Study on the Anti-Aging Physiological Characteristics and Molecular Mechanism of Camellia Oleifera

Yongzhong Chen Hunan Academy of Forestry Jianjun Chen (  jjchen@u .edu ) University of Florida Zhen Zhang Hunan Academy of Forestry Yanming Xu Hunan Academy of Forestry Zhilong He Hunan Academy of Forestry Caixia Liu Hunan Academy of Forestry Rui Wang Hunan Academy of Forestry Xiangnan Wang Hunan Academy of Forestry Yinghe Peng Hunan Academy of Forestry Shaofeng Peng Hunan Academy of Forestry Longshen Chen Hunan Academy of Forestry Li Ma Hunan Academy of Forestry Zhigang Li Hunan Academy of Forestry Wei Tang Hunan Academy of Forestry


Introduction
Tree senescence refers to the process of decline occurring in plants or some organs during growth and development, which leads to the natural termination of life activities 1,2 . Usually, during tree growth and development, the changes of internal factors, such as hormones, metabolic levels, aging-related genes, transcription factors and the effects of external environment factors, such as drought, high temperature, pests may lead to chlorophyll degradation, protein, lipids, nucleic acid, starch and other biological macromolecules hydrolysis 3,4 , and cause, accelerate aging, and even cause tree death. At present, the research on the mechanism of tree senescence includes stomatal regulation theory, senescence gene regulation theory, active oxidation theory, nutrition de cit theory, plant hormone regulation theory, external stress and pest theory 5,6 . The commonness of these theories is to acknowledge that plant senescence and death are the universal laws of life development, and are an active and necessary process in plant growth and development, morphogenesis and environmental response. Moreover, these theories also show that tree senescence is formed under the combined action of many different factors. At the same time, studies have found that different tree senescence processes are different due to different mechanisms and inducing factors of senescence 7 . When encountering many external stress and internal senescence related factors, some plants could form the corresponding resistance as well as the defense mechanism by some antioxidase, the transcription factor, and the signal transduction factor and thus prolonged its senescence process.
Camellia oleifera which were plants of Camellia family, Camellia plants and perennial small trees or shrubs was a speci c oleiferous tree species of China which usually distributed at low mountains and hills in South of China 8 . Elaeis guineensis, Olea europaea, Cocos nucleifera as well as Camellia oleifera were four edible oleiferous tree species of the world. Camellia oleifera possessed characteristics of poor soil tolerance, strong resistance, and exuberant vitality. It was common to nd the growth of Camellia oleifera with more than 100 year old age in natural forests. For example, there was a Camellia oleifera tree with more than 120 year old age grown in Hendong Country, Henyang City in China (N27 o 05', E112 o 56'). This single tree produced annual average Camellia fresh fruit for more than 200 kg during the continuous eld observation from 2014 to 2016. What were the factors which maintained the exuberant vitality of those ancient Camellia oleifera? Nowadays, studies about the transcription level of plants antisenescence mainly focused on the model plants such as Arabidopsis thaliana 9,10 . However, research about antisenescence of Camellia oleifera was scarce. In this study, focusing on antisenescence of Camellia oleifera, leaves of Camellia oleifera with different ages were selected as targets. ROS and antioxidase activities in leaves of mature tree (30 a) and ancient tree (> 100 a) were determined. By conducting the transcriptome sequencing analysis, the ROS clearance system of Camellia oleifera with different ages as well as the expression of differential genes was studied. Furthermore, antisenescence physiological characteristics and molecular mechanism of ancient Camellia oleifera were studied preparing for the further studies of stress resistance molecular biology and genetic improvement.

Experimental materials
The experimental site was located in the experimental forest farm of Hunan Academy of Forestry (east longitude 113 o 01 , 20 ,, , north latitude 28 o 06 , 40 ,, ). On April 18th, 2017, good single plant of Camellia oleifera of mature tree (30 a) and ancient tree (> 100 a) with strong growth and good development were sampled (Fig. 1). Annual leaves which received consistent illumination, were strong and had no pests and diseases in those single plants at the middle and upper part of the periphery of crown were selected as experimental material, which had three biological replicates. The collected leaves were put into the plastic bags, numbered, quickly put into the ice box and taken back to the laboratory. After rinsing by deionized water and treatment with liquid nitrogen, leaves were put into − 70 o C refrigerator for further analysis.

Determination methods
Contents of chlorophyll (Chl), malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), SOD, POD and CAT were determined by the extraction of acetone, the thiobarbituric acid method, the trichloroacetic acid colorimetry, the ribo avin-NBT photoreduction, the guaiacol method and the permanganate titration method, respectively.
Samples were sent to Beijing Genomics Institution in Guangzhou to conduct the transcriptome sequencing. The paired end sequencer of Illumina Hiseq TM 2000 was used for sequencing. Raw data were processed by Beijing Genomics Institution in Guangzhou.

Data analysis
Excel 2003 was used for the data processing and the gure drawing. SPSS 16.0 was used for data statistics and the variance analysis. The differential signi cance of the physiological and biochemical indexes was analyzed by one-way ANOVA test. Multiple comparisons used the least signi cant difference test (LSD).

Statement
We con rm that the use of plants in the present study complies with international, national and/or institutional guidelines.

Results
Antisenescence physiological indexes analysis of Camellia oleifera trees with different ages There was a signi cant difference of chlorophyll content among Camellia oleifera leaves with different ages. Chl a + Chl b content in leaves of ancient Camellia oleifera was 0.44 mg/g, which was higher than 0.27 mg/g in mature tree. Ratio of Chl a to Chl b in leaves of ancient Camellia oleifera were higher than mature tree, which were 10.32 6.34, respectively. MDA and H 2 O 2 contents in leaves of ancient Camellia oleifera were also higher than mature tree, which were 8.83 nmol/g and 126.77 ug/g. Antioxidase activities of SOD, POD and CAT were the highest in ancient Camellia oleifera leaves, namely 671.00 (U/g), 22.58 U/(g min) and 498.42 U/(g min), respectively (Table 1). CAT was speci c-functional enzyme for the clearance of H 2 O 2 . POD was also very important for the clearance of surplus H 2 O 2 in plants. The changing trends of these two enzymes were negatively correlated with the change of H 2 O 2 to some extent, illustrating that these two could promote the defense of Camellia oleifera against the attack of H 2 O 2 and thus prolong senescence. SOD mainly cleared superoxide anion In all, with the increase of the tree age, MDA and H 2 O 2 contents of Camellia oleifera leaves increased, which resulted in excessive or unbalanced ROS in plants and nal senescence. Meantime, chlorophyll in ancient Camellia oleifera leaves maintained at a relatively higher level. Besides, ancient Camellia oleifera leaves possessed relatively higher antioxidase activities, which not only effectively guaranteed photosynthesis but also defended the possibly adverse effect caused by the increase of MDA and H 2 O 2 contents. Finally, the normal growth and development was promoted and the senescence process was prolonged. Note: Different letters illustrated the difference of physiological and biochemical indexes among different tree ages (P<0.05). Data in brackets were standard deviation.
The differential expression of antisenescence related genes of Camellia oleifera leaves in stages of different tree ages The differential expression of chlorophyll degradation related genes Chlorophyll a/b binding protein gene was very important for the maintenance of the chlorophyll content and the photosynthetic ability of the plant. In this study, 10 differentially expressed related genes of chlorophyll a/bbinding protein were found in ancient Camellia oleifera leaves, wherein 9 genes were up regulated expressed and 1 gene was down regulated expressed ( Table 2). The expression increase of Chlorophyll a/b binding protein gene possibly prolonged the senescence process by enhancing the photosynthetic ability of ancient Camellia oleifera tree. The differential expression of antioxidase related genes SOD, POD, CAT and APX were common antioxidases, which were usually very important for the clearance of surplus ROS in plants and the prolongation of senescence. In this study, 22 antioxidase genes were found signi cantly differentially expressed in ancient Camellia oleifera leaves, wherein 15 antioxidase genes were up regulated expressed and 7 genes were down regulated expressed (Table 3).
These antioxidase genes mainly included SOD, POD, CAT and APX. POD had the most differential genes amount, followed by those of APX, SOD and CAT. The increase of the expression of antioxidase genes could prolong the senescence process by enhancing the antioxidase activity of ancient Camellia oleifera trees. The differential expression of hormone related genes Hormone levels were closely related to plant senescence. Usually, Auxin and cytokinin (CTK) could prolong plant senescence, whereas abscisic acid (ABA) could accelerate plant senescence. In this study, dramatic differential expression of 40 hormone related genes were found in ancient Camellia oleifera leaves, wherein 21 genes were up regulated and 19 genes were down regulated expressed (Table 4). From categories, hormone related genes mainly included ABA, auxin, CKX and SAUR, wherein auxin related differential gene number was the highest, which was followed by that of ABA. Due to the combined effects of those hormone related genes, ancient Camellia oleifera could maintain hormones at a relatively stable level and further its exuberant vitality. The differential expression of stress resistance related genes The transcription factor, the signal transduction factor, the resistance gene and the defense related gene could signi cantly enhance the resistance of plants and further effectively answer the external adverse growth factors, respectively. 496 stress resistance related genes were signi cantly differentially expressed in ancient Camellia oleifera leaves, wherein 301 genes were up regulated expressed and 195 genes were down regulated expressed. Those stress resistance genes mainly included transcription factors of MYB, NAC, MRKY and zinc Finger etc., signal transduction factors of GTP-binding, receptor protein kinase etc., resistance protein of pathogenesis related protein, disease resistance protein etc., and defense-related gene. The amount of differential genes related to Zinc Finger and disease resistance protein was the highest, followed by other genes of LRR receptorlike serine/threonine-proteinkinase, HSP, MYB, Leucine zipper, bHLH and ERF (Table 5). With the combined effects of those stress resistance related genes, ancient Camellia oleifera could maintain normal growth through the corresponding adjustment and adaption mechanism to response the external growth stress. The differential expression of protein degradation related genes Protein degradation which was also an important reason for plant senescence usually occurred due to the function of some proteinase and F-box family protein genes. In this study, 72 functional genes related to protein degradation were found in ancient Camellia oleifera leaves (Table 6). In detail, those included 17 aspartyl protease genes, 11 cysteine proteinase genes and 44 F-box family protein genes. Among cysteine proteinase, 7 genes were up regulated and 4 were down regulated. Among F-box family protein genes and aspartyl protease genes, the expression of most differential genes demonstrated a down regulated trend. With the combined effects, protein degradation of ancient Camellia oleifera leaves may be effectively delayed, which further was possibly helpful for the prolongation of senescence. Veri cation of transcriptome differential genes Real time quantitative PCR was used to further verify the reliability of transcriptome sequencing results and the expression patterns of differential genes. The selected veri cation genes include NAC, F-box, aspertyl protection, cystaine protonase, chlorophyll a / B and others. ETIF3H was selected as the internal reference gene. The results showed that the expression of c184012_g3 (F-box) c178940_g2 (Aspartyl protease) c163401_g1 (Cysteine proteinase) were down regulated in ancient trees, up regulated in mature tree, c167863_g1 (NAC) c184170_g1 (Chlorophyll a/b) were up regulated in ancient trees and down regulated in mature tree ( Figure 2). In general, the expression patterns of these genes are consistent with the results of transcriptome sequencing, which further veri es the reliability of transcriptome sequencing results.

Discussion
The Chlorophyll content of Camellia oleifera leaves in ancient trees was signi cantly higher than those in mature tree. 9 chlorophyll a/b binding proteins in ancient Camellia oleifera leaves demonstrated an up regulated trend. Chlorophyll guaranteed high e cient photosynthesis of plants. Thus, the degradation of chlorophyll was also a main marker of senescence of plant leaves. In Zea mays 11 , Sorghum bicolor 12 , and Arabidopsis thaliana 13 , chlorophyll contents were closely related to leaf senescence. Besides, during leaf senescence, some genes involved in photosynthesis would be down regulated expressed, resulting in the decrease of the photosynthetic ability of leaves 14,15 . Chlorophyll binding protein genes were in a down regulated trend during the senescence process of Oryza Sativa 16 and Gossypium 17 . In this study, the chlorophyll content and the expression of chlorophyll a/b binding protein gene of ancient Camellia oleifera leaves demonstrated an increasing trend, illustrating that ancient Camellia oleifera leaves still had a high level of chlorophyll, which was very important for the maintenance of the photosynthesis of the whole tree, the promotion of the growth and development of tree and the prolongation of senescence.  30,31 . In Gossypium, the increase of the expression of auxin related gene could inhibit leaf senescence 17,32 . In this study, the expression of 1 SAUR related gene increased in ancient Camellia oleifera leaves. SAUR gene played an important role in the maintenance of auxin level, the regulation of auxin transport and cell ampli cation 33,34 . With the combined effects of those hormone genes, hormone levels in Camellia oleifera leaves could be maintained in balance, which could promote the benign growth and prolong senescence.
External growth stresses of high temperature, low temperature, drought, diseases and pests were usually closely related to plant senescence 35 . Due to many external growth stresses, the transcription factors related to stress in plants would be induced to be expressed. The stress transcription factors could regulate the expression of the stress resistance gene in plants, thus enhancing the stress resistance of plants. In Arabisopsis thaliana 36 , Gossypium 17 , Oryza sativa 37 , the increase of the expression of the NAC transcription factor was very useful for prolonging plant senescence, and answering drought as well as salt and marsh stress. The MYB transcription factor expression could enhance the resistance to growth stress of drought, salt, coldness and high temperature in Lycopersicon esculentum 38 , Arabidopsis thaliana 39,40,41 and Salicornia brachiata 42 . In Oryza sativa 43 and Arabidopsis thaliana 44 , it has been found that WRKY could in uence leaf senescence by enhancing the resistance to the adverse factors of growth including drought, high temperature, pathogenic bacteria etc.
In Trifoliate Orange, the increase of the expression of the bHLH transcription factor could enhance POD activity of plants, clear ROS, and further enhance coldness resistance of plants 45  In the growth process, plant would not only be in uenced by external growth stress but also encountered the invasion of many pathogenic bacteria, which nally exacerbated the senescence process. Chitinase was veri ed to effectively inhibit activities of some pathogens, intensify the defense system of plants and enhance the disease resistance of plants to many pathogenic bacteria 66,67 . when encountering stress, plant cells could also increase the defense gene expression of resistance protein, defense-related gene etc. to prevent cells from harm.
In ancient trees, the whole gene expression of disease resistance protein and pathogenesis related protein demonstrated an increasing trend.
In this study, the expression of multiple signal transduction factors, transcription factors and disease resistance related genes were up regulated. With the combined effect of those functional genes, Camellia oleifera could response to external stress and some pathogen invasion in time, enhance resistance to various growth stresses and pathogenic bacteria through those factors and nally prolong senescence.
There were many differential genes related to stress resistance of Camellia oleifera, which was related to the growing environment of Camellia oleifera itself. Camellia oleifera was a speci c economic tree species in China, which mainly distributed at low mountains and hills in different provinces of South China. In those areas, during the growth process, Camellia oleifera encountered stresses of low temperature in winter, lots of rain in spring, high temperature in summer and drought. Besides, Camellia oleifera was susceptible to anthracnose and soft rot. By preliminary speculation, due to the in uence of those growth stresses and pathogenic bacteria, the expression of many resistance related genes in Camellia oleifera including the transcription factors, the resistance factors, the defense factors and the signal transduction genes, which could better defend against those adverse growth factors and nally promote normal growth and development of plants.
In ancient Camellia oleifera leaves, the overall expression of functional genes related to protein degradation were down regulated. Similar with the situation of chlorophyll degradation, protein would also be degraded during plant senescence 15 . Cysteine proteinase was regarded as the most abundant enzyme which was related to plant leaf senescence 68 . and in protein hydrolysis 69 . The expression of aspartic protease which took part in petal senescence as well as pathogen related protein degradation 69 would be increased during senescence in Glycine max 69 and Arabidopsis thaliana 70 . F-box gene was one of the largest gene families which took part in cell protein degradation in plants. Encountering stresses and during leaf senescence, F-box gene was up regulated expressed in chickpea 71 and Oryza sativa 72 . With the degradation of protein, the senescence process of plants would be exacerbated. In ancient Camellia oleifera leaves, the expression of aspartyl protease gene, cysteine proteinase gene and F-box family protein gene were decreased, which effectively inhibited the degradation of protein and thus prolonged senescence.

Conclusions
This study shows that: (1) With the increase of the tree age of Camellia oleifera, the chlorophyll content in ancient Camellia oleifera leaves was signi cantly higher than those in mature and young trees, which was also veri ed by the chlorophyll related gene expression in Camellia oleifera leaves. Multiple chlorophyll a/b-binding proteins in ancient Camellia oleifera leaves demonstrated an up regulated trend, illustrating more active vital movement in ancient Camellia oleifera leaves.
(2) The whole expression of related genes of POD, CAT and APX in ancient Camellia oleifera leaves demonstrated an up regulated trend, which was veri ed by antioxidase contents of SOD, POD and CAT in ancient Camellia oleifera leaves, illustrating that the clearance ability of ROS in ancient Camellia oleifera leaves could be enhanced by the increase of antioxidase activities.
(3) The expression of multiple antisenescene hormone related genes increased in Camellia oleifera leaves, wherein auxin related gene number was the most.
(4) Multiple stress resistance and pathogenic bacteria resistance related genes were found in ancient Camellia oleifera leaves. With the combined effects of those genes, Camellia oleifera could answer various external growth stress and pathogenic bacteria in time and further response correspondingly by the corresponding transcription factor, signal transduction, resistance protein and defense related gene.
(5) The whole expression of the functional differential genes of Aspartyl protease, Cysteine proteinase and F-box family protein, which in uenced protein degradation, demonstrated a decreasing trend in ancient Camellia oleifera leaves. Thus, protein degradation would be effectively inhibited.
(6) The results of real-time quantitative PCR were consistent with those of transcriptome sequencing, which further veri ed the reliability of transcriptome sequencing results.
(7) With the comprehensive effects of those factors, ancient Camellia oleifera leaves still kept an exuberant vitality, which had a high reference value for the stress resistance biological study and genetic improvement research of Camellia oleifera. Figure 1 The growth status of Camellia oleifera trees with different ages Note: Those from left to night were ancient Camellia oleifera (> 100 a), mature tree (30 a).