pflp轉殖水稻對白葉枯病的抗病性來自於增強蔗糖轉化酶與六磷酸激酶活性

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(1)國立高雄大學生物科技研究所碩士論文. pflp 轉殖水稻對白葉枯病的抗病性來自於增強蔗糖轉化酶與六磷酸激酶活性. Disease resistance of pflp-transgenic rice against bacterial blight results from enhancement of invertase and hexokinase activities. 研究生：張雅雲撰指導教授：葛孟杰博士. 中華民國 102 年 7 月 I.

(2) Contents Contents ................................................................................................................................. I List of Figures ...................................................................................................................... III 摘要 ...................................................................................................................................... 1 Abstract .................................................................................................................................. 3 Chapter I Introduction ........................................................................................................... 5 Chapter II Materials and methods ......................................................................................... 9 2.1 Plant material and growth condition........................................................................ 9 2.2 Bacterial blight inoculation ..................................................................................... 9 2.3 RNA extraction and Reverse transcription PCR ................................................... 10 2.4 Quantitative reverse transcription PCR ................................................................. 10 2.5 H 2 O 2 content ......................................................................................................... 11 2.6 DAB stain .............................................................................................................. 11 2.7 Soluble sugars and starch contents assay .............................................................. 11 2.8 Statistical analysis ................................................................................................. 12 Chapter III Results ............................................................................................................... 13 3.1 Characterization of pflp transgenic lines ............................................................... 13 3.2 Overexpression of pflp enhances tolerance to bacterial blight in rice ................... 13 3.3 Highly contents of glucose accumulate in transgenic plants ................................. 13 3.4 Relatively high expression pattern of starch and sucrose metabolism relative genes in transgenic plants ............................................................................................ 14 3.5 Relatively high expression pattern of hexokinase gene in transgenic plants ........ 15. I.

(3) 3.6 pflp transgenic plants accumulate less ROS concentrations in early phase of pathogen infection ....................................................................................................... 16 3.7 Relatively high expression pattern of pathogenesis-related gene in transgenic plants after inoculation with Xoo ................................................................................ 17 Chapter IV Discussion ......................................................................................................... 18 Chapter V References .......................................................................................................... 36 Supplemental data Ⅰ: List of abbreviation ........................................................................... i Supplemental data Ⅱ: PCR primers................................................................................... iii Supplemental data Ⅲ: The kimura B solution ..................................................................... v. II.

(4) List of Figures Fig. 1 Characterization of pflp transgenic plants ................................................................. 21 Fig. 2 Resistance to Xoo of TNG67 and transgenic plants ................................................. 23 Fig. 3 Soluble sugars and starch content in TNG67 and transgenic plants after inoculation with Xoo ....................................................................................................................... 25 Fig. 4 Starch metabolism and synthesis relative gene expression in TNG67 and transgenic plants after inoculation with Xoo.................................................................................. 27 Fig. 5 The expression pattern of invertase related genes in TNG67 and transgenic plants after inoculation with Xoo ............................................................................................ 28 Fig. 6 The expression pattern of hexokinase related genes in TNG67 and transgenic plants after inoculation with Xoo ............................................................................................ 29 Fig. 7 Comparisons of DAB stain and H 2 O 2 content between TNG67 and transgenic plants after inoculation with Xoo ............................................................................................ 32 Fig. 8 The expression pattern of pathogenesis-related genes in TNG67 and transgenic plants after inoculation with Xoo.................................................................................. 34 Fig. 9 Proposed model for the role of PFLP in sugar signal-regulation during defense response in transgenic plants ........................................................................................ 35. III.

(5) pflp 轉殖水稻對白葉枯病的抗病性來自於增強蔗糖轉化酶與六磷酸激酶活性指導教授:葛孟杰博士國立高雄大學生物科技研究所. 學生:張雅雲國立高雄大學生物科技研究所摘要醣類為植物生長發育過程的重要物質，不僅可作為代謝能量來源，同時也是一種訊息傳遞分子。當植物遭受病原入侵時，蔗糖轉化酶(invertase，INV)可將蔗糖水解成果糖與葡萄糖，葡萄糖即可透過六磷酸激酶(hexokinase，HXK)調控粒線體中的過氧化物(reactive oxygen species，ROS)生成。ROS作為一訊息傳遞分子，可誘發植物體內包括細胞壁增厚，產生植物防禦素 (phytoalexins) 與病程相關蛋白 (pathogenesis-related proteins，PR)等抗病反應。植物硫鐵蛋白相似蛋白(Plant ferredoxin like protein，PFLP)分離自甜椒，與阿拉伯芥之第一型硫鐵蛋白有高度相似性。第一型硫鐵蛋白廣泛分布在植物綠色組織中，其主要在光合作用電子傳遞鏈中扮演傳遞電子的角色。先前研究已知過表現PFLP之轉殖水稻具有較高的電子傳遞能力與光合作用效率，使轉殖水稻含有較高的醣類累積與HXK基因表現量。許多研究也指出PFLP 轉殖植物可透過ROS的產生使植物對抗病原性的侵害，但醣類相關酵素在此抗病反應中所參與的作用機制尚未明瞭。因此本篇研究將探討蔗糖轉化酶與六磷酸激酶在pflp 轉殖水稻於白葉枯病抗病上所扮演的角色。實驗結果顯示轉殖水稻對白葉枯病有較高的抗病性，而其在感病初期PR蛋白的誘發程度亦較台農67號水稻(TNG67)高。在感染白葉枯病病原菌時轉殖水稻能表現較TNG67高2倍量的cwINV，以此累積比TNG67高 1.6倍的葡萄糖含量，並透過葡萄糖分別提升HXK2與HXK6約2倍與2.5倍的基因表現量，. 1.

(6) 在ROS生成方面，轉殖水稻在前期可產生較TNG67約2.5倍高的ROS，使轉殖水稻引發較高量的ROS誘導抗病系統抑制病原進一步發展。綜合上述結果，本篇研究指出 PFLP轉殖水稻可以透過INV累積較高量的葡萄糖並透過此來活化HXK，以在病原感染初期即產生ROS使之作訊號傳遞，表現PR蛋白等相關抗病基因來增進轉殖水稻對白葉枯病的抗性。. 關鍵字：植物硫鐵蛋白、白葉枯病、蔗糖轉化酶、六磷酸激酶. 2.

(7) Disease resistance of pflp-transgenic rice against bacterial blight results from enhancement of invertase and hexokinase activities Advisor: Dr. Mang-Jye Ger Institute of Biotechnology National University of Kaohsiung. Student: Ya-Yun Chang Institute of Biotechnology National University of Kaohsiung Abstract Sugar is an important biological compound for plant growth and development. Not only serves as important energy source but also acts as signaling molecule in plants. Invertase (INV) can be hydrolysis of sucrose to fructose and glucose during pathogen infection. Glucose signal regulates reactive oxygen species (ROS) generation through mitochondrial hexokinase (HXK) during pathogen infection. As signals, ROS induces several defense responses including cell wall thickening, phytoalexin synthesis and pathogenesis-related (PR) proteins transcription. The plant ferredoxin - like protein (PFLP), isolated from sweet peppers, shows high homology to sequence of Fd-I, an electron carrier in photosynthesis. Previous studies reported that constitutive expression of pflp in transgenic plants exhibited highly photosynthesis efficiency, increased soluble sugars contents, and altered HXK expression pattern. Many studies also have been reported that continuously expression of PFLP in transgenic plants encourages the. 3.

(8) generation of ROS and participates in pathogen-resistant mechanism. However, the function of sugar associated enzyme involved in pathogen-resistant mechanism is unclear. This study is to investigate the role of INV and HXK in pflp transgenic plants resistant to bacterial blight. In this report, we demonstrated that PFLP can enhance disease resistance to bacterial blight and quickly induce PR protein transcription in early phase of pathogen infection. Additionally, the expressions of cwINV in transgenic plants showed 2-folds higher than TNG67 after Xoo infection, and accumulated glucose content 1.6 times higher than TNG67 in transgenic plants. This result was due to increased HXK2 and HXK6 2-fold and 2.5-fold gene expression pattern. In ROS generation, the transgenic rice generated ROS about 2.5 times higher than TNG67 in early Xoo infection phase. Generated ROS induced defense responses of against bacterial blight. Altogether, we demonstrated PFLP transgenic rice plants accumulated higher levels of glucose via INV and tolerance against bacterial blight was improved through glucose activation HXK, which regulated ROS production in pathogen infection early phase.. Keywords: plant ferredoxin-like protein (PFLP), bacterial blight, invertase, hexokinase. 4.

(9) Introduction Sugars are not only important energy sources and structural components, they also serve as central regulatory molecules controlling physiology, metabolism, cell cycle, development, and gene expression in prokaryotes and eukaryotes (Jang et al. 1997; Bolouri-Moghaddam et al. 2010). Both sucrose and glucose are recognized as pivotal integrating regulatory molecules that control gene expression related to plant stress resistance, growth and development (Pego et al. 2000; Rolland et al. 2006; Kano et al. 2010; Kano et al. 2011). Previous study has been reported that sucrose was catalyzed to glucose which could play a role as signal to regulate Pathogenesis-Related (PR) gene expression, via hexokinase (HXK)-dependent pathway and glycolysis-dependent pathway (Xiao et al. 2000). HXK is a dual-function enzyme that can phosphorylate hexose to form hexose 6-phosphate and play an important role in sugar sensing and signaling (Cho et al. 2006). Studies have showed that hexokinases interact with membranes of various cellular organelles (Frommer et al. 2003), such as chloroplasts, mitochondria, Golgi complexes, endoplasmic reticula, and plasma membranes. Hexokinases interacting with mitochondria may be coupled to ATP production for cellular metabolism (Pastorino et al. 2002). In addition to organelle-bound hexokinases, cytosolic hexokinase plays an important role in removal of free hexoses from the cytosol depending on cellular demands (Moore et al. 2003). There are ten HXK genes in rice. Sugars induced the expression of three OsHXK genes, OsHXK2, OsHXK5, and OsHXK6 (Cho et al. 2006). Cho et al. (2009) reported that OsHXK5 and OsHXK6 play a role as glucose sensors and both were located in mitochondria. Invertase (INV) is the enzyme that cleave sucrose into glucose and fructose, which is involved in carbohydrate partitioning and the regulation of sucrose:hexose ratios. INV contributes to maintaining cellular glucose contents and plays roles in signaling under. 5.

(10) stress (Weber et al. 2005; Pugh et al. 2010; Ruan et al. 2010). INV are grouped into three based on their subcellular localization and pH optima. There are cytoplasmic invertase (cyt-INV), vacuolar invertase (VIN), and cell wall invertase (cwINV) (Roitsch et al. 2004; Wang et al. 2010). cwINV is an extracellular enzyme catalyzing the cleavage of the transport sugar sucrose into glucose and fructose, which is a key enzyme for supplying sink tissues with carbohydrates (Goetz et al. 2000). Expression of cwINV leads to the accumulation of carbohydrates and a metabolic source-to-sink shift and also increased gene expression of PR proteins and enhanced resistance against pathogen infection in tobacoo (Herbers et al. 2000). Based on INV is one of glucose accumulation source, another source may from starch degradation. Amylase is the key enzyme in starch degradation (Smith et al., 2005; Zeeman et al., 2010), AMY4A, AMY5A and putative chloroplast-targeted β-amylase (CT-BMY) transcript activity were monitored. ADP-glucose pyrophosphorylase (AGPase) that is the key enzyme in starch synthesis (Orzechowski, 2008). AGPase have several isoforms in different tissue. In the plastid of leaf, AGPS2α and AGPL3 are responsible for starch synthesis (Lee 2007). Plants always subject to stresses, those stresses include abiotic stress such as extremes of temperature, drought and high salinity, or biotic stress such as fungi, bacteria, viruses and insects (Chaves et al. 2009). For successful survival strategies, plants have to adapt to stressful condition. Reactive Oxygen Species (ROS) is an important molecule of plant adaptation to stress. ROS, including superoxide radical (O 2 -), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH-) and singlet oxygen (1O 2 ), can serve as signaling molecules to triggered defense mechanism in response to stress (Sharma et al. 2012). Pathogenesis-related (PR) proteins play an important role in the disease resistance response (Hou et al. 2012). Several types of proteins are common to many plants and these have been classified into 17 families of PR proteins (van Loon et al. 2006). The PR. 6.

(11) proteins are often used as markers of the plants enhanced defense response in pathogen infection (Ryals et al. 1996). In rice, PR genes are induced by biotic stresses like bacterial blight (Chittoor et al. 1997; Wu et al. 2011). PR proteins include various types such as SCP-like extracellular protein (PR1), Thaumatin (TLP, PR5), subitilisin-chymotrypsin inhibitor (Sci1, PR6), probenazole-inducible protein (PBZ1. PR10) and phenylalanine ammonia-lyase (PAL) (Hou et al. 2012). PR1 (PRla, PRlb, PR1c) is a glycine-rich protein, may be involved in anti-infection effect of plant cell walls (Mitsuhara et al. 2008). PR5 can activate some resistance of serine endopeptidase enzyme protein (van Loon et al. 2006); PR10 has nuclease and antibacterial activity, which play an important role in plant defense resistance (Kim et al. 2008; Liu and Ekramoddoullah 2006). PFLP (Sweet pepper ferredoxin-like protein) was isolated from sweet pepper (Capsicum annuum L.), which shows highly homology with Ferredoxin-I (Fd-I) from Arabidopsis thaliana (54%), Lycop-ersicon esculentum (72%), Oryza sativa (56%), Spinacia oleracea (52%) and maize (48%) (Dayakar et al. 2003). Fd-I protein, known as an electron carrier in photosynthesis, generally exists in green tissue (Zurbriggen et al. 2008). PFLP contains an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain (Lin et al. 1997). Expression of the pflp in transgenic plants, such as tobacco, arabidopsis, calla lily and oncidium orchid, which could enhance host resistance to virulent bacterial pathogens through ROS (Reactive oxygen species) (Chan et al. 2005; Dayakar et al. 2003; Huang et al. 2004; Liau et al. 2003; Tang et al. 2001; Yip et al. 2007). Chang (2010) utilized PFLP expression in rice and the transgenic plant grown were grown 2 months in soil. This study demonstrated that expression pflp in transgenic plants can increase photosynthesis efficiency, capacity of sugar synthesis and crop yield.The pflp transgenic rice plants contained higher contents of glucose, fructose and sucrose compared with wild-type plants (Oryza sativa, TNG67).. 7.

(12) Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important diseases of rice in Africa and Asia (Wang et al. 1996). Plant ferredoxin-like protein (pflp)-transgenic plants are generated and showed resistance against bacterial blight through unknown mechanism (Tang et al. 2001). One of the defense responses of pflp-transgenic plants upon pathogen attack is the production of ROS that can signal to active defense response (Huang et al. 2004). Moreover, because sugar accumulations were higher in leaves of pflp transgenic plants, our hypothesis is that transgenic plants rice expressing pflp gene might increase resistance to bacterial blight though sugar signal transduction pathway. Two transgenic lines, pflp-1 and pflp-2, show higher tolerance against bacterial blight than that of WT (TNG67). Transgenic rice plants accumulated glucose content which is through the function of invertase and amylase. Glucose increased expression of HXK, regulation of mitochondrial ROS production. That would quickly induce PR protein transcription in early phase of pathogen infection and increase disease resistance to bacterial blight in transgenic plants.. 8.

(13) Materials and methods Plant material and growth condition TNG67 (Oryza sativa japonica cv. Tainung 67, Taiwan) and pflp transgenic plants were used. The constructed pflp cDNA for transformation was designated as pBI121-PFLP (Huang et al. 2004). The transgenic plants seeds were obtained from the laboratory of Dr. Feng T.Y. (Institute of Plant and Microbial Biology, Academia Sinica, Taiwan). Two pflp transgenic lines are indicated as pflp-1 and pflp-2. Rice seeds were germinated for 3-5 days in plates with water at 28°C and further transferred to plastic pots containing hydroponic Kimura B solution. Plants were grown in growth chamber at 28°C/ 25°C, 16 h light/8 h dark photoperiod. Kimura B solution was followed according to Ma et al. (2001). The solution contained 172 μM (NH 4 ) 2 SO 4 , 92 uM KNO 3 , 134 μM MgSO 4 ．7 H 2 O, 91 μM KH 2 PO 4 , 127 μM Ca(NO 3 ) 2 ．4 H 2 O, 2.5 μM H 3 BO 3 , 0.201 μM MnSO 4 ．4 H 2 O, 0.201 μM ZnSO 4 ．7 H 2 O, 0.052 μM CuSO 4 ．5 H 2 O, 0.044 μM H 2 MoO 4 , and 31 μM Fe-citrate. The pH value of Kimura B solution was adjusted between 5.5-5.8, and renewed every 3 days.. Bacterial blight inoculation Xanthomonas campestris pv. oryzae XF-89b (Xoo) was used in this study. The isolate was grown at 28°C for 72 hours on nutrient broth (NB) agar medium (BD Difco, France). Xoo cultures were maintained in NB medium and 24 hours shaking at 150 rpm. One-month-old TNG67 and pflp transgenic plants with merging flag leaves were sampled. Two cm from the tip of fully expanded leaves were cut using scissors dipped in a bacterial suspension (≈ 1×10 9 CFU mL-1). Rice plants incubated at 28°C/ 25°C (16 h light and 8 h dark) with Kimura B solution for 20 days and then the length of blight lesions were measured.. 9.

(14) RNA extraction and Reverse transcription PCR Total RNA was isolated from plant leaf tissues of rice (0.07 - 0.1 g fresh weight) using Trizol reagent according to the RNA extraction manual (Molecular research center, Cincinnati, USA). The total RNA was reverse-transcripted (rt) into cDNA using ImProm-IITM Reverse Transcription System (Promega, Madison WI, USA) with an oligo-dT primer. For cDNA amplification, 1 μg RNA was used for 20 μl reverse transcription reaction. Followed by using gene-specific primers, 2 μl of cDNA was used for PCR amplification. The PCR condition was the first cycle at 95°C for 3 min, and then the optimized cycle numbers for each gene product followed 1 min at 95°C, 30 second at appropriate temperature and 30 second at 72°C. The final extension was at 72°C for 5 min. PCR products were subjected to electrophoresis at 80 volt for 60 min by using 2 % agarose gel. After which, the gels were stained in 0.5x TAE solution containing ethidium bromide for 20 min. Gels were photographed under UV illumination using UV light box (DGIS-8 Digital Gel Image System, TopBio, Taiwan).. Quantitative reverse transcription PCR For qRT-PCR analysis, RNA was used as templates and an Applied Biosystem StepOne Real Time PCR System (Applied Biosystems Inc., Foster, USA) was performed. 70 ng RNA was used for 20 μl reaction per tube and using the Real-Time SYBR Green RT-PCR Kit (Yeastern Biotech, Taiwan). The PCR program was the first cycle at 48°C for 30 min and 95°C for 10 min, then followed 30 sec at 94°C, 30 sec at 60°C underwent 40 cycles. Melting curve program was 15 sec at 95°C, 30 sec at 55°C. The transgenic plants gene expression level was showed as related values compared with TNG67 plants and using OsActin as internal control. The expression levels of these genes are presented as values relative to that of TNG67 at 0 h.. 10.

(15) H2O2 content The plant leaf tissues were homogenized with liquid nitrogen, and suspended lysate in 1 ml of 50 mM phosphate buffer (pH=7.0) that containing 10 mM 3-amino-1, 2, 4-triazole. The homogenates were centrifuged at 9000 g for 25 min. The amount of 200 μl of the supernatant was added to 900 μl reaction mixture that contained 100 μM Xylenol orange, 250 μM FeSO 4 ．7 H 2 O, 100 mM sorbitol and 25 mM H 2 SO 4 . The reaction mixture was incubated in dark for 40 min at room temperate, and measured the absorption of supernatant at 560 nm (Model number, Maker, Country) to determine the level of H 2 O 2 (Jiang et al. 1990).. DAB stain Leaf tissues of TNG67 and transgenic plants were removed, and 1 mg/mL 3, 3’diaminobenzidine (DAB)-HCl, pH=3.8 (Sigma, MO, USA) was used to incubate in growth chamber in dim light for 24 h. After 24 h, each sampling was washed twice with ddH 2 O and then placed in 95% ethanol for 30 min. After that, samples were stored in 70% ethanol (Thordal-Christensen et al. 1997).. Soluble sugars and starch contents assay For determination of soluble sugar, the samples of 0.1 g leaf tissue were homogenized and extracted by a hot 80% EtOH as previously described (Wang et al. 2000). Sugars were separated on an HPAEC-PAD (Dionex Corporation, Sunnyvale, CA, USA) with a Dionex CarboPac PA10 column (2 mm diameter) using 18 mM NaOH as eluent and quantified by a Dionex Pulsed Amperometric Detector (Wang et al. 2008). For leaf starch content determination are according to Chen et al. (2008). The 2,. 11.

(16) 2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) (Sigma, Steinheim, Germany) is used to determination of starch content and potato starch (Sigma, Steinheim, Germany) as standard for the color reaction and then measuring the absorbance of the reaction at 405 nm.. Statistical analysis When applying statistics a one-way ANOVA was used with a 5% LSD Means Test in cases of multiple comparisons. The statistical analysis was carried out using Cohort statistical analysis software (Ver. 6.4, Cohort). Values based on the means ± SE of 3to10 experimental replicates.. 12.

(17) Results Characterization of pflp transgenic lines The transgenic rice seeds were kindly provided from the laboratory of Dr. Feng TY (Institute of Plant and Microbial Biology, Academia Sinica). Two transgenic lines, pflp-1 and pflp-2, were subjected to reverse-transcription PCR (RT-PCR) analysis to detect pflp gene expression levels. It was found that the pflp transgene was strongly expressed in two transgenic plants (Fig. 1).. Overexpression of pflp enhances tolerance to bacterial blight in rice Xoo was used in inoculation tests on TNG67 and transgenic plants. Lesion length analysis showed both pflp-1 and pflp-2 transgenic plants exhibited significant levels of resistance against Xoo, compared to TNG67 after challenge Xoo 21 days (Fig. 2A). Resistance was elevated more than 50% in bothpflp-1 and pflp-2 plants (Fig. 2B). This result further suggested that PFLP contributes to defense against virulent pathogens.. Highly contents of glucose accumulate in transgenic plants Soluble sugars like glucose, fructose and sucrose are recognized as signaling molecules under stress in plants (Bolouri-Moghaddam et al. 2010). The sugar contents were detected in both pflp transgenic lines and TNG67. The contents of glucose in transgenic plant were 2.1 and 2.0-folds higher than TNG67 at basal level. Contents of glucose accumulate drastically increased in TNG67 and two transgenic plants, and the glucose content of pflp-1 and pflp-2 were stilled 1.5 and 1.7-folds higher than TNG67 after 24 hr of Xoo treatment. However, it decreased drastically in TNG67 and two transgenic plants after 48 hr of Xoo treatment (Fig. 3A). The contents of fructose were no significant. 13.

(18) difference between TNG67 and two pflp transgenic plants at basal level. However, it increased significantly in TNG67 and two transgenic plants after Xoo treatment. The fructose contents of TNG67 were ~60% respectively less than two transgenic plant at 24 hr of Xoo infection. TNG67 fructose contents were 1.5 and 1.3-folds higer than pflp-1 and pflp-2 at 48 hr of Xoo infection (Fig 3B). The sucrose contents were no significant difference between TNG67 and two transgenic plants at basal level. Sucrose contents decreased over time in TNG67 after Xoo infection (Fig 3C). As shown in Fig. 3D, starch contents were no significant difference between TNG67 and two transgenic plants at basal level. It increased drastically in TNG67 and two transgenic plants after Xoo treatment. The contents of starch in pflp-1 and pflp-2 plants were 34% and 27% respectively less than that of TNG67 after Xoo infection for 48h. In ddH 2 O treatment conditions, starch contents increased in TNG67 and two transgenic plants, but no significant difference between TNG67 and two transgenic plants. In normal growth conditions, starch contents were no significant difference between basal level, 24 hr and two 48 hr.. Relatively high expression pattern of starch and sucrose metabolism relative genes in transgenic plants To understand the mechanism of starch accumulation in plant after Xoo infection, genes of starch degradation, including AMY4A, AMY5A as well as CT-BMY, and gene of starch synthesis including AGPL3 and AGPS2α were monitored at 0 hr, 8 hr, 24 hr and 48 hr after treatment. The transcriptional activities of AMY4A were high expression pattern after Xoo infection for 8 hr and returned to basal level after 24 hr treatment of TNG67 and two transgenic plants, whereas TNG67 plants showed strongly induction after 48 hr treatment (Fig. 4A). The expressions of AMY4A in two transgenic plants were 2.0 and 2.4. 14.

(19) times higher than that of TNG67 at basal level (Fig. 4B). The AMY5A expression pattern was no significant difference between basal level, after 8hr, 24hr and 48 hr Xoo infection (Fig. 4A). After 8 hr for Xoo infection, the CT-BMY was high expression pattern in TNG67 and two transgenic plants and returned to basal level after 24 hr and 48 hr treatment (Fig. 4A). Both AGPL3 and AGPS2α were down regulation in TNG67 and two transgenic plants after Xoo infection (Fig. 4A). The data showed AGPS2α expression pattern were 5.3 and 4.6-fold higher than TNG67 (Fig 4B). INV is the enzyme that cleaves sucrose into glucose and fructose, which involved in carbohydrate partitioning and the regulation of sucrose:hexose ratios (Weber et al. 2005). Cell well INV, encodes by cwIN1 in rice (Hirose et al. 2002), were also monitor by RT-PCR and qRT-PCR analysis. At basal level, the transcriptional activities of cwIN1 in two transgenic plants were 2.1 and 2.3 times higher than that of TNG67. Expression pattern of cwIN1 was down regulation in TNG67 but not found in two transgenic plans at 8 hr and 24 hr Xoo treatment (Fig. 5A and 5B). Alkaline/neutral INV, encodes by NIN1 in rice which localization on mitochondria (Murayama et al. 2007). The NIN1 expression patterns in two transgenic plants were 4.7 and 5.1-fold higher than that of TNG67 without Xoo treatment, but NIN1 expression level was reduced in two transgenic plants during Xoo infection (Fig. 5A and 5B). Cytosol INV encodes by cyt-INV in rice which is localized on cytosol (Jia et al. 2008). The expressions of cyt-INV in two transgenic plants were 5.6 and 6.6 times higher than that of TNG67 in basal level (Fig. 5A and 5B).. Relatively high expression pattern of hexokinase gene in transgenic plants Both pflp-1 and pflp-2 transgenic plants exhibited increased ROS levels, stronger induction of the defense-related genes and enhanced disease resistance. To determine these. 15.

(20) results were related to the sugar signaling pathway or not, qRT-PCR analysis was used to monitor transcriptional activities of HXK genes in two transgenic plants and TNG67 plants. HXK1 and HXK2, encodes by HXK1 and HXK2 in rice, were both located in cytosol and involved mainly in the glycolysis process (Cho et al. 2006). The HXK1 expressions patterns in two transgenic plants were 2.4 and 1.7-fold higher than that of TNG67 in basal level. However, HXK1 expression levels were reduced after 8 hr Xoo infection and below detectable after 24 hr Xoo treatment (Fig. 6A and 6B). The expressions of HXK2 in two transgenic plants were 2.2 and 2.1 times higher than that of TNG67 at 8 hr inoculation, which were down regulated in TNG67 and two transgenic plants after 24 hr infection (Fig. 6A and 6B). HXK5 and HXK6, encodes by HXK5 and HXK6 in rice, both played roles as glucose sensors and located in mitochondria (Cho et al. 2009). Regardless of inoculation with Xoo, HXK5 showed no significant difference between TNG67 and two transgenic plants. The expressions of OsHXK6 in two transgenic plants were 2.5 and 2.2 times higher than that of TNG67 at 24 hr inoculation (Fig. 6A and 6B).. pflp transgenic plants accumulate less ROS concentrations in early phase of pathogen infection Rapid generation of ROS is an early unique feature of the disease resistance following perception of pathogen avirulence signals. Inoculation of pathogen causes a rapid but weak transient accumulation of oxidants (phase I). Massive and prolonged oxidative burst after 24 hr pathogen inoculation (phase II) (Lamb and Dixon 1997; Bolwell and Daudi 2009; Fluhr 2009). TNG67 and two transgenic plants were inoculated with Xoo, and then ROS content in both transgenic plants and TNG67 were subsequently observed. The H 2 O 2 content was no significant difference between TNG67 and two transgenic plants before inoculation. After 8 hr treatment, the contents of H 2 O 2 in pflp-1 and pflp-2 transgenic. 16.

(21) plants showed 2 folds higher than that of TNG67. After 24 and 48 hr treatment, TNG67 increased H 2 O 2 more 10-folds than 8 hr (Fig. 7A). Same result in DAB stain data was observed . TNG67 plantsshowed higher levels of DAB staining than transgenic plants after 48 hr Xoo inoculation. This implied that TNG67 accumulated high levels of ROS with cell damage (Fig. 7B).. Relatively high expression pattern of pathogenesis-related gene in transgenic plants after inoculation with Xoo The expression of PR genes is a widely used indicator of induction of plant defense responses. These expressions proposed to be considered as. markers in defense or stress. responses in rice were analyzed (Agrawal et al. 2001). The transcriptional activities of PR1a of two transgenic plants were rapidly induced after inoculation with Xoo for 8 hr, whereas TNG67 plants showed slightly induction after inoculation with Xoo. The other genes such as PR1b, TLP, Sci1, PBZ1 and OsPAL were analyzed in this work gave similar results (Fig. 8A). The expressions of PR1a in two transgenic plants were 2.3 and 2.9 times higher than that of TNG67, and PR1b expression pattern were 2.3 and 2.9 times higher than TNG67 without Xoo inoculation. The expressions of PR1a in two transgenic plants were 2.5 and 2.6 times higher than that of TNG67, and PR1b expression pattern were 1.9 and 2.1 times higher than TNG67 at 8 hr inoculation. The TLP expression patterns in two transgenic plants were 1.5-fold higher than that of TNG67, Sci1 expression patterns in two transgenic plants were 1.9 and 1.8-fold higher than that of TNG67, PBZ1 expression patterns in two transgenic plants were 3.5 and 4.2-fold higher than that of TNG67, and OsPAL expression pattern were 2.5 and 2.4 times higher than TNG67 after inoculation with Xoo 24 hr (Fig. 8B).. 17.

(22) Discussion In this study, we demonstrated that transgenic pflp rice plants exhibited higher resistance to bacterial blight though sugar signal transduction pathway. PFLP can promote accumulation of glucose content (Fig. 3A) through through cwIN1 and CT-BMY highly expressions (Fig. 4 and Fig. 5). Glucose increased expression of HXK2 and HXK6 (Fig. 6), regulation of mitochondrial ROS generation (Fig. 7). ROS as signal that quickly and strongly induced PR genes transcription in early phase of pathogen infection (Fig. 8). These gene expressions enhanced disease resistance to bacterial blight. Chang (2010) demonstrated that expression pflp in transgenic plants can increase photosynthesis efficiency, and the transgenic plants contained higher soluble sugar contents such as glucose, fructose and sucrose with TNG67. However, in this study, transgenic plants only glucose content was increased, where as sucrose and fructose contents remained the same as TNG67. We believe that the different result was due to different growing conditions and plant age (Fig. 2). There is a possible mechanisms of glucose source in transgenic plants. A rapid accumulation of glucose was caused by induction of cwINV activity. That activity is thought to promote their utilization for host defense reactions, supporting the successful establishment of resistance (Scharte et al. 2005 ; Swarbrick et al. 2006 ; Essmann et al. 2008 ). Glucose contributes to the disease resistance of transgenic plants. Most deference related compounds like callose deposition and the production of phenolic compounds (including salicylic acid) are both known outputs of plant defense and require large amounts of metabolizable sugars (Herbers et al. 1996 ; Scharte et al. 2005 ). Kano (2010) demonstrated that one of the rare sugars, d-allose, non-metabolizable glucose analogues, conferred resistance to rice bacterial blight, a vascular wilt disease that is one of the most serious rice-plant diseases worldwide. Kano (2010) using a rice microarray revealed that. 18.

(23) d-allose treatment causes a high upregulation of many defense-related PR-protein genes. In this report, transgenic PFLP rice plants demonstrated that expression patterns of PR proteins include PR1a, PR1b, TLP and PBZ1 were higher than TNG67 without pathogen infection (Fig. 7A and 7B). That may due to accumulated Glucose contents in basal level of transgenic plants (Fig. 3A). Intracellular glucose can activate hexokinase activity. In addition to its familiar role as a metabolic enzyme, mitochondria-associated hexokinases (mtHXK) activity could be involved in the regulation of both mitochondrial respiration and ROS production in plants (Claeyssen and Rivoal 2007). The expressions of OsHXK6 in two transgenic plants are higher than that of TNG67 either with or without pathogen inoculation (Fig. 6). Control Fig. 7 transgenic plants can quickly produce ROS in the early phase of pathogen infection, and acts as plant deference system signal. In conclusion, HXK6 might act through glucose catalytic activity, as a crucial regulator of ROS levels in mitochondria. The glucose and starch contents were increased after pathogen infection, whereas sucrose contents were no significant difference between basal level and after pathogen infection. The source of increased glucose might come from the unknown sugar pools (Go´mez-Ariza et al. 2007). However, the insolubilization of sugars into starch may allow continuation of assimilate import by decreasing the hexose concentration, avoiding feed-back inhibition (Balibrea 1999). By comparing with Fig. 3D and Fig. 4, the inconsistency of starch contents and starch synthesis relative genes (AGPL3 and AGPS2α) was observed. Starch contents drastically increased in TNG67 and two transgenic plants after Xoo treatment (Fig. 3D).However, both AGPL3 and AGPS2α were down regulated in TNG67 and two transgenic plants after Xoo infection (Fig. 4A). We believe that starch synthesis may be controlled by anothers enzymes such as starch synthase. PFLP can increase photosynthesis efficiency and sugar metabolism that accumulated. 19.

(24) glucose in pflp transgenic plants. PFLP would also affect the production of ROS under stress. Sugar pools act as a sucrose source during pathogen infection (Bolouri-Moghaddam et al. 2010; Bolouri-Moghaddam and Van-den-Ende 2012). cwINV1 catalyzes the hydrolysis of sucrose into glucose and fructose in apoplast, and CT-BMY catalyzes the breakdown of starch into glucose in chloroplast. Glucose contents accumulated in plant cell that enhance HXK2 and HXK6 expression in the process of pathogen infection. It has been demonstrated that mitochondria-associated HXK can contribute to the steady-state recycling of ADP (ADP production by mitochondria-associated HXK, bound to the mitochondrial outer membrane; ADP consumption through oxidative phosphorylation) which regulates H 2 O 2 formation in the electron transport chain on the mitochondrial inner membrane (Kim etal. 2006; Bolouri-Moghaddam et al. 2010; Xiang et al. 2011). ROS is as a signaling active antioxidant system forPR gene expression (Fig 8). Pflp-transgenic plants showed resistance to bacterial blight though HXK-dependent sugar signal transduction pathway.. 20.

(25) TNG67. pflp-1. pflp-2. pflp α-tubulin. Fig. 1 Characterization of pflp transgenic plants. RT-PCR analysis for detecting pflp expression from 1-month-old TNG67 and 2 transgenic plants were performed and expression of α-tubulin as an internal control.. 21.

(26) (A) TNG67. pflp-1. (B). Length of lesion/leaf (mm). 70 60. a. 50 40 30. b. 20. b. 10 0 TNG67. pflp-1. pflp-2. 22. pflp-2.

(27) Fig. 2 Resistance to Xoo of TNG67 and transgenic plants. One-month-old plants with merging flag leaves were inoculated with Xoo (107 cfu ml-1). a Photograph of rice leaves taken 21 days after inoculation of cut leaves with Xoo. Scale bars are 1 cm. b The lesion length was scored on three leaves of each independent transgenic plant and nine leaves of three untransformed, control plants 21 days after inoculation. The solid bar values of two pflp transgenic plants differ significantly from the TNG67 according to one-way ANOVA LSD Means Test (P < 0.05). The results are given as means ± SD (n=9 individual plants).. 23.

(28) (A). Glucose 0.6 a. mg/gFw. 0.5. a. 0.4 a a a. 0.2 0.1. TNG67 pflp-1 pflp-2. b. 0.3. a. a. b. 0 0 hr. 24 hr. 48 hr. (B). Fructose 1.4. a. 1.2 mg/gFw. 1. ab. b b. a. 0.8 0.6. TNG67 pflp-1 pflp-2. b. 0.4 0.2. a a a. 0 0 hr. 24 hr. 48 hr. (C). Sucrose 10 a a. mg/gFw. 8. a a a. a a b. 6. TNG67 pflp-1 pflp-2. b. 4 2 0 0 hr. 24 hr. 48 hr. 24.

(29) (D). Starch 1.8. a. 1.6 1.4 a. mg/gFw. 1.2. a a. b b. TNG67. 1. pflp-1. 0.8. pflp-2. 0.6 0.4 0.2. a a a. a a a. a a a. a a a a a a. a a a. a a a. 0 hr. 24 hr. 48 hr. 0 0 hr. 24 hr xoo. 48 hr. 0 hr. 24 hr ddH2O ddH2O. 48 hr. normal. Fig. 3 Soluble sugars and starch content in TNG67 and transgenic plants after Xoo inoculation (109 cfu ml-1). Detection of a Glucose; b fructose and c sucrose contents in leaf tissues of TNG67 and transgenic plants which grown for 1 month after inoculation with Xoo (109 cfu ml-1) 0 hr, 24 hr and 48 hr. d starch content were determined tissue from one-month-old TNG67 and pflp transgenic plants which inoculation with Xoo (109 cfu ml-1) or treated with ddH 2 O or grown in normal condition. The solid bar values of two pflp transgenic plants differ significantly from the TNG67 according to one-way ANOVA LSD Means Test (P < 0.05). The results are given as means ± SD (n=3 individual plants).. 25.

(30) (A) 0 hr. 8 hr. 24 hr. 48 hr. AMY4A AMY5A CT-BMY AGPS2α AGPL3 actin. a a. 2. a a. a. c 1. 0.5. b. 0. a. 6. a. 5 4 3 2. a. b 1. ab. 0 0 hr. 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0. 8 hr. b. ab. 4. a. 3. a. 8 hr. 2 b. 0 hr. AGPL3 a ab a. b. ab b. 0 hr. 8 hr. 26. TNG67 pflp-1 pflp-2. b. 0 0 hr. AGPS2α. 5. 1. 8 hr. 7. a. 6 a. b. CT-BMY. 7 a. a. 1.5. 0 hr. relative expression. a. AMY5A. 2.5. relative expression. AMY4A. relative expression. 10 9 8 7 6 5 4 3 2 1 0. relative expression. relative expression. (B). 8 hr.

(31) Fig. 4 Starch metabolism and synthesis relative gene expressions in TNG67 and transgenic plants after inoculation with Xoo (109 cfu ml-1). RNA was isolated from 1 month old seedings of TNG67 and two transgenic plants under 0 hr, 8 hr, 24 hr and 48 hr Xoo treatment for a RT-PCR and b qRT-PCR analysis to detect expression patterns of AMY4A (α-amylase isozyme 4A), AMY5A (α-amylase isozyme 5A), CT-BMY (β-amylase), AGPS2α (ADP-glucose pyrophosphorylase small subunit), AGPL3 (ADP-glucose pyrophosphorylse large subunit) and expression of actin as internal control.. 27.

(32) (A) 0 hr. 8 hr. 24 hr. 48 hr. cwIN1 NIN1 cyt-INV actin. (B). a a. 2 1.5 b 1. b b. 0.5. NIN1. 6. a. a. a. 10. 4 a. 3. a. 2 1. a a a. b b. 0 hr. 8 hr. 24 hr. a 8. a a. TNG67 pflp-1. a. pflp-2. 6 4 2. a. b. ab b. 0. 0. 0. cyt-INV. 12. a. 5 relative expression. a a. 2.5 relative expression. a. relative expression. cwIN1. 3. 0 hr. 8 hr. 24 hr. 0 hr. 8 hr. 24 hr. Fig. 5 The expression pattern of invertase related genes in TNG67 and transgenic plants after inoculation with Xoo (109 cfu ml-1). RNA was isolated from one month old seeding of TNG67 and two transgenic plants at 0 hr, 8 hr, 24 hr and 48 hr Xoo treatment for a RT-PCR and b qRT- PCR analysis to detect gene expression patterns of cwINV1 (cell-wall invertase), NIN1 (alkaline/neutral invertase), cyt-INV (cytosol invertase) and actin as internal control respectively.. 28.

(33) (A) 0 hr. 8 hr. 24 hr. 48 hr. HXK1 HXK2 HXK5 HXK6 actin. (B) HXK1. 3.5. relative expression. 2.5. relative expression. a. 3. ab. 2 1.5. b. 1. a. a a. 0.5 0 0 hr. 8 hr. 2. a a. a a TNG67 pflp-1. a a. a. pflp-2. b. 0 hr. a a a. 8 hr. 24 hr. HXK6. 6. a. a. 1.5 a. b. ab. 1. a. 5. a. b 0.5. relative expression. relative expression. a. HXK2. 24 hr. HXK5. 2.5. 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0. 4 a a. 3. a a. 2. b. b. b 1. 0. 0 0 hr. 8 hr. 24 hr. 0 hr. 29. 8 hr. 24 hr.

(34) Fig. 6 The expression patterns of hexokinase related genes in TNG67 and transgenic plants after inoculation with Xoo (109 cfu ml-1). RNA was isolated from 1 month old seeding of TNG67 and two transgenic plants under 0 hr, 8 hr, 24 hr and 48 hr Xoo treatment for a RT-PCR and b qRT- PCR analysis to detect HXK1 (hexokinase 1), HXK2 (hexokinase 2), HXK5 (hexokinase 5), HXK6 (hexokinase 6) gene expression patterns and the expression of actin as internal control respectively.. 30.

(35) (A) ddH2O. Xoo TNG67. pflp-1. pflp-2. TNG67. pflp-1. pflp-2. 0 hr 8 hr 24 hr 48 hr phenotype 5 day. (B). H2O2 content 80 a. 70. nmol/gFw. 60 50 40. b. a. 30. pflp-1 b b. a a. 20 10. TNG67. b. pflp-2. b a a a. a a a. a a a. a a a. a a a. 0 hr. 8 hr. 24 hr. 48 hr. 0 0 hr. 8 hr. 24 hr. 48 hr. ddH2O ddH2O. xoo. 31.

(36) Fig. 7 Comparisons of DAB stain and H 2 O 2 content between TNG67 and transgenic plants after inoculation with Xoo (109 cfu ml-1). a Localization of ROS in leaf tissues by using DAB stain assay after treatment for 0 hr, 8 hr, 24 hr and 48 hr. DAB (1mg/ml) polymer by reddish-brown coloration indicates the presence of ROS. Scale bars are 1 cm. b Detection of H 2 O 2 content in leaf tissues of TNG67 and transgenic plants that grown for 1 month after inoculation Xoo for 0 hr, 8 hr, 24 hr and 48 hr. The values of solid bar for two pflp transgenic plants were significantly different from TNG67 according to one-way ANOVA LSD Means Test (P < 0.05). The results are given as means ± SD (n=3 individual plants).. 32.

(37) (A) 0 hr. 8 hr. 24 hr. 48 hr. PR1a PR1b TLP Sci1 PBZ1 OsPAL actin. (B). a. 10 8 6. b. 4 2. a. a. b. 0 0 hr. relative expression. relative expression. 10 8. b. 4 2. a. a a. 0 0 hr. 24 hr. 10 9 8 7 6 5 4 3 2 1 0. a. a. b b. a. 7. a TNG67. 6. b. 5. pflp-1 pflp-2. 4 3 2 1. b. a ab. 0 0 hr. a a. 12. 6. a. TLP. 8. a. 8 hr. Sci1. 14. PR1b relative expression. a. relative expression. relative expression. 12. 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0. 8 hr. PBZ1. 0 hr. 24 hr. OsPAL. 8. a. a. 7. a. b. a a b. relative expression. PR1a. 14. 6. TNG67. 5. pflp-1 pflp-2. 4. b. 3 2 1. a a. a. 0 0 hr. 24 hr. 33. a. 0 hr. 24 hr.

(38) Fig. 8 The expression patterns of pathogenesis-related genes in TNG67 and transgenic plants after inoculation with Xoo (109 cfu ml-1). RNA was isolated from one month old seeding of TNG67 and two transgenic plants under 0 hr, 8 hr, 24 hr and 48 hr Xoo treatment for a RT-PCR and b qRT- PCR analysis to detect gene expression patterns of PR1a (Pathogenesis-related protein 1a), PR1b (Pathogenesis-related protein 1b), TLP (thaumatin-like protein), Sci1 (subitilisin-chymotrypsin inhibitor), PBZ1 (probenazole-inducible protein), PAL (phenylalanine ammonia lyase) and actin as internal control respectively.. 34.

(39) Fig. 9 Proposed model for the role of PFLP in sugar signal-regulation during defense response in transgenic plants. (1) PFLP increases photosynthesis efficiency and glucose production in normal growth. (2) PFLP enhances ROS generation during pathogen infection. (3) Sugar pools act as a sucrose source during pathogen infection. (4) cwINV1 catalyzes the hydrolysis of sucrose into glucose and fructose in apoplast. (5) CT-BMY catalyzes the breakdown of starch into glucose in chloroplast. (6) HXK2 and HXK6 expressions were enhanced by glucose accumulation in plant cell in the process of pathogen infection. (7) Glucose accumulation leads to increase of pyruvate content, which affects the TCA cycle, one of the ROS generation site in plant. (8) NIN1 catalyzes the hydrolysis of sucrose into glucose and fructose in mitochondria. (9) HXK6 is linked to mitochondrial ROS production. (10) ROS is as a signaling active antioxidant system and PR gene expression. sucrose (S); glucose (G); fructose (F); glucose 6-phosphate (G6p); pyruvate (Pyr); photosynthetic electron transport chain (PETC); tricarboxylic acid cycle (TCA); reactive oxygen species (ROS); invertase (INV); hexokinase (HXK); β-amylase (BMY); hexose transporters (blue circles).. 35.

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(47) Supplemental data Ⅰ: List of abbreviation AGPase: ADP-glucose pyrophosphorylase AGPL3: ADP-glucose pyrophosphorylase large subunit AGPS2α: ADP-glucose pyrophosphorylase small subunit AMY4A: α-amylase isozyme 4A AMY5A: α-amylase isozyme 5A CT-BMY: putative chloroplast-targeted β-amylase cwINV1: cell-wall invertase cyt-INV:. cytosol invertase. DAB: 3, 3’- diaminobenzidine G6p: glucose 6-phosphate Fd-1: ferredoxin 1 H 2 O 2 : hydrogen peroxide HXK: hexokinase INV: invertase NB: nutrient broth NIN1: alkaline/neutral invertase PAL: phenylalanine ammonia-lyase PETC: photosynthetic electron transport chain PFLP : plant ferredoxin - like protein PR protein : pathogenesis-related protein PR1: SCP-like extracellular protein PR5 (TLP): thaumatin like protein PR6 (Sci1): subitilisin-chymotrypsin inhibitor i.

(48) PR10 (PBZ1): probenazole-inducible protein Pyr: pyruvate ROS: Reactive Oxygen Species RT-PCR: reverse-transcription PCR qRT-PCR: quantitative reverse transcription PCR TCA: tricarboxylic acid cycle Xoo: Xanthomonas oryzae pv. oryzae. ii.

(49) Supplemental data Ⅱ: PCR primers Gene. Primer sequences. Tm. Cycles. 55℃. 26. 55℃. 26. 55℃. 28. 55℃. 27. 55℃. 27. 55℃. 28. 55℃. 24. 55℃. 24. 55℃. 28. 55℃. 26. (F) 5’-GGTGGTTGGGCTGTCGTCTA-3’ AMY4A (R) 5’-GGTGAAACCCATCGATGACA-3’ (F) 5’-TCTGGTGGAAAGGGCAACGA-3’ AMY5A (R) 5’-CGCCACAACTCACCTTTGAC-3’ (F) 5’-CCGCCTCCAGATGGTCATGT-3’ CT-BMY (R) 5’-CGACGCCCTCATGTACTTGT-3’ (F) 5’-TGTGCTTGGAATCATTCTTG-3’ AGPS2a (R) 5’-TTCAACGAACCCTTCATTCT-3’ (F) 5’-CTCCCTTATCCAACCAGAAA-3’ AGPL3 (R) 5’-TTTCTTCGCCTGCAGATTAC-3’ (F) 5’-GACCGTTCGGTGGTTGAGA-3’ cwIN1 (R) 5’-GTATATCGCAGATGGCGAGTG-3’ (F) 5’-GTGCTCCATGACTCCAAGA-3’ Cyt-INV (R) 5’-GTACCCATAAACACCCATTCT-3’ (F) 5’-GCACCAGTTGATTCGGGTCT-3’ NIN1 (R) 5’-GACCATTTCACGGGCACAAC-3’ (F) 5’-AGCAGACCTACGAGAAGCTCAT-3’ HXK1 (R) 5’-CCTCCCGATCTTCTTCAGGAT-3’ (F) 5’-TATACTGGGAACAGGTACTAATGC-3’ HXK2 (R) 5’-CCATCTTTAATAGGACTCTACGAA-3’. iii.

(50) (F) 5’-AAAACTGTTGGAGCTAAGCTAAAG-3’ HXK5. 55℃. 25. 55℃. 26. 54℃. 26. 54℃. 25. 54℃. 25. 56℃. 26. 56℃. 24. 55℃. 24. 55℃. 29. 57℃. 22. 55℃. 22. (R) 5’-CAACTGCTGAACTTCTTGTAATGT-3’ (F) 5’-GATACCTCACATGATCTGAAACAC-3’ HXK6 (R) 5’-GTAATGCTCATAGAGACCACCATC-3’ (F) 5’-GGAAGTACGGCGAGAACATC-3’ PR1a (R) 5’-GGCGAGTAGTTGCAGGTG-3’ (F) 5’-CTTGGCGAGAACCTCTTCTG-3’ PR1b (R) 5’-GCCGGCTTATAGTTGCATGT-3’ (F) 5’-CAGTACTGCTGCACCGGCTC-3’ TLP (R) 5’-ACATCGATCAGATGCCAGCTAA-3’ (F) 5’-GGCCAAGAAGGTGATTCTCAAGGAC-3’ Sci1 (R) 5’-ACAGCAGCATCGCTACTAACCA-3’ (F) 5’-CCGGGCACCATCTACACC-3’ PBZ1 (R) 5’-CCTCGATCATCTTGAGCATGC-3’ (F) 5’-TTCTATACAACAACGGGCTTCC-3’ OsPAL (R) 5’-CCTGGAGGAGATGAGACCAA-3’ (F) 5’-CAAAgTCACTTgCATggCTT-3’ pflp (R) 5’-CgAgTTCTgCCTCTTTgTgA-3’ (F) 5’-GCATCTCTCAGCACATTCCA-3’ actin (R) 5’-GCGATAACAGCTCTCTTGG-3’ α-tubulin. (F) 5’-TCAGATGCCCAGTGACAGGA-3’ (R) 5’-TTGGTGATCTCGGCAACAGA-3’. iv.

(51) Supplemental data Ⅲ: The kimura B solution. Compounds (NH 4 ) 2 SO 4 KNO 3 MgSO 4 ．7 H 2 O KH 2 PO 4. Working concentration 172 μM 92 uM 134 μM 91 μM. Ca(NO 3 ) 2．4 H 2 O. 127 μM. H 3 BO 3. 2.5 μM. MnSO 4 ．4 H 2 O. 0.201 μM. ZnSO 4 ．7 H 2 O. 0.201 μM. CuSO 4 ．5 H 2 O. 0.052 μM. H 2 MoO 4. 0.044 μM. Fe-citrate. 31 μM. * pH: 5.5~5.8. v.

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