress response. Here, we summarize recent research advances on the roles of J-proteins in the chloroplast, and discuss the open questions that remain in this field.

Research Progress in J-Proteins in the Chloroplast.

Heat shock proteins 70 (HSP70s) could cooperate with structurally diverse HSP40s (J proteins) to generate diverse chaperone networks in various cellular compartments, performing multiple housekeeping and stress-related functions in the organisms. There are two kinds of chloroplast heat shock protein 70 (cpHsc70-1, cpHsc70-2) and multiple J proteins in the Arabidopsis chloroplasts, while the interaction between cpHsc70s and J proteins and the function of most J proteins are largely unknown. In the present study, we found that AtDJC78 interacts with cpHsc70-1 through its C terminal, according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Bioinformatics analysis showed that DJC78 is one of the widespread and highly conserved J proteins in plants, AtDJC78 could be transported into chloroplasts, and the expression of AtDJC78 was significantly up-regulated under heat stress. Furthermore, we found that AtDJC78 may be associated with regulating hydrogen peroxide levels under heat stress in plants. These findings suggest that AtDJC78 is a new cochaperone interacting with cpHsc70-1 in the chloroplasts.

Xueyun Hu

College of Bioscience and Biotechnology

yangzhou university

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

Xueyun Hu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}College of Bioscience and Biotechnology

yangzhou university

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

College of Bioscience and Biotechnology