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(1)國立高雄大學生物科技研究所碩士論文. 轉殖阿拉伯芥中過量表現 AtFd3 可藉由過氧化物生成促進根部發育 Overexpression AtFd3 in transgenic Arabidopsis promotes root development through ROS generation. 研究生：詹喬瑋撰指導教授：葛孟杰博士. 中華民國九十七年七月.

(2) Table of content The list of figure .............................................................................................. II Chinese abstract............................................................................................... III English abstract ...............................................................................................IV Introduction ....................................................................................................... 1 Materials and Methods ...................................................................................... 8 Plant Materials ............................................................................................ 8 Genomic PCR Analysis .............................................................................. 8 RNA Isolation and Reverse Transcription of RNA .................................... 8 Western Blotting Analysis ........................................................................ 10 Measurement of H2O2 .............................................................................. 10 DAB Staining ........................................................................................... 11 Peroxidase Isoelectric Focusing Gel Electrophoresis and Peroxidase Activity Staining....................................................................................... 11 Salt Treatment........................................................................................... 11 Results ............................................................................................................. 13 Overexpression of AtFd3 in Arabidopsis ................................................. 13 Two transgenic Arabidopsis contain more and longer root hair .............. 14 The ROS generation increase in transgenic lines ..................................... 14 PODs isozymes were decreased in two transgenics ................................. 15 AtFd3 induce the root development genes and the ROS related genes expression ................................................................................................. 16 AtFd3 enhance the resistance to salt stress in Arabidopsis ...................... 17 AtFd3 does not induce the salt stress-related genes expression............... 17 Discussions ...................................................................................................... 19 References ....................................................................................................... 37. I.

(3) The list of figure Figure 1. Map of the relevant portions of the transformation plasmid. .......... 25 Figure 2. The expression of AtFd3 in the transgenic plants ............................ 26 Figure 3. The root hairs images of WT and transgenic lines .......................... 27 Figure 4. Quantitative analysis of WT and AtFd3 transgenic root hairs ......... 28 Figure 5. AtFd3 enhance the H2O2 production in the transgenic plant ........... 29 Figure 6. The distribution of H2O2 in WT and the transgenic roots ................ 30 Figure 7. The POD isozymes expression pattern of WT and two transgenic Arabidopsis roots on IEF gel .................................................................... 31 Figure 8. RT-PCR analysis of development genes .......................................... 32 Figure 9. RT-PCR analysis of ROS relative genes .......................................... 33 Figure 10. Photograph of WT and transgenic lines after salt treatment.......... 34 Figure 11. The quantity analysis of the faded leaves stressed by salt ............. 35 Figure 12. RT-PCR analysis of salt stress genes ............................................. 36. II.

(4) 轉殖阿拉伯芥中過量表現 AtFd3 可藉由過氧化物生成促進根部發育指導教授：葛孟杰博士國立高雄大學生命科學系學生：詹喬瑋國立高雄大學生物科技研究所摘要硫鐵蛋白廣泛存於植物體內，並且是參與各不同部位光合作用系統或非光合作用系統的電子攜帶者。阿拉伯芥光合組織相關的硫鐵蛋白可分為 AtFd1 及 AtFd2 兩種(亦稱為葉型態硫鐵蛋白)。葉型態的硫鐵蛋白可以將光合作用中光系統 I 的電子傳遞給線性電子流(linear electron flow, LEF)的 NADPH 或 thioredoxin，亦可以將電子傳遞給循環電子流(cyclic electron flow, CEF)的 Cytochrome b6f。而非光合作用相關硫鐵蛋白則分成 AtFd3 及 AtFd4(亦稱為根型態硫鐵蛋白)，通常存在於儲存組織，如種子、果實及根部的澱粉體(amyloplast)中。在先前的研究中，我們發現轉殖甜椒的 Fd1 可以促進阿拉伯芥根部累積過氧化物並進而促進根毛的發育。因此，為了研究根型態硫鐵蛋白是否也參與根部發育，本論文利用花椰菜鑲嵌病毒的 35S 啟動子持續表現 AtFd3，觀察轉殖阿拉伯芥其根部發育以及相關的基因表現。結果顯示兩種轉植株的根毛平均長度皆比野生型根毛的長度長，數量也比野生型的根毛多。轉殖株根部中的過氧化物含量皆高於野生型將近兩倍的含量。而透過反轉錄聚合酶連鎖反應的結果得知，在兩種轉殖株的根部中的根毛發育相關基因與氧化還原調節基因都因為促使過氧化物的累積而被誘導並進一步的促進了根毛的發育。根據以上的結果，我們認為植物的根在發育時期需要藉由 AtFd3 產生的過氧化物作為訊息傳遞的分子而促使其根毛及根的發育。. 關鍵字：硫鐵蛋白, 過氧化物, 根部發育. III.

(5) Overexpression AtFd3 in transgenic Arabidopsis promotes root development through ROS generation Advisor: Dr. Mang-Jye Ger Department of Life Science National University of Kaohsiung Student: Chiao-Wei Chan Institute of Biotechnology National University of Kaohsiung ABSTRACT Ferredoxin generally exists in whole plant tissue known as an electron carrier in the photosynthetic system and non-photosynthetic system in various organisms. The photosynthesis-associated Fds in Arabidopsis consist of AtFd1 and AtFd2, known as Leaf-type Fd (LT-Fd), that exist in all green tissues. LT-Fd catalyzes electron transfer from photosystem I (PSI) to NADH or thioredoxin for linear electron flow (LEF), and Cytochrome b6f for cyclic electron flow (CEF) in chloroplasts. The non-photosynthesis associated Fds consist AtFd3 and AtFd4 that usually exist in the amyloplast of storage tissue, such as seed, fruit and root, known as Root-type Fd (RT-Fd). In previous studies, the plant ferredoxin like protein (pflp) gene belonged to the LT-Fd of sweet pepper was introduced into the Arabidopsis and then promoted root hair growth through the enhanced production of reactive oxygen species (ROS). In this thesis, CaMV 35S promoter was used to drive the RT-Fd gene of Arabidopsis thaliana, AtFd3, in the Arabidopsis, and studied the effect of AtFd3 in the root development. The root hair length and the content of ROS in the root of two transgenic lines were both apparently increased. In the roots of two transgenic lines, the root hair development-related and redox-regulated gene transcriptional activities were mediated by the higher levels of the ROS to influence root hair growth. In this study, we present a possible role of AtFd3 in the plant root development. Keywords: Ferredoxin, ROS, Root development. IV.

(6) Introduction Ferredoxin (Fd) is an acidic, low-molecular weight, soluble iron-sulfur protein and acts as a multifunctional electron carrier in various organisms. The Fds in Arabidopsis are subdivided into two different groups: photosynthesis-associated. and non-photosynthesis-associated Fds. The. photosynthesis-associated Fds consist of AtFd1 and AtFd2, known as Leaf-type Fd (LT-Fd), that exist in all green tissue (Hanke et al., 2004). LT-Fd contains high negative reductive potential (about −450 mV) and catalyzes electron transfer from photosystem I (PSI) to NADH or thioredoxin for linear electron flow (LEF), and Cytochrome b6f for cyclic electron flow (CEF) in chloroplasts. In plant chloroplast, Fd1 reduces NADP+ to NADPH via ferredoxin-NADP(H) reductase (Hanke et al., 2004). Occasionally, the photosynthetic electron transport chain is over-reduced, Fd transfers elections to O2 but not to NADP+ resulting in the generation of ROS (Krause, 1994). In contrast, the non-photosynthesis associated Fds consist AtFd3 and AtFd4 that usually exist in the amyloplast of storage tissue, such as seed, fruit and root, known as Root-type Fd (RT-Fd). RT-Fd harbors lower negative reductive potential (about −350 to −150 mV) than LT-Fd (Hanke et al., 2004). The function of RT-Fd might regard to receive electrons from NADPH and deliver them to thioredoxin in the amyloplast (Balmer et al., 2006). The expression profile of some RT-Fd could be regulated by ammonia and nitrate treatments (Matsumura et al., 1997). Fd3 in root can serve as an electron 1.

(7) donor for the reduction of nitrite to ammonia and to glutamate synthesis (Mattana et al., 1997). Addition to the carbon and nitrogen assimilation, Fd3 also possesses the ability to regulate the redox state (Ruelland and Miginiac-Maslow, 1999). According to this reason, Fd can be regarded as a regulator of ROS in plants. The ROS, particularly superoxide and H2O2 at the plasma membrane, is one of the major ways in which higher plants transmit information concerning changes in the changing environment (Barnes and Mayfield, 2003; Terman and Brunk, 2006). It also play as secondary messengers in many processes associated with plant growth and development (Delledonne et al., 2003; Link, 2003; Rouhier et al., 2003; Schurmann, 2003; Borland et al., 2006; Crawford, 2006; Grun et al., 2006). A substantial body of research shows that ROS signal are positioned ether downstream or upstream of the higher plant hormones, such as that H2O2 can induce the accumulation of stress hormones, salicylic acid (SA) and ethylene (Foyer and Noctor, 2003; Yabuta et al., 2004; Kiffin et al., 2006; Li and Jin, 2007; Shao et al., 2008b). SA is an important endogenous signal transduction molecule that activates disease resistance mechanisms in plants. Overexpression of nahG gene (salicylate hydroxylase) or mutation of npr1 (non-expresser of PR genes) in Arabidopsis interrupted the SA-mediated signal transduction pathway(Friedrich et al., 1995; Shah, 2003). In tobacco, the reduction of catalase and ascorbate peroxidase activities resulted in plants hyperresponsive to pathogens (Mittler et al., 1999),. 2.

(8) whereas the overexpression of catalase leads to more disease sensitive plants (Polidoros et al., 2001). Collectively, these results suggest that the ROS-scavenging systems can have an important role in managing ROS generated in response to pathogens. Further, compartmentalization of both ROS production and activation of ROS-scavenging systems could contribute to fine-tuning of ROS levels and their signaling properties. Ethylene is a gaseous plant hormone that affects myriad developmental processes and fitness responses, including germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responsiveness to stress and pathogen attack (Johnson and Ecker, 1998; Bleecker and Kende, 2000). Responses associated with ROS may also interact with ethylene signaling. Ethylene can induce programmed cell death and senescence (De Jong et al., 2002). Both ROS and ethylene have been implicated in signaling in response to viral infection (Love et al., 2005). Interestingly, the ethylene receptor ETR1 can function as an ROS sensor, mediating stomatal closure in response to H2O2 (Desikan et al., 2005). In addition to the hormone signal pathway, ROS also participate in the plant abiotic stress responses, such as salt stress response (Banu et al., 2008; Boursiac et al., 2008). The plethora of salt makes the low water potential in soil and ion imbalance, and then destroys the normal plant physiology reactions such as photosynthesis system. (Zhu, 2001). Under salt stress, plants maintain a high concentration of K+ and a low concentration of Na+ in the. 3.

(9) cytosol. High Na+ stress is sensed either externally or internally and leads to an increase of cytosolic free Ca2+ concentration (Tracy et al., 2008). SOS3, the first protein that involved in the salt stress, binds to this Ca2+ and activates the protein kinase SOS2. The protein kinase complex then phosphorylates and activates various ion transporters, such as the plasma membrane Na +/H+ antiporter SOS1 and the vacuole membrane Na+/H+ antiporter AtNHX1 under salt stress. These transporters drive the surfeit of Na+ ions out to the extracellular or drive into the vacuole by the force from their H + pump (Zhu, 2003). These findings determined that the ROS not only act as a participant in the plant hormone signal transduction but also a regulator in the abiotic stress responses (Banu et al., 2008; Boursiac et al., 2008; Shao et al., 2008a). ROS are also involved in the plant pathogen defense system. To date research has suggested that Fd may be activated in response to pathogen invasion, which then modifies HR sensitivity for disease resistance in plants (Huang et al., 2004). HR is one form of programmed cell death (PCD) that is activated upon invasion by non-pathogenic bacteria (Heath, 2000; Greenberg and Yao, 2004). Activation of PCD proximal to the infection area, which is dependent on the biphasic burst of ROS, immediately induces systemic acquired resistance in neighboring tissues (Gechev et al., 1911; Apel and Hirt, 2004). Dayakar et al., (2003) demonstrated that both generation of ROS and activation of HR induced by harpin are intensified in a transgenic plant overexpressing redundant LT-Fd (Huang et al., 2006). Pathogen infection in. 4.

(10) this transgenic plant resulted in typical HR necrosis and enhanced disease resistance (Liau et al., 2003; Huang et al., 2004; Huang et al., 2007; Yip et al., 2007). These investigations indicated that LT-Fd enhances disease resistance in transgenic plants via activation of HR. According to these, the disease resistance in plants is regulated by LT-Fd through ROS to activate HR. Many environmental and genetic factors can influence the root hair development, such as light, nutrients, calcium, pH, free radicals, ethylene, auxin, Transparent Testa Glabra (TTG) and GLABRA2, GTPase and phosphorylation events (Carol and Dolan, 2002; Jones et al., 2002). Recently, it was shown that H2O2 produced by NADPH oxidase (NOX) can regulate Arabidopsis root hair growth (Foreman et al., 2003). The ROS mediate cell-wall loosening that occurs both in vitro and in vivo (Schopfer, 2001), which in turn leads to elongation growth of maize coleoptiles (Schopfer et al., 2002). Cell-wall loosening occurred when the polysaccharides in the cell-wall matrix were cleaved by HO- produced from the oxidase bound to the cell-wall matrix (Chen and Schopfer, 1999; Schweikert et al., 2000; Schopfer, 2001). In Arabidopsis roots, H2O2 induce Ca2+ signals and activation of G-protein, and then the activities of phospholipase C/D are expressed, which results in the accumulation of phosphatidic acid to increase AtPDK1 activities (Coelho et al., 2002; Ohashi et al., 2003; Anthony et al., 2004). Further, Oxidative signal-inducible. 1. (OXI1). is. activated. by. AtPDK1. through. the. phosphatidic-acid pathway and induce Arabidopsis MAPK3 (AtMPK3) and. 5.

(11) AtMPK6 activities, which are all involved in rearrangements of the actin cytoskeleton for vesicular trafficking in root hair (Hirt, 2002). OXI1 is a serine/threonine protein kinase that has been shown to be necessary for normal root hair development (Rentel et al., 2004). Except for OXI1, the proteins thereinafter are also highly related to the root development. Peroxidases (PODs) could mediate H2O2 production in barley roots grown under stress conditions (Šimonovičová et al., 2004). AtCSLD3 is a cellulose synthase-like protein and synthesize polymers for the fast-growing primary cell wall at the root hair tip in Arabidopsis (Wang et al., 2001). AtEXP7 are expansins, which are cell wall-loosening proteins capable of mediating cell wall extension in acidic conditions without hydrolytic breakage of major structural components of the cell wall (Cho and Cosgrove, 2002). GLABRA2 encodes a WRKY transcription factor, which is required for normal trichome development in Arabidopsis roots (Johnson et al., 2002). Therefore, regulating the genes of these proteins proves to be central to root hair development. In previous studies, we introduced a sweet pepper LT-Fd into Arabidopsis and found that the transgenic plant promotes root hair growth through the enhanced production of ROS (unpublished). In addition, a numerous research was demonstrated that the LT-Fd is involved in the plant pathogen defense system and some stress responses (Liau et al., 2003; Huang et al., 2004; Huang et al., 2006; Huang et al., 2007). Although substantial. 6.

(12) studies have been performed on the novel functions of the LT-Fd, those of RT-Fd are still critical lacking. In this study, two AtFd3 transgenic Arabidopsis were used to investigate whether RT-Fds are involved in the root development. In order to determine the transgenic plants we got were usable plant materials, the genomic PCR analysis was used to determine if AtFd3 was inserted into the Arabidopsis genome. The transcription level of AtFd3 was determined by the RT-PCR analysis and the protein level was analyzed by western blotting. After affirming the transgenic plants were usable, the statistic analysis of root hair shows that two transgenic plants contained longer and more root hair than WT did. The content of H2O2 between transgenic and WT plants was indicated by xylenol orange. The DAB staining analysis was used to detect the accumulation area of H2O2. Some root development and ROS related genes were selected to reveal how the transcription level was regulated, and some of the select genes are indeed upregulated. Here we present a possible function of AtFd3 in the plant root growth.. 7.

(13) Materials and Methods Plant Materials Wild type and two AtFd3 transgenic Arabidopsis lines used in this study were derived from the Columbia backgrounds. Plants were grown in a growth chamber. Growth chamber condition was at 22℃with 16hr light /8hr dark regime on 1.8％ agar-MS medium that vertical on growth-chamber grounds with a light intensity of about 100μmol quanta/m2s. Plants used for salt stress studying were grown in a steam-sterilized soil mix of commercial potting soil at 22℃with 16hr light /8hr dark with a light intensity of about 100μmol quanta/m2s.. Genomic PCR Analysis Arabidopsis leaf ground with liquid nitrogen and the FavorPrep Plant Genomic DNA Extraction Kit (Favorgen, USA) was used to extracted the genomic DNA. The extracted genomic DNA was quantified by UV absorption at 260 nm. PCR was performed with Taq polymerase using primers to NPTII (forward, 5’-TGCTCCTGCCGAGAAAGTAT-3’; reverse, 5’-AATATCACGGGTAGCCAACG-3’).. RNA Isolation and Reverse Transcription of RNA Total RNA was prepared from frozen roots (-80℃) by the Plant Total RNA Miniprep System (Viogene, USA) and quantified by UV absorption at 260 nm. First-strand cDNA synthesis was carried out with ImProm-ⅡTM Reverse Transcription System (Promega, USA). Amplification of β-tubulin 8.

(14) was used to equalize levels of cDNA from Wt and transgenic lines roots tissue. PCR was performed with Taq polymerase using primers to OXI1 (forward, 5’-ACGACGCTAAATTGCTTGCT-3’; reverse, 5’-AACTGGTGA AGCGGAAGAGA- 3’), AtEXP7 (forward, 5’-TTTAACAGCGGCTACGGA CT-3’; reverse, 5’-GGAAATTAGCGGTGCTCTTG-3’), AtCSLD3 (forward, 5’-ATGTGGGTTCCTTTCTGTCG-3’; reverse, 5’-ATCGAGTCAGGCAAG CTGTT-3’), GL2 (forward, 5’-TGCTGCATCAAGCTACCATC-3’; reverse, 5’-TGGCTGTTTCTGTCGTCTTG-3’), GST (forward, 5’-CCACAAAATCC AATTCTCCCTC-3’; reverse, 5’-CCTTCTCCAAATTCCTAACCC-3’), NOX (forward, 5’-CACCATCACTCCTCAATCAC-3’; reverse, 5’-GAAGAACAC GAGCAGAGAC-3’), SOD (forward, 5’-GCACATTATCCACAGCACTTAC -3’; reverse, 5’-CTTACAGCTTCCCAAGACAC-3’), β-tubulin (forward, 5’-CAACTCTGACCTCCGAAAGC-3’; reverse, 5’-TGTGAATTCCATCTC GTCCA-3’), POD 7.7 (forward, 5’-CTAATGACATTGGGTTGTCTTCTG-3’; reverse, 5’-CACGAAGTGTCTGGAGGTATGTAG -3’). The PCR for development related genes were at 94℃, 3 minutes for 1 cycle, then 94℃, 1 minute, 60℃, 1 minute, 72℃, 1 minute for 30cycles, and followed by the extension step at 72℃for 10 minutes. The PCR for ROS related genes were at 94℃, 3 minutes for 1 cycle, then 94℃, 1 minute, 50℃, 1 minute, 72℃, 1 minute for 30cycles, and followed by the extension step at 72℃for 10 minutes.. 9.

(15) Western Blotting Analysis Proteins were extracted by homogenizing 100 mg of fresh root tissue in 100 μl phosphate buffer (50 mM Tris, pH 7.5). The protein concentrations of the samples were detected with Coomassie brilliant blue dye using a microassay method as recommended by the manufacturer (BioRad, Hercules, CA, U.S.A.). A samples (5 μg) of each protein was electrophoresed through gel containing 12% polyacrylamide plus sodium dodecyl sulfate. These gels were then either stained with electro-transferred onto PVDF membranes using the BioRad blue tank method. AtFd3 proteins were detected on Western blots using anti-AtFd3 antibodies followed with anti-rabbit IgG-peroxidase conjugate.. Measurement of H2O2 Arabidopsis thaliana wild type and transgenic lines roots (70 mg) were homogenized with liquid nitrogen, and suspended lysate in 0.6 mL of 50 mM phosphate buffer (pH 6.5) contain 10 mM 3-amino-1, 2, 4-triazole. The homogenates were centrifuged at 6,000 g for 25 min. 100 μL of the supernantant was added to 900 μL reaction mixture that contained 100 μM Xylenol orange, 250 μM FeSO4, 100 mM sorbitol and 25 mM H2SO4. The reaction mixture was incubated for 45 min at room temperature, and centrifuged at 12,000 g for 2 min, measured the absorption of supernatant at 560 nm to determine the level of H2O2.. 10.

(16) DAB Staining The root from 7-days old were cut, place in 1mg/ml 3,3’diaminobenzidine (DAB)-HCl, pH 3.8 (Sigma, MO, USA) and incubated in the growth chamber in dim light for 8 hr prior sampling. After 8 hr incubation, the roots were washed twice with ddH2O and then placed in 96％ ethanol for 30min. The latter samples was stored and examined in 96％ ethanol. H2O2 is visualized as reddish-brown colorations.. Peroxidase Isoelectric Focusing Gel Electrophoresis and Peroxidase Activity Staining The extracted PODs were subjected to analytical flat bed isoelectric focusing on polyacrylamide gels containing ampholines in the pH range of 3.5 to 9.5. The samples were subjected to electrophoresis for 1.5 hours with 30W at 12℃. After focusing, the gels were soaked in 500 mL of PBS for 30 minutes with shaking to remove ampholines and equalize the pH. The PODs isoenzymes were detected by soaking the gel for 10 minutes in 200 mL of the phosphate buffer containing 0.6 mg/mL 4-chloro-1-naphthol and 0.16% H2O2.. Salt Treatment Plants were grown for 4 weeks in control condition, and then treated with 200 mM NaCl solution for first, third, and fifth days. The RNA was extracted from the root tissue treated with NaCl solution for 3 days and 5 days. First-strand cDNA synthesis was carried out with ImProm-ⅡTM Reverse Transcription System (promega, USA). Amplification of β-tubulin. 11.

(17) was used to equalize levels of cDNA from WT and transgenic lines roots tissue. PCR was performed with Taq polymerase using primers to AtNHX1 (forward, 5’-AGTGTCGAAACTGCCTTCGT-3’; reverse, 5’-TGCGGAAAA ACTGCTTCTTT-3’), SOS3 (forward, 5’- TTGATGTGAAGCGAAATGGA3’; reverse, 5’-TTGATGAGCGATGGATTCAA-3’), rd29a (forward, 5’-CCG GTGGGCTTTGGTGAC-3’; reverse, 5’-CTCCTCCGATGCTGCCTTCT-3’). The PCR for salt stress related genes were at 94℃, 3 minutes for 1 cycle, then 94℃, 1 minute, 55℃, 1 minute, 72℃, 1 minute for 25cycles, and followed by the extension step at 72℃for 10 minutes.. 12.

(18) Results Overexpression of AtFd3 in Arabidopsis The transgenic Arabidopsis were taken from the Laboratory of Molecular Plant-Pathogen Interactions of Institute of Plant and Microbial Biology of Academia. Sinica. The. AtFd3. constructed. cDNA for. transformation was designated from pBI121 (Figure 1). In order to identify the distribution of the AtFd3 transgene in transgenic Arabidopsis genomes, the genomic DNA was extracted from the transgenic lines (Fd3-7-6 and Fd3-8-14) and subjected to genomic PCR. Given that wild-type Arabidopsis contains the AtFd3 gene, neomycin phosphotransferase II (NPT II) cDNA was used as a marker to identify whether the transgene was well accessed. As shown in figure 2a, the NPT II gene was only detected in the two transgenic lines, this indicated that the transgene was well inserted into the transgenic plants. After confirming the AtFd3 genes were well inserted into the transgenic Arabidopsis genomes, we went on determining the transcription level of AtFd3 in the transgenic plants. The total RNA was extracted from the 7 days old Arabidopsis root and Reverse Transcription PCR performed with the AtFd3 specific primers. Two transgenic lines showed 1.4 and 1.8 fold of AtFd3 mRNA levels comparable to that of WT (Figure 2b). The RT-PCR analysis also indicates that the transcription levels were both increased in the two transgenic lines roots. As AtFd3 was overexpression in both the DNA and RNA levels. The total protein was extracted from the 7 days-old plants to 13.

(19) determine the protein level of AtFd3 in transgenic lines by western blotting analysis. As shown in figure 2c, the AtFd3 proteins of two transgenic lines are 1.05 and 1.21 fold compared to WT. These results showed that the two transgenic lines are suitable to carry the following experiment out.. Two transgenic Arabidopsis contain more and longer root hair To determine whether AtFd3 would affect the root hair growth, we visualized observation the root hair growth of Arabidopsis grown on the MS plates. As shown in figure 3, the root hair length of two transgenic lines was significant longer than that of WT. To quantities the growing status of Arabidopsis, two transgenic lines and WT were inoculated on the 1.8% MS-plates for four days to measure the length and quantities of root hairs. The 70% root hairs of two transgenics were longer than 300 μm, in contrast, the most (over 50%) root hairs of WT were shorter than 300 μm (Figure 4). In addition, the mean amounts of root hairs of Fd3-7-6 and Fd3-8-14 were 79.42 and 97.14 respectively. The amounts of root hair of both transgenic lines were significant more than WT (53.28; P<0.05). According to these results, AtFd3 can increase the length and amount of root hair.. The ROS generation increase in transgenic lines To date research, the LT-Fd can increase the production of ROS. ROS production is the most important factor during root hair emergence and elongation (Foreman et al., 2003). It was reported that LT-Fd would increase the ROS generation in tobacco suspension cells in response to stress. 14.

(20) conditions (Dayakar et al., 2003). In order to identify if RT-Fd promotes root hair development through the enhancement of ROS generation, WT and two transgenic lines roots were sampled and the contents of ROS were measured. Both two transgenic lines roots showed significant increase in ROS generation than in the WT (Figure 5). The ROS content in Fd3-7-6 and Fd3-8-14 roots were 204.72 μM and 210.25 μM rose to 1.87 and 1.92 fold respectively, compared to WT (109.41 μM). This result shows that AtFd3 can significant enhance ROS generation in two transgenic lines roots (P<0.05). In order to trace the ROS accumulation in roots, DAB staining assay was used to detect the H2O2 accumulation area. The reddish-brown polymerizations of DAB were more intense in the two transgenic lines roots than that of WT at the exuberance area of root hair (Figure 6). These results suggest that AtFd3 in the two transgenic lines roots is related to enhance the ROS content. PODs isozymes were decreased in two transgenics In order to demonstrate which POD isozymes activities changed between WT and two transgenic lines, the total proteins extracted from 4 and 7 days old plants were analyzed by peroxidase isoelectric focusing (IEF) gel electrophoresis. The activities of PODs those pI are 8.8, 7.7 and 4.3 can be detected in WT Arabidopsis. However, only pI 8.8 PODs can be detected in two transgenics, the activities of other PODs were barely detected in two transgenic lines (Figure 7). This result showed that AtFd3 decreases the activities of PODs.. 15.

(21) AtFd3 induce the root development genes and the ROS related genes expression Plants maintain the appropriate cellular redox state and translate ROS signals to modulate the cellular response to prevent plethora ROS harming itself. It has shown that AtFd3 enhanced ROS generation (Figure 5) and promotes root hair growth (Figure 3) in two transgenic lines. To investigate which root hair development-related and oxidative signal-regulated genes transcriptional activities would influence the two transgenic lines roots, RT-PCR analysis was used for detecting these gene transcriptional activities at seven days, respectively. The gene expression of OXI1, AtEXP7, AtCSLD3 and GL2 influence root hair growth. As shown in Figure 8, OXI1 transcriptional levels in transgenic lines roots were slightly higher than that of WT. Moreover, there were 1.35 and 1.27 fold of AtCSLD3, as well as 1.44 and 1.18 fold of GL2 in mRNA level of two transgenic lines higher than that of WT at day 7 (Figure 8). Similar individual levels of AtEXP7 were observed among WT and two transgenic lines roots. These results suggest a lighter expression of root hair development-related genes were appeared in two transgenic lines roots. These above different gene expression pattern, involved in AtFd3 enhance two transgenic lines root hair growth, owing to more ROS generation. The expression of SOD, GST, and POD were be detected to check the degree that ROS accumulation influence the redox state in WT and two transgenic lines roots (Figure 9). Extremely high levels of. 16.

(22) NOX expression was also measured in two transgenic lines roots. The expression of SOD was slightly higher than that in WT. The transcriptional levels of gene encoding the pI 7.7 POD were similar to WT in two transgenic lines roots. AtFd3 enhanced root ROS generation and mediated oxidative signal related genes expression. According to these results of the RT-PCR analysis, AtFd3 can promote transcriptional activity of some root hair development-related and redox-regulated genes to promote root hair growth.. AtFd3 enhance the resistance to salt stress in Arabidopsis As ROS are involved in the stress resistant, a salt treatment was applied to the transgenic plants to see if AtFd3 can also enhance the resistance to salt stress. As shown in figure 10, the stressed leaves of two transgenic lines are significant less than the WT. To quantitate the stressed status of Arabidopsis, two transgenics and WT were treated with 200 mM NaCl solution. Only 30% and 21% leaves were faded respectively to Fd3-7-6 and Fd3-8-14. In contrast, up to 50% leaves of WT were faded (figure 11). These results suggested that the AtFd3 transgenic plants could enhance the resistance to salt stress.. AtFd3 does not induce the salt stress-related genes expression To determine how AtFd3 enhance the salt resistance, a RT-PCR analysis was used to detect these genes related to salt stress pathways at 3 days and 5 days after salt treatment. Three salt stress related genes were selected as markers to reveal if AtFd3 would affect these genes to enhance the salt resistance of Arabidopsis. AtNHX1 is the vacuole membrane Na+/H+. 17.

(23) antiporter; and SOS3 is thought to be the first protein that involved in the salt stress through binding to Ca2+ and activates the protein kinase SOS2 to pass down the salt stress signal. Rd29a encodes hydrophilic proteins as an ABA pathway marker. As shown in figure 12, the genes expression of AtNHX1, SOS3, and rd29a displayed no significant difference at either 3 DAT or 5 DAT. According to these results, AtFd3 seems not to affect these selected genes.. 18.

(24) Discussions In previous studies, LT-Fd increases ROS generation to enhance the activity of disease resistance in PFLP transgenic plants was reported (Dayakar et al., 2003). In addition, the ROS generation can promote the root development in the PFLP transgenic plants (Shin, 2006). Based on the above findings, LT-Fd not only enhances disease resistance, but also promotes root hair growth in transgenic plants. Although substantial studies have been performed on the novel functions of the LT-Fd, those of RT-Fd are still lacking. In this study, both the transgenic plants contain longer root hair and higher ROS content than that of WT. Some transcript activities of root hair development-related and redox-regulated genes were modulated in two transgenic lines roots to promote the root hair growth. Thus, we have demonstrated that the AtFd3 may also involve in the root development. At least six different ferredoxin isoforms existing in various photosynthetic and non-photosynthetic tissues have been reported and these are divided based on the characteristics of organ distribution in their gene expression (Onda et al., 2000). Fd1 and Fd3 are the major ferredoxin isoproteins in leaves and in roots, respectively (Kimata and Hase, 1989; Hase et al., 1991; Suzuki et al., 1991). In previous study, the Fd1 was determined that it can promote Arabidopsis root hair growth through ROS generation. Similarly in this study, we demonstrated the AtFd3 could also enhance ROS generation and then promote root hair growth. However, Fd1 mRNA contains 19.

(25) an internal light response element (iLRE) and accumulates obstruction when grown in dark conditions (Hansen et al., 2001). In contrast, Fd3, the non-photosynthetic ferredoxin, exhibited distinct import patterns under light and dark conditions. In the presence of light, the precursor form accumulated in the intermembrane space of plastid envelope membranes, whereas in the dark, the protein was processed correctly (Hirohashi et al., 2001). This may explain why the result of western blotting was no significant differential. Under normal situation, the roots are usually under the dark condition in the soils. Thus, the AtFd3 transgenic plants seem to be more practical than the PFLP transgenic plants. However, the AtFd3 transgenic plants roots were regime under the light condition in this study, it’s may be an interesting issue to study the phenomenon of AtFd3 transgenic plants roots under dark condition. Many environmental and genetic factors affect the root hair development; ROS is a most important factor and essential for root hair development (Foreman et al., 2003). Evidently, two transgenic lines, Fd3-7-6 and Fd3-8-14, showed more ROS generation than that of WT. The distribution of DAB reddish brown polymer along the entire roots of WT and two transgenic lines also supports the view that ROS is required for both root and root hair growth. ROS produced by NOX was necessary during root hair growth (Foreman et al., 2003). The increasing of ROS accumulation in transgenic plants was dependent on the activation of individual enzymes, such. 20.

(26) as plasma membrane–bound NADPH oxidases (PHOX), cell wall–bound peroxidases, and amine oxidases in the apoplast and chloroplast (Grant and Loake, 2000; Mittler, 2002; Apel and Hirt, 2004; Liu et al., 2007). However, in this study, the activities of peroxidases were much decreased in two transgenic lines. The accumulated AtFd3 in roots of two transgenic lines roots influence their ROS local generation and root hair morphogenesis. In Arabidopsis, ten different NOX have been detected and three NOX (AtRBOHD–AtRBOHF) were expressed in the leaf and the root (Keller et al., 1998; Torres et al., 1998; Overmyer et al., 2003), and AtRBOHD, F are involved in pathogen-induced oxidative burst (Torres et al., 2001; Simon-Plas et al., 2002). Another report showed that AtrbohC regulates cell expansion during root hair formation (Foreman et al., 2003). In previous study, we hypothesized that the ectopic expression Fd1 in the root was an electron donor to an NADPH that catalyzes the oxidative reaction via NOX in transgenic plants (Shin, 2006). However, AtFd3 need not transport the electron to NOX by means of NADPH; it was the electron donor to NOX. This may be an explanation to explain why the AtFd3 transgenic plants contain higher ROS content than PFLP transgenic plants. Although, the activities of PODs were inhibited in two transgenics, ROS was still accumulated in the transgenic lines. Such explanations for this may lay in that the NOXs were more influential than PODs in the transgenics ROS accumulation.. 21.

(27) The oxidative burst induces some antioxidant enzymes expression to reduce the levels of ROS for protection plant itself. The expression of SOD, GST and POD are regulated and known for constructing apparent redox state in plants (Asada, 1999; Mittler, 2002). The ROS content in the transgenic were taken up to almost 2 fold than WT. However, the transcriptional levels of these genes in two transgenic lines roots were no significant increasing compared to WT at neither 4 days old nor 7 days old. Such explanations for this may lay in that the timing of detect theses genes is not appropriate. Expecting ROS is necessary for root hair growth, and expressions of OXI1, AtEXP7, AtCSLD3 and GL2 were known to be essential factors during root hair development (Cho and Cosgrove, 2002; Johnson et al., 2002; Rentel et al., 2004). However, RT-PCR analysis showed that these genes expression in two transgenic lines roots was just little higher than that in WT. Rentel et al. (2004) reported that OXI1 expression was regulated more strongly in response to elicitor, such as cellulose rather than to ROS, because ROS treatment does not fully mimic a genuine oxidative burst generated by stresses. It is suggested that the signal of OXI1 induced by ROS has conveyed and then alleviated after few days. The AtCSLD3 and GL2 transcript activities were more intense in two transgenic lines than that of WT. It shows that the expressions of AtCSLD3 maybe acutely activated in two transgenic lines for primary cell wall synthesis in the tip-growing zone of root hair. The expression of GL2 suggests that the root hair differentiation and initiation. 22.

(28) were more inhibited in the Fd3-7-6 compared to the Fd3-8-14; however, the amounts of root hairs of both transgenics were still more than WT. This result may indicate that AtFd3 may affect the differentiation and the initiation of root hair directly, but the effect of ROS to promote root hairs development weas much influential. The expression pattern of AtEXP7 is similar between WT and two transgenic lines roots. The RT-PCR analysis of AtEXP7 corresponding with the amount and length of root hair is conspicuously different between them. As the longer and more root hair, the expansins should be strongly expression in our hypothesis. These results of AtEXP7 expression suggested that AtFd3 can enhance root hair elongation but what gene or protein that activated by AtFd3 to enhance the root hair are still controversial. As the result of AtFd3 transgenic lines applied with 200mM NaCl solution did enhance the resistance to salt stress. However, the RT-PCT analysis did not support the phenotype we observed. The NaCl solution with such high concentration may result in this outcome. When we see the resistant to high salt stress at leaves at 3DAT or 5 DAT, the root underground is becoming to death, and the selected genes in root was already become degradation. That’s why the transcripts of AtNHX1 and SOS3 were no significant difference between transgenic lines and WT. Another possibility is the AtFd3 induced salt stress resistance is enhanced through another pathway that we still unknown. There are still lots of works to work on.. 23.

(29) We demonstrated that AtFd3 enhances ROS generation through NOX to promote elongation of root hairs. AtFd3 is now known to promote root hair growth in transgenic plants even though it seems to be a side effect.. 24.

(30) Figure 1. Map of the relevant portions of the transformation plasmid. NOS-pro = the promoter region of Agrobacterium tumefaciens nopalin synthase gene, NOS-ter = the terminator region of the same gene, NPTII = the coding sequence of the neomycin phosphotransferase II gene, CaMV 35S-pro = the CaMV 35S promoter sequence, and AtFd3 = the coding sequence for Arabidopsis ferredoxin3. 25.

(31) a.. WT. Fd3-7-6. Fd3-8-14. WT 1. Fd3-7-6 1.4. Fd3-8-14 1.8. WT. Fd3-7-6. Fd3-8-14. NPTII. b.. AtFd3 β-tubulin. c. 16.5 kD. Figure 2. The expression of AtFd3 in the transgenic plants a. WT Arabidopsis contains the AtFd3 gene, neomycin phosphotransgerase II (NPT II) cDNA was used as a probe to identify whether the 35S prompter promoted AtFd3 is well accessed. b. The RT-PCR analysis indicated the expression patterns of AtFd3 in two transgenic plants were higher than in the WT. β-tubulin was used as an internal control. c. Western blot analyses was used to detect PFLP translational level in two transgenic lines. The antibodies recognized AtFd3 of 16.5kD on PVDF membrane.. 26.

(32) WT. Fd3-7-6. Fd3-8-14. Figure 3. The root hairs images of WT and transgenic lines AtFd3 influence the root hair growth of transgenic plants. Both the transgenic lines contain significant longer root hair. Scale bar indicates 100 μm.. 27.

(33) a. b. Figure 4. Quantitative analysis of WT and AtFd3 transgenic root hairs The statistics on the root hair length of 4 days WT (white bar, n=7) and two transgenic lines, Fd3-7-6 (grey bar, n=7) and Fd3-8-14 (dark grey bar, n=7), grown on the 1.8% agar MS-medium. The majority root hair length of WT is shorter than 300μm. In the contrast, the majority root hair length of two transgenic lines is longer than 300μm. 28.

(34) Figure 5. AtFd3 enhance the H2O2 production in the transgenic plant The H2O2 overproduction was detected in the AtFd3 transgenic plants grown for 4 days. The ROS content of WT is 109.41 ± 2.78 μM, 204.72 ± 5.54 μM for Fd3-7-6, and 210.25 ± 8.76 μM for Fd3-8-14.. 29.

(35) Fd3-7-6. WT. Fd3-8-14. Figure 6. The distribution of H2O2 in WT and the transgenic roots DAB (1mg ml-1) was used to trace the H2O2 localization in the roots. The DAB polymer was more accumulation in the transgenic roots. The black bar indicated 100 μM.. 30.

(36) 4 days. 7days. pH 8.8. 7.7. 6.4. 4.3. Figure 7. The POD isozymes expression pattern of WT and two transgenic Arabidopsis roots on IEF gel Total protein 7 μg of 4 and 7 days plants were assay by isoelectric focusing gel. Isoelectric points of peroxidases are marked on the right.. 31.

(37) 4 days. 7 days. OXI1 GL2 AtCSLD AtEXP7 β-tubulin. Figure 8. RT-PCR analysis of development genes RNA was extracted from WT and two transgenic lines roots under grown for 4-days and 7-days. The expressional levels were being calculated and the expression of β-tubulin is as the loading control. The numbers over each diagram of RT-PCR analysis of Fd3-7-6 (grey bar, n=3) and Fd3-8-14 (dark grey bar, n=3) are stand for its intrinsic intensifies and the expression of WT (white bar, n=3) was defined as 1.. 32.

(38) 4 days. 7 days. NOX SOD GST POD7.7 β-tubulin. Figure 9. RT-PCR analysis of ROS relative genes RNA was extracted from WT and two transgenic lines roots under grown for 7-days conditions. RT-PCR analysis of selected ROS related genes were performed with their specific primers. The expressional levels were being calculated and the expression of β-tubulin is as the loading control. The numbers over each diagram of RT-PCR analysis of Fd3-7-6 (grey bar, n=3) and Fd3-8-14 (dark grey bar, n=3) are stand for its intrinsic intensifies and the expression of WT (white bar, n=3) was defined as 1.. 33.

(39) WT. Fd3-7-6. Fd3-8-14. Figure 10. Photograph of WT and transgenic lines after salt treatment. AtFd3 enhance the resistance to salt stress in Arabidopsis. The stressed leaves of two transgenic lines are significant less than the WT.. 34.

(40) Figure 11. The quantity analysis of the faded leaves stressed by salt The percentage of faded leaves of WT and two transgenic lines (n=9) with 7 days after treat. The percentage of faded leaves of WT is 50.1 ± 2.6%, 29.9 ± 1.7% for Fd3-7-6, and 21.2 ± 1.4% for Fd3-8-14.. 35.

(41) 3DAT. 5DAT. SOS3. rd29a. AtNHX1. β-tubulin. Figure 12. RT-PCR analysis of salt stress genes RNA was extracted from WT and two transgenic lines roots under 3 and 5 days after salt treatment conditions. RT-PCR analysis of selected salt stress related genes were performed with their specific primers. The expressional levels were being calculated and the expression of β-tubulin is as the loading control. The numbers over each diagram of RT-PCR analysis of Fd3-7-6 (grey bar, n=3) and Fd3-8-14 (dark grey bar, n=3) are stand for its intrinsic intensifies and the expression of WT (white bar, n=3) is defined as 1.. 36.

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