Senescence Induced Serotonin Biosynthesis and Its Role in Delaying Senescence in 2 Rice Leaves

1 Serotonin, which is well known as a pineal hormone in mammals, plays a key role in 2 conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin 3 has been suggested to be involved in several physiological roles, including flowering, 4 morphogenesis, and adaptation to environmental changes, its regulation and functional 5 roles are as yet not characterized at the molecular level. In this study, we found that 6 serotonin is greatly accumulated in rice leaves undergoing senescence induced by either 7 nutrient deprivation or detachment, and its synthesis is closely coupled with 8 transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as 9 tryptophan decarboxylase ( TDC ). Transgenic rice plants that overexpressed TDC 10 accumulated higher levels of serotonin than the wild type and showed delayed 11 senescence of rice leaves. However, transgenic rice plants, in which expression of TDC 12 was suppressed through an RNAi system, produced less serotonin and senesced faster 13 than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. 14 The senescence-retarding activity of serotonin is associated with its high antioxidant 15 activity compared to either tryptophan or chlorogenic acid. Results of TDC 16 overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for 17 serotonin accumulation, but the synthesis of serotonin depends on an absolute amount 18 of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic 19 genes. In addition, immunolocalization analysis revealed that serotonin was abundant in 20 the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, 21 suggestive of its involvement in maintaining the cellular integrity of these cells for 22 facilitating efficient nutrient recycling from senescing leaves to sink tissues during 23 senescence. transgenic of the transgenic line; first of serotonin synthesis upon senescence in these results indicate that serotonin plays a practical role in delaying senescence by scavenging ROS efficiently. Although our results exhibited a clear correlation between serotonin and senescence symptoms, it clear if these are the result of the action of whether


INTRODUCTION
1 Serotonin (5-hydroxytryptamine) is a ubiquitous monoamine that plays multiple roles as 2 a neurotransmitter, hormone, and mitogenic factor, and mediates a series of activities in 3 various animal cells (Frazer and Hensler, 1999). In plants, serotonin has been found in a 4 wide range of plant species (Roshchina, 2001) since its discovery was first reported in 5 the fruit of the cowhage plant (Bowden et al., 1954). Similar to the multiple roles played 6 by serotonin in animal cells, serotonin has also been implicated in an array of 7 physiological functions in plants that are purportedly related to growth regulation, 8 flowering, xylem sap exudation, ion permeability, and plant morphogenesis (Csaba and 9 Pal, 1982;Odjakova and Hadjiivanova, 1997;Murch et al., 2001;Roshchina, 2001).

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In addition, serotonin levels are known to increase as fruits ripen in many species 1 4 including tomato, although the inverse is true of the fruit of Ananas comosus 1 5 (pineapple) (Udenfriend et al., 1959;Foy and Parrat, 1961). Apart from enriched 1 6 serotonin accumulation in fruits, serotonin accumulates in the stinging nettle Urtica 1 7 dioica (Collier and Chesher, 1956) and in the pods of Mucuna pruriens (Bowden et al., 1 8 1954) in which serotonin is suggested to play a protective role against predators.

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One interesting study on serotonin synthesis and its possible biological function 2 0 was reported for walnut seeds in which serotonin is mainly accumulated during the 2 1 process of fruit abscission (Bergmann et al., 1970). This abscission period is molecular level in our in planta system, we performed Northern blot analysis using 1 representative senescence-associated genes such as Osl2 and Osl139, which were 2 induced in rice leaves upon senescence (Lee et al., 2001). As shown in Figure 2D, the 3 Osl2 transcript was rarely detectable before senescence, but was gradually induced in 4 response to senescence. Higher levels of Osl2 transcripts were detected after 21 d. 5 Compared to Osl2, the level of Osl139 transcripts was relatively low, but increased as 6 senescence proceeded. Taking all the data together, rice seedlings underwent senescence 7 in our in planta rice seedling system. Serotonin is consecutively synthesized from tryptophan by two enzymes. conversion of tryptophan to tryptamine, followed by catalysis of tryptamine to produce 1 4 serotonin by tryptamine 5-hydroxylase (T5H) (Kang et al., 2007a). TDC is the rate-1 5 limiting enzyme for serotonin biosynthesis and exists in at least two functional copies in 1 6 the rice genome (Kang et al., 2007b). To determine whether serotonin accumulation 1 7 upon nutrient-deficient induced senescence is closely associated with induction of TDC 1 8 mRNA, we performed independent Northern blot analyses with two TDC cDNAs as 1 9 probes (Fig. 3A, B). The TDC1 mRNA transcript was not detected in healthy tissues 2 0 even after 6 d of senescence treatment. By day 11, the level of the TDC1 transcript 2 1 began to increase, and rapid increase was observed in leaves after 16 d. The increase in 2 2 TDC1 transcripts was proportional to the accumulation of serotonin upon senescence.

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Unlike leaves, stems and roots showed no significant induction of the TDC1 transcript 1 0 in response to senescence, which was consistent with low serotonin levels and the lesser 1 degree of senescence symptoms compared to leaves. In marked contrast, the TDC2 2 transcript was rarely detectable in all senescing tissues including leaves, suggesting that 3 TDC1 played the major role in serotonin biosynthesis when rice plants were challenged 4 with senescence. To see whether the transcriptional induction of TDC1 is related to high 1 1 were closely associated with the level of serotonin. The tip contained the highest level 1 of serotonin at 270 μg/g fw, whereas the middle and base parts contained 152 and 30 2 μg/g fw, respectively. In particular, tryptophan, the substrate of the TDC enzyme, 3 coordinately increased up to 400 μg/g fw in the fully senesced tip, which corresponded 4 to a level 1.5 fold higher than serotonin. These data clearly suggested that TDC 5 expression is abundant in the senesced tissues of rice leaves and is strongly induced in 6 parallel with the high production of serotonin as well as tryptophan as the rice leaves 7 undergo senescence. In addition, the effects of exogenous applications of serotonin on 8 leaf senescence were investigated by measuring chlorophyll, ROS, and MDA. As shown 9 in Supplemental Figure S1, treatment with 500 µM serotonin showed two-fold higher 1 0 chlorophyll content than the untreated leaves at 26 d. Both ROS and MDA levels 1 1 decreased significantly in serotonin-treated leaves compared to untreated leaves. In 1 2 addition, TDC enzyme activity was three-fold lower in serotonin-treated leaves (500 In Figures 1-4, we show the course of serotonin synthesis as well as its effects on 2 0 senescence (Supplemental Figs. S1, S2) using an in planta system. To further verify the 2 1 mechanisms of serotonin biosynthesis and its physiological roles and to simplify the 2 2 experiment, we next used leaves detached from 4-week-old rice seedlings and measured 2 3 the levels of serotonin in response to senescence. As shown in Figure 5, serotonin began 1 2 to be synthesized on day 4, produced 984 μg/g fw on day 6, and peaked on day 8 with 1 1,634 μg/g fw (Fig. 5A). The maximum level of serotonin in the detached leaves was 2 4.7-fold higher than that found in the attached leaves upon senescence, indicating that 3 the detachment of the leaves had a more dramatic effect on serotonin synthesis than was 4 observed for the attached leaves.

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The effects of plant hormones such as zeatin and abscisic acid (ABA) on 6 serotonin synthesis in response to senescence were investigated. ABA treatment 7 accelerated serotonin synthesis, producing 450 μg/g fw after 4 d, while control leaves 8 produced only 16 μg/g fw serotonin and showed a maximum synthesis of serotonin (720 9 μg/g fw) at 6 d, followed by gradual decrease. In contrast to ABA, zeatin treatment 1 0 delayed serotonin synthesis and its levels were far lower compared to those of the 1 1 untreated control leaves. These data on the changes in serotonin levels upon hormonal 1 2 treatment were consistent with the known roles of zeatin and ABA, which play 1 3 inhibitory and stimulatory roles in senescence, respectively. In addition, the tryptophan   The results from the detached leaves show that serotonin accumulation in 1 1 response to senescence is accompanied by the induced accumulation of free tryptophan.

2
To test whether tryptophan biosynthetic genes are induced upon senescence, five 1 3 tryptophan biosynthetic genes were selected for characterization. Figure 6A shows that 1 4 the mRNAs encoding the enzymes anthranilate synthase (ASα1 and ASβ2), and TSα were shown to be induced at 6 d. In addition, glutamine synthetase (GS) was wounded leaves of rice (Tozawa et al., 2001). However, the induction of ASα1 was not 1 observed at all, and the ASα1 transcript was suppressed upon pathogen infection. Thus, 2 ASα1 seems to play a key role either in senescence-induced tryptophan or serotonin 3 synthesis in rice plants. To determine whether the induction of AS mRNA is functionally 4 associated with the induction of the AS protein, we measured AS enzyme activity 5 during the time course of senescence. As shown in Figure 6B, AS enzyme activity was 6 very low prior to senescence, but was induced after 4 d upon commencement of 7 senescence and showed a maximum activity of 6 pkat/mg protein followed by a gradual 8 decrease after 8 d. Note that the maximum AS activity preceded the peak synthesis of 9 serotonin.  The role of serotonin in senescence was further investigated by performing a  concentrations in leaves were 16 µg/g fw in the wild type, whereas the transgenic lines 2 2 (lines 10 and 14) produced more than 250 µg/g fw after 4 d (Fig. 7B). During 2 3 senescence, the serotonin concentration increased in both the wild type and transgenic 1 5 lines as senescence proceeded, but the relative levels of serotonin were always higher in 1 transgenic lines than in the wild type due to the overexpression of TDC. In contrast, 2 tryptophan levels were lower in the transgenic lines (10 and 14) than in the wild type 3 because the tryptophan was converted into serotonin more efficiently in the transgenic 4 lines than in the wild type by TDC overexpression (Fig. 7C). During the period of 5 senescence, transgenic lines 10 and 14 showed delayed senescence compared to the 6 wild type, except line 18, when judged by phenotype (Fig. 7D). These phenotypic 7 differences were further confirmed by biochemical analyses measuring MDA and 8 chlorophyll content. The transgenic lines with the high levels of serotonin exhibited less 9 MDA production than the wild type (Fig. 7E). Accordingly, the loss of chlorophyll upon 1 0 senescence was slower in the transgenic lines than in the wild type (Fig. 7F). The results 1 1 were consistent with those obtained with the exogenous serotonin treatment. To further examine the function of serotonin in vivo, we employed RNAi 1 6 interference to silence the expression of TDC1, which is a rate-limiting enzyme for 1 7 serotonin synthesis. A transgene TDC1 was controlled by a maize (Zea mays) ubiquitin 1 8 promoter, and 20 independent transgenic lines were generated through Agrobacterium- control produced around 900 μg g -1 fw serotonin 6 d after senescence, whereas the 1 RNAi lines (T 0 ), such as RNAi-11 and RNAi-16, only produced 125 and 207 μg g -1 fw 2 serotonin, respectively (Fig. 8B). Accordingly, these RNAi lines exhibited a rapid 3 senescence relative to the wild type. Chlorophyll concentrations were 1.5-fold less in 4 the RNAi lines than in the wild type after 8 d, confirming that serotonin itself plays a 5 direct role in delaying senescence in rice leaves (Fig. 8D). In contrast to serotonin levels, 6 tryptophan levels did not dramatically change in the RNAi lines, although the RNAi 7 lines had lower levels of tryptophan than the wild type or vector control, especially after 8 6 d (Fig. 8C). Note that the RNAi lines (T 1 ) were not different phenotypically from the 9 wild type in both the vegetative and reproductive stages, suggesting that serotonin is not 1 0 directly involved in primary metabolism, but rather in secondary metabolism acting as a symptoms. As shown in Figure 9, the SHT transgenic line produced high levels of 2 0 serotonin derivatives, whereas the levels of serotonin and tryptophan were similar to 2 1 those of the wild type. The resulting senescence severity was slightly lower in the SHT 2 2 transgenic line than in the wild type, possibly due to the high levels of serotonin 2 3 derivatives in the SHT transgenic line; this suggests that serotonin derivatives are also 1 7 slightly involved in delaying senescence, but are not a determining factor for delaying 1 senescence compared to serotonin in rice plants. The levels of serotonin derivatives in 2 the TDC transgenic line were lower than those of the wild type, whereas serotonin 3 levels were the inverse. The reason for the low levels of serotonin derivatives in the 4 TDC line appears to be due to the low levels of endogenous rice SHT enzyme activity as 5 a result of the delayed senescence of the TDC transgenic line. To test whether the 6 endogenous rice SHT enzyme is also induced upon senescence, we measured SHT 7 enzyme activity. As expected, the SHT enzyme activity was two-fold lower in the TDC 8 transgenic line than in the wild type (data not shown), suggesting the induction of rice 9 SHT enzyme activity upon senescence as observed for TDC. In addition, the 1 0 preferential production of CS rather than FS was manifested in the wild type as well as showing two-fold higher radical scavenging activity (RSA) of serotonin rather than between serotonin accumulation and senescence retardation.  The spatial distribution of the TDC protein and serotonin in the cross section of 1 rice leaves was examined by immunohistochemical localization using TDC and 2 serotonin polyclonal antibodies (Fig. 10). TDC protein and serotonin were not stained in 3 control leaves, but were clearly observed 7 d after senescence, which was consistent 4 with the results of previous analyses (Fig. 5). Although all of the mesophyll cells except 5 the epidermal cells were thoroughly stained, signals for the TDC protein were abundant 6 in vascular parenchyma cells, whereas bundle sheath cells and the metaxylem were not 7 stained (Fig. 10C, F). The companion cells were also clearly stained, but the signal 8 intensity was not high compared to that in the xylem parenchyma cells. In contrast, 9 serotonin was strongly stained in companion cells at a level of intensity similar to the 1 0 vascular parenchyma cells (Fig. 10D, G). These data suggest that serotonin may play an 1 1 important role in maintaining the cellular integrity of vascular bundles with its high 1 2 antioxidant activity during the process of senescence. This study characterized the mechanisms by which senescence triggers and coordinates 1 7 serotonin synthesis through the biosynthetic machinery of the tryptophan pathway. of organic nitrogen (Hayashi and Chino, 1990). In addition to these amino acids, other 1 amino acids, including tryptophan, were reported to increase in concentration during 2 senescence in tobacco flowers and in the detached leaves of oats and Arabidopsis, and  As expected, the induction of TDC took place at the same time as the tryptophan feast 7 upon senescence. In addition to the induction of tryptophan biosynthetic genes, 8 glutamine synthetase was also induced upon senescence, but its induction occurred at a 9 later stage of senescence compared to that of other tryptophan biosynthetic genes. In The report on tryptophan-overproducing transgenic rice demonstrated that 2 1 tryptophan seems to be a stable primary metabolite suitable for either nutrient serotonin synthesis are as compared to plants unable to make serotonin. Although 1 several different roles have been proposed, the function of serotonin is not yet clear. In 2 this study, we found that tryptophan levels were significantly induced upon senescence 3 and that the increased tryptophan was readily converted into serotonin by the induction 4 of TDC. Thus, it seems reasonable to think that serotonin, rather than tryptophan, is a 5 preferable mediator of senescence. This hypothesis is further supported by the sl mutant 6 in rice, which is known to be controlled by a single recessive mutation and lacks 7 serotonin synthesis (Ueno et al., 2003;Ishihara et al., 2008). During senescence, the   with serotonin production may be coupled with the enriched tryptophan that was 1 4 induced upon senescence, although tryptophan biosynthesis occurs in the plastids 1 5 (Radwanski and Last, 1995). In particular, companion cells and xylem parenchyma cells showing strong signals for serotonin were also reported to be the major site of the 1 7 glutamine synthetase enzyme, which catalyzes the conversion of glutamate to glutamine, 1 8 a major long-distance transport form of organic nitrogen during the process of 1 9

Immunohistochemical Localization of the TDC Protein and Serotonin in Senesced
senescence (Sakurai et al., 1996). Companion cells and xylem parenchyma cells play 2 0 key roles in the regulation of phloem loading (Van Bel, 1993). Therefore, it is highly 2 1 likely that serotonin, with its high antioxidant activity, may play an important role in conical tubes with their roots exposed to water containing no nutrients. Senescence was 2 0 visible after 16 d. Rice tissues were harvested at specified time points and subjected to 2 1 further analysis. The data were analyzed by two to five replicates and then the Duncan's 2 2 multiple range was carried out to find the significant differences at P < 0.05.