{'fraction_non_alphanumeric': 0.03399448738042479, 'fraction_numerical': 0.025617467437712805, 'mean_word_length': 4.757311761045426, 'pattern_counts': {'":': 0, '<': 7, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
PHYSIOLOGICAL AND MOLECULAR COMPARATIVE ANALYSIS OF TWO CONTRASTING RICE VARIETIES UNDER JASMONIC ACID TREATMENT

Huong Thi Mai To*, Nga T. P. Mai, Minh Anh Ngo, Ngan Huyen Nguyen, Kieu Thi Hanh, Thi Trang Nguyen, Hien Thi Thu Vu, Dang Khanh Tran, Anh Thi Van Le, Trang Hieu Nguyen, Antony Champion University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST), LMI-RICE, 18 Hoang Quoc Viet, Cau Giay District, Hanoi, Vietnam Plant Protection Department, MARD, 149 Ho Dac Di, Dong Da District, Hanoi, Vietnam Agricultural Genetic Institute (AGI), Km2 Pham Van Dong, Hanoi, Vietnam Institute of Research for Development, University of Montpellier, UMR DIADE, UMR IPME, UMR LSTM, Montpellier, France


INTRODUCTION
Rice (Oryza sativa L.) is one of the most abundant food crops around the world, especially in Asia. Nowadays, the demand on rice increase significantly due to the rising of the world population. Along with the escalation in rice requirement, rice production is being forced to cope with numerous obstacles, for example, climate change, reduction of cultivable land and natural disasters. This challenge impulses an urge solution for creating new varieties of rice which can withstand harsh condition and also maintain high productivity.
Jasmonic acid, its derivatives and conjugates (referred to JAs), are known to be important regulators of plant responses to wounding, insects, herbivores and environmental stresses. In interaction with other hormones pathways, JAs are involved in various aspects from plant growth and development regulation to the adaptation to environmental stimuli (Devoto, Turner, 2005;Goossens et al., 2016). JA was also known as hormone involving in the response of plants to phosphate starvation condition. In Arabidopsis, Pi starvation enhanced synthesis of JA and JA-isoleucine, and therefore led to the expression JA-related genes (Khan et al., 2016). In 2019, under Pi starvation, four candidate genes involving in the JA signaling pathway, including three Jasmonate-zim-domain (JAZ) genes and one Coronatine insensitive 1 (COI1) gene were also induced in the sensitive rice accessions (Zhang et al., 2019). The JA-signaling pathway is regulated by the key model COI1-JAZs-MYC2 (Chini et al., 2009). CORONATINE INSENSITIVE 1 (COI1) is an F-BOX E3 LIGASE protein which only interacts with JA-Ile. JASMONATE ZIM DOMAIN protein (JAZ) is a repressor of MYC2 transcription factor of JAsignaling. JA-Ile binds to COI1 and JAZ protein thus leads to JAZ degradation. MYC2 would not be repressed anymore; it is active to conduct the transcription process (Chini et al., 2007;Thines et al., 2007). Lying downstream in the JA-signaling pathway, Pathogenesis-Related (PR) genes are regulated by MYC2 and response to cold signals with the pathogen in plants (Seo et al., 2010).
The whole scenario however is not that clear in rice. Since this research tends to focus on JA and its effect on the molecular and morphological characterization of contrasted rice varieties in Vietnamese rice collection, we want to evaluate firstly expression of JA biosynthesis related genes under JA treatment. The expression of JA-related genes would be analyzed and associated with the phenotyping results of root to understand the differences in the physiological aspect and molecular aspect between two contrasted rice varieties. The thorough knowledge of the JA pathway involved in plant resistance and development will assist breeders in selecting cultivars possessing an improved stress tolerance capacity in the future.

Plant materials
The seeds of G11 or Tam_tron (Hai Duong) and G38 or Nep_nuong (Ha Giang) were provided by Plant Resource Center, Hanoi, Vietnam. Both of two varieties are traditional landraces, G11 represent for Indica group and G38 represents for Japonica group. These accessions were selected according to the contrasted results of JA response presenting in (To et al., 2019).

Primers used in this study
A total of 12 JA biomarkers including 3 markers related to the JA biosynthesis, 3 markers related to the JA signaling pathway, 6 markers related to the JA defense response were used in this study in order to investigate the difference between two contrasting rice varieties. Actin was used as housekeeping gene. The primer list of these 13 genes was summarized in Table 1.

Seed germination and growth plants
Seeds of each variety were selected based on their typical and consistent size, shape and color, without symptoms neither damaged embryos nor contamination. The seeds were kept in the drying chamber at 45°C for 3 days to break the seed dormancy.
Sterilization process was conducted as 2 minutes shaking in ethanol 70% then soaked in Javel water (NaClO 3.8%) including 2 drops of Tween 20% (China). After 25 minutes, discard the liquid and rinse the seeds with sterilized distilled water at least 7 times to remove the Javel water. The tubes were incubated at 28°C for overnight to maximize the water absorption. In the next day, the rice grains were transferred to the petri dishes containing 6g/L Agar. The petri dishes were incubated at 28°C for 2 days in the dark for germination. Qualified germinated seeds were transferred into glass test tubes of half Murashige and Skoog medium (MS/2) at pH= 5.8, containing 2.7 g/L Phytagel (Sigma Aldrich) with or without 5 µM of JA (TCI-Japan) for JA stress phenotype experiments. For low Pi experiment, the qualified germinated seeds were grown in glass test tubes of half Murashige and Skoog medium (MS/2) at pH= 5.8, containing 2.7g/L Phytagel (Sigma Aldrich) with full phosphate (625 µM) or low phosphate (40 µM) content by modifying the amount of KH 2 PO 4 . The tubes were left in a growth chamber (DAIHAN Scientific, Thermo Stable GC-450) under a 12 h daily light cycle at a humidity of 80%. The light intensity was 12,000 lux. The temperature was adjusted to 26°C in the dark phase and 28°C during light phases. After 7 days, the glass tubes were taken out to measure phenotype parameters: number of crown root (NCR), root length (RTL), shoot length (SHL), root weight (RTW), shoot weight (SHW), total plant weight (TTW). For qPCR experiments, tissues were taken from at least 4 plants.

RNA extraction and cDNA synthesis
RNA was extracted using Trizol® Reagent (Thermo Scientific) following the manufacturer's protocol. Briefly, the sample was ground in liquid nitrogen using a mortar and pestle, 0.75 mL Trizol was added, and the mixture was incubated for 15 minutes at room temperature. After centrifuging for 10 minutes at 12000 rpm, 4ºC, the aqueous phase was transferred into 2 mL Eppendorf tube, 0.3 mL of chloroform was added; the tube was shaken vigorously for 15 seconds, and then incubated at room temperature for 2 -3 minutes. The sample was centrifuged for 10 minutes at 12000 rpm, 4ºC; 0.75 mL of the clear upper aqueous layer containing RNA was transferred into a new tube containing 0.75 mL isopropanol (-20ºC) followed by incubation at 4ºC for 1 h. RNA was precipitated by centrifugation at 12000 rpm, 4ºC for 15 min and washed twice with 1mL of 75% Ethanol. Pellet was dried in air for 30 min -1 h and dissolved in 30 µL of MilliQ water, stored at 4ºC for 15 h. RNA integrity was checked on 1% agarose gel. Potential co-purified genomic DNA was digested using DNAse I (Thermo Scientific) according to the manufacturer's recommendation. Extracted RNA was assessed for quality and quantity using NanoDrop-2000 spectrophotometer (Thermo Scientific) before and after DNAse I treatment.
cDNA was synthesized from 2 µg of total RNA (after DNAse I treatment) with Maxima® First Strand cDNA Synthesis Kit (Thermo Scientific), using Oligo(dT) according to the manufacturer's instruction. The reaction was conducted with the thermal cycle of 15 minutes at 25ºC, followed by 45 minutes at 50ºC and terminated by 5 minutes at 85ºC. cDNA quality was checked by PCR with housekeeping gene (OsACT) followed by running the PCR products on the agarose gel.

qPCR (Real-time PCR)
Quantitative PCR reactions were performing in the Mx3005P qPCR system (Agilent Technologies) with GoTaq® qPCR master mix (Promega) followed the manufacturer's instruction. The thermal profile was: 95°C for 2 min, followed by 38 cycles of 95°C for 30s, 56°C for 30s and 72 o C for 1 min. Data was interpreted by MxPro ET qPCR software ver 4.10 (Agilent Technology). Gene expression level was normalized to the reference gene (Actin) and calibrated with the J0 condition without JA added. In order to determine the relative gene expression in different conditions, mean Ct values were obtained and fold change values were calculated using the 2-ΔΔCT method (Livak and Schmittgen, 2001).

Statistical analysis
The data represented mean ± SD of three replicates and the differences between the data were examined by Student's t-test at p < 0.05. Statistical analysis (Student t-test) was conducted using Graphpad Prism 6 (https://www.graphpad.com/).

The contrasting effect of JA treatment on two genotypes
The remarkable differences in term of phenotype under 5 µM of JA treatment between the two varieties were selected throughout the study of screening from 156 Vietnamese rice collection against exogenous (To et al., 2019). The percentage of reduction of each trait was used to analyze the effect of JA while the two varieties were exposed to 5 µM of JA compare to non-treatment condition. Figure 1 showed clearly the differences in phenotypes between G38 plants and G11 plants. G38 plants reduced the SHL, RTW, SHW and TTW negatively in response to JA treatment compare to medium without JA, whereas G11 plants change their phenotype slightly in the same condition except for the RTL trait. Based on this analysis, the contrasting response in term of JA treatment between G11 and G38 can be confirmed.
Gene expression study of JA-related genes in two contrasting genotypes Figure 2 and 3 summarized the difference in the genes expression level of 12 JA markers genes between the sensitive variety G38 and from the tolerant variety G11. Changes are more obvious in plant's root than in the leaf might indicate that external JA mostly alters the root's metabolism as its first exposed organ with JA in the medium. Interestingly, there was a significant increase of expression level of some biosynthesis genes group such as OsHILOX, OsAOS, OsAOC and OsJMT in the root variety G11 plants compare to that in G38a sensitive genotype.
In the pool of examined genes, JASMONATE ZIM-domain OsJAZ5 and OsJAZ8 shown to be clearly up-regulated by JA in root: OsJAZ5 in G11 was upregulated over 40 times and also in G38, the upregulation was nearly 20 times; OsJAZ8 is over 10 folds greater in the presence of JA for G11 and around 13 folds greater for G38. We observed the activation of OsPR1a and OsPR10 (around 9 times and 40 times more expressed when plants were treated with JA, respectively) in G11 while it was nearly the same as the control level in the case of G38 variety for each.

Effect of low phosphorus condition (40 µm) on two JA-contrasted-response rice varieties
The percentage of change was used to analyze the effect of low phosphorus condition (40 µM) plants compared to full-phosphorus condition (625 µM) for each trait. Effect of phosphorus starvation in G11 was significantly different compared to that in G38 plants for all 5 traits, especially increase more than 30% in root weight (Figure 4).  A minimum 2-fold change is considered as significant relative quantification within the technical variation. Presented data are means ± SD of three samples obtained from independent plates, with three technical replicates for each sample. Each sample consists of a pool of tissues isolated from 4 plants for shoots and roots. Statistical significance (* for P < 0.05; ** for P < 0.01; and *** for P < 0.001) according to Student's t-test.  . Ratio (%) of variation for 5 traits including root length (RTL), root weight (RTW), shoot length (SHL), shoot weight (SHW) and total weight (TTW) was calculated by the offset of means of the treated trait to means of non-treated trait then dividing by means of the non-treated trait. Presented data is means ± SE from 10 plants grown in the independent test tube. Statistical significant (*, P < 0.05, **, P < 0.01, and ***, P < 0.01) according to Student's t-test.
Root hairs play an important role in facilitating phosphorus acquisition when phosphorus availability is low by increasing the absorptive surface area of the root and allowing the root to explore a greater soil volume (Jungk., 2001). This role has been demonstrated in wheat (Triticum aestivum L.) and barley cultivars (Hordeum vulgare L.) (Singh Gahoonia et al., 1997). The increase up to 0.7 mm of root hair length was also seen in tomato (Lycopersicon esculentum), rape (Brassica oleracea), spinach (Spinacia oleracea) when phosphate concentration decreased to 2 µM (Foehse, Jungk, 1983) and in Arabidopsis thaliana (Bates, Lynch, 2000); (Song et al., 2016). Longer root hairs were also observed in our two selected rice lines in Pi deficiency condition compared to full Pi condition ( Figure 5). Moreover, G11 seems to react stronger than G38 by increasing more root hair length than G38. It shows that the genetic variation for root hair length in response to P deficiency also exists.

DISCUSSION
Abiotic stress is becoming more and more intense in Vietnam, which reduces rice production severely. Therefore, it is necessary to identify the "tolerant variety to stress as well as to understand the mechanism underlying the stress response in rice. In this study, we found that G11 plants can tolerate better in JA treatment condition in term of phenotyping index compare to G38 -a "sensitive" one. Combined physiological and molecular approaches through the JA pathway help us to understand better how these contrasting varieties response differently. The tolerance of G11 plants can be explained through different aspects; the contribution of JA-signaling pathway in term of its defense was investigated in this paper. It is generally accepted that exogenous JAs causes a significant upregulation of some genes, especially in root part. However, the level of expression of some key markers in JA biosynthesis and JA transcriptional regulation genes were remarkably different between the two varieties. Figure 6 shows the simplified schema of the JA biosynthesis and response pathway for 12 JA genes markers used in this study. G11 had a higher expression level of some JA-biosynthesis genes and transcriptional regulation genes than G38 under JA treatment. Following the pathway of JA-signaling, the group of JA-biosynthesis players which includes OsHI-LOX, OsAOS and OsAOC are the first set of enzymes that needed to be analyzed and discussed. The involvements of these genes into biosynthesis pathway are simplified and highlight in the following schema (Fig. 6). From that, OsHI-LOX is the first gene (involved in JA biosynthesis pathway. The upregulation (20-folds) of OsHI-LOX in G11 is significantly higher compared to that of G38. Following up is the dramatic increase of OsAOS transcript in G11 (60 times), which is the next key component in the synthesis process of JAs. Subsequently, the expression level of OsAOC in G11 is still nearly 2 times higher than that of G38.
In stress condition, after JA has been synthesized, JA was derived in some distinguished branches: the making of hormonally active metabolite JA-Isoleucine (JA-Ile) and the production of a non-active yet very active locally signaling compound, for example: MeJA (Wasternack, Hause, 2013). JA-Ile is one of the active branches of JAs metabolites that can interact with SCF COI1-JAZs co-receptor to promote the transcription of JAinduced genes. This mechanism happened strictly under the control of some OsJAZs protein network family. OsJAZ5 and OsJAZ8 were used as representatives of JAZs encoding genes in the gene set markers. The increase in expression of these two genes is one of the most significant features in the results indicated the binding of JA-Ile to SCF COI1-JAZs complex, which requires multiple JAdependent physiological responses.
The expression level of OsPR1a in G11 was 9 times induced, whereas there was almost no induction in G38. Regards to OsbHLH148, a gene which is involved in JA-signaling, the expression level of this gene in G11 is significantly different compare to that in G38. These results give support for the hypothesis of JA is synthesized in larger quantity in G11 compared to G38, which leads to the high expression of some stress-responsive genes such as OsPR1a and OsPR10, then conferring its tolerance in G11.
The diverse response of Vietnamese rice to Pi starvation has been studied (Mai et al., 2018). Here, we also show the different response to Pi starvation of 2 Vietnamese rice accessions which response contrastly to JA. The link between JA pathway and Pi starvation has been investigated in various studies. In 2016, Khan and his colleagues found that phosphorus starvation induces the JA pathway by an accumulation of JA, JA-Ile and other derivatives level in both root and leaves in Arabidopsis thaliana. Moreover, the Pi starvation also activates the AtJAZ10 and AtVSP2 expression, resulting in the resistance to insect herbivory (Khan et al., 2016).
In 2016, Huang and his colleagues found that the overexpression of OsPR10 under Pi condition confers to rice and Arabidopsis the resistance to pathogen Xoo and Xoc (Huang et al., 2016). In this study, we also found that the genes OsPR10 is more than 40 times up-regulates in G11 than in G38 in root tissues and 5 times in shoot tissues. Consistence to the phenotype effect, these results obtained suggest a strong involvement of JA signaling pathway and regulation pathway in order to deal with the Pi starvation response in rice.

CONCLUSION
The obtained results above demonstrated that shoot growth of G11 accession is less inhibited by exogenous JA treatment and even "stimulated" by phosphorus starvation than G38 plant. Pi deficiency has a negative impact to G38 root length by inhibiting its growth while positively affected G11 root length suggesting that G11 did not suffer much effect of Pi stress, which was the similar effect with JA treatment. This study highlights the differences in JA growth response in 2 contrasting rice genotypes. Also, it suggests the link between JA developmental response and the tolerance to the Pi starvation condition in rice.