Chapter 10 Application of the Artificial Annual Environmental Cycle and Dormancy-Induced Suppression of Cesium Uptake in Poplar

In perennial woody plants, dormancy-induced alteration of potassium (K) localization is assumed one of the mechanisms for adapting and surviving the severe winter environment. To establish if radio-cesium (137Cs) localization is also affected by dormancy initiation, the artificial annual environmental cycle was applied to the model tree poplar. Under the short day-length condition, the amount of 137Cs in shoots absorbed through the roots was drastically suppressed, but the amount of 42K was unchanged. Potassium uptake from the rhizosphere is mainly mediated by KUP/HAK/KT and CNGC transporters. However, in poplar, these genes were constantly expressed under the short-day condition and there were no upor down-regulation. These results indicated the suppression of 137Cs uptake was triggered by the short-day length, however, the key transporter and the mechanism remains unclear. We hypothesized that Cs and K transport systems are separately regulated in poplar.


Introduction
In 2011, radionuclides were released into the environment due to the Fukushima Daiichi Nuclear Power Plant accident. Among the released radionuclides, radiocesium ( 137 Cs) has been considered the main environmental contaminant. A large part of the contaminated land was forested and, after the accident, the 137 Cs deposition to tree canopies, uptake through leaves and/or bark, and the translocation to growing branch were actively investigated (Kato et al. 2012;Takata 2013;Kanasashi et al. 2015). However, 137 Cs uptake from the contaminated soil through the root was not well investigated because of methodological challenges. Therefore, the physiological knowledge of Cs transfer from the soil and its distribution among the tree organs is limited.
Cesium has a chemical property similar to potassium (K), but it is not an essential nutrient for plants. Generally, the rhizosphere Cs concentration (almost all is stable 133 Cs) is less than approx. 200 μM and not toxic to plant growth (White and Broadley 2000). Cesium uptake and translocation within the plant body are considered to be mediated by K + transport systems (White and Broadley 2000). Arabidopsis HAK5 (AtHAK5) is one of the best investigated root K + uptake transporters, and its mutant, athak5, showed a tolerance against 300 μM Cs treatment under low K conditions (Ahn et al. 2004;Gierth et al. 2005;Qi et al. 2008). AtCNGCs are Voltage-Independent Cation Channels (VICCs) type K + permeable channels and AtCNGC1 is the candidate gene for Cs uptake identified by quantitative trait locus analysis in Arabidopsis (Leng et al. 1999;Kanter et al. 2010).
To understand 137 Cs behavior within woody plants, Cs content in each organ should be revealed under each seasonal environment, because K distribution among tree organs is changed by the seasonal transition. To combine the seasonal change of Cs distribution and the genetic information related to Cs transport, we used poplar as an experimental plant, although it is not the major tree species in the Fukushima region. Poplar is a perennial deciduous tree and the seasonal cycle of growth and dormancy is distinct. The genome of Populus trichocarpa is available (Tuskan et al. 2006) and the findings related to the Cs and K transport mechanisms obtained from crop research are applicable. Moreover, the high transfer rate of Cs from soil to poplar leaves has been observed under 137 Cs contaminated regions in Europe (International Atomic Energy Agency (IAEA) 2010).
In trees, the phase shift from growth to dormancy is a basic winter adaptation mechanism (Jansson et al. 2010) and the transition of meristems into dormant buds is crucial for cold adaptation to protect the meristems against hazardous frosts. By perceiving the change in photoperiod and temperature, woody plants can shift their growth stage (Welling et al. 2002), and the initiation of cold acclimation under the short-day length increases endogenous abscisic acid levels and reduces endogenous gibberellic acid levels (Olsen et al. 1997;Welling et al. 1997;Mølmann et al. 2005). In beech (Fagus sylvatica L.), leaf K content is rapidly decreased before shedding and the retrieved K is deposited in the cortex and pith tissues of the stem (Eschrich et al. 1988). Japanese native poplar (Populus maximowiczii) can decrease leaf K concentration after dormant bud formation (Furukawa et al. 2012). In addition, the increase of K concentration in xylem sap was observed during the winter period in field grown Populus nigra (Furukawa et al. 2011). These K behaviors implied the existence of a re-translocation mechanism and it is assumed that K and Cs are transported to K required organs for growth regulation and/or survival.
In this chapter, we will outline the experimental method of the artificial annual environmental cycle for cultivating small scale sterilized poplar and the investigation of 137 Cs and 42 K uptake through their roots under long and short photoperiod conditions.

Application of the Artificial Annual Environmental Cycle to Poplar
In poplar, dormancy is primarily initiated in response to short-day lengths (Sylven 1940;Nitsch 1957), and the recent genetic and physiological understandings of dormancy initiation and break make poplar a highly suitable model tree for investigating growth rhythms. And the Populus trichocarpa genome was sequenced and expressed sequence tags of Populus were also identified (Kohler et al. 2003;Sterky et al. 2004). To utilize these advantages, we used poplar Hybrid aspen T89 (Populus tremula x tremuloides) as our experimental plants for investigating the shift of K and Cs distribution.  (Table 10.1). Poplars can break its dormant bud after the return to Stage-1 within 2 weeks. In our artificial annual environmental cycle, in contrast to Kurita et al. (2014), the temperature in Stage-2 was set to 23 °C. Our preliminary experiments revealed that the dormant bud formation can be triggered by 4 weeks of short day-length treatment when the temperature is less than 23 °C. To minimize the environmental factors related to the dormancy initiation, the temperature was kept constant in Stage-1 and -2.

Measurement of 137 Cs and 42 K Distributions in Poplar
Under the artificial annual environmental cycle using Populus alba L., the shift of growth condition from Long Day (LD) to Short Day (SD) condition decreased the phosphate contents in lower leaves (Kurita et al. 2014). This change of phosphate content suggested the existence of a mechanism for the re-translocation of phosphate from lower leaves to younger upper leaves with seasonal changes. Regarding calcium (Ca) translocation, Furukawa et al. (2012) indicated the Ca transport from root to shoot is also regulated by the shift from LD to SD in Populus maximowiczii. To investigate if a similar shift occurs for 137 Cs, Cs uptake through roots and its behavior within the plant body was compared in LD and SD conditions (Noda et al. 2016).
Poplars were grown under Stage-1 (LD) condition for 3 or 9 weeks (LD3 and LD9) in a light-and temperature-controlled environment. For investigating the effect of seasonal transition, a part of the LD3 plants were grown under Stage-2  (Aramaki et al. 2015;Kobayashi et al. 2015) solution was added to the growth medium to observe root absorption. Cesium-137 distribution was investigated with the autoradiography technique in LD3, LD9 and SD6 plants and the harvested plants were cut into four parts, apex (shoot apex and top three leaves), leaf (remaining leaves and petioles), stem and root. Figure 10.2a displays the localization of 137 Cs through root absorption under LD3, LD9 and SD6 conditions. In LD3 plants, 137 Cs was localized entirely and the radiation intensity around the apex was higher than other organs. Similarly, LD9 which was the same age as SD6 plants and grown under LD condition also showed the same 137 Cs behavior as LD3 plants. In SD6 plants, 137 Cs was mainly localized in the stem and root, furthermore, the total 137 Cs quantity seemed to have decreased. Comparing 137 Cs in the shoot (all shoot organs) and root under LD3, LD9 and SD6, the quantity of 137 Cs in shoots under SD6 condition was the lowest (Fig. 10.2b). However, 137 Cs quantity in roots was similar under each condition.
In respect to the amount of 137 Cs in each organ, 137 Cs mainly accumulated in leaves under LD3 condition (48.8% of applied 137 Cs) ( Fig. 10.3a); in the LD9 condition, accumulation pattern was similar to LD3. However, under SD6 condition, leaf 137 Cs content was the second highest (32.1%) and 137 Cs mostly accumulated in the stem (39.7%). As for the 137 Cs concentration, the effect of SD transition on 137 Cs distribution mainly resulted in suppressing Cs transport into shoot apices and leaves ( Fig. 10.3b). From the result of 137 Cs uptake assays, it was expected that the amount of 42 K absorbed through the root was also down-regulated by SD transition. To explain the K uptake activity with seasonal change, poplar was treated with exogenous 42 K application to the root and the quantity of K in shoot and root under LD3, SD2, SD4 and SD6 conditions were measured after 24 h incubation (Fig. 10.4). Contrary to our expectations, there was no difference in the amount of 42 K in either root or shoot among the four conditions. These data suggest that the K demand for the rhizosphere was not changed by SD transition in poplar up to 6 weeks. Comparing the data presented in Figs. 10.2 and 10.4,137 Cs accumulation decreased under SD6 condition but 42 K accumulation remained constant through the SD transition. This inconsistency suggests that Cs accumulation might be separately controlled, and not part of the major K uptake systems in poplar. In addition, it is also implied that the responsible transporter for poplar Cs uptake might be poorly involved in K uptake because no decrease of K accumulation was observed under the low Cs accumulation condition.

Expression of Potassium Influx Transporters in Poplar Root
To identify the Cs uptake responsible transporter, we investigated the expression patterns of some candidate genes under SD condition. Among various K + uptake transporters, we focused on high-affinity K + transporters, KUP/HAK/KT family (Rubio et al. 2000;Mäser et al. 2001;Gupta et al. 2008), and low-affinity channel, cyclic-nucleotide-gated channel (CNGC) (Hua et al. 2003;Harada and Leigh 2006;Ahmad and Maathuis 2014). As for one of the KUP/HAK/KT family genes, we focused on Populus tremula K + uptake transporter 1, PtKUP1 (POPTR_0003s13370), identified from hybrid aspen. PtKUP1 was used for the complementation test using K uptake-deficient E. coli mutant. The addition of toxic level Cs to the culture media inhibited the growth of E. coli expressing PtKUP1 strongly, suggesting PtKUP1 can transport both K and Cs (Langer et al. 2002). In addition to PtKUP1, there are eight AtHAK5 orthologue genes in Populus trichocarpa. Arabidopsis HAK5 (AtHAK5) is a well-known root K + uptake transporter and it has been reported that the induction of AtHAK5 is enhanced by K + deficiency (Ahn et al. 2004;Gierth et al. 2005) or by Cs + applications when there is sufficient K + (Adams et al. 2013). Based on the similarity of those nine putative KUP/HAK/KT family K + transporters, POPTR_0010s10450 and POPTR_0001s00580 were chosen as highly homologous genes with AtHAK5. We identified POPTR_0010s10450 and POPTR_0001s00580 homologues in hybrid aspen T89 and named those Populus tremula x tremuloides HAK-like1 (PttHAK-like1) and PttHAK-like2, respectively. Similar to HAK-like genes, two CNGC-like genes were selected from nine AtCNGC1 homologue genes in P. trichocarpa. POPTR_0012s01690 and POPTR_0015s02090 had a higher similarity to ATCNGC1 and those homologues in hybrid aspen T89 were named PttCNGC1-like1 and PttCNGC1-like2, respectively. The expression patterns of these genes were measured during the transition to the SD condition (Fig. 10.5). There was no significant change in PtKUP1 expression. PttHAK-like1 showed steady expression until SD4 condition and was slightly upregulated by about 1.5-fold under SD6 condition. PttHAK-like2 exhibited the decreasing tendency in SD2 and SD4 plants, but remained statistically constant through the SD transition. Two PttCNGC1-like genes also expressed relatively constantly under SD condition.
In this experimental condition, dormant buds were formed up to 4 weeks after starting the short-day treatment and, therefore, the re-translocation of K should have already commenced at SD6. However, the results showed that the 42 K accumulation through root uptake and gene expression related to the root K uptake were almost constant. Steady-state of K requirement under SD condition seems to indicate abundant K was stored in the plant body and the change of K demand might be covered by the internal re-translocation.

Perspectives in Cs + Transporter Research
Despite the constant K accumulation patterns under SD conditions, Cs accumulation was drastically decreased in SD6 plant (Figs. 10.2 and 10.4). Cesium uptake and translocation is considered to be regulated by plant K transport system, however, no down-regulation in candidate genes was observed at SD6 (Fig. 10.5). However, it is not known if elemental transport is only regulated by gene expression and not by protein activity (White and Broadley 2000). But the inconsistence of 42 K + and 137 Cs + localization patterns indicates a possible existence of an unknown Cs uptake system which preferentially transports Cs rather than K.
Based on our experimental design to identify permeable Cs transporters, it would appear that the mechanisms regulating the specificity of these transporters are very complex and it might be difficult to identify the responsible genes through reverse genetics. Our studies have also demonstrated that Cs uptake in poplar is regulated by the photoperiod, therefore, the mechanisms of the dormancy initiation might be involved in the suppression of Cs uptake. Using our data and the increasing Fig. 10.5 Effect of short-day (SD) transition on transcriptional expression of HAK and CNGC orthologue genes in poplar root. Total RNA was isolated from root and these gene transcript levels were analyzed by qRT-PCR. UBIQUTIN was used as the reference gene. All gene expression levels were normalized by expression level of LD3 condition. Error bars represent standard deviation. * indicated significant difference to the level of LD3 expression level (* <0.01). (Modified from Noda et al. (2016)) knowledge of dormancy, the mechanisms of Cs uptake from contaminated soil to forest trees should be revealed and we hope that the understanding of Cs circulation within the forest ecosystem will be utilized in the prediction of Cs transfer among the terrestrial environment in the near future.
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