CsARGs involved in different occurrence stages of autophagy in tea plant
Autophagy is a catabolic degradation pathway essential for degrading long-lived proteins, protein aggregates, and damaged organelles . It has been proved that autophagy is highly conserved from yeast to humans, which is the result of the interaction of many proteins. So far, there have more than 30 ATG-related genes been identified in many eukaryotes, and those ATGs encode many core proteins that involved in the entire process of autophagy from the induction to the degradation, recovery and recycling of autophagosome. As a type of evergreen wooden plant, the recycling of some broken or discarded macromolecular substances plays an important role in the special development period or in the resistance to stresses in tea plant. At present study, a total of 35 CsARGs were identified in tea plant genome. Some CsARGs, like CsATG8 and CsATG18, exist multiple copies in tea plant genome, and the same results were also observed in many other species, such as Oryza sativa , Nicotiana tabacum , Vitis vinifera , Musa acuminate, and Setaria italic , etc. In addition, the results of phylogenetic and protein domain analysis further confirmed that ATGs are highly homologous among different plant species. In yeast, ATG proteins were divided into four functional groups based on their roles involved in autophagy process . Similarly, the identified CsARGs also constituted a relatively complete autophagic machinery, where they function in forming ATG1 kinase complex (CsATG1s/13, CsTOR), Class ⅠⅠⅠ PI3K complex (CsATG6/14, CsVPS15/34), ATG9 recycling complex (CsATG2/9/18 s), and Atg8-lipidation system (CsATG3/4/7/8 s) and Atg12-conjugation system (CsATG5/7/10/12/16).
Until now, there have lots of reports on the functional analysis of ATG14 in mammals, but few in plants. In mammals, the human homologs ATG14, hAtg14/Barkor/Atg14L, has been shown to be the sole specific subunit in phosphatidylinositol 3-kinase (PI3-kinase) complex. Atg14L could be interacted with Beclin-1/2 through their coiled-coil domains, and proved to be the targeting factor for PI3KC3 to autophagosome membrane [63, 64]. Similarly, ATG14 is only integrated into phosphatidylinositol 3-kinase complex I to direct association of complex I to the pre-autophagosomal structure (PAS) in yeast . Here, we identified an ATG14 homologous protein, referred to as CsATG14, in tea plant. Bioinformatics analysis results predicted that CsATG14 is a hydrophilic protein, and contains a coiled-coil motif at the N-terminus region from 10 to 367 aa, suggesting that CsATG14 may be interacted with Beclin-1 (named as CsATG6 in our study) to serve as a scaffold for recruiting the class III phosphatidylinositol-3-kinase (PIK3C3).
As a highly conserved ubiquitin-like protein, ATG8 is activated by conjugation to the lipid phosphatidylethanolamine (PE) to form ATG8-PE adduct, thereby participates to the autophagosome formation and phagophore expansion [66, 67]. During conjugation, the C-terminal of ATG8 must be cleaved by a cysteine protease, Atg4, to expose a glycine residue [68, 69]. In present study, five ATG8 isoforms were identified, and they displayed high sequence similarity to AtATG8s, NtATG8s and OsATG8s. However, a conserved glycine residue for lipidation was directly exposed at the C-terminal of CsATG8a/g respectively, similar phenomena were also observed in MdATG8g/i, AtATG8h/i and OsATG8e, suggesting that ATG4 may be not necessary to the conjugation of ATG8-PE. Besides, ATG8 also can interact with their specific substrates or receptors via an Atg8 Interacting Motif (AIM) in the target proteins during selective autophagy. Recently, two plant-specific proteins in Arabidopsis, termed ATI1 and ATI2, were identified and proved to be interact with AtATG8f and AtATG8h . In our study, however, only one unique ATI homologous gene, CsATI, was identified. Sequences alignment analysis found that CsATI also contains two putative AIMs (17–20, 267–270) and a predicted transmembrane domain (242–259) (Additional file2), suggesting that CsATI also can bind one of five CsAtg8 isoforms in tea plant.
During macroautophagy, a v-SNARE complex, including v-SNARE VTI1, Rab-like GTP-binding protein (YKT6), and syntaxin (VAM3), was contributed to the maturation and fusion of autophagosome to lysosome/vacuole . In plants, there are three VTI1-type SNAREs members (VTI11, VTI12, VTI13) have been identified . Sanmartin et al. (2007) suggested that VTI12 and VTI11 might involve in trafficking to storage and lytic vacuoles in vegetative and seed tissues in Arabidopsis, respectively . Moreover, VTI13 participates in trafficking of cargo to the vacuole within root hairs and also plays an essential role in maintenance of cell wall organization in Arabidopsis . In our study, CsVTI12 and CsVTI13a/b were all predicted contain the typical v-SNARE domains (Fig. 2b), and phylogenetic analysis result showed a close relationship with other VTI1s (Fig. 1), suggesting that CsVTI1s are essential for autophagy and may mediate different protein transport pathways.
CsARGs mediated the growth and development of tea plant
Under normal growth conditions, autophagy served as a housekeeping process to degrade unwanted proteins, organelles and damaged cytoplasmic contents. In Arabidopsis, to explore the functions of ATG genes, the corresponding ATG-mutants and ATG-overexpression lines were popularly used. Numerous studies have showed that almost all of ATG-mutants could complete their entire life cycles but coupled with early senescence phenotypes under normal growth condition, suggesting that autophagy mediates plant senescence . Indeed, autophagy is necessary to anther and seed development [26, 30, 76], root elongation , chloroplast recycling . In present study, we found the transcription abundances of most CsARGs were higher in stem and seed than other tissues, which indicate that autophagy may mediates the nutrients allocation or recycling from source tissues to sink tissues in tea plant. Specifically, CsATG8s subfamily genes showed high transcription levels in all detected tissues, which demonstrate they play a great role in modulating tea plant growth and development. A similar result was also observed in Arabidopsis, where AtATG8s were distinctly expressed throughout the plant . As core ATG proteins, ATG8s have been used as very convenient markers to monitor autophagic activity, and also play vital role in regulating the nitrogen remobilization efficiency and grain quality in plants . For instance, overexpression of OsATG8b increased the nitrogen recycling efficiency to grains in transgenic plants, while reduced nitrogen recycling efficiency and grain quality in OsATG8b-RNAi transgenic plants . Similarly, ATG8a, ATG8e, ATG8f and ATG8g overexpressed in Arabidopsis could promote autophagic activity and improve nitrogen remobilization efficiency and grain filling in transgenic plants . In our study, both CsATG8c and CsATG8i were strongly expressed in tea seed, suggesting that the high mRNA levels of these two genes may promote nitrogen remobilization efficiency to tea seeds. From this point of view, selecting tea plant germplasms with higher CsATG8s transcription abundances may attribute to improving the tea seed quality so that guarantee the seedling emergence rate and survival rate.
Chloroplasts are specific energy converters of higher plants and photoautotrophs, which are not only participating in photosynthesis, but also performing plant metabolism. It has been well established that nearly 80% of the total leaf nitrogen was stored in chloroplasts in C3 plants . In addition, more recent evidences found that chloroplasts degraded by autophagy in RCBs and whole organelles forms respectively during leaf senescence, so that chloroplasts could be served as a principal nitrogen source for recycling and remobilization. Indeed, it has been reported that autophagy also contributed to leaf starch degradation . Tea plant is evergreen and C3 plants, the numbers of chloroplasts in leaves are gradually increased from the tender leaves to mature leaves, and then decreased with leaf senescence. In the present study, we analyzed CsARGs expressions both in mature and tender leaves, the results found that there have 11 CsARGs, especailly CsATG1c/4/5/8c/8f/10/13/18 h, exhibited more than 3-folds higher expression levels in mature leaves than in tender leaves, indicating that autophagic activity is changed following the maturation of leaves, a higher autophagic activity in mature leaves may be attribute to prolonging leaf longevity so that maintaining the evergreen of tea plant for a long time.
CsARGs improved abiotic stress tolerance in tea plant
In addition to mediate plant growth and development, autophagy also plays a critical role in plant resistance to various stresses, such as nutrient deficiency, oxidation stress, cold, drought, salt, wounding, heavy metal, pathogen attack, etc. Under favorable condition, autophagy is maintained at basal level, but it is relative quickly stimulated under stress conditions. Overexpression and mutation methods have been widely used to explore the functions of ATG genes in different species. For example, AtATG18a was dramatically induced after a few hours of NaCl and mannitol treatments, and the roots of AtATG18apro: GUS transgenic plants were evenly stained under nutrient starvation, oxidative stress (MV), NaCl, mannitol stress conditions. However, the growth of RNAi-AtATG18a plants were retarded and the seed germination was also delayed as compared to WT under different stress conditions, indicated that AtATG18a may function in the response of plants to these stresses . Similarly, a homologous gene, MdATG18a, overexpressed in apple plants could enhance drought resistance probably by inducing a greater autophagosome production and a higher autophagic activity . Overexpression of MdATG18a also regulated the expressions of many genes that involved in anthocyanin biosynthesis, sugar metabolism, and nitrate uptake and assimilation, and finally promoted the soluble sugar and anthocyanin accumulation, starch degradation and nitrate utilization improvement in response to N-depletion . Apart from ATG18a, ATG3/5/7/8 s/10 were also been reported had critical roles in dealing with different stresses. Overexpression of MdATG3s enhanced the tolerance to multiple abiotic stresses both in transgenic Arabidopsis and apple plants . Overexpression of AtATG5 or AtATG7 in transgenic Arabidopsis activated AtAtg8-PE conjugation, autophagosome formation, and autophagic flux, thus increased the tolerance of necrotrophic pathogens and oxidative stress, also retarded aging and improved growth and seed yields . Indeed, MdATG8i proved to interact with MdATG7a and MdATG7b, overexpression of MdATG8i enhanced tolerance to nutrient-starvation both in transgenic Arabidopsis and apple plants . In the present study, the expressions of CsATG3/5/7/8 g/8i/18a/18 g were quickly induced by cold, drought, NaCl treatments. Furthermore, the promoters of these genes contains series of cis-acting elements that potentially involved in responding to environmental stresses or hormones, indicating that autophagy would be induced in tea plants under adverse environmental conditions, and lots of CsATG genes participate in dealing with different stresses.
It is well known that sugars are not only important osmoprotectants and ROS scavengers, but also act as core signaling molecules in plants under adverse conditions . The occurring of autophagy also closely related to sugar signaling. The central energy-sensing SnRK1 acts as a positive regulator, which acts upstream of TOR on sugar-phosphate perception to activate autophagy, and TOR kinase acts as a negative factor to inhibit autophagy . Accumulating evidences proved that sugar contents increased, and many genes involved in sugar metabolism, transport and signaling were differentially expressed in tea plant during abiotic stress conditions [53, 89]. Among of these genes, CsSnRK1.2 was induced, but CsSnRK1.1was not influenced and CsSnRK1.3 was sharply reduced during CA periods . Combined with our results, the expression profile of CsTOR was not strictly showed a contrary tendency to CsSnRK1, where we found the expression of CsTOR was slightly induced under cold condition, but reduced under drought and NaCl conditions, suggesting that autophagy could be activated by TOR-independent pathways, and SnRK1 could also mediate autophagy through a TOR independent mechanism in tea plant under certain stress conditions. Autophagy is also regulated by phytohormones. Under normal conditions, TOR kinase phosphorylates PYLs receptors and represses ABA signaling, whereas ABA signaling represses TOR kinase activity through the phosphorylation of Raptor B mediated by SnRK2 under stress conditions . In the present study, however, we found the expression of CsTOR was slightly induced under ABA treatment condition, which indicate that the inhibition of TOR is not simply affected by the transcription level, but mainly influenced at post-translation level during stress conditions. At present, there are few studies explored the relationship between autophagy and GA. Kurusu et al. (2017) found that OsATG7 mutated in rice could reduce the endogenous level of active-forms of gibberellins (GAs) in anthers of autophagy-defective mutant, Osatg7-1 during flowering stage, which suggested that autophagy mediated the biosynthesis of GAs in rice . In the present study, we found two-thirds of CsARGs were induced after 2 d of GA treatment, which indicates that there is a close relationship between the GA metabolism and autophagy, but the specific regulatory mechanism needs to be further investigated.
Cold acclimation (CA) is an indispensable process to increase cold tolerance of tea plant. During CA, gene expressions, protein activities, and metabolic contents are altered and varied between cold-resistant cultivar ‘Longjing43’ and cold-susceptible cultivars ‘DaMianBai’ . Specifically, a higher ROS contents and a lower SOD activity were observed in ‘DaMianBai’ as compared to ‘Longjing43’, and many genes related to ROS production and scavenging were also induced and deduced, respectively, in ‘DaMianBai’ under CA condition, demonstrated that the stimulation of ROS-scavenging genes was a principal strategy for tea plants in response to cold stress. In addition to ROS-scavenging genes, there has also been reported that the production of ROS could induce autophagy, which in turn inhibits ROS production . In our study, we performed 11 CsATGs expressions during CA periods (from Dec.13 to Jan.17), and their expressions were higher in cold-resistance cultivar ‘Longjing43’ than the cold-susceptible cultivar ‘DaMianBai’, except for CsATG101, which indicate that autophagic activity may higher in cold-resistance cultivar ‘Longjing43’ than the cold-susceptible cultivar ‘DaMianBai’ during CA periods. Strangely, we found the transcription of CsATG101 was higher in cold-susceptible cultivar ‘DaMianBai’ than the cold-resistance cultivar ‘Longjing43’ throughout CA periods in winter season. It is well known that CsATG101 is a component of the ULK1 complex, which served as a stabilizer of ATG13 in cells. In mammals, ATG101 is required for maintaining tissue homeostasis in both adult brains and midguts. In plants, however, the physiological role of ATG101 has not been fully understood. The specific regulatory mechanism of CsATG101 responding to CA needs to be further studied in future. In a word, based on the differential expressions patterns in different cultivars, we believe that these 11 CsATG genes would be served as putative molecular markers for cold-resistance breeding of tea plant in future.