Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants Deoxymugineic acid -LRB- DMA -RRB- is a member of the mugineic acid family phytosiderophores -LRB- MAs -RRB- , which are natural metal chelators produced by graminaceous plants . Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe -LRB- III -RRB- -- MAs complex . In contrast with barley , the roots of which secrete MAs in response to Zn deficiency , the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply . There was a concomitant increase in endogenous DMA in rice shoots , suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants . The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots . The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots ; correspondingly , HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots . The expression of OsDMAS1 was increased in Zn-deficient shoots . Analyses using the positron-emitting tracer imaging system -LRB- PETIS -RRB- showed that Zn-deficient rice roots absorbed less 62Zn-DMA than 62Zn2 + . Importantly , supply of 62Zn-DMA rather than 62Zn2 + increased the translocation of 62Zn into the leaves of Zn-deficient plants . This was especially evident in the discrimination center -LRB- DC -RRB- . These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil . Introduction Zn , an essential element for plant growth , is absorbed from the rhizosphere by higher plants through specific transporters . The Zinc-regulated and Iron-regulated transporter Proteins -LRB- ZIP -RRB- have been isolated from both graminaceous -LRB- Ramesh et al. 2003 ; Ishimaru et al. 2005 -RRB- and non-graminaceous plants -LRB- Grotz et al. 1998 ; Lasat et al. 2000 ; Moreau et al. 2002 -RRB- . Several ZIP transporters have been shown to be transcriptionally upregulated in Zn-deficient roots , but their contribution to the absorption of Zn from the rhizosphere into roots has not yet been demonstrated . Zn is also absorbed from the rhizosphere as complexes of Zn -LRB- II -RRB- -- mugineic acid family phytosiderophores -LRB- MAs -RRB- . The maize protein YS1 transports Fe -LRB- III -RRB- -- MAs complexes -LRB- Curie et al. 2001 -RRB- , as well as other micronutrient-MAs complexes , including Zn -LRB- II -RRB- -- MAs -LRB- Schaaf et al. 2004 -RRB- . von Wirén et al. -LRB- 1996 -RRB- reported that the ys1 mutant absorbed less 65Zn -LRB- II -RRB- - MAs than did the wild type , and Suzuki et al. -LRB- 2006 -RRB- showed that Zn-deficient barley absorbed more 62Zn -LRB- II -RRB- - DMA than 62Zn2 + . These two studies also showed that free Zn2 + ions are absorbed into roots to some extent , suggesting that graminaceous plant roots absorb both Zn-DMA and Zn2 + . Graminaceous plants secrete MAs into the rhizosphere from their roots , although the amount differs among species in the order of : barley > wheat = rye > oat > maize > sorghum > rice -LRB- Takagi 1993 -RRB- . While the amount of MAs secreted is increased dramatically by Fe deficiency , it is not clear if Zn deficiency increases the secretion of MAs . Zn deficiency has been reported to increase -LRB- Cakmak et al. 1994 ; Walter et al. 1994 ; Zhang et al. 1989 -RRB- or have no effect on the secretion of MAs from wheat and barley roots into the rhizosphere -LRB- Gries et al. 1995 ; Pedler et al. 2000 -RRB- . The biosynthesis of MAs and their corresponding genes have been characterized -LRB- Fig. 3b -RRB- as being synthesized from methionine -LRB- Mori and Nishizawa 1987 -RRB- . Three molecules of S-adenosyl-l-methionine -LRB- SAM -RRB- are combined to form one molecule of nicotianamine -LRB- NA -RRB- by nicotianamine synthase -LRB- NAS -RRB- . NA is then converted to the 3 ′ ′ - keto acid by NA aminotransferase -LRB- NAAT -RRB- , and 2 ′ - deoxymugineic acid -LRB- DMA -RRB- is synthesized by DMA synthase -LRB- DMAS -RRB- . In some graminaceous species , including barley , DMA is further hydroxylated by two dioxygenases , IDS2 and IDS3 -LRB- Kobayashi et al. 2001 ; Nakanishi et al. 2000 -RRB- . The genes encoding the enzymes involved in DMA synthesis have been well-characterized in Fe-deficient rice and barley . The expression of HvNAS1 , HvNAAT-A , HvNAAT-B , HvDMAS1 , HvIDS2 , and HvIDS3 is increased in Fe-deficient barley roots -LRB- Nakanishi et al. 1993 ; Okumura et al. 1994 ; Higuchi et al. 1999 ; Takahashi et al. 1999 ; Bashir et al. 2006 -RRB- . In rice , the expression of OsNAS1 , OsNAS2 , OsNAAT1 , and OsDMAS1 is increased in both roots and shoots by Fe deficiency -LRB- Inoue et al. 2003 ; Bashir et al. 2006 -RRB- . The expression of OsNAS3 is increased in Fe-deficient roots , but decreased in Fe-deficient shoots -LRB- Inoue et al. 2003 -RRB- . Promoter-GUS analysis suggests that OsNAS1 and OsNAS2 are involved in DMA secretion , because those genes are expressed in all root cells . On the other hand , OsNAS3 may not be involved in DMA secretion because its expression is restricted to the pericycle and companion cells of the roots -LRB- Inoue et al. 2003 -RRB- . Recently , we showed that the expression of NASHOR2 , a NAS gene in barley -LRB- Herbik et al. 1999 -RRB- , and HvNAAT-B was increased in Zn-deficient barley shoots , but that the expression of IDS2 and IDS3 was not detected in Zn-deficient shoots -LRB- Suzuki et al. 2006 -RRB- . On the other hand , the expression of HvNAS1 , HvNAAT-A , HvNAAT-B , IDS2 , and IDS3 was increased in both Zn - and Fe-deficient barley roots , while the expression of these genes was not evident in Fe-deficient shoots -LRB- Suzuki et al. 2006 -RRB- . This suggests that in barley Zn deficiency induces DMA synthesis in shoots , while both Zn and Fe deficiency induce MAs synthesis and secretion in roots . The aims of the present study were to examine if the stimulation of MAs secretion by Zn deficiency is common in graminaceous plants and roles of MAs in Zn homeostasis of rice plants . In this report , we shows that DMA secretion is decreased in rice roots , while the concentration of endogenous DMA is increased in Zn-deficient rice shoots . Materials and methods Plant material and growth conditions Rice seeds -LRB- Oryza sativa L. cv . Nipponbare -RRB- were germinated for 7 days at room temperature on paper towels soaked with distilled water . After germination , the seedlings were transferred to a Saran net floating on nutrient solution -LRB- Higuchi et al. 2001 -RRB- and grown in a glasshouse . After 4 days , 270 seedlings were transferred to six 20-l plastic containers containing nutrient solution . After a further 5 days , 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Zn followed 7 days later when a further 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Fe . All samples were harvested 5 days later for analysis . Consequently , the Zn-deficient treatment was performed for 12 days , and Fe-deficient treatment for 5 days since Fe deficiency has a much more rapid deleterious effect . The nutrient solutions were renewed weekly with adjustment to pH 5.5 every 2 days with KOH . Measurement of DMA secretion and endogenous levels of DMA and NA DMA secretion was measured as described by Suzuki et al. -LRB- 2006 -RRB- and the concentrations of endogenous DMA and NA were measured as described by Higuchi et al. -LRB- 2001 -RRB- . Briefly , the roots of 12 plants were rinsed with deionized water and then placed in 600 ml of deionized water at sunrise . Root exudates were collected for two periods of 2 and 3 h . After collection , Micropur -LRB- Katadyn , Switzerland -RRB- , an antimicrobial agent , was added to prevent microbial degradation of the MAs Both solutions containing root exudates were combined and filtered -LRB- Advantec 5C , Toyo Roshi Kaisha , Ltd. , Japan -RRB- . The shoots of the plants from which root exudates were collected were ground in liquid nitrogen using a mortar and pestle . Approximately 1 g of material was suspended in 40 ml of distilled water at 80 °C for 30 min and filtered . The cationic fraction of the samples was prepared as a 2-M NH4OH eluate from Amberlite IR -LRB- H + -RRB- 120 -LRB- Rohm and Haas Co. , USA -RRB- . The condensed and microfiltered samples were subjected to HPLC analysis , as described previously -LRB- Mori et al. 1987 ; Shojima et al. 1989 -RRB- . To ensure better separation of DMA , the pH of 0.15-N Li-citrate buffer was decreased to 2.97 and the pH of 0.2-N Li-citrate buffer increased to 3.30 for better separation of NA . Northern blot analysis The samples for Northern blot analysis were harvested 3 h after sunrise . The roots and shoots were separated immediately and stored at − 80 °C . Total RNA was extracted from the roots and shoots using SDS-phenol . Ten micrograms of total RNA were electrophoresed in 1.2 % -LRB- w/v -RRB- agarose gels containing 0.66 M formaldehyde , then transferred to a Hybond-N + membrane -LRB- Amersham , USA -RRB- and hybridized with probes at 65 °C . The blots were analyzed using a Bio-imaging Analyzer System -LRB- BAS -RRB- 3000 -LRB- Fuji Film , Japan -RRB- . The following specific primers were used to prepare specific probes : OsNAS1 , 5 ′ - GTCTAACAGCCGGACGATCGAAAGG-3 ′ and 5 ′ - TTTCTCACTGTCATACACAGATGGC-3 ′ ; OsNAS2 , 5 ′ - TGAGTGCGTGCATAGTAATCCTGGC-3 ′ and 5 ′ - CAGACGGTCACAAACACCTCTTGC-3 ′ ; OsNAS3 , 5 ′ - GACTGCTTCCATCGCTTGCTACCTC-3 ′ and 5 ′ - CGCAACAGAGACAATGGTTGATTGT-3 ′ ; OsNAAT1 , 5 ′ - TAAGAGGATAATTGATTTGCTTAC-3 ′ and 5 ′ - CTGATCATTCCAATCCTAGTACAAT-3 ′ ; and OsDMAS1 , 5 ′ - GCCGGCATCCCGCAGCGGAAGATCA-3 ′ and 5 ′ - CTCTCTCTCTCGCACCTGCTAGCGT-3 ′ . The accession numbers of the genes are AB021746 -LRB- OsNAS1 -RRB- , AB023818 -LRB- OsNAS2 -RRB- , AB023819 -LRB- OsNAS3 -RRB- , AB206814 -LRB- OsNAAT1 -RRB- , and AB269906 -LRB- OsDMAS1 -RRB- . PETIS analysis of 62Zn translocation PETIS experiment was established by Kume et al. -LRB- 1997 -RRB- . 62Zn -LRB- half life : 9.13 h -RRB- was produced by the method of Watanabe et al. -LRB- 2001 -RRB- . After adjustment of the 62Zn2 + solution to about pH 4 with 1 M KOH , 62Zn2 + was chelated with DMA in darkness for more than 3 h. Prior to the analysis , Zn-deficient rice plants were grown as described above but in a growth chamber -LRB- day : 30 °C , 14 h of light at 320 μmol photons m − 2 sec − 1 ; night : 10 h at 25 °C -RRB- . The Zn-deficient rice plants were each supplied with 15 ml of culture solution lacking Zn in a polyethylene bag . The plants and bags were fixed between two acrylic boards and placed between a pair of PETIS detectors in a chamber set at 30 °C , 65 % humidity , and a light density of 320 μmol m − 2 s − 1 . In the root absorption experiment , 62Zn -LRB- 0.6 nmol -RRB- with DMA -LRB- 79 nmol -RRB- was dissolved in 1 ml of distilled water and added to the culture solution . In the leaf absorption experiment , 62Zn -LRB- 0.5 nmol -RRB- with DMA -LRB- 33 nmol -RRB- was dissolved in 4.5 ml of nutrient solution containing cold Zn -LRB- 2.5 μmol -RRB- and additional DMA -LRB- 308 μmol -RRB- , and supplied at the cut end of the second newest leaf . The individual leaves were cut to ensure a constant distance from discrimination center -LRB- DC -RRB- which is the junction of shoot and root -LRB- Kiyomiya et al. 2001 -RRB- . The γ-rays emitted from decaying positrons from 62Zn were counted over time using the coincident method with the paired detectors . The data were automatically corrected using 9.13 h as the half-life of 62Zn . The radioactivity of selected region was measured as region of interest -LRB- ROI -RRB- analysis . After PETIS analysis for 8 h , the plants were removed from the polyethylene bags , and the roots were gently washed for 1 min in 50 ml of 0.01 mM EDTA . Next , each plant was placed under a Bio-Imaging plate inside a cassette . The plate was scanned using an image analysis system -LRB- BAS-1500 , Fuji Film , Japan -RRB- . The plants were then cut into pieces , and the absolute amount of radioactivity was analyzed using γ-ray spectrometry with an ORTEC HPGe detector -LRB- Seiko EG & G Co. , Ltd. , Japan -RRB- . Each experiment was conducted in triplicate . Due to the very short half-life of 62Zn , the radioactivity was different in each experiment . Therefore , images of individual plants and PETIS results are representative of those obtained . Results Plant growth , DMA synthesis and gene expression In contrast with the good growth of the control rice plants , Zn or Fe deficiency had a markedly detrimental effect when plants were grown for 12 d or 5 d in solutions containing no Zn or Fe , respectively . Shoot growth was reduced by 50 % in the absence of Zn and by 10 % without Fe -LRB- Fig. 1 -RRB- . The corresponding figure for the Zn-deficient roots was a reduction of 20 % , while the absence of Fe supply for 5 d had no significant effect on root growth . As previously established in our studies -LRB- Suzuki et al. 2006 -RRB- , there was no measurable effect of no Zn supply during the first week of growth -LRB- data not shown -RRB- , but thereafter plants developed symptoms of Zn deficiency and growth declined . Chlorotic symptoms of Fe deficiency developed after 5 days of treatment while the fresh weights of the shoots and roots were almost the same as in the control plants . Fig. 1Fresh weights of rice shoots and roots . 31-days old rice seedlings grown in hydroponically were harvested . Zn-deficient treatment -LRB- - Zn -RRB- was performed for 12 days , and Fe-deficient treatment -LRB- - Fe -RRB- was performed for 5 days . Values are mean ± SD -LRB- n = 4 -RRB- The amount of DMA secreted from the roots of Fe-deficient rice was much higher than that of the control plants -LRB- Fig. 2 -RRB- . On the other hand , Zn deficiency slightly decreased the level of DMA secretion . In fact , the amount of DMA secreted per plant in Zn-deficient rice was significantly decreased compared to that in the control plants -LRB- P < 0.05 -RRB- . Fig. 2Amount of DMA secreted from rice roots . The growth condition and harvest time are the same in Fig. 1 . Root exudates were collected for 5 h after sunrise . - Zn , zinc deficiency ; - Fe , iron deficiency . Values are mean ± SD -LRB- n = 4 -RRB- We next analyzed the expression pattern of the genes participated in DMA synthesis in roots and shoots under Zn or Fe deficiency -LRB- Fig. 3a , b -RRB- . As reported previously , the transcription of OsNAS1 , OsNAS2 , OsNAS3 , OsNAAT1 , and OsDMAS1 in roots was increased by Fe deficiency -LRB- Inoue et al. 2003 , 2008 ; Bashir et al. 2006 -RRB- . On the other hand , Zn deficiency enhanced the transcription of OsNAS3 and OsNAAT1 , but not that of OsNAS1 , OsNAS2 , and OsDMAS1 . The expression of OsNAS2 in roots was slightly decreased by Zn deficiency . The expression patterns of OsNAS1 and OsNAS2 were well correlated with the amount of DMA secreted from the roots of Fe-deficient or Zn-deficient rice plants -LRB- Figs. 2 , 3a -RRB- . Fig. 3Expression patterns of genes involved in DMA synthesis -LRB- a -RRB- Northern blot analysis of each gene involved in DMA synthesis . Each lane contained 10 μg of total RNA . C , control ; - Zn , zinc deficiency ; - Fe , iron deficiency . -LRB- b -RRB- DMA synthesis from S-adenosyl-l-methionine -LRB- SAM -RRB- In the shoots , Fe deficiency increased the expression of OsNAS1 , OsNAS2 , OsNAAT1 , and OsDMAS1 , but decreased that of OsNAS3 . In comparison , Zn deficiency increased the expression of OsNAS3 and especially that of OsNAAT1 . The expression of OsDMAS1 was slightly increased in Zn-deficient shoots . Transcripts of OsNAS1 and OsNAS2 were not detected in control or Zn-deficient shoots -LRB- Fig. 3a -RRB- . Higuchi et al. -LRB- 2001 -RRB- reported that the concentration of endogenous DMA was increased in Fe-deficient rice shoots . We confirmed that the concentration of endogenous DMA in Fe-deficient shoots was about three times higher than that in control shoots -LRB- Fig. 4a -RRB- . The concentration of endogenous DMA in Zn-deficient shoots was about two times higher than that in control shoots -LRB- Fig. 4b -RRB- . Although the concentration of endogenous DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots , the expression of OsNAAT1 , which encodes the enzyme that converts nicotianamine to its 3 ′ ′ - keto acid , in Zn-deficient shoots was much higher than that in Fe-deficient shoots -LRB- Fig. 3a -RRB- . Therefore , we measured the endogenous NA , which is an intermediate of DMA . Corresponding to the expression pattern of OsNAAT1 , endogenous NA in Zn-deficient shoots was under the detection limit by HPLC , while in Fe-deficient shoots NA was lower than in the control shoots -LRB- Fig. 4b -RRB- . Fig. 4Concentration of endogenous DMA and NA in rice shoots -LRB- a -RRB- Concentration of endogenous DMA . -LRB- b -RRB- Concentration of endogenous NA . The plants were harvested after collection of root exudates -LRB- Fig. 2 -RRB- , then DMA or NA was measured using HPLC . - Zn , zinc deficiency ; - Fe , iron deficiency ; N.D. , not detected . Values are mean ± SD -LRB- n = 4 -RRB- Contribution of DMA to Zn translocation in Zn-deficient rice To investigate whether DMA is involved in Zn absorption and translocation , a PETIS experiment was performed using Zn-deficient rice -LRB- Fig. 5 ; supplemental movie 1 , supplemental Fig. 1 -RRB- . 62Zn chelated with DMA -LRB- 62Zn-DMA -RRB- or 62Zn without DMA -LRB- 62Zn2 + -RRB- was supplied to Zn-deficient rice roots . Images of the plants and the localization of 62Zn after 6 h of absorption are shown in Fig. 5a . In both plants , most of the 62Zn absorbed into the plant was observed at the DC and in the bottom of the leaf sheath -LRB- Fig. 5a -RRB- . The radioactivity of the plant supplied with 62Zn2 + was higher than that of the plant supplied with 62Zn-DMA both in the roots and shoots -LRB- Fig. 5b , c -RRB- . This indicates that compared to Zn-DMA , Zn2 + is the preferred form for root absorption in Zn-deficient rice . Real-time imaging of the 62Zn absorption was observed by PETIS analysis -LRB- Fig. 5d -RRB- . More 62Zn was observed in the bottom of the leaf sheath supplied with 62Zn2 + than in that supplied with 62Zn-DMA . This observation was quantified by ROI analysis -LRB- Fig. 5e -RRB- On the other hand , beginning at 90 min after the start of the experiment , more 62Zn was observed in the newest leaf of the plant supplied with 62Zn-DMA than in that supplied with 62Zn2 + -LRB- Fig. 5d , f -RRB- . This indicates that Zn-DMA is preferred over Zn2 + for translocation within Zn-deficient rice plants . Continuous PETIS imaging is shown in supplemental movie 1 . We performed three experiments of 62Zn absorption . Due to the very short half-life of 62Zn , however , the radioactivity was different in each experiment . Therefore , we showed the relative radioactivity of each experiment in supplemental Fig. 1 . In each experiment , 62Zn2 + was more absorbed than 62Zn-DMA . Fig. 562Zn movement from roots in Zn-deficient rice using PETIS -LRB- a -RRB- Plant image -LRB- left -RRB- and BAS image -LRB- right -RRB- . -LRB- b -RRB- Radioactivity in roots after 6 h of absorption . -LRB- c -RRB- Radioactivity in shoots after 6 h of absorption . -LRB- d -RRB- Time course of the accumulation of radioactivity as determined by PETIS analysis . The data were scored every 3 min , and the images shown were taken at 90-min intervals for 6 h after supplying 62Zn through the roots . -LRB- e -RRB- Region of interest -LRB- ROI -RRB- analysis of 62Zn movement in the bottom of the sheath white boxed in -LRB- d -RRB- . -LRB- f -RRB- ROI analysis in the newest leaf yellow boxed in -LRB- d -RRB- We conducted an additional PETIS experiment in which Zn-DMA or Zn2 + was supplied to Zn-deficient rice at the cut second newest leaf -LRB- Fig. 6 ; supplemental movie 2 , supplemental Fig. 2 -RRB- . Plant images and the localization of 62Zn after 6 h of absorption are shown in Fig. 6a . Most of the 62Zn absorbed from the second newest leaf remained in that leaf , but some 62Zn was translocated to the other leaves and roots as well as to the DC and leaf sheath . The level of radioactivity in the entire plant , except for the second newest leaf , was higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2 + -LRB- Fig. 6b -RRB- , while that of the second newest leaf was not higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2 + -LRB- supplemental Fig. 3 -RRB- . PETIS analysis showed that more 62Zn appeared continuously in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2 + . This suggests that 62Zn-DMA is translocated more rapidly than 62Zn2 + within the plant . Fig. 662Zn movement from cut leaf in Zn-deficient rice using PETIS -LRB- a -RRB- Plant image -LRB- left -RRB- and BAS image -LRB- right -RRB- . Arrows indicate the point where 62Zn was absorbed . -LRB- b -RRB- Radioactivity in the entire plant except for the second newest leaf after 8 h of absorption . -LRB- c -RRB- Time course of the accumulation of radioactivity as determined by PETIS analysis . The data were scored every 3 min , and the images shown were taken at 120-min intervals for 8 h after supplying 62Zn through the shoots Discussion The concentration of endogenous DMA is increased in Zn-deficient shoots , but DMA secretion from the roots is not increased Previously we reported increased MAs secretion in Zn-deficient barley -LRB- Suzuki et al. 2006 -RRB- ; however , in rice , Zn deficiency did not increase the secretion of DMA , but rather slightly decreased it -LRB- Fig. 2 -RRB- . Fe deficiency increased MAs secretion in both rice and barley -LRB- Suzuki et al. 2006 ; Fig. 2 -RRB- . In barley , expression of the genes encoding the MAs biosynthetic enzymes was increased in the roots of both Zn - and Fe-deficient plants -LRB- Suzuki et al. 2006 -RRB- . On the other hand , in rice , the expression pattern of the genes involved in DMA synthesis in roots was increased in Fe-deficient roots , while the expression of OsNAS1 , OsNAS2 , and OsDMAS1 was not increased in Zn-deficient roots -LRB- Fig. 3a -RRB- . Inoue et al. -LRB- 2003 -RRB- suggested that OsNAS1 and OsNAS2 are involved in DMA secretion . Supporting this idea , the expression pattern of OsNAS1 and OsNAS2 in roots was in good correlation with the amount of DMA secreted -LRB- Figs. 2 , 3a -RRB- . On the other hand , OsNAS3 is thought not to be involved in DMA secretion , because the expression of OsNAS3 is restricted to the pericycle cells adjacent to the protoxylem and companion cells but not in other cortical cells -LRB- Inoue et al. 2003 -RRB- . The expression of OsNAS3 is observed in whole leaf tissues in the control plants -LRB- Inoue et al. 2003 -RRB- . The expression of OsNAS3 was increased in Zn-deficient roots and shoots -LRB- Fig. 3a -RRB- ; therefore , OsNAS3 may function to synthesize NA or subsequent components , such as DMA , for the distribution of Zn within Zn-deficient plants . It is noteworthy that Zn deficiency enhanced the expression of OsNAS3 in shoots , while Fe deficiency enhanced the expression of OsNAS1 and OsNAS2 . This suggests that NA and DMA are involved both in Fe and Zn homeostasis , but that their synthesis under conditions of Fe and Zn deficiency is catalyzed by different isozymes . The concentration of endogenous DMA in the shoots was increased by Zn and Fe deficiencies -LRB- Fig. 4a -RRB- . Although the induction of OsNAAT1 in Zn-deficient shoots was much higher than that in Fe-deficient shoots , the concentration of DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots . In accordance with the high level of OsNAAT1 expression in Zn-deficient shoots , the concentration of NA in Zn-deficient shoots was low -LRB- Fig. 4b -RRB- . This result led to us hypothesize that OsDMAS1 is a rate-limiting enzyme for DMA synthesis in Zn-deficient rice . Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice Recently , we showed that Zn-deficient barley absorbs more Zn-DMA than Zn2 + from its roots -LRB- Suzuki et al. 2006 -RRB- . In the present paper , however , less Zn-DMA than Zn2 + was absorbed from the roots of Zn-deficient rice -LRB- Fig. 5 -RRB- . These findings are correlated with the change in the amount of MAs secreted under Zn deficiency . In barley , MAs secretion is increased by Zn-deficiency , and this contributes to the absorption of Zn from the soil . On the other hand , in rice , the level of DMA secretion was slightly decreased -LRB- Fig. 2 -RRB- . This suggested that rice may prefer to absorb Zn2 + rather than Zn-DMA . Our PETIS experiment clearly showed that more 62Zn was translocated to the newest leaf when 62Zn-DMA was supplied than when 62Zn2 + was supplied , while the opposite tendency was observed at the bottom of the leaf sheath -LRB- Fig. 5 , supplemental movie 1 -RRB- . This finding suggests that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice . The preferential translocation of 62Zn-DMA was also observed in the PETIS experiment in which 62Zn-DMA and 62Zn2 + were supplied from the Zn-deficient leaf -LRB- Fig. 6 , supplemental movie 2 -RRB- . These data also support the idea that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice . Since DMA synthesis might be promoted by Zn deficiency in barley shoots -LRB- Suzuki et al. 2006 -RRB- , graminaceous plants have a common system for Zn translocation within the plant , while the contribution of MAs to Zn uptake from the soil differs among species . Further studies will enable us to understand the mechanism of Zn transport , to manipulate the distribution of Zn in order to produce crops that tolerate Zn-deficient stress , and to enhance the Zn content of crop seeds . Electronic supplementary material -LRB- EPS 348 kb -RRB- -LRB- EPS 362 kb -RRB- -LRB- EPS 353 kb -RRB- -LRB- MOV 2400 kb -RRB- -LRB- MOV 2257 kb -RRB-

B I O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

deoxymugineic acid
iron
nicotianamine
rice
transport
zinc


Tomato

linalool

synthase

is

induced

in

trichomes

by

jasmonic

acid

Tomato

-LRB-

Lycopersicon

esculentum

-RRB-

plants

emit

a

blend

of

volatile

organic

compounds

which

mainly

consists

of

terpenes

Upon

herbivory

or

wounding

the

emission

of

several

terpenes

increases

We

have

identified

and

characterized

the

first

two

tomato

monoterpene

synthases

and

Although

these

proteins

were

highly

homologous

recombinant

protein

produced

-LRB-

R

-RRB-

linalool

from

geranyl

diphosphate

-LRB-

GPP

-RRB-

and

-LRB-

E

-RRB-

nerolidol

from

farnesyl

diphosphate

-LRB-

FPP

-RRB-

while

recombinant

produced

β-phellandrene

β-myrcene

and

sabinene

from

GPP

In

addition

these

genes

were

expressed

in

different

tissues

was

expressed

in

flowers

young

leaves

stems

and

petioles

while

was

strongest

expressed

in

stems

and

roots

expression

in

leaves

was

induced

by

spider

mite-infestation

wounding

and

jasmonic

acid

-LRB-

JA

-RRB-

treatment

while

did

not

respond

to

these

stimuli

The

expression

of

in

stems

and

petioles

was

predominantly

detected

in

trichomes

and

could

be

induced

by

JA

Because

JA

treatment

strongly

induced

emission

of

linalool

and

overexpression

of

in

tomato

resulted

in

increased

production

of

linalool

we

propose

that

is

a

genuine

linalool

synthase

Our

results

underline

the

importance

of

trichomes

in

JA-induced

terpene

emission

in

tomato

Introduction

Plants

produce

a

wide

variety

of

terpenoids

that

have

primary

functions

as

hormones

-LRB-

gibberellins

abscisic

acid

brassinolide

-RRB-

sterols

pigments

-LRB-

carotenoids

phytol

-RRB-

and

as

parts

of

electron

carrier

moieties

-LRB-

ubiquinone

plastoquinone

-RRB-

-LRB-

McGarvey

and

Croteau

-RRB-

Most

terpenoids

however

are

secondary

metabolites

over

different

terpenoid

structures

from

plants

have

been

described

-LRB-

Sacchettini

and

Poulter

-RRB-

Some

terpenoids

function

in

the

direct

defence

strategy

of

plants

as

phytoalexins

which

accumulate

upon

pathogen

infection

Sesquiterpene

phytoalexins

-LRB-

e.g.

capsidiol

-RRB-

are

characteristic

for

the

Solanaceae

-LRB-

Chappell

and

Nable

Egea

et

al.

-RRB-

Volatile

monoterpenes

and

sesquiterpenes

can

function

in

the

interaction

of

plants

with

other

organisms

They

are

present

in

floral

scents

that

attract

pollinators

-LRB-

Knudsen

et

al.

Langenheim

-RRB-

and

are

emitted

by

many

plant

species

in

response

to

herbivory

by

insects

or

spider

mites

Induced

terpene

emission

can

attract

predators

or

parasitoids

of

the

herbivores

a

mechanism

commonly

referred

to

as

the

indirect

defense

strategy

of

plants

This

has

been

studied

in

for

instance

Arabidopsis

-LRB-

Arabidopsis

thaliana

-RRB-

-LRB-

Van

Poecke

et

al.

-RRB-

lima

bean

-LRB-

Phaseolus

lunatus

-RRB-

-LRB-

Dicke

Takabayashi

and

Dicke

-RRB-

tobacco

-LRB-

Nicotiana

tabacum

N.

attenuata

-RRB-

maize

-LRB-

Zea

mays

-RRB-

and

cotton

-LRB-

Gossypium

hirsutum

-RRB-

-LRB-

De

Moraes

et

al.

Pare

and

Tumlinson

-RRB-

and

tomato

-LRB-

Kant

et

al.

Takabayashi

et

al.

-RRB-

These

volatile

terpenes

are

produced

by

sesqui

and

monoterpene

synthases

of

which

several

are

induced

by

herbivory

For

instance

nerolidol

synthase

activity

is

induced

in

lima

bean

cucumber

-LRB-

Cucumis

sativus

-RRB-

and

maize

-LRB-

Bouwmeester

et

al.

Degenhardt

and

Gershenzon

-RRB-

whereas

transcript

levels

of

various

other

sesquiterpene

synthases

increase

in

amongst

others

maize

-LRB-

Shen

et

al.

-RRB-

cucumber

-LRB-

Mercke

et

al.

-RRB-

and

wormwood

-LRB-

Artemisia

annua

-RRB-

-LRB-

Cai

et

al.

-RRB-

In

Arabidopsis

caterpillars

-LRB-

Pieris

rapae

-RRB-

induce

the

myrcene/ocimene

synthase

and

the

β-ocimene

synthase

which

coincides

with

increased

myrcene

emission

-LRB-

Van

Poecke

et

al.

-RRB-

Moreover

a

β-ocimene

synthase

-LRB-

LjEβOS

-RRB-

has

been

identified

from

Lotus

-LRB-

Lotus

japonicus

-RRB-

which

is

induced

by

spider

mite-feeding

resulting

in

increased

emission

of

β-ocimene

-LRB-

Arimura

et

al.

-RRB-

However

artificial

wounding

or

jasmonic

acid

-LRB-

JA

-RRB-

treatment

are

often

also

sufficient

to

induce

terpene

synthases

The

wound

and

JA-induced

synthesis

of

terpenes

by

coniferous

plants

is

well

described

-LRB-

Martin

et

al.

Miller

et

al.

Steele

et

al.

-RRB-

and

these

treatments

also

mimic

herbivore-induced

responses

in

several

angiosperms

-LRB-

Arimura

et

al.

Gomez

et

al.

Schnee

et

al.

Shen

et

al.

-RRB-

Here

we

describe

the

identification

and

characterization

of

the

first

two

monoterpene

synthases

from

a

solanaceous

species

-LRB-

tomato

-RRB-

and

We

provide

evidence

that

these

genes

are

differentially

regulated

and

more

importantly

that

expression

of

linalool

synthase

is

restricted

to

trichomes

and

induced

by

JA

Results

Identification

of

tomato

terpene

synthases

and

-LRB-

and

-RRB-

In

order

to

identify

tomato

monoterpene

synthases

that

are

induced

upon

spider

mite

herbivory

we

first

queried

tomato

EST

databases

-LRB-

and

-RRB-

with

known

monoterpene

synthase

sequences

from

other

plant

species

Retrieved

tomato

EST

sequences

were

used

to

design

a

primer

specific

for

monoterpene

synthases

-LRB-

Fig.

-RRB-

We

then

used

cDNA

derived

from

spider

mite-infested

plants

to

amplify

a

single

bp

fragment

with

this

primer

and

an

oligo

dT

-LRB-

-RRB-

primer

This

fragment

was

subsequently

used

as

a

probe

to

screen

available

tomato

cDNA

libraries

Two

different

full-length

cDNAs

were

identified

-LRB-

-RRB-

was

isolated

from

tomato

leaves

-LRB-

sequence

identical

to

the

probe

-RRB-

and

-LRB-

-RRB-

from

tomato

roots

and

stems

The

and

open

reading

frames

encode

proteins

of

and

amino

acids

-LRB-

Fig.

-RRB-

which

have

predicted

masses

of

and

kDa

respectively

and

are

identical

and

contain

amino

acid

motifs

conserved

in

angiosperm

-LRB-

mono

-RRB-

terpene

synthases

These

are

based

on

amino

acid

positions

R

-LRB-

-RRB-

R

-LRB-

-RRB-

W

-LRB-

-RRB-

required

for

the

initial

diphosphate

migration

-LRB-

Williams

et

al.

-RRB-

D

-LRB-

-RRB-

D

-LRB-

-RRB-

D

-LRB-

-RRB-

required

for

binding

of

a

or

ion

which

is

used

as

cofactor

-LRB-

Cane

et

al.

Starks

et

al.

-RRB-

D

-LRB-

-RRB-

T

-LRB-

-RRB-

E

-LRB-

-RRB-

required

for

binding

of

a

second

or

cofactor

and

D

-LRB-

-RRB-

in

the

active

site

cleft

-LRB-

Schwab

et

al.

Whittington

et

al.

-RRB-

and

contain

both

putative

-LRB-

predicted

with

TargetP

Emanuelsson

et

al.

-RRB-

plastid

targeting

signal

peptides

which

should

be

processed

in

the

region

upstream

of

the

conserved

R

-LRB-

-RRB-

R

-LRB-

-RRB-

arginine

pair

Fig.

of

deduced

amino

acid

sequences

of

and

with

three

terpene

synthases

A

ClustalW

alignment

is

shown

of

Mentha

aquatica

linalool

synthase

a

monoterpene

synthase

-LRB-

-RRB-

MaLin

-LRB-

-RRB-

Lycopersicon

esculentum

monoterpene

synthase

-LRB-

-RRB-

L.

esculentum

monoterpene

synthase

-LRB-

-RRB-

Mentha

longifolia

limonene

synthase

MlLim

-LRB-

-RRB-

and

L.

esculentum

germacrene

C

synthase

a

typical

sesquiterpene

synthase

-LRB-

-RRB-

LeGerC

-LRB-

-RRB-

Amino

acids

are

shaded

dark

if

identical

in

at

least

three

of

the

five

sequences

and

light

if

identical

in

and

only

Conserved

amino

acid

motifs

typical

for

terpene

synthases

are

boxed

-LRB-

Williams

et

al.

-RRB-

DDxxD

-LRB-

Cane

et

al.

Starks

et

al.

-RRB-

and

D

-LRB-

Schwab

et

al.

Whittington

et

al.

-RRB-

The

presence

of

putative

N-terminal

plastid

targeting

peptides

in

the

four

monoterpene

synthases

and

the

primer

used

for

amplifying

the

initial

LeMTS

fragment

from

cDNA

of

spider

mite-infested

leaves

are

indicated

Remarkably

contains

an

extra

sequence

stretch

of

amino

acids

starting

at

-LRB-

Fig.

-RRB-

This

sequence

is

not

found

in

any

other

terpene

synthase

nor

does

it

have

homology

to

any

protein

in

the

NCBI

database

-LRB-

-RRB-

Its

presence

can

also

not

be

explained

by

alternative

splicing

since

the

flanks

do

not

contain

canonical

splice

sites

This

extra

internal

element

is

not

an

artefact

of

the

isolated

cDNA

since

it

was

present

in

four

independent

cDNAs

isolated

from

different

organs

-LRB-

data

not

shown

-RRB-

and

belong

to

the

TPSb

subfamily

of

terpene

synthases

and

are

more

similar

to

typical

monoterpene

synthases

from

e.g.

mint

and

citrus

than

to

a

typical

tomato

sesquiterpene

synthase

-LRB-

Fig.

-RRB-

Based

on

protein

sequence

relatedness

and

can

be

classified

in

the

TPSb

subfamily

-LRB-

Fig.

-RRB-

a

distinct

group

of

angiosperm

monoterpene

synthases

according

to

the

original

classification

proposed

by

Bohlmann

et

al.

-LRB-

-RRB-

So

unlike

the

snapdragon

-LRB-

Antirrhinum

majus

-RRB-

monoterpene

synthases

from

subfamily

TPSg

-LRB-

Dudareva

et

al.

-RRB-

including

an

Arabidopsis

and

a

strawberry

terpene

synthase

the

monoterpene

synthases

from

the

solanaceous

family

do

not

seem

to

form

a

new

subfamily

Fig.

and

belong

to

the

TPSb

subfamily

of

terpene

synthases

The

TPS

family

is

divided

into

subfamilies

according

to

the

nomenclature

of

Bohlmann

et

al.

-LRB-

-RRB-

TPSa

angiosperm

sesquiterpene

synthases

TPSb

angiosperm

monoterpene

synthases

TPSc

angiosperm

diterpene

synthases

TPSd

gymnosperm

mono-sesqui

and

diterpene

synthases

TPSe

a

second

group

of

angiosperm

diterpene

synthases

TPSf

a

distinct

group

of

-LRB-

putative

-RRB-

linalool

synthases

mainly

from

Clarkia

-LRB-

Dudareva

et

al.

-RRB-

and

TPSg

a

recently

identified

group

of

monoterpene

synthases

mainly

from

Antirrhinum

-LRB-

Dudareva

et

al.

-RRB-

A

limited

amount

of

representative

terpene

synthases

is

shown

The

bootstrapped

phylogenetic

tree

was

constructed

using

PAUP

and

TreeView

software

A

representative

tree

of

replicates

is

shown

Genbank

protein

accessions

of

terpene

synthases

shown

are

top

to

bottom

TPSf

TPSe

TPSc

TPSd

TPSg

TPSa

TPSb

Terpene

synthase

activity

of

recombinant

and

Recombinant

and

proteins

were

produced

in

Escherichia

coli

in

order

to

characterize

their

enzymatic

activities

The

plastid

targeting

peptide

was

deleted

up

to

one

amino

acid

upstream

of

the

motif

-LRB-

Fig.

-RRB-

to

produce

a

His-tagged

pseudo-mature

form

of

the

proteins

since

it

has

been

reported

that

this

can

improve

protein

expression

without

affecting

activity

and

product

specificity

of

the

enzymes

-LRB-

Bohlmann

et

al.

Williams

et

al.

-RRB-

The

soluble

protein

fractions

were

assayed

for

terpene

synthase

activity

and

products

were

analyzed

on

a

GC

coupled

to

a

Time

Of

Flight-MS

Using

the

monoterpene

precursor

geranyl

diphosphate

-LRB-

GPP

-RRB-

as

substrate

generated

only

linalool

whereas

generated

several

monoterpene

products

β-phellandrene

β-myrcene

and

sabinene

-LRB-

Fig.

-RRB-

Mass

spectra

and

relative

retention

indices

-LRB-

Adams

-RRB-

or

authentic

standards

confirmed

the

identification

of

terpene

reaction

products

Extracts

of

E.

coli

cells

expressing

the

empty

vector

control

generated

minor

amounts

of

linalool

-LRB-

Fig.

-RRB-

and

occasionally

geraniol

Thermally

induced

autoconversion

or

solvolysis

of

GPP

to

linalool

has

been

observed

before

-LRB-

Crowell

et

al.

Jia

et

al.

-RRB-

as

well

as

conversion

to

geraniol

by

aspecific

hydrolase

or

phosphatase

activity

in

protein

extracts

-LRB-

Crowell

et

al.

-RRB-

However

product

levels

are

several

hundred-fold

higher

than

the

background

levels

of

the

control

-LRB-

Fig.

-RRB-

Fig.

activity

of

recombinant

and

proteins

-LRB-

A

-RRB-

GC/MS

chromatograms

of

the

and

monoterpene

products

and

the

empty

vector

control

with

GPP

as

substrate

-LRB-

B

-RRB-

GC/MS

chromatogram

of

the

sesquiterpene

product

and

the

empty

vector

control

with

FPP

as

substrate

The

chromatograms

show

detector

responses

for

the

terpene-specific

ion

mass

Products

were

identified

as

linalool

-LRB-

-RRB-

sabinene

-LRB-

-RRB-

β-myrcene

-LRB-

-RRB-

β-phellandrene

-LRB-

-RRB-

and

-LRB-

E

-RRB-

nerolidol

-LRB-

-RRB-

Representative

chromatograms

are

shown

from

assays

of

at

least

two

independent

experiments

Enantiomer

separation

on

an

enantiomer-selective

column

showed

that

produced

only

-LRB-

R

-RRB-

linalool

-LRB-

data

not

shown

-RRB-

was

also

able

to

utilize

the

sesquiterpene

precursor

farnesyl

diphosphate

-LRB-

FPP

-RRB-

as

substrate

to

produce

-LRB-

E

-RRB-

nerolidol

-LRB-

Fig.

-RRB-

whereas

had

no

detectable

sesquiterpene

synthase

activity

Both

enzymes

were

unable

to

form

terpene

products

from

the

diterpene

precursor

geranylgeranyl

diphosphate

and

are

differentially

expressed

in

various

plant

organs

We

investigated

the

organ

specificity

of

and

expression

in

a

mature

-LRB-

weeks

old

-RRB-

tomato

plant

-LRB-

Fig.

-RRB-

Transcripts

of

were

detected

in

young

fruit

flower

buds

petals

sepals

stems

petioles

and

in

young

leaves

The

expression

pattern

of

was

very

different

no

transcripts

were

detected

in

petals

sepals

and

leaves

However

was

expressed

in

roots

Fig.

expression

patterns

of

and

in

tomato

-LRB-

A

-RRB-

Expression

of

and

in

different

organs

of

an

adult

tomato

plant

-LRB-

B

-RRB-

Expression

in

trichomes

or

complementary

tissue

of

petioles

and

stems

Expression

was

analyzed

by

RT-PCR

The

data

from

one

of

two

independent

tissue

sets

that

gave

similar

results

is

shown

conjugating

enzyme

-LRB-

-RRB-

was

used

as

constitutive

control

Ethidium

bromide-stained

agarose

gels

are

shown

For

plants

that

contain

glandular

trichomes

monoterpene

production

is

considered

to

be

localized

exclusively

in

these

organs

-LRB-

Gershenzon

et

al.

Iijima

et

al.

Kutchan

Turner

et

al.

Turner

and

Croteau

-RRB-

Tomato

plants

possess

several

types

of

trichomes

including

glandular

trichomes

that

are

present

in

high

density

on

leaves

petioles

and

stems

and

that

contain

terpenes

-LRB-

Maluf

et

al.

Snyder

and

Carter

-RRB-

Therefore

we

investigated

the

location

of

and

expression

in

trichomes

of

petioles

and

stems

This

showed

that

is

expressed

in

trichomes

whereas

is

expressed

in

the

complementary

tissue

-LRB-

Fig.

-RRB-

but

not

is

induced

by

spider

mite

feeding

artificial

wounding

and

jasmonic

acid

To

study

whether

and

were

induced

upon

spider

mite

feeding

changes

in

transcript

levels

of

intact

plants

were

analyzed

by

RT-PCR

since

LeMTS

gene-expression

was

too

low

to

determine

by

RNA

gel-blot

analysis

Expression

of

was

indeed

induced

in

leaves

by

spider

mite-feeding

but

which

expression

was

very

low

in

leaves

was

not

-LRB-

Fig.

-RRB-

Spider

mites

damage

plant

tissue

and

induce

JA

and

SA-responsive

genes

such

as

a

wound-induced

proteinase

inhibitor

-LRB-

WIPI-II

-RRB-

and

a

pathogenesis

related

protein

-LRB-

-RRB-

respectively

-LRB-

Kant

et

al.

-RRB-

Therefore

we

investigated

the

effects

of

artificial

wounding

and

exogenous

application

of

JA

and

SA

-LRB-

Fig.

-RRB-

Both

artificial

wounding

and

JA

induced

In

contrast

was

not

induced

by

these

treatments

-LRB-

Fig.

-RRB-

SA-treatment

induced

the

SA-marker

but

neither

nor

was

increased

Induction

of

and

WIPI-II

was

validated

by

real-time

Q-RT-PCR

This

showed

consistent

and

clear

induction

of

both

genes

by

JA

artificial

wounding

and

spider

mites

although

there

was

large

variation

in

the

level

of

induction

between

independent

experiments

-LRB-

Fig.

-RRB-

We

routinely

used

the

tomato

cultivar

Moneymaker

for

our

experiments

However

in

our

earlier

work

-LRB-

Ament

et

al.

Kant

et

al.

-RRB-

the

cultivar

Castlemart

-LRB-

CM

-RRB-

was

used

because

it

has

the

same

genetic

background

as

the

-LRB-

-RRB-

mutant

-LRB-

Howe

et

al.

-RRB-

Here

we

show

that

spider

mite

and

JA-induced

expression

occurred

both

in

CM

and

Moneymaker

-LRB-

Fig.

-RRB-

Fig.

of

in

tomato

leaves

in

response

to

spider

mites

wounding

and

jasmonic

acid

-LRB-

JA

-RRB-

-LRB-

A

-RRB-

Gene

expression

analysis

by

semi-quantitative

RT-PCR

of

and

after

various

treatments

compared

to

the

well

known

JA

and

SA-marker

genes

WIPI-II

-LRB-

Graham

et

al.

-RRB-

and

-LRB-

van

Kan

et

al.

-RRB-

respectively

Treatments

JA

SA

and

artificial

wounding

-LRB-

W

-RRB-

after

h

and

spider

mite-infestation

-LRB-

M

-RRB-

after

h

A

representative

result

from

three

independent

experiments

is

shown

conjugating

enzyme

-LRB-

-RRB-

was

used

as

constitutive

control

Ethidium

bromide-stained

agarose

gels

are

shown

-LRB-

B

-RRB-

Validation

of

and

WIPI-II

induction

by

real-time

Q-RT-PCR

Two

different

tomato

cultivars

were

used

to

verify

induction

by

mites

and

JA

Values

represent

expression

levels

relative

to

controls

calculated

after

correction

for

expression

of

the

control

gene

-LRB-

-RRB-

Average

values

and

standard

errors

are

shown

from

three

to

five

independent

replicates

To

investigate

whether

spider

mite-induced

expression

of

was

dependent

on

JA

or

SA

we

made

use

of

the

mutant

which

is

impaired

in

induced

JA-accumulation

-LRB-

Howe

et

al.

-RRB-

and

the

SA-deficient

transgenic

NahG

tomato

line

-LRB-

Brading

et

al.

-RRB-

In

plants

with

the

mutation

was

not

induced

by

spider

mites

whereas

the

expression

could

be

induced

by

exogenous

JA

-LRB-

Fig.

-RRB-

In

contrast

the

NahG

plants

showed

higher

and

clearly

induced

expression

-LRB-

Fig.

-RRB-

This

shows

that

induction

of

expression

is

dependent

on

JA

but

not

on

SA

Fig.

mite-induced

expression

is

dependent

on

jasmonic

acid

-LRB-

JA

-RRB-

-LRB-

A

-RRB-

Expression

of

in

wild

type

-LRB-

wt

-RRB-

or

JA

deficient

mutant

plants

infested

with

spider

mites

-LRB-

M

-RRB-

or

treated

with

JA

for

days

-LRB-

B

-RRB-

expression

in

wild

type

and

SA

deficient

NahG

plants

in

response

to

days

of

spider

mite-feeding

Gene

expression

was

analyzed

by

RT-PCR

a

representative

result

from

one

out

of

two

independent

experiments

is

shown

conjugating

enzyme

-LRB-

-RRB-

was

used

as

constitutive

control

Ethidium

bromide-stained

agarose

gels

are

shown

JA

induces

expression

of

in

trichomes

Since

basal

expression

in

stems

and

petioles

seemed

to

be

restricted

to

trichomes

-LRB-

Fig.

-RRB-

we

addressed

the

question

whether

JA

would

induce

expression

of

solely

in

trichomes

or

also

in

other

tissues

Indeed

JA-induced

expression

was

strongest

in

trichomes

-LRB-

Fig.

-RRB-

Interestingly

WIPI-II

expression

showed

the

opposite

pattern

WIPI-II

expression

is

very

low

in

trichomes

and

basal

as

well

as

strongly

induced

WIPI-II

expression

occurred

mainly

in

the

complementary

stem

tissues

Fig.

is

induced

in

trichomes

by

JA

Expression

analysis

of

and

WIPI-II

by

real-time

Q-RT-PCR

Intact

plants

were

treated

with

JA

and

subsequently

trichomes

were

separated

from

remaining

tissues

of

stems

The

results

from

four

independent

experiments

are

shown

as

mean

values

and

standard

errors

Expression

levels

are

indicated

as

expression

relative

to

the

control

gene

Linalool

accumulates

in

glandular

trichomes

and

linalool

emission

is

induced

by

wounding

jasmonic

acid

and

by

ectopic

overexpression

Figure

showed

that

in

petioles

and

stems

was

expressed

specifically

in

the

trichomes

Extraction

of

terpenes

from

the

same

tissues

of

that

experiment

demonstrated

that

linalool

was

produced

predominantly

in

trichomes

-LRB-

Fig.

-RRB-

-LRB-

E

-RRB-

nerolidol

and

β-phellandrene

also

accumulated

in

trichomes

Conversely

the

volatile

benzenoid

methyl-salicylate

was

not

produced

in

trichomes

and

was

also

clearly

detected

in

roots

in

contrast

to

most

terpenes

Fig.

accumulation

in

trichomes

Relative

levels

of

linalool

a

selection

of

other

terpenes

and

methyl

salicylate

in

extracts

of

petioles

and

stems

before

and

after

removal

of

trichomes

compared

to

total

leaf

and

root

extracts

Equal

amounts

of

the

different

tissues

were

used

-LRB-

freshweight

g

-RRB-

and

trichome

material

was

corrected

for

freshweight

of

the

original

petiole

or

stem

Averages

of

two

measurements

and

min/max

are

shown

Because

was

induced

by

spider

mite-infestation

wounding

and

JA

we

investigated

whether

the

emission

of

its

in

vitro

products

was

induced

concomitantly

Analysis

of

volatile

production

during

days

of

wounding

or

JA-treatment

conditions

that

induce

expression

-LRB-

Fig.

-RRB-

revealed

that

plants

emitted

more

linalool

after

both

treatments

-LRB-

Fig.

-RRB-

while

nerolidol

emission

was

not

induced

Fig.

linalool

emission

-LRB-

A

-RRB-

Linalool

and

-LRB-

E

-RRB-

nerolidol

emission

by

intact

tomato

plants

that

were

wounded

-LRB-

W

-RRB-

or

treated

with

mM

jasmonic

acid

-LRB-

JA

-RRB-

twice

a

day

for

days

The

bar-graph

shows

the

average

-LRB-

n

-RRB-

emission

over

days

compared

to

the

control

and

standard

errors

Data

-LRB-

corrected

for

unequal

variances

-RRB-

were

analyzed

by

means

of

ANOVA

followed

by

a

Fishers-LSD

post

hoc-test

Linalool

emission

for

both

JA

and

wounding

treatments

was

significantly

increased

compared

to

the

control

-LRB-

P

-RRB-

whereas

-LRB-

E

-RRB-

nerolidol

emission

was

not

-LRB-

B

-RRB-

Linalool

emission

by

a

transgenic

tomato

line

overexpressing

Headspaces

were

sampled

from

desiccators

containing

two

plants

each

and

compared

to

that

of

plants

transformed

with

the

empty

vector

The

graph

expresses

average

values

and

standard

deviations

of

four

measurements

Emission

of

-LRB-

E

-RRB-

nerolidol

β-phellandrene

and

methyl-salicylate

did

not

significantly

differ

between

plants

overexpression

in

leaves

was

confirmed

by

RNA

gel-blot

analysis

The

expression

of

conjugating

enzyme

-LRB-

-RRB-

and

ribosomal

RNA

levels

-LRB-

ethidium

bromide-stained

gel

-RRB-

are

shown

to

indicate

equal

loading

of

the

gel

Next

we

tried

to

overexpress

the

full

length

cDNA

-LRB-

including

the

putative

targeting

peptide

-RRB-

in

tomato

plants

under

control

of

the

CaMV

promoter

Four

transformation

experiments

yielded

only

two

transformants

of

which

one

overexpressed

Compared

to

empty

vector-transformed

plants

overexpressing

plants

emitted

several

hundred-fold

more

linalool

into

the

plants

headspace

-LRB-

Fig.

-RRB-

while

emission

of

other

terpenes

including

nerolidol

did

not

differ

significantly

from

control

plants

This

suggests

that

functions

in

planta

as

a

true

linalool

synthase

Discussion

Monoterpenes

are

abundantly

emitted

by

tomato

plants

For

instance

β-pinene

β-myrcene

β-phellandrene

limonene

and

terpinolene

are

constitutively

emitted

while

linalool

and

β-ocimene

emission

is

induced

after

spider

mite

feeding

-LRB-

Kant

et

al.

-RRB-

In

this

paper

we

describe

the

first

two

monoterpene

synthases

from

tomato

and

and

show

that

is

induced

by

JA

in

trichomes

Identification

of

two

tomato

monoterpene

synthases

In

vitro

assays

with

the

recombinant

and

proteins

show

that

has

both

-LRB-

R

-RRB-

linalool

synthase

and

-LRB-

E

-RRB-

nerolidol

synthase

activity

and

that

is

a

β-phellandrene/β-myrcene/sabinene

synthase

-LRB-

Fig.

-RRB-

activity

in

planta

seems

restricted

to

linalool

synthase

activity

since

increased

expression

by

wounding

JA-treatment

and

ectopic

overexpression

led

to

increased

linalool

emission

but

did

not

affect

nerolidol

levels

-LRB-

Figs.

-RRB-

Generally

speaking

monoterpene

synthases

are

plastid

targeted

and

use

GPP

while

sesquiterpene

synthases

use

FPP

in

the

cytosol

There

are

however

some

exceptions

to

this

rule

For

instance

a

cytosolic

strawberry

nerolidol

synthase

-LRB-

-RRB-

has

both

-LRB-

-RRB-

-LRB-

E

-RRB-

nerolidol

synthase

and

S-linalool

synthase

activity

and

is

likely

responsible

for

both

linalool

and

nerolidol

synthesis

in

planta

-LRB-

Aharoni

et

al.

-RRB-

Vice

versa

when

was

targeted

to

the

plastid

in

Arabidopsis

it

also

produced

both

linalool

and

nerolidol

-LRB-

Aharoni

et

al.

-RRB-

The

authors

suggested

that

GPP

and

FPP

substrate

pools

are

not

strictly

separated

between

the

cytosol

and

plastid

which

has

recently

been

supported

by

similar

engineering

experiments

in

tobacco

-LRB-

Wu

et

al.

-RRB-

These

observations

might

be

explainable

by

for

instance

cytosolic

as

well

as

plastidial

targeting

of

GPP

synthase

-LRB-

GPS

-RRB-

in

Arabidopsis

and

Litospermum

erythrorizon

-LRB-

Bouvier

et

al.

Sommer

et

al.

-RRB-

In

addition

FPP

might

be

transported

from

the

cytosol

into

plastids

-LRB-

Lichtenthaler

-RRB-

and

finally

the

prenyltransferases

GPS

FPS

or

GGPS

might

generate

multiple

products

However

since

induced

expression

does

not

correlate

with

increased

nerolidol

emission

but

only

with

increased

linalool

emission

-LRB-

Figs.

-RRB-

we

consider

to

be

a

linalool

synthase

Furthermore

we

hypothesize

that

the

plastidial

FPP

concentrations

in

tomato

are

negligible

Despite

their

high

sequence

homology

-LRB-

Fig.

-RRB-

and

have

different

biochemical

activities

It

is

commonly

found

that

different

terpene

synthases

within

one

plant

species

often

have

higher

sequence

similarity

to

one

another

than

to

functionally

related

terpene

synthases

of

other

plant

species

For

example

β-pinene

synthase

from

citrus

-LRB-

Citrus

limon

-RRB-

is

more

homologous

to

the

citrus

γ-terpinene

synthase

than

to

β-pinene

synthase

from

wormwood

-LRB-

Fig.

-RRB-

It

is

also

known

from

work

with

other

terpene

synthases

that

product

specificity

can

depend

on

only

a

few

amino

acids

-LRB-

Kollner

et

al.

Rising

et

al.

Yoshikuni

et

al.

-RRB-

and

thus

product

specificity

of

terpene

synthases

is

not

predictable

on

basis

of

protein

sequence

or

specific

amino

acid

residues

By

comparing

terpene

synthase

structures

attempts

have

been

made

to

ascribe

substrate

binding

and

substrate

modifications

to

certain

residues

Modeling

of

the

active

sites

of

a

Salvia

-LRB-

Salvia

officinalis

-RRB-

sabinene

synthase

a

mint

-LRB-

Mentha

citrata

-RRB-

limonene

synthase

and

a

mint

linalool

synthase

led

to

the

following

observation

-LRB-

Crowell

et

al.

-RRB-

compared

to

the

other

two

enzymes

the

linalool

synthase

has

a

three

amino

acid

deletion

in

the

active

site

pocket

-LRB-

two

amino

acids

after

the

conserved

see

Fig.

-RRB-

resulting

in

a

more

open

structure

of

the

so

called

J/K

loop

and

providing

an

easier

access

of

water

during

substrate

ionization

Water

access

results

in

the

premature

release

of

the

intermediate

monoterpenol

carbo-cation

before

cyclization

to

a

monoterpene

olefin

-LRB-

non-hydrated

-RRB-

can

occur

The

tomato

linalool

synthase

also

has

a

deletion

in

the

active

site

pocket

after

the

conserved

-LRB-

Fig.

-RRB-

Although

the

deletion

is

eight

instead

of

three

amino

acids

it

indicates

that

might

utilize

a

similar

structural

mechanism

to

generate

a

monoterpene

alcohol

expression

correlates

with

induced

linalool

emission

The

induction

of

expression

correlated

with

the

increased

emission

of

linalool

by

tomato

plants

Wounding

and

JA

application

induced

-LRB-

Fig.

-RRB-

as

well

as

linalool

emission

-LRB-

Fig.

-RRB-

Induction

of

upon

spider

mite-feeding

-LRB-

Fig.

-RRB-

coincided

with

linalool

emission

-LRB-

Kant

et

al.

-RRB-

in

the

tomato

cultivar

CM

Moreover

overexpression

of

resulted

in

enhanced

production

of

linalool

-LRB-

Fig.

-RRB-

and

no

other

volatile

terpenes

providing

evidence

for

in

planta

linalool

synthase

activity

of

RNAi

lines

suppressing

expression

in

trichomes

would

provide

additional

evidence

that

acts

as

a

linalool

synthase

in

planta

It

is

generally

known

that

successful

overexpression

of

terpene

synthases

can

be

problematic

-LRB-

reviewed

by

Chappell

-RRB-

Substrate

limitation

and

product

conversions

can

explain

low-terpene

production

levels

-LRB-

Aharoni

et

al.

Lucker

et

al.

Ohara

et

al.

-RRB-

In

addition

ectopic

terpene

synthase

overexpression

might

lead

to

product

toxicity

-LRB-

Aharoni

et

al.

Besumbes

et

al.

-RRB-

and

selection

for

plant

lines

with

low

transgene

expression

or

low

terpene

accumulation

-LRB-

Diemer

et

al.

Krasnyanski

et

al.

-RRB-

We

managed

to

regenerate

only

one

overexpressing

plant

line

after

four

successive

independent

transformations

The

empty

vector

and

unrelated

constructs

gave

at

least

higher

transformation

frequencies

Similarly

overexpression

was

unsuccessful

seven

transgenic

plant

lines

showed

only

expression

of

truncated

transcripts

-LRB-

data

not

shown

-RRB-

is

expressed

in

roots

stems

and

petioles

-LRB-

Fig.

-RRB-

In

roots

only

minute

amounts

of

the

main

product

β-phellandrene

were

detected

hardly

visible

in

Fig.

since

levels

are

lower

than

in

leaves

The

role

of

monoterpenes

in

roots

remains

unclear

although

they

might

play

a

role

in

the

defense

response

of

plants

The

release

of

the

monoterpene

from

Arabidopsis

roots

is

induced

upon

bacterial

pathogen

infection

-LRB-

Steeghs

et

al.

-RRB-

although

transcriptional

regulation

of

the

corresponding

synthase

-LRB-

Chen

et

al.

-RRB-

has

not

been

shown

yet

Although

is

hardly

expressed

in

tomato

leaves

-LRB-

Fig.

-RRB-

β-phellandrene

and

β-myrcene

are

constitutively

emitted

by

tomato

plants

-LRB-

Buttery

et

al.

Kant

et

al.

-RRB-

This

suggests

that

the

constitutive

low

expression

of

might

be

sufficient

to

generate

these

monoterpenes

However

like

the

majority

of

volatile

terpenes

-LRB-

data

not

shown

-RRB-

β-phellandrene

is

mostly

located

in

trichomes

-LRB-

Fig.

-RRB-

where

is

not

expressed

-LRB-

Fig.

-RRB-

This

indicates

that

there

are

other

β-phellandrene/β-myrcene

synthases

in

tomato

that

synthesize

these

terpenes

JA-induced

expression

occurs

in

trichomes

Although

the

presence

of

terpene

synthases

in

trichomes

has

been

well

documented

-LRB-

Bertea

et

al.

Iijima

et

al.

Lange

et

al.

-RRB-

the

regulation

of

their

expression

in

trichomes

remains

obscure

Here

we

show

that

JA

treatment

of

intact

plants

leads

to

higher

transcript

levels

of

mainly

in

trichomes

-LRB-

Fig.

-RRB-

In

contrast

WIPI-II

expression

is

induced

by

JA

in

the

complementary

tissues

indicating

that

there

is

a

tissue-specific

differential

activation

of

JA-regulated

genes

Basal

expression

of

WIPI-II

was

very

low

in

tomato

trichomes

although

constitutive

expression

of

a

WIPI-II

in

glandular

trichomes

was

recently

demonstrated

in

nightshade

-LRB-

Solanum

americanum

-RRB-

-LRB-

Liu

et

al.

-RRB-

It

is

unknown

whether

trichome-expressed

WIPI-II

plays

a

role

in

the

defense

against

herbivores

Induction

of

WIPI-II

in

non-glandular

tissue

-LRB-

mesophyll/parenchyma

-RRB-

meets

the

expectation

that

it

should

be

present

in

those

cells

that

suffer

from

herbivory

Previously

JA

has

been

shown

to

increase

trichome

density

on

newly

formed

leaves

of

for

instance

Arabidopsis

and

tomato

-LRB-

Boughton

et

al.

Traw

and

Bergelson

-RRB-

Also

JA

might

induce

trichome-based

defenses

directly

The

production

of

acylsugars

on

the

leaf

surface

-LRB-

probably

in

trichomes

-RRB-

of

Datura

wrightii

plants

increased

without

affecting

trichome

density

-LRB-

Hare

and

Walling

-RRB-

More

clearly

nornicotin

production

on

the

Nicotiana

repanda

plant

surface

increased

twofold

within

h

of

JA

treatment

-LRB-

Laue

et

al.

-RRB-

Although

it

should

be

kept

in

mind

that

in

the

two

above-mentioned

studies

it

was

perhaps

unjustly

assumed

that

or

extraction

releases

only

trichome

contents

it

indicates

that

trichome-based

secondary

metabolite

biosynthesis

can

be

inducible

Results

from

our

study

show

for

the

first

time

a

specific

transcriptional

regulation

of

a

gene

involved

in

secondary

metabolite

biosynthesis

in

trichomes

Isolation

of

trichomes

revealed

that

linalool

synthase

expression

in

tomato

trichomes

was

induced

more

than

twofold

by

JA

after

h

In

this

paper

we

report

the

identification

of

the

first

two

monoterpene

synthases

from

a

plant

of

the

solanaceae

family

As

it

has

recently

been

described

that

increased

volatile

terpene

production

in

transgenic

plants

can

benefit

the

indirect

defense

-LRB-

Kappers

et

al.

Schnee

et

al.

-RRB-

it

will

be

interesting

to

investigate

the

biological

effect

of

overexpression

in

tomato

Furthermore

the

role

of

terpenes

in

roots

and

stems

remains

to

be

investigated

Finally

the

specific

induction

of

in

trichomes

by

JA

can

provide

an

excellent

opportunity

for

identification

of

novel

JA-related

promoter

elements

and

transcription

factors

Materials

and

methods

Isolation

of

and

cDNAs

Tomato

plants

Lycopersicon

esculentum

cv

Moneymaker

-LRB-

weeks

old

-RRB-

were

infested

with

spider

mites

-LRB-

Tetranychus

urticae

-RRB-

as

described

by

Kant

et

al.

-LRB-

-RRB-

Leaves

were

collected

and

pooled

after

days

of

infestation

RNA

was

isolated

using

Trizol

-LRB-

Invitrogen

Carlsbad

CA

USA

-RRB-

cDNA

was

made

using

SuperscriptII

RNAse

H

-LRB-

Invitrogen

-RRB-

A

LeMTS

fragment

was

amplified

from

this

cDNA

using

the

primer

in

combination

with

an

oligo

dT

-LRB-

-RRB-

primer

The

primer

was

designed

based

on

putative

tomato

monoterpene

synthases

-LRB-

and

-RRB-

as

such

that

it

should

not

anneal

to

sesquiterpene

synthase

cDNAs

The

bp

PCR

product

was

cloned

into

pGEM-T

easy

-LRB-

Promega

Madison

WI

USA

-RRB-

and

sequenced

using

the

ABI

PRISM

BigDye

terminator

kit

-LRB-

Applied

Biosystems

Foster

city

CA

USA

-RRB-

cDNA

was

synthesized

from

leaves

of

Moneymaker

plants

and

from

hypocotyls

and

roots

of

Moneymaker

plants

with

a

ZAP-cDNA

synthesis

kit

cloned

into

the

Lambda-ACT

vector

-LRB-

Elledge

et

al.

-RRB-

and

packaged

with

a

ZAP-cDNA

Gigapack

II

gold

cloning

kit

-LRB-

Stratagene

La

Jolla

CA

USA

-RRB-

plaques

of

each

library

were

screenend

with

the

bp

RT-PCR

product

as

probe

radioactively

labeled

by

the

ALL-IN-ONE

Random

prime

labeling

method

-LRB-

Sigma

Saint

Louis

MI

USA

-RRB-

Filters

were

hybridized

at

°C

in

BLOTTO

hybridization

buffer

-LRB-

Sambrook

and

Russell

-RRB-

and

washed

three

times

for

min

in

SSC

SDS

at

°C

DNA

from

positive

plaques

was

converted

to

plasmids

and

their

inserts

were

sequenced

was

obtained

from

the

leaf

cDNA

library

from

the

hypocotyl

and

root

cDNA

library

Sequences

can

be

found

in

Genbank

as

and

respectively

The

presence

of

plastid

targeting

signals

was

predicted

using

PREDOTAR

-LRB-

Small

et

al.

-RRB-

and

TargetP

-LRB-

Emanuelsson

et

al.

-RRB-

Expression

of

recombinant

proteins

in

E.

coli

enzyme

assays

and

product

analysis

and

were

subcloned

into

the

expression

vector

-LRB-

Novagen

San

Diego

CA

USA

-RRB-

after

removal

of

the

plastid

targeting

signal

sequences

up

to

one

amino

acid

upstream

of

the

conserved

arginine

pair

-LRB-

Fig.

-RRB-

by

generating

truncated

and

PCR

products

with

Pfu

DNA

polymerase

-LRB-

Stratagene

-RRB-

Forward

primers

contained

NcoI

and

reverse

primers

XhoI-restriction

sites

primers

-LRB-

-RRB-

GATCCATGGACACAAGGCGTTCAGGGAATTAC

and

GTACTCGAGCAAAGTAATAAAATGAAGCCTACG

primers

GATCCATGGGTATCCGACGTTCAGGAAATTAC

and

GTACTCGAGAATAAAAGGTAATAATTCCTTGTC

constructs

were

transformed

into

E.

coli

-LRB-

-RRB-

which

were

selected

on

Luria-Bertani

-LRB-

LB

-RRB-

plates

containing

mg/l

ampicillin

A

single

colony

was

grown

overnight

at

°C

on

a

plate

and

transferred

to

ml

of

LB

with

ampicillin

Cultures

were

grown

at

°C

for

min

to

of

before

addition

of

mM

and

grown

at

°C

for

an

additional

h.

Cells

were

harvested

by

centrifugation

and

resuspended

in

ml

monoterpene

synthase

assay

buffer

containing

mM

HEPES

pH

glycerol

mM

DTT

mM

mM

proteinase

inhibitor

cocktail

Complete

-LRB-

Roche

Basel

Switzerland

-RRB-

and

mM

ascorbic

acid

Cells

were

disrupted

by

addition

of

mg/ml

lysozym

-LRB-

Sigma

-RRB-

incubation

on

ice

for

min

and

sonication

After

centrifugation

-LRB-

g

-RRB-

for

min

at

°C

ml

of

the

soluble

enzyme

fraction

was

assayed

directly

or

after

purification

of

the

His-tagged

recombinant

fusion

protein

on

Ni-agarose

beads

-LRB-

Novagen

-RRB-

for

monoterpene

sesquiterpene

or

diterpene

synthase

activity

using

μM

of

the

substrates

GPP

FPP

or

GGPP

respectively

The

assay

mix

was

incubated

in

a

closed

ml

vial

at

°C

for

h

under

gentle

shaking

-LRB-

rpm

-RRB-

Reaction

products

were

sampled

with

a

Solid

Phase

Micro

Extraction

fiber

-LRB-

SPME

-RRB-

for

min

during

which

the

vial

was

agitated

and

heated

to

°C

The

SPME

fiber

was

desorbed

min

in

an

Optic

injector

port

-LRB-

ATAS

GL

Int.

Zoeterwoude

The

Netherlands

-RRB-

which

was

kept

at

°C

Compounds

were

separated

on

a

column

-LRB-

m

μm

μm

film

thickness

Hewlett

Packard

Palo

Alto

CA

USA

-RRB-

in

an

N

gas

chromatograph

-LRB-

Agilent

Amstelveen

The

Netherlands

-RRB-

with

a

temperature

program

set

to

°C

for

min

ramp

to

°C

at

°C

min

and

°C

for

an

additional

min

Helium

was

used

as

carrier

gas

the

column

flow

was

set

to

ml/min

for

min

and

to

ml/min

thereafter

Mass

spectra

were

generated

with

the

ion

source

set

to

V

at

°C

and

collected

with

a

Time-of-Flight

MS

-LRB-

Leco

Pegasus

III

St.

Joseph

MI

USA

-RRB-

at

V

with

an

acquisition

rate

of

scans/s

Terpene

products

were

identified

using

authentic

standards

and

comparison

of

ion

specra

and

relative

retention

times

-LRB-

Adams

-RRB-

For

separation

of

terpene

enantiomers

a

Cyclosil-B

column

-LRB-

m

mm

μm

film

thickness

Agilent

-RRB-

was

used

The

temperature

program

was

isothermal

at

°C

for

min

with

a

final

ramp

to

°C

at

°C

min

and

°C

for

min

Column

flow

was

ml/min

Plant

treatments

plant

headspace

sampling

trichome

isolation

and

measurement

of

extracts

For

determining

tissue

specific

gene

expression

a

weeks

old

untreated

tomato

plant

was

dissected

to

obtain

material

of

each

of

the

described

tissues

For

separation

of

trichomes

from

petioles

and

stems

plants

were

used

Trichomes

were

collected

on

the

bottom

of

a

ml

tube

after

vortexing

several

-LRB-

l

-RRB-

frozen

petiole

or

stem

segments

standing

in

upright

position

The

trichome

fraction

was

used

directly

whereas

the

remaining

cleaned

petiole

or

stem

segments

were

thoroughly

brushed

to

remove

all

remaining

trichome

material

Wounding

spider

mite

and

hormone-treatments

-LRB-

for

both

volatile

sampling

and

gene

expression

analysis

-RRB-

were

done

using

tomato

plants

Wounding

was

inflicted

by

squeezing

leaflets

several

times

with

a

hemostat

a

total

of

spider

mites

were

put

on

leaflets

per

plant

for

days

JA

and

salicylic

acid

were

applied

to

plants

by

spraying

mM

solutions

made

with

tap

water

containing

Wounding

and

hormone-treatments

were

done

-LRB-

cumulative

-RRB-

at

h

and

at

h

and

leaves

were

sampled

at

h.

Headspace

sampling

of

plants

was

performed

as

described

by

Kant

et

al.

-LRB-

-RRB-

Tenax

sampling

tubes

were

eluted

with

pentane

diethyl

ether

-LRB-

-RRB-

The

Eluted

fractions

were

concentrated

times

under

a

stream

of

and

μl

was

injected

into

the

GC

injector

port

For

root

leaf

trichome

stem

or

petiole-extracts

g

tissue

was

used

The

amount

of

trichome

material

was

adjusted

to

be

equivalent

to

the

trichomes

present

on

g

of

the

original

stem

or

petiole

Tissues

were

ground

in

-LRB-

l

-RRB-

and

transferred

to

glass

ml

vials

containing

ml

saturated

-LRB-

M

-RRB-

buffered

in

mM

sodium

acetate

-LRB-

pH

-RRB-

which

were

capped

immediately

and

kept

at

°C

Extracts

were

pre-incubated

for

min

at

°C

under

agitation

-LRB-

rpm

-RRB-

and

sampled

for

min

at

°C

on

a

μM

PDMS

SPME

fiber

-LRB-

Supelco

Zwijndrecht

The

Netherlands

-RRB-

The

SPME

fiber

was

desorbed

min

in

the

injector

port

of

the

GC/MS

which

was

kept

at

°C

GC/MS

analysis

was

done

as

described

earlier

-LRB-

Kant

et

al.

-RRB-

Gene

expression

analysis

by

RT-PCR

and

real-time

Q-RT-PCR

For

semi-quantitative

RT-PCR

the

tomato

conjugating

enzyme

-LRB-

-RRB-

was

used

as

constitutively

expressed

control

gene

-LRB-

-RRB-

Initial

PCR

was

performed

with

primers

product

levels

were

compared

and

individual

cDNA

samples

were

diluted

accordingly

to

ensure

equal

template

concentrations

PCRs

with

primers

of

genes

of

interest

were

subsequently

performed

for

non-saturating

number

of

amplification

cycles

primers

were

always

included

in

each

experiment

Used

primers

-LRB-

-RRB-

for

forward

-LRB-

F

-RRB-

GATTCTCTCTCATCAATCAATTCG

reverse

-LRB-

R

-RRB-

GCATCCAAACTTTACAGACTCTC

WIPI-II

F

GACAAGGTACTAGTAATCAATTATCC

R

CACATAACACACAACTTTGATGCC

F

TCAGTCCGACTAGGTTGTGG

R

TAGATAAGTGCTTGATGTGCC

F

GATGACATTTATGATGTTTATGGC

R

GGCCATCTCGAGACTTGAGAGCGAATGCAACATTAG

F

GATGACATTTATGATGTTTATGGC

R

GGGTAATAATTCCTTGTCTTATTTC

Expression

differences

were

validated

by

varying

the

amount

of

PCR

cycles

For

real-time

Q-RT-PCR

total

RNA

was

isolated

using

Trizol

-LRB-

Invitrogen

-RRB-

and

DNA

was

subsequently

removed

with

DNAse

-LRB-

Ambion

Huntingdon

UK

-RRB-

cDNA

was

synthesized

from

μg

RNA

using

SuperscriptII

-LRB-

Invitrogen

-RRB-

in

μl

reaction

volume

that

was

diluted

to

μl

prior

to

using

it

for

PCR

PCRs

were

performed

in

the

ABI

Real-Time

PCR

System

-LRB-

Applied

Biosystems

-RRB-

using

the

Platinum

SYBR

Green

qPCR

SuperMix-UDG

kit

-LRB-

Invitrogen

-RRB-

About

μl

PCR

reactions

contained

μM

of

each

primer

μl

ROX

reference

dye

and

μl

template

The

cycling

program

was

set

to

min

°C

min

°C

cycles

of

s

at

°C

and

min

°C

and

a

melting

curve

analysis

Primer

pairs

were

tested

for

specificity

and

for

linearity

with

a

standard

cDNA

dilution

curve

Primers

used

QF

QR

-LRB-

-RRB-

QF

QR

-LRB-

-RRB-

QF

QR

Expression

levels

were

normalized

using

mRNA

levels

Generation

of

transgenic

tomato

plants

The

complete

cDNA

including

the

putative

signal

peptide

was

cloned

into

the

EcoRI-digested

and

Klenow-blunted

binary

vector

-LRB-

Brandwagt

et

al.

-RRB-

resulting

in

a

nos

cassette

The

construct

was

transferred

to

Agrobacterium

tumefaciens

strain

carrying

the

pSoup

helper

plasmid

-LRB-

Hellens

et

al.

-RRB-

Tomato

plants

cv

Moneymaker

were

transformed

essentially

as

described

by

Vanroekel

et

al.

-LRB-

-RRB-

Heterozygous

and

homozygous

overexpressing

plants

were

used

to

measure

linalool

production

by

sampling

the

headspace

of

intact

plants

-LRB-

four

sets

of

two

plants

for

both

overexpressors

and

control

plants

-RRB-

B B O O O O B O B I B O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O O B O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O B O O O O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O

tomato
jasmonic acid
trichomes
linalool
terpenes
defence