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Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO2

Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO2 www.nature.com/scientificreports opeN sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated Received: 19 October 2018 Co Accepted: 8 May 2019 Published: xx xx xxxx 1,2 2 2 1,2 t akatoshi Kiba , Yumiko t akebayashi , Mikiko Kojima & Hitoshi sakakibara Carbon availability is a major regulatory factor in plant growth and development. Cytokinins, plant hormones that play important roles in various aspects of growth and development, have been implicated in the carbon-dependent regulation of plant growth; however, the details of their involvement remain to be elucidated. Here, we report that sugar-induced cytokinin biosynthesis plays a role in growth enhancement under elevated CO in Arabidopsis thaliana. Growing Arabidopsis seedlings under elevated Co resulted in an accumulation of cytokinin precursors that preceded growth enhancement. In roots, elevated CO induced two genes involved in de novo cytokinin biosynthesis: an adenosine phosphate-isopentenyltransferase gene, AtIPT3, and a cytochrome P450 monooxygenase gene, CYP735A2. The expression of these genes was inhibited by a photosynthesis inhibitor, DCMU, under elevated Co , and was enhanced by sugar supplements, indicating that photosynthetically generated sugars are responsible for the induction. Consistently, cytokinin precursor accumulation was enhanced by sugar supplements. Cytokinin biosynthetic mutants were impaired in growth enhancement under elevated CO , demonstrating the involvement of de novo cytokinin biosynthesis for a robust growth response. We propose that plants employ a system to regulate growth in response to elevated Co in which photosynthetically generated sugars induce de novo cytokinin biosynthesis for growth regulation. Being sessile, plants integrate environmental and internal cues and regulate physiological and morphological processes accordingly to optimize growth and development. Because multicellular higher plants consist of organs with different functions, for example photosynthesizing leaves and roots that absorb water and inorganic nutri- ents, the responses must be coordinated at the whole plant level. Local as well as long-distance signalling between 1–6 cells and organs via signalling molecules such as sugars and plant hormones are vital for this coordination Cytokinins (CKs) are a class of plant hormones that play a central role in the regulation of numerous aspects 7–11 of plant growth and development acting as local and long-distance signals . Naturally occurring CKs are 6 6 2 mostly N -prenylated adenine derivatives; N -(∆ -isopentenyl)adenine (iP), trans-zeatin (tZ) and their conju- 10–12 gates (iP-type and tZ-type CKs, respectively) are the major forms in Arabidopsis thaliana . CK activity is con- trolled at diverse levels, including CK quantity and modification. CK quantity is regulated mostly at the levels of de novo biosynthesis and degradation catalysed by adenosine phosphate-isopentenyltransferase (IPT) and CK 13–16 oxidase/dehydrogenase (CKX), respectively . Side-chain modification to form tZ-type CKs by cytochrome 17,18 P450 monooxygenase CYP735A specie fi s CK activity toward shoot growth . Recently, CK translocation via the 19–21 vascular system was reported to also be important . Shoot-to-root translocation of CK via phloem is critical for root vascular patterning, whereas root-to-shoot translocation via xylem mediated by ABCG14 regulates shoot growth and development. Regulation of CK activity is relevant to various plant developmental processes and 22–28 environmental responses such as shoot apical meristem activity, branching, stress and nutritional responses . Because plants are autotrophs that rely on photosynthesis to gain most of their building materials and 29–32 energy, carbon availability is a major factor defining plant growth and development . To maximize t fi ness, Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, nagoya University, n agoya, 464-8601, Japan. RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi, Yokohama, 230-0045, Japan. Correspondence and requests for materials should be addressed to T.K. (email: kiba@agr.nagoya-u.ac.jp) or H.S. (email: sakaki@agr.nagoya-u.ac.jp) Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 1 www.nature.com/scientificreports www.nature.com/scientificreports/ long-distance communication is required for plants to balance the growth of photosynthesizing leaves and that 6,33 of carbon consuming roots in response to carbon availability . In various plant species, elevated CO (i.e. high carbon availability) generally results in growth acceleration of both shoots and roots, although the root-to-shoot 25,26,29,34–37 mass ratios are variable depending on species and environmental conditions . CKs have been impli- cated in growth acceleration because cell division and cell differentiation in the meristem are influenced by CKs 26,38 and are often accompanied by CK accumulation . In addition, an increase in tZ-type CKs was detected in the xylem sap of cotton and tobacco plants grown under elevated CO , implying that tZ-type CKs have a role as 25,39 root-to-shoot signals under elevated CO conditions . However, how CKs accumulate and whether the accu- mulation and root-to-shoot translocation of CK is relevant to growth acceleration under elevated CO (i.e. high carbon availability) remains to be determined. In this study, we revealed that enhancement of de novo biosynthesis is responsible for CK accumulation under elevated CO and that the enhancement is triggered by sugars derived from photosynthesis. Detailed growth analyses of mutants defective in cytokinin de novo biosynthesis (ipt3 ipt5 ipt7 and cyp735a1 cyp735a2) revealed that accumulation of tZ-type cytokinins through de novo biosynthesis plays a role in a robust growth response to elevated CO by both shoots and roots. Altogether, these results suggest that the de novo tZ-type CK biosynthe- sis triggered by photosynthetically generated sugars contributes to growth enhancement under elevated CO in Arabidopsis. Results elevated Co increases cytokinin precursor concentrations in shoots and roots. To examine the effects of elevated CO on growth and CK levels, plants were grown under low [280 parts per million by volume (ppmv)] and high CO (780 ppmv) on soil. Two hundred and eighty ppmv is the pre-industrial atmospheric concentration and 780 ppmv is a value close to the median of values predicted at the end of this century . When wild-type Arabidopsis Col-0 were germinated and grown under low or high CO with a 12 h light/12 h dark pho- toperiod for four weeks, high CO -grown plants deposited more biomass and developed more leaf area and 32,41,42 rosette leaves than low CO -grown plants did, as described previously (Supplementary Fig. S1 ). Using the same growth conditions, we analysed changes in the CK concentration following exposure to high CO . Sixteen- day-old Col-0 plants grown in low CO were transferred to low or high CO , and CK concentrations in the whole 2 2 shoot were followed for four days. Under these conditions, significant die ff rences in shoot fresh weight between high and low CO -treated plants became evident from day 4 onward (Fig. 1a). The levels of iP-type CK precursors (iPR and iPRPs) and tZ-type CK precursors (tZR and tZRPs) in high CO -treated shoots increased aer o ft ne day and stayed high until day 4 compared with those of low CO -treated plants (Fig. 1b,c). On the other hand, con- centrations of other CK metabolites including inactivated iP-type CKs (iP7G and iP9G), and tZ-type CKs (tZ7G, tZ9G, tZOG, tZROG, and tZRPsOG) did not change consistently during the period of observation (Fig. 1b,c; Supplementary Table S1). Furthermore, the high CO -treatment did not significantly affect the levels of other plant hormones, including a gibberellin precursor (GA ), IAA, and ABA (Fig. 1d,e; Supplementary Table S1). es Th e results showed that iP-type and tZ-type CK precursors accumulate in the shoot prior to growth enhance- ment at high CO under our experimental conditions. Next, we employed a growth system in which Col-0 seedlings were germinated and grown on half-strength MS (1/2 MS) agar plates placed vertically to allow the analysis of both shoots and roots. Twelve-day-old wild-type seedlings grown under continuous light in low CO were transferred to low or high CO , and the CK concentra- 2 2 tions in shoots and roots were measured aer 6 ft h and 24 h. The basal level of tZ, tZRPs and tZ-N-conjugates in this measurement (Supplementary Table S2) was very different from that in soil-grown plants (Supplementary Table S1). This is possibly due to differences in growth conditions and plant ages, as a similar trend has been observed previously . Accumulation of tZ, and iP-type and tZ-type precursors became evident in shoots and roots as early as 6 h after commencing the high CO -treatment and continued until 24 h, whereas the levels of other CK metabolites did not consistently change (Fig. 2a,b; Supplementary Table S2). 7,43 It is known that the accumulation of CK precursors generally results in increased CK activity . To verify that CK signalling is activated in parallel with precursor accumulation, the expression of immediate-early CK responsive type-A ARR genes was analysed in whole seedlings treated as in Fig. 2a. As expected, ARR4, ARR6, and ARR15 were induced, with the timing of induction similar to that of CK precursor accumulation (Fig. 2c). Since recent studies on plant membrane binding and crystal structure analysis showed that precursors do not bind to 44,45 Arabidopsis CK receptors , one would expect that active CKs (iP and tZ) are accumulated in response to an increase in CK precursor levels. However, active CKs were not always increased significantly in our experiments (for example, Supplementary Tables S1, S2). This lack of significant change in active CK levels has been reported 46,47 previously and we assume that it is because only a fraction of active CKs exists in a compartment where they can be perceived by CK receptors. Taken together, these results indicated that elevated CO resulted in increased CK activity, which is triggered by CK precursor accumulation in shoots and roots. Cytokinin biosynthetic genes, AtIPT3 and CYP735A2, are up-regulated in roots under elevated 7,43 Co . Generally the accumulation of CK precursors reflects increased de novo biosynthesis . The initial step 13,14 of de novo CK biosynthesis is catalysed by IPT , and the key step of tZ-type de novo CK biosynthesis requires 17,18 CYP735A . We examined the expression levels of seven IPT (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, AtIPT8) and two CYP735A (CYP735A1 and CYP735A2) genes in Arabidopsis shoots and roots of Col-0 seedlings incubated at low or high CO from 3 h, an earlier time point than when CK precursor accumulation was observed (up to 9 h). AtIPT4, AtIPT6, and AtIPT8 were not detected in shoots nor roots in our experimental conditions. In shoots, none of the genes examined were affected by high CO except for AtIPT5 that was down-regulated at 9 h (Fig. 3a). In roots, the transcript level of CYP735A2 increased aer 3 ft h and stayed high till 9 h and that of AtIPT3 steadily accumulated aer t ft he onset of high CO treatment (Fig. 3b). On the other hand, the levels of the Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 2 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 1. Effects of high CO on growth and hormone concentrations in soil-grown plants. Shoot fresh weight (a), concentrations of iP-type cytokinin (CK) precursors and inactivated iP-type CKs (b), concentrations of tZ-type CK precursors and inactivated tZ-type CKs (c), IAA concentration (d), and ABA concentration (e) of Col-0 shoots incubated at 280 ppmv or 780 ppmv CO for the indicated periods. Error bars represent standard deviations (a, n = 10; b–e, n = 8). Asterisks indicate statistically significant differences between 280 ppmv CO - and 780 ppmv CO -treated samples at the same exposure time (*p < 0.05; **p < 0.01; Student’s t-test). FW, fresh weight; tZ, trans-zeatin; iP, N -(∆2-isopentenyl)adenine; iP-type CK precursor, sum of iPR and iPRPs; inactivated iP-type CK, sum of iP7G and iP9G; tZ-type CK precursor, sum of tZR and tZRPs; inactivated tZ- type CK, sum of tZ7G, tZZ9G, tZOG, tZROG, and tZRPsOG. The concentrations of all quantified hormones are shown in Supplementary Table S1. other transcripts remained unchanged or showed transient fluctuations (Fig.  3b). Down-regulation of AtIPT5 in both shoot and root might be caused by accumulated CK because AtIPT5 has been reported to be repressed by CK . Since CK levels are determined by the balance between de novo biosynthesis and degradation, we also analysed the expression of genes encoding CK-degrading enzymes, CKX. Among seven CKXs in Arabidopsis, the expression of six genes was detected but none of these genes were down-regulated in shoots and roots under high CO treatment (Supplementary Fig. S2). Rather, the expression of CKX1, CKX4, CKX6, and CKX7 was transiently enhanced, possibly in response to CK accumulation (Supplementary Fig. S2). Similar CK precursor accumulation and induction of AtIPT3 and CYP735A2 were observed when seedlings were grown and treated under 12-h-light/12-h-dark cycles (Supplementary Fig. S3; Supplementary Table S3). These results suggested that the induction of AtIPT3 and CYP735A2 in roots plays a role in iP- and tZ-type CK precursor accumulation under elevated CO . photosynthetically generated sugars induce AtIPT3 and CYP735A2 in roots. Next, we tested the involvement of photosynthesis in the induction of AtIPT3 and CYP735A2 by incubating wild-type seedlings in the dark or by applying the photosynthesis inhibitor DCMU, which blocks electron flow from photosystem II. When seedlings were incubated in the dark or in the light with DCMU at 280 ppmv for 6 h, expression lev- els of AtIPT3 and CYP735A2 were reduced compared to the control (Fig. 4a,b). The induction of AtIPT3 and Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 3 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 2. Cytokinin levels and activity in seedlings exposed to high CO . (a,b) Cytokinin (CK) levels in shoots and roots of Col-0 seedlings exposed to low and high CO . iP-type CK precursor levels (a) and tZ- type CK precursor levels (b) in shoots and roots are presented. (c) Expression levels of type-A ARR genes in Col-0 seedlings exposed to low and high CO . Transcript levels of ARR4, ARR6, and ARR15 were analysed by quantitative RT-PCR. Expression levels were normalized using At4g34270 as an internal control. Twelve-day- old seedlings grown on 1/2 MS agar plates at 280 ppmv were exposed to 280 ppmv (280) or 780 ppmv (780) CO for the indicated periods. Error bars represent standard deviations of three biological replicates. Asterisks indicate statistically significant differences between 280 ppmv CO - and 780 ppmv CO -treated samples at 2 2 6 2 the same exposure time (*p < 0.05; **p < 0.01; Student’s t-test). FW, fresh weight; tZ, trans-zeatin; iP, N -(∆ - isopentenyl)adenine. The concentrations of cytokinin molecular species are shown in Supplementary Table S2. CYP735A2 in response to elevated CO was completely abolished by these treatments (Fig. 4a,b), indicating that photosynthetic activity is required for the maintenance and induction of AtIPT3 and CYP735A2 expression. Elevated CO -treatment reportedly increases endogenous sugar concentrations (e.g. fructose, glucose, and 31,37,49 sucrose), whereas DCMU treatment reduces sugar levels . To examine whether the DCMU-triggered attenuation of AtIPT3 and CYP735A2 induction were caused by lowered levels of sugars, we supplemented DCMU-treated seedlings with sucrose. Sucrose reversed the ee ff ct of DCMU on AtIPT3 and CYP735A2 expres- sion (Fig. 4c,d). We also tested the effects of other sugars on AtIPT3 and CYP735A expression. Seedlings were transferred to agar plates containing metabolizable sugars (sucrose and glucose) or non-metabolizable sugars (sorbitol and mannitol) and were incubated at 280 ppmv CO in the dark. Metabolizable sugars were able to induce AtIPT3 and CYP735A expression (Fig. 4e,f ). On the other hand, non-metabolizable sugars were ineffective in inducing AtIPT3 expression (Fig. 4e). CYP735A2 expression was induced by non-metabolizable sugars but to a much lower extent compared with metabolizable sugars (Fig. 4f ). Since CYP735A2 seems to be moderately acti- vated by osmotic stress , the induction by non-metabolizable sugars is probably due to osmotic effects. 18,21 CYP735A2 is known to be a CK-inducible gene . Thus we tested whether CYP735A2 induction by elevated CO and sugars is the result of accumulated CKs by employing ipt3 ipt5 ipt7 (ipt357) and the cytokinin receptor 51–53 mutants ahk2 ahk3 and ahk3 ahk4 that are defective in CK biosynthesis and signalling, respectively. Elevated CO and sugars induced CYP735A2 expression in the mutants at a level comparable to Col-0 (Fig. 5a,b), indicat- ing that sugars induce this gene independently of CK. These results suggested that AtIPT3 and CYP735A2 are induced in roots under elevated CO by sugars gen- erated in shoots by photosynthesis. Consistent with this, sucrose treatment resulted in an accumulation of CK precursors in shoots and roots (Fig. 4g,h; Supplementary Table S4). Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 4 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 3. Expression of genes involved in cytokinin biosynthesis in shoots and roots upon exposure to high CO . Transcript levels of AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, AtIPT8, CYP735A1, and CYP735A2 were analysed in shoots (a) and roots (b) of Col-0 seedlings by quantitative RT-PCR. Expression levels of AtIPT4, AtIPT6, and AtIPT8 were below the detection limit in shoots and roots. Expression levels were normalized using At4g34270 as an internal control. Twelve-day-old seedlings grown on 1/2 MS agar plates at 280 ppmv were exposed to 280 ppmv (280) or 780 ppmv (780) CO for the indicated periods. Error bars represent standard deviations of three biological replicates. Asterisks indicate statistically significant differences between 280 ppmv CO - and 780 ppmv CO -treated samples at the same exposure time (**p < 0.01; *p < 0.05; 2 2 Student’s t-test). Photosynthetically generated sugars induce cytokinin precursor accumulation irrespective of the nitrate status. It is known that de novo CK biosynthesis is regulated by nitrate in Arabidopsis . Since 55,56 carbon availability is reported to influence nitrate transporter gene expression and nitrate uptake , it is possible that carbon availability ae ff cts CK biosynthesis indirectly through nitrate-related pathways. To test this possibility, we measured CK levels in wild-type seedlings treated with high CO or sucrose, and with and without nitrate. Twelve-day-old seedlings were treated with high CO or sucrose on agar plates containing 10 mM KNO , 10 mM 2 3 NH Cl, or no nitrogen source, and the CK concentrations in the whole seedling were measured after 24 h. The levels of CK precursors increased in all nitrogen conditions tested in response to high CO or sucrose treatment (Fig. 6; Supplementary Tables S5 and S6), suggesting that sugars induce CK precursor accumulation independent of nitrate-related pathways. the ipt3 cyp735a2 mutant still accumulates cytokinin precursors in response to elevated CO . Having shown the relevance of AtIPT3 and CYP735A2 in the elevated CO -enhanced de novo CK biosynthesis, we investigated whether these processes contribute to growth enhancement under elevated CO by generating an ipt3 cyp735a2 double mutant. To this end, 12-day-old seedlings grown on agar plates were incubated at low or high CO for seven days. Fresh weight (FW) was measured before and aer t ft he treatment, and relative growth rate (RGR) was calculated. Growth differences of Col-0 between low CO - and high CO -incubated seedlings 2 2 were clearly observed; the FW and RGR of both the shoot and the root were significantly increased by the high CO treatment (Supplementary Fig. S4a–c). However, no signic fi ant die ff rence in the FW and RGR was observed between the double mutant and WT (Supplementary Fig. S4a–c). To understand this lack of growth phenotype, we analysed changes in iP- and tZ-type precursor CK levels in shoots and roots of the double mutant following exposure to high CO . Under low CO , the double mutant contained significantly reduced levels of iP-type CK 2 2 precursors in shoots (Supplementary Fig. S4d; Supplementary Table S7). However, it accumulated both CK pre- cursors in both organs in response to high CO -treatment, though the levels of accumulation were generally lower compared with WT (Supplementary Fig. S4d,e; Supplementary Table S7), indicating that AtIPT3 and CYP735A2 are not the only factors mediating the elevated CO -induced CK precursor accumulation. Together, these results suggest that the double mutant lacks a growth phenotype because it still is able to accumulate enough CKs for elevated CO -triggered growth enhancement. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 5 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 4. Effects of photosynthesis and sugars on the expression of AtIPT3 and CYP735A2, and cytokinin levels. (a,b) Effects of dark and DCMU on AtIPT3 (a) and CYP735A2 (b) expression in Col-0 roots. Seedlings were exposed to 280 ppmv or 780 ppmv CO under light (Light), under light with 40 µM DCMU (Light + DCMU), or in the dark (Dark). (c,d) AtIPT3 (c) and CYP735A2 (d) expression in Col-0 seedlings treated with 40 µM DCMU in the presence (+) or absence (−) of 90 mM sucrose (Suc) and/or DCMU for six hours. (e,f) Effects of sugars on the expression of AtIPT3 (e) and CYP735A2 (f ) in Col-0 roots. Seedlings were incubated on plates with 90 mM sorbitol (Sorb), mannitol (Man), sucrose (Suc), glucose (Glc), with 45 mM sucrose (Suc45), or without sugar (-sugar) for six hours at 280 ppmv CO in the dark. (g,h) Changes in cytokinin levels in seedlings treated with sucrose. iP-type CK precursor levels (g) and tZ-type CK precursor levels (h) in shoots and roots are presented. Twelve-day-old seedlings grown on 1/2 MS agar plates at 280 ppmv were treated with 45 mM sucrose (+Suc) or without sucrose (−Suc) at 280 ppmv for 24 h. The concentrations of cytokinin molecular species are shown in Supplementary Table S3. Asterisks indicate statistically significant Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 6 www.nature.com/scientificreports www.nature.com/scientificreports/ differences (*p < 0.05; Student’s t-test). Error bars represent standard deviations of four biological replicates. Asterisks indicate statistically significant differences (*p < 0.05; Student’s t-test). Different lower-case letters indicate statistically significant differences as indicated by Tukey’s HSD test (p < 0.05). Expression levels were analysed by quantitative RT-PCR and normalized using At4g34270 as an internal control. t he ipt3 ipt5 ipt7 and cyp735a1 cyp735a2 mutants are impaired in elevated CO -triggered growth enhancement. Since the ipt3 cyp735a2 double mutant still accumulated CKs in response to high CO (Supplementary Fig. S4d,e), we employed higher order CK-biosynthetic mutants, ipt357 and cyp735a1 cyp735a2 (cypDM). The ipt357 mutant lacks three major IPT genes and, thus, has a dramatically reduced ability to de novo synthesize both iP- and tZ-type CKs. The cypDM mutant lacks all CYP735A genes and, thus, is expected to accumulate iP-type CKs but not tZ-type CKs under elevated CO . To verify that elevated CO -induced de novo CK biosynthesis is attenuated in ipt357 and cypDM, the CK concentrations in shoots and roots were measured. Seedlings were grown and treated as in Fig. 2 (24 h high CO treatment). In the ipt357 mutant, the accumulation level of all CKs was relatively low compared with the wild type. The iP-type CK precur - sor concentrations were unae ff cted in shoots and roots, but the levels of tZ-type precursor CKs increased slightly in shoots with a high CO treatment (Fig. 7a,b; Supplementary Table S8). In the cypDM mutant, iP-type precursor CKs accumulated but tZ-type precursor CKs levels were consistently low in shoots and roots under elevated CO (Fig. 7a,b; Supplementary Table S8). These observations confirmed the inability of the mutants to accumulate CKs of the expected types under elevated CO . These results showed that de novo CK biosynthesis, most likely medi- ated by IPT3, IPT5, IPT7, CYP735A1 and CYP735A2, plays an important role in CK accumulation in response to high CO . We then investigated whether these mutants are impaired in growth enhancement under elevated CO . Seedlings were grown and treated on agar plates as in Supplementary Fig. S4, and the FW was measured before and aer t ft reatment, and the RGRs were calculated. In Col-0, the FW and RGR of both the shoot and the root were dramatically increased in response to high CO (Fig. 7c,d; Supplementary Fig. S5). The ipt357 mutant also gained more FW both in shoots and roots in high CO compared with the low CO treatment, but the extent of the 2 2 increase was smaller compared with that of Col-0 (Fig. 7c). RGR analysis revealed that shoots and roots of ipt357 grew faster in high CO than in low CO but at a lower rate compared with those of Col-0, whereas the RGR in 2 2 low CO was similar among all genotypes in this growth system (Fig. 7d). Interestingly, the cypDM mutant dis- played essentially the same growth response defects to elevated CO as the ipt357 mutant (Fig. 7c,d), showing that accumulation of tZ-type CKs is critical for the response. We also analysed the growth response of soil-grown plants. The mutants were germinated and grown on soil together with Col-0 under low or high CO , and shoot growth was analysed at 17 and 31 days aer ft germination (DAG) by measuring dry weight (DW). Note that it was not possible to evaluate root growth in this system. Although Col-0 plants grown in high CO had significantly higher shoot biomass compared with those grown in low CO at the beginning of analysis (17 DAG), they gained more biomass by further growth in high CO (31 2 2 DAG, Supplementary Fig. S6). RGRs between 17 and 31 DAG were significantly higher in high CO -grown Col-0 plants than in the mutants (Fig. 7e). The number of rosette leaves counted on 31 DAG also significantly increased (Fig. 7f ). Although the cypDM mutant gained more biomass under high CO (Supplementary Fig. S6), no sig- nificant change in RGR in response to high CO treatment was observed (Fig. 7e). The RGR of the ipt357 mutant was slightly enhanced by high CO treatment (Fig. 7e). Rosette leaf numbers did not change in the cypDM mutant and were only marginally increased in the ipt357 mutant (5.4 more leaves in the wild type compared with 1.9 in ipt357) in response to high CO (Fig. 7f ). These results show that the ipt357 and cypDM mutants are impaired in the acceleration of shoot growth and development under elevated CO during the growth period examined and that the cypDM mutant, which cannot accumulate tZ-type CKs, is severely compromised. Together, these growth analyses suggest that CK accumulation, especially of the tZ-type, through de novo biosynthesis contributes to robust growth enhancement under elevated CO . photosynthetically generated sugars induce ABCG14 in roots. It has been reported that tZ-type 17,20,21,58 CKs are translocated from root to shoot by the ABCG14 protein to act as shoot growth signals . To get insight into whether root-to-shoot translocation of CKs is relevant to the observed CK accumulation in shoots, ABCG14 expression in roots was investigated (Fig. 8). Interestingly, ABCG14 expression responded to high CO and sugars in a similar manner to that of AtIPT3 and CYP735A2 (Fig. 8). Since ABCG14 has been reported to be CK-inducible , we tested whether the CKs that accumulate in response to elevated CO and sugars are rele- 51–53 vant to ABCG14 induction. The ipt357 and the cytokinin receptor mutants ahk2 ahk3 and ahk3 ahk4 were analysed as in Fig. 5a,b. ABCG14 induction in response to elevated CO and sugars was maintained in these mutants (Fig. 5c,d), indicating that sugars induce this gene independent of CK. Together, these results suggest that root-to-shoot translocation of CKs via ABCG14 might be involved in robust growth enhancement under elevated CO by mediating tZ-type CK accumulation in the shoot. Discussion 7,43,48,59,60 9,61–63 9,64 e a Th vailability of macronutrients such as nitrogen , phosphate and sulphate affects IPT expres- sion as well as CK levels. er Th efore, macronutrient availability has been proposed to regulate CK levels through 9,27,65 de novo biosynthesis to control plant growth and development . Our investigation has revealed another pathway in which photosynthesis-derived sugars regulate de novo CK biosynthesis to control plant growth and development. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 7 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 5. Expression of CYP735A2 and ABCG14 in cytokinin biosynthetic and signaling mutants treated with high CO or sugars. (a,c) Wild-type (Col-0), ipt3 ipt5 ipt7 (ipt357), ahk2 ahk3 (ahk23) and ahk3 ahk4 (ahk34) seedlings grown on 1/2MS agar plates at 280 ppmv CO for 12 days were exposed to 280 ppmv or 780 ppmv for six hours and then roots were harvested. (b,d) ipt357 seedlings were transferred to new plates containing 90 mM of sorbitol (Sorb), mannitol (Man), sucrose (Suc) or glucose (Glc), or without any sugar (-sugar). Roots were harvested aer six h ft ours. Expression levels were analysed by quantitative real-time PCR and normalized using At4g34270 as an internal control. Error bars represent standard deviation of three biological replicates. Different lower-case letters indicate statistically significant differences as indicated by Tukey’s HSD test (p < 0.01). Our study suggests that de novo CK biosynthesis is triggered by photosynthetically generated sugars (Figs 1–6). There are several other reports indicating that sugars induce the expression of genes involved in the de novo 66 67,68 synthesis of CKs. Transcriptome analyses show that glucose and sucrose treatments up-regulate AtIPT3 and CYP735A2. However, how sugars are perceived (as signalling molecules, energy sources or building blocks) to induce the expression of these genes is still not understood. u Th s, it is possible that sugars act indirectly through the signalling pathways of macronutrients because the metabolism of carbon and macronutrients are tightly inter- twined. Although our data suggests that sugars induce CK precursor accumulation independent of nitrate-related pathways (Fig. 6), Kamada-Nobusada et al. reported a pathway in which the internal nitrogen status regulates CK biosynthesis. Since the internal nitrogen status can also be modulated by carbon availability, we cannot rule out the possibility that sugars ae ff ct CK biosynthesis through this pathway. Further studies on sugar and internal nitrogen sensing and signalling mechanisms are required to resolve this problem. In any case, we propose that sugars generated by photosynthesis in shoots directly or indirectly promotes de novo CK biosynthesis. Under our experimental conditions, AtIPT3 was the only gene of the AtIPT family induced by elevated CO and sugars (Figs 3, 4). AtIPT3 expression is also regulated by various environmental signals to control CK levels; 9,43,48,63 increases in nitrogen, phosphate, and sulphate availability induce AtIPT3 expression , whereas drought and Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 8 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 6. Cytokinin levels in wild-type seedlings exposed to high CO or treated with sucrose under different nitrogen nutrient conditions. (a) Cytokinin (CK) levels in wild-type (Col-0) whole seedlings exposed to low or high CO under different nitrogen nutrient conditions. Seedlings were exposed to 280 ppmv (280) or 780 ppmv (780) CO for 24 h on modified 1/2 MS agar plates containing 10 mM KNO (NO ) or 10 mM NH Cl 2 3 3 4 (NH ), or without any nitrogen source (-N). The concentrations of cytokinin molecular species are shown in Supplementary Table S5. (b) Cytokinin levels in wild-type (Col-0) whole seedlings treated with (+Suc) or without (−Suc) 45 mM sucrose for 24 h on modified 1/2 MS agar plates containing 10 mM KNO (NO ) or 3 3 10 mM NH Cl (NH ), or without any nitrogen source (-N). The concentrations of cytokinin molecular species 4 4 are shown in Supplementary Table S6. Twelve-day-old seedlings grown at 280 ppmv were used. Error bars represent standard deviations of four biological replicates. Asterisks indicate statistically significant differences 6 2 (*p < 0.05; Student’s t-test). FW, fresh weight; tZ, trans-zeatin; iP, N -(∆ -isopentenyl)adenine. salt stress repress AtIPT3 expression . Although it remains unclear – with the exception of nitrate – whether these 70,71 signals regulate AtIPT3 expression directly , the available evidence suggests that AtIPT3 functions to integrate and translate various signals in the root into de novo CK biosynthetic activity. We also found that CYP735A2 and ABCG14 are high CO - and sugar-inducible (Figs 3, 4, 5, 8). Although CYP735A2 and ABCG14 are known to be 18,21 CK-inducible genes , we showed that sugars induce these genes independent of CK (Fig. 5). These results indi- cate that CYP735A2 and ABCG14 are controlled by two independent signals: shoot-derived signals (sugars) and root internal cues (root-synthesized CKs) in the response to elevated CO . Thus, CYP735A2 and ABCG14 might act to integrate signals from shoots and roots and translate these signals into tZ-type CKs translocated from root to shoot. However, it remains to be determined whether ABCG14-mediated root-to-shoot translocation activity is regulated at the level of expression or by some other means. In our expression analysis, AtIPT3 and CYP735A2 were the only genes induced under elevated CO among the de novo CK biosynthetic genes (Fig. 3). However, the ipt3 cyp735a2 double mutant still accumulated CKs, although at a lower level than WT (Supplementary Fig. S4d,e). The ipt357 and cypDM mutants were unable to accumulate iP-type and tZ-type CKs, respectively, in response to high CO suggesting that not only AtIPT3 and 2, CYP735A2 but also AtIPT5, AtIPT7, and CYP735A1 are involved in the accumulation. Since these genes were not found to be regulated at the level of transcript accumulation, post-transcriptional regulation might be involved. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 9 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 7. Cytokinin levels and growth of wild-type, ipt3 ipt5 ipt7 and cyp735a1 cyp735a2 seedlings exposed to high CO . (a,b) The concentration of iP-type cytokinin (CK) precursors (a) and tZ-type CK precursors (b) in shoots and roots of wild-type (Col-0), ipt3 ipt5 ipt7 (ipt357) and cyp735a1 cyp735a2 (cypDM) plants exposed to 280 ppmv (280) or 780 ppmv (780) CO for 24 h. Asterisks indicate statistically significant differences (**p < 0.01; *p < 0.05; Student’s t-test). The concentrations of cytokinin molecular species are shown in Supplementary Table S7. (c,d) Fresh-weight (c) and relative growth rate (RGR) (d) of 19-day-old wild type (Col-0), ipt3 ipt5 ipt7 (ipt357), and cyp735a1 cyp735a2 (cypDM) seedlings treated under 280 ppmv (280) or 780 ppmv (780) CO for seven days. (d) RGR was calculated using the fresh weight (FW) data obtained previously (Supplementary Fig. S5) and aer (c) lo ft w or high CO treatment. (e,f) Shoot growth of soil-grown wild-type, ipt3 ipt5 ipt7 and cyp735a1 cyp735a2 plants under low or high CO . (e) Relative growth rates (RGR) of shoots of Col-0, ipt357, and cypDM grown under 280 or 780 on soil. Dry weights of shoots shown in Supplementary Fig. 6b were used to calculate the RGR. Asterisks indicate statistically significant differences (**p < 0.001; Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 10 www.nature.com/scientificreports www.nature.com/scientificreports/ *p < 0.01; not significant (ns), p > 0.01; two-way ANOVA). (f) Rosette leaf number of Col-0, ipt357, and cypDM counted at 31 DAG. Error bars represent standard deviations (a, n = 3; b, n = 3; c, n = 9; f, n = 10) and standard error (d, n = 9; e, n = 9). Lower-case letters denote statistically significant classes (Tukey’s HSD test, p < 0.05). Consistently, it has been reported that AtIPT3 farnesylation modulates this protein’s subcellular localization and enzymatic properties . It should be noted that we cannot exclude that other genes involved in CK biosynthesis, modification, and/or degradation, and/or post-transcriptional regulation might be relevant to the accumulation of CKs. In this study, the role of CKs in growth enhancement under elevated CO was evaluated by analysing the growth of ipt357, a mutant deficient in iP- and tZ-type CKs, and cypDM , a mutant deficient in tZ-type CKs. Both mutants displayed similar growth response defects (Fig. 7), indicating that tZ-type CKs are required for robust growth enhancement of shoots and roots under elevated CO . A reduction in shoot growth acceleration in these mutants is consistent with previous reports that tZ-type CKs and their root-to-shoot translocation act to promote 17,20,21 shoot growth . However, a reduction in root growth acceleration cannot be explained by CK action because 73,74 CKs generally act to repress root growth . This result suggests that CK is not the major determinant of root growth rate. It is plausible that slowed root growth is a consequence of reduced photosynthesis (as sources of energy and building blocks) by smaller shoots, but it is also possible that complex crosstalk might exist between CK and sugars. Here, we revealed that sugar-induced de novo biosynthesis of CKs plays a role in the robust growth enhance- ment under elevated CO . This finding provides some insight into the mechanisms that plants employ to optimise growth in a fluctuating environment. Taking into account that AtIPT3, CYP735A2, and ABCG14 are induced in the root by photosynthetically generated sugars (Figs 3, 4, 5, 8), it is tempting to speculate that there is a systemic growth regulatory mechanism in which photosynthetically generated sugars induce de novo tZ-type CK biosyn- thesis in the root and root-to-shoot translocation of the CK via ABCG14 for growth regulation of the shoot. Materials and Methods Plant material and growth conditions. Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type. The cytokinin biosynthetic triple mutants ipt3 ipt5 ipt7 , the cytokinin receptor double mutants ahk2 53 17 ahk3 and ahk3 ank4 , and the cyp735a1-2 cyp735a2-2 double mutant were characterized previously. The ipt3 cyp735a2-1 and ipt3 cyp735a2-2 double mutants were generated by crosses between the ipt3 ipt5 ipt7 and the cyp735a1-2 cyp735a2-1 mutant, and cyp735a1-2 cyp735a2-2 mutants. For studies on soil-grown plants, strati- fied seeds were sown directly on nutrient-rich soil (Supermix A, Sakata, Japan), and grown in a CO -controlled growth chamber (LPH-0.5P-SH; Nippon Medical & Chemical Instrument) at 280 ppmv or 780 ppmv CO under −2 −1 150 µmol m s fluorescent light (12 h light/12 h dark) at 22 °C. For studies on seedlings, plants were grown on half-strength MS (1/2 MS) agar plates (pH 5.8; 1% agar) placed vertically at 22 °C in the CO -controlled growth −2 −1 chamber at 280 ppmv or 780 ppmv CO under continuous light (120 µ mol m s ) unless otherwise noted. To avoid any chamber effects, we used two growth chambers simultaneously with different CO concentrations and repeated each experiment at least twice with different chamber and CO concentration combinations. Although the data presented are from one representative experiment, similar results were obtained from different chamber and CO concentration combinations. Quantification of plant hormones. Cytokinin level was determined using an ultra-performance liquid chromatograph coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface as described previously . IAA and ABA levels were determined using an ultra-high-performance liquid chromatog- raphy (UHPLC)-electrospray interface (ESI) and a quadrupole-orbitrap mass spectrometer (UHPLC/Q-Exactive; er Th mo Scientific) as described previously . In the results reported, the category iP-type CK precursors comprise iPR and iPRPs; inactivated iP-type CK comprise iP7G and iP9G; tZ-type CK precursors comprise tZR and tZRPs; and inactivated tZ-type CK comprise tZ7G, tZ9G, tZOG, tZROG, and tZRPsOG. Gene expression analysis. Total RNA was extracted from root and shoot samples using the RNeasy Plant Mini kit (QIAGEN) in combination with the RNase-Free DNase set (QIAGEN). Total RNA was used for first strand cDNA synthesis by the SuperScript III First-Strand Synthesis System (Life Technologies) with oligo(dT) primers. Quantitative reverse transcription-PCR (RT-PCR) was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems) with the KAPA SYBR Fast qPCR kit (KAPA Biosystems). At4g34270 was used as an 77,78 internal control because this gene has been shown to be one of the most stably expressed genes in Arabidopsis . Similar results were obtained using other internal control genes (At1g13320 and At2g28390) as described by Czechowski et al. . Primer sets are listed in Supplementary Table S9. DCMU and sugar treatment. For 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) treatment, 8-day-old Col-0 seedlings grown on 1/2 MS agar plates (1% agar) placed vertically under continuous fluorescent light (120 −2 −1 µ mol m s ) at 22 °C in a CO -controlled growth chamber at 280 ppmv CO were sprayed with 40 µ M DCMU 2 2 −2 −1 or mock solution (0.05% ethanol) and exposed to 280 ppmv or 780 ppmv CO under 120 µ mol m s light or in the dark. The DCMU stock solution was 40 mM in 50% ethanol. For DCMU and sucrose co-treatment, seedlings were treated with 40 µ M DCMU or mock solution (0.05% ethanol) and then transferred to 1/2 MS agar plates (1% agar) containing 90 mM sucrose. For sugar treatment, seedlings were transferred to 1/2 MS agar plates (1% agar) containing 90 mM of sorbitol, mannitol, sucrose, glucose, or 45 mM sucrose. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 11 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 8. Effects of high CO , photosynthesis and sugars on the expression of ABCG14 in roots. (a) Expression levels of ABCG14 in roots of Col-0 seedlings exposed to 280 ppmv (280) or 780 ppmv (780) CO for the indicated periods. Treatment was conducted as in Fig. 3. (b) Effects of dark and DCMU on ABCG14 in Col-0 roots. Treatments were conducted as in Fig. 4. (c,d) Effects of sugars on ABCG14 in Col-0 roots. Treatments were conducted as in Fig. 4. Expression levels were analysed by quantitative RT-PCR and normalized using At4g34270 as an internal control. Error bars represent standard deviations of four biological replicates. Asterisks indicate statistically significant differences (*p < 0.05; Student’s t-test). Different lower-case letters indicate statistically significant differences as indicated by Tukey’s HSD test (p < 0.05). High Co and sugar treatment under different nitrogen conditions. Wild-type seedlings were pre-grown for 11 days on modified 1/2 MS agar plates (1% agar) containing 10 mM KNO , 10 mM NH Cl or 3 4 5 mM NH NO as the sole nitrogen source in the CO -controlled growth chamber at 280 ppmv. Seedlings grown 4 3 2 with 10 mM KNO , 10 mM NH Cl or 5 mM NH NO were then transferred to new 1/2 MS agar plates (1% agar) 3 4 4 3 containing 10 mM KNO , 10 mM NH Cl or no nitrogen source, respectively. Aer 24 ft h incubation at 280 ppmv, 3 4 seedlings were subjected to high CO and sugar treatments under the same nitrogen conditions. Growth analysis under low or high CO . For growth analysis of soil-grown plants, stratified seeds were sown directly on nutrient-rich soil (Supermix A, Sakata, Japan), and grown in a CO -controlled growth chamber −2 −1 at 280 ppmv or 780 ppmv CO under 150 µ mol m s fluorescent light (12 h light/12 h dark) at 22 °C and 60% relative humidity. Shoots were harvested at 17 and 31 days aer g ft ermination (DAG) and their dry weights were determined ae ft r drying them in an oven set at 80 °C for three days. Rosette leaf number was counted on 31 DAG. For seedling growth analysis, surface sterilized seeds were sown on 1/2 MS agar plates (1% agar) containing 1% −2 −1 sucrose. Aer s ft tratification, plates were placed vertically in a CO -controlled growth chamber (120 µmol m s continuous fluorescent light, 22 °C) at 280 ppmv. Five-day-old seedlings were transferred to 1/2 MS agar plates (1% agar without sucrose) and grown vertically for another 7 days at 280 ppmv. Then, the 12-day-old seedlings were exposed to 280 ppmv or 780 ppmv CO for seven days. The shoots and roots were separated and their fresh Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 12 www.nature.com/scientificreports www.nature.com/scientificreports/ weights were measured before (Supplementary Figs S4a; S5) and after exposure (Fig.  7c). Relative growth rate (RGR) was calculated from the dry and fresh weights as described elsewhere . To avoid any chamber ee ff cts, we used two growth chambers simultaneously with die ff rent CO concentra- tions and repeated each experiment at least twice with different chamber and CO concentration combinations. Although the data presented are from one representative experiment, similar results were obtained from different chamber and CO concentration combinations. Statistical analysis. Data are given as means ± standard error (SE) or means ± standard deviation (SD) of one representative experiment. In order to examine whether hormone concentration, gene expression, or shoot growth were significantly different between treatments, Student’s t-test, two-way ANOVA, and Tukey’s honest significant difference (HSD) test were performed using KaleidaGraph ver. 4.1 software (Synergy Software). Accession numbers. Sequence data for the genes described in this article can be found in The Arabidopsis Information Resource database (see http://www.arabidopsis.org) under the following accession numbers: CYP735A1 (At5g38450), CYP735A2 (At1g67110), AtIPT1 (At1g68460), AtIPT3 (At3g63110), AtIPT4 (At4g24650) AtIPT5 (At5g19040), AtIPT6 (Ay1g25410), AtIPT7 (At3g23630), AtIPT8 (At3g19160), CKX1 (At2g41510), AtCKX2 (At2g19500), CKX3 (At5g56970), CKX4 (At4g29740), CKX5 (At1g75450), CKX6 (At3g63440), CKX7 (At5g21482), ARR4 (At1g10470), ARR6 (At5g62920), ARR15 (At1g74890), ABCG14 (At1g31770). 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Shinozaki, Y. et al. Ethylene suppresses tomato (Solanum lycopersicum) fruit set through modification of gibberellin metabolism. Plant J. 83, 237–251 (2015). 77. Dekkers, B. J. et al. Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds. Plant Cell Physiol. 53, 28–37 (2012). 78. Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. & Scheible, W. R. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17 (2005). 79. Hoffmann, W. A. & Poorter, H. Avoiding bias in calculations of relative growth rate. Ann. Bot. 90, 37–42 (2002). Acknowledgements We are grateful to Prof. Tatsuo Kakimoto (Osaka Univ.) for providing the ipt3 ipt5 ipt7, ahk2 ahk3, and ahk3 ahk4 mutants. We thank Dr. Ko Noguchi (Tokyo Univ.) for advice concerning statistical analysis. Hormone analyses were supported by the Japan Advanced Plant Science Network. This work was, in part, supported by the Grant- in-Aid for Scientific Research on Innovative Areas (No. 21114005, JP16H01477, JP17H06473, JP18H04793) from the Ministry of Education, Culture, Sports, Science & Technology of Japan. Author Contributions T.K. and H.S. conceived the research. T.K., Y.T. and M.K. conducted the experiments. T.K., Y.T. and M.K. and H.S. analysed and discussed the data. T.K. and H.S. wrote the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44185-4. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO2

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Abstract

www.nature.com/scientificreports opeN sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated Received: 19 October 2018 Co Accepted: 8 May 2019 Published: xx xx xxxx 1,2 2 2 1,2 t akatoshi Kiba , Yumiko t akebayashi , Mikiko Kojima & Hitoshi sakakibara Carbon availability is a major regulatory factor in plant growth and development. Cytokinins, plant hormones that play important roles in various aspects of growth and development, have been implicated in the carbon-dependent regulation of plant growth; however, the details of their involvement remain to be elucidated. Here, we report that sugar-induced cytokinin biosynthesis plays a role in growth enhancement under elevated CO in Arabidopsis thaliana. Growing Arabidopsis seedlings under elevated Co resulted in an accumulation of cytokinin precursors that preceded growth enhancement. In roots, elevated CO induced two genes involved in de novo cytokinin biosynthesis: an adenosine phosphate-isopentenyltransferase gene, AtIPT3, and a cytochrome P450 monooxygenase gene, CYP735A2. The expression of these genes was inhibited by a photosynthesis inhibitor, DCMU, under elevated Co , and was enhanced by sugar supplements, indicating that photosynthetically generated sugars are responsible for the induction. Consistently, cytokinin precursor accumulation was enhanced by sugar supplements. Cytokinin biosynthetic mutants were impaired in growth enhancement under elevated CO , demonstrating the involvement of de novo cytokinin biosynthesis for a robust growth response. We propose that plants employ a system to regulate growth in response to elevated Co in which photosynthetically generated sugars induce de novo cytokinin biosynthesis for growth regulation. Being sessile, plants integrate environmental and internal cues and regulate physiological and morphological processes accordingly to optimize growth and development. Because multicellular higher plants consist of organs with different functions, for example photosynthesizing leaves and roots that absorb water and inorganic nutri- ents, the responses must be coordinated at the whole plant level. Local as well as long-distance signalling between 1–6 cells and organs via signalling molecules such as sugars and plant hormones are vital for this coordination Cytokinins (CKs) are a class of plant hormones that play a central role in the regulation of numerous aspects 7–11 of plant growth and development acting as local and long-distance signals . Naturally occurring CKs are 6 6 2 mostly N -prenylated adenine derivatives; N -(∆ -isopentenyl)adenine (iP), trans-zeatin (tZ) and their conju- 10–12 gates (iP-type and tZ-type CKs, respectively) are the major forms in Arabidopsis thaliana . CK activity is con- trolled at diverse levels, including CK quantity and modification. CK quantity is regulated mostly at the levels of de novo biosynthesis and degradation catalysed by adenosine phosphate-isopentenyltransferase (IPT) and CK 13–16 oxidase/dehydrogenase (CKX), respectively . Side-chain modification to form tZ-type CKs by cytochrome 17,18 P450 monooxygenase CYP735A specie fi s CK activity toward shoot growth . Recently, CK translocation via the 19–21 vascular system was reported to also be important . Shoot-to-root translocation of CK via phloem is critical for root vascular patterning, whereas root-to-shoot translocation via xylem mediated by ABCG14 regulates shoot growth and development. Regulation of CK activity is relevant to various plant developmental processes and 22–28 environmental responses such as shoot apical meristem activity, branching, stress and nutritional responses . Because plants are autotrophs that rely on photosynthesis to gain most of their building materials and 29–32 energy, carbon availability is a major factor defining plant growth and development . To maximize t fi ness, Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, nagoya University, n agoya, 464-8601, Japan. RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi, Yokohama, 230-0045, Japan. Correspondence and requests for materials should be addressed to T.K. (email: kiba@agr.nagoya-u.ac.jp) or H.S. (email: sakaki@agr.nagoya-u.ac.jp) Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 1 www.nature.com/scientificreports www.nature.com/scientificreports/ long-distance communication is required for plants to balance the growth of photosynthesizing leaves and that 6,33 of carbon consuming roots in response to carbon availability . In various plant species, elevated CO (i.e. high carbon availability) generally results in growth acceleration of both shoots and roots, although the root-to-shoot 25,26,29,34–37 mass ratios are variable depending on species and environmental conditions . CKs have been impli- cated in growth acceleration because cell division and cell differentiation in the meristem are influenced by CKs 26,38 and are often accompanied by CK accumulation . In addition, an increase in tZ-type CKs was detected in the xylem sap of cotton and tobacco plants grown under elevated CO , implying that tZ-type CKs have a role as 25,39 root-to-shoot signals under elevated CO conditions . However, how CKs accumulate and whether the accu- mulation and root-to-shoot translocation of CK is relevant to growth acceleration under elevated CO (i.e. high carbon availability) remains to be determined. In this study, we revealed that enhancement of de novo biosynthesis is responsible for CK accumulation under elevated CO and that the enhancement is triggered by sugars derived from photosynthesis. Detailed growth analyses of mutants defective in cytokinin de novo biosynthesis (ipt3 ipt5 ipt7 and cyp735a1 cyp735a2) revealed that accumulation of tZ-type cytokinins through de novo biosynthesis plays a role in a robust growth response to elevated CO by both shoots and roots. Altogether, these results suggest that the de novo tZ-type CK biosynthe- sis triggered by photosynthetically generated sugars contributes to growth enhancement under elevated CO in Arabidopsis. Results elevated Co increases cytokinin precursor concentrations in shoots and roots. To examine the effects of elevated CO on growth and CK levels, plants were grown under low [280 parts per million by volume (ppmv)] and high CO (780 ppmv) on soil. Two hundred and eighty ppmv is the pre-industrial atmospheric concentration and 780 ppmv is a value close to the median of values predicted at the end of this century . When wild-type Arabidopsis Col-0 were germinated and grown under low or high CO with a 12 h light/12 h dark pho- toperiod for four weeks, high CO -grown plants deposited more biomass and developed more leaf area and 32,41,42 rosette leaves than low CO -grown plants did, as described previously (Supplementary Fig. S1 ). Using the same growth conditions, we analysed changes in the CK concentration following exposure to high CO . Sixteen- day-old Col-0 plants grown in low CO were transferred to low or high CO , and CK concentrations in the whole 2 2 shoot were followed for four days. Under these conditions, significant die ff rences in shoot fresh weight between high and low CO -treated plants became evident from day 4 onward (Fig. 1a). The levels of iP-type CK precursors (iPR and iPRPs) and tZ-type CK precursors (tZR and tZRPs) in high CO -treated shoots increased aer o ft ne day and stayed high until day 4 compared with those of low CO -treated plants (Fig. 1b,c). On the other hand, con- centrations of other CK metabolites including inactivated iP-type CKs (iP7G and iP9G), and tZ-type CKs (tZ7G, tZ9G, tZOG, tZROG, and tZRPsOG) did not change consistently during the period of observation (Fig. 1b,c; Supplementary Table S1). Furthermore, the high CO -treatment did not significantly affect the levels of other plant hormones, including a gibberellin precursor (GA ), IAA, and ABA (Fig. 1d,e; Supplementary Table S1). es Th e results showed that iP-type and tZ-type CK precursors accumulate in the shoot prior to growth enhance- ment at high CO under our experimental conditions. Next, we employed a growth system in which Col-0 seedlings were germinated and grown on half-strength MS (1/2 MS) agar plates placed vertically to allow the analysis of both shoots and roots. Twelve-day-old wild-type seedlings grown under continuous light in low CO were transferred to low or high CO , and the CK concentra- 2 2 tions in shoots and roots were measured aer 6 ft h and 24 h. The basal level of tZ, tZRPs and tZ-N-conjugates in this measurement (Supplementary Table S2) was very different from that in soil-grown plants (Supplementary Table S1). This is possibly due to differences in growth conditions and plant ages, as a similar trend has been observed previously . Accumulation of tZ, and iP-type and tZ-type precursors became evident in shoots and roots as early as 6 h after commencing the high CO -treatment and continued until 24 h, whereas the levels of other CK metabolites did not consistently change (Fig. 2a,b; Supplementary Table S2). 7,43 It is known that the accumulation of CK precursors generally results in increased CK activity . To verify that CK signalling is activated in parallel with precursor accumulation, the expression of immediate-early CK responsive type-A ARR genes was analysed in whole seedlings treated as in Fig. 2a. As expected, ARR4, ARR6, and ARR15 were induced, with the timing of induction similar to that of CK precursor accumulation (Fig. 2c). Since recent studies on plant membrane binding and crystal structure analysis showed that precursors do not bind to 44,45 Arabidopsis CK receptors , one would expect that active CKs (iP and tZ) are accumulated in response to an increase in CK precursor levels. However, active CKs were not always increased significantly in our experiments (for example, Supplementary Tables S1, S2). This lack of significant change in active CK levels has been reported 46,47 previously and we assume that it is because only a fraction of active CKs exists in a compartment where they can be perceived by CK receptors. Taken together, these results indicated that elevated CO resulted in increased CK activity, which is triggered by CK precursor accumulation in shoots and roots. Cytokinin biosynthetic genes, AtIPT3 and CYP735A2, are up-regulated in roots under elevated 7,43 Co . Generally the accumulation of CK precursors reflects increased de novo biosynthesis . The initial step 13,14 of de novo CK biosynthesis is catalysed by IPT , and the key step of tZ-type de novo CK biosynthesis requires 17,18 CYP735A . We examined the expression levels of seven IPT (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, AtIPT8) and two CYP735A (CYP735A1 and CYP735A2) genes in Arabidopsis shoots and roots of Col-0 seedlings incubated at low or high CO from 3 h, an earlier time point than when CK precursor accumulation was observed (up to 9 h). AtIPT4, AtIPT6, and AtIPT8 were not detected in shoots nor roots in our experimental conditions. In shoots, none of the genes examined were affected by high CO except for AtIPT5 that was down-regulated at 9 h (Fig. 3a). In roots, the transcript level of CYP735A2 increased aer 3 ft h and stayed high till 9 h and that of AtIPT3 steadily accumulated aer t ft he onset of high CO treatment (Fig. 3b). On the other hand, the levels of the Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 2 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 1. Effects of high CO on growth and hormone concentrations in soil-grown plants. Shoot fresh weight (a), concentrations of iP-type cytokinin (CK) precursors and inactivated iP-type CKs (b), concentrations of tZ-type CK precursors and inactivated tZ-type CKs (c), IAA concentration (d), and ABA concentration (e) of Col-0 shoots incubated at 280 ppmv or 780 ppmv CO for the indicated periods. Error bars represent standard deviations (a, n = 10; b–e, n = 8). Asterisks indicate statistically significant differences between 280 ppmv CO - and 780 ppmv CO -treated samples at the same exposure time (*p < 0.05; **p < 0.01; Student’s t-test). FW, fresh weight; tZ, trans-zeatin; iP, N -(∆2-isopentenyl)adenine; iP-type CK precursor, sum of iPR and iPRPs; inactivated iP-type CK, sum of iP7G and iP9G; tZ-type CK precursor, sum of tZR and tZRPs; inactivated tZ- type CK, sum of tZ7G, tZZ9G, tZOG, tZROG, and tZRPsOG. The concentrations of all quantified hormones are shown in Supplementary Table S1. other transcripts remained unchanged or showed transient fluctuations (Fig.  3b). Down-regulation of AtIPT5 in both shoot and root might be caused by accumulated CK because AtIPT5 has been reported to be repressed by CK . Since CK levels are determined by the balance between de novo biosynthesis and degradation, we also analysed the expression of genes encoding CK-degrading enzymes, CKX. Among seven CKXs in Arabidopsis, the expression of six genes was detected but none of these genes were down-regulated in shoots and roots under high CO treatment (Supplementary Fig. S2). Rather, the expression of CKX1, CKX4, CKX6, and CKX7 was transiently enhanced, possibly in response to CK accumulation (Supplementary Fig. S2). Similar CK precursor accumulation and induction of AtIPT3 and CYP735A2 were observed when seedlings were grown and treated under 12-h-light/12-h-dark cycles (Supplementary Fig. S3; Supplementary Table S3). These results suggested that the induction of AtIPT3 and CYP735A2 in roots plays a role in iP- and tZ-type CK precursor accumulation under elevated CO . photosynthetically generated sugars induce AtIPT3 and CYP735A2 in roots. Next, we tested the involvement of photosynthesis in the induction of AtIPT3 and CYP735A2 by incubating wild-type seedlings in the dark or by applying the photosynthesis inhibitor DCMU, which blocks electron flow from photosystem II. When seedlings were incubated in the dark or in the light with DCMU at 280 ppmv for 6 h, expression lev- els of AtIPT3 and CYP735A2 were reduced compared to the control (Fig. 4a,b). The induction of AtIPT3 and Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 3 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 2. Cytokinin levels and activity in seedlings exposed to high CO . (a,b) Cytokinin (CK) levels in shoots and roots of Col-0 seedlings exposed to low and high CO . iP-type CK precursor levels (a) and tZ- type CK precursor levels (b) in shoots and roots are presented. (c) Expression levels of type-A ARR genes in Col-0 seedlings exposed to low and high CO . Transcript levels of ARR4, ARR6, and ARR15 were analysed by quantitative RT-PCR. Expression levels were normalized using At4g34270 as an internal control. Twelve-day- old seedlings grown on 1/2 MS agar plates at 280 ppmv were exposed to 280 ppmv (280) or 780 ppmv (780) CO for the indicated periods. Error bars represent standard deviations of three biological replicates. Asterisks indicate statistically significant differences between 280 ppmv CO - and 780 ppmv CO -treated samples at 2 2 6 2 the same exposure time (*p < 0.05; **p < 0.01; Student’s t-test). FW, fresh weight; tZ, trans-zeatin; iP, N -(∆ - isopentenyl)adenine. The concentrations of cytokinin molecular species are shown in Supplementary Table S2. CYP735A2 in response to elevated CO was completely abolished by these treatments (Fig. 4a,b), indicating that photosynthetic activity is required for the maintenance and induction of AtIPT3 and CYP735A2 expression. Elevated CO -treatment reportedly increases endogenous sugar concentrations (e.g. fructose, glucose, and 31,37,49 sucrose), whereas DCMU treatment reduces sugar levels . To examine whether the DCMU-triggered attenuation of AtIPT3 and CYP735A2 induction were caused by lowered levels of sugars, we supplemented DCMU-treated seedlings with sucrose. Sucrose reversed the ee ff ct of DCMU on AtIPT3 and CYP735A2 expres- sion (Fig. 4c,d). We also tested the effects of other sugars on AtIPT3 and CYP735A expression. Seedlings were transferred to agar plates containing metabolizable sugars (sucrose and glucose) or non-metabolizable sugars (sorbitol and mannitol) and were incubated at 280 ppmv CO in the dark. Metabolizable sugars were able to induce AtIPT3 and CYP735A expression (Fig. 4e,f ). On the other hand, non-metabolizable sugars were ineffective in inducing AtIPT3 expression (Fig. 4e). CYP735A2 expression was induced by non-metabolizable sugars but to a much lower extent compared with metabolizable sugars (Fig. 4f ). Since CYP735A2 seems to be moderately acti- vated by osmotic stress , the induction by non-metabolizable sugars is probably due to osmotic effects. 18,21 CYP735A2 is known to be a CK-inducible gene . Thus we tested whether CYP735A2 induction by elevated CO and sugars is the result of accumulated CKs by employing ipt3 ipt5 ipt7 (ipt357) and the cytokinin receptor 51–53 mutants ahk2 ahk3 and ahk3 ahk4 that are defective in CK biosynthesis and signalling, respectively. Elevated CO and sugars induced CYP735A2 expression in the mutants at a level comparable to Col-0 (Fig. 5a,b), indicat- ing that sugars induce this gene independently of CK. These results suggested that AtIPT3 and CYP735A2 are induced in roots under elevated CO by sugars gen- erated in shoots by photosynthesis. Consistent with this, sucrose treatment resulted in an accumulation of CK precursors in shoots and roots (Fig. 4g,h; Supplementary Table S4). Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 4 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 3. Expression of genes involved in cytokinin biosynthesis in shoots and roots upon exposure to high CO . Transcript levels of AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, AtIPT8, CYP735A1, and CYP735A2 were analysed in shoots (a) and roots (b) of Col-0 seedlings by quantitative RT-PCR. Expression levels of AtIPT4, AtIPT6, and AtIPT8 were below the detection limit in shoots and roots. Expression levels were normalized using At4g34270 as an internal control. Twelve-day-old seedlings grown on 1/2 MS agar plates at 280 ppmv were exposed to 280 ppmv (280) or 780 ppmv (780) CO for the indicated periods. Error bars represent standard deviations of three biological replicates. Asterisks indicate statistically significant differences between 280 ppmv CO - and 780 ppmv CO -treated samples at the same exposure time (**p < 0.01; *p < 0.05; 2 2 Student’s t-test). Photosynthetically generated sugars induce cytokinin precursor accumulation irrespective of the nitrate status. It is known that de novo CK biosynthesis is regulated by nitrate in Arabidopsis . Since 55,56 carbon availability is reported to influence nitrate transporter gene expression and nitrate uptake , it is possible that carbon availability ae ff cts CK biosynthesis indirectly through nitrate-related pathways. To test this possibility, we measured CK levels in wild-type seedlings treated with high CO or sucrose, and with and without nitrate. Twelve-day-old seedlings were treated with high CO or sucrose on agar plates containing 10 mM KNO , 10 mM 2 3 NH Cl, or no nitrogen source, and the CK concentrations in the whole seedling were measured after 24 h. The levels of CK precursors increased in all nitrogen conditions tested in response to high CO or sucrose treatment (Fig. 6; Supplementary Tables S5 and S6), suggesting that sugars induce CK precursor accumulation independent of nitrate-related pathways. the ipt3 cyp735a2 mutant still accumulates cytokinin precursors in response to elevated CO . Having shown the relevance of AtIPT3 and CYP735A2 in the elevated CO -enhanced de novo CK biosynthesis, we investigated whether these processes contribute to growth enhancement under elevated CO by generating an ipt3 cyp735a2 double mutant. To this end, 12-day-old seedlings grown on agar plates were incubated at low or high CO for seven days. Fresh weight (FW) was measured before and aer t ft he treatment, and relative growth rate (RGR) was calculated. Growth differences of Col-0 between low CO - and high CO -incubated seedlings 2 2 were clearly observed; the FW and RGR of both the shoot and the root were significantly increased by the high CO treatment (Supplementary Fig. S4a–c). However, no signic fi ant die ff rence in the FW and RGR was observed between the double mutant and WT (Supplementary Fig. S4a–c). To understand this lack of growth phenotype, we analysed changes in iP- and tZ-type precursor CK levels in shoots and roots of the double mutant following exposure to high CO . Under low CO , the double mutant contained significantly reduced levels of iP-type CK 2 2 precursors in shoots (Supplementary Fig. S4d; Supplementary Table S7). However, it accumulated both CK pre- cursors in both organs in response to high CO -treatment, though the levels of accumulation were generally lower compared with WT (Supplementary Fig. S4d,e; Supplementary Table S7), indicating that AtIPT3 and CYP735A2 are not the only factors mediating the elevated CO -induced CK precursor accumulation. Together, these results suggest that the double mutant lacks a growth phenotype because it still is able to accumulate enough CKs for elevated CO -triggered growth enhancement. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 5 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 4. Effects of photosynthesis and sugars on the expression of AtIPT3 and CYP735A2, and cytokinin levels. (a,b) Effects of dark and DCMU on AtIPT3 (a) and CYP735A2 (b) expression in Col-0 roots. Seedlings were exposed to 280 ppmv or 780 ppmv CO under light (Light), under light with 40 µM DCMU (Light + DCMU), or in the dark (Dark). (c,d) AtIPT3 (c) and CYP735A2 (d) expression in Col-0 seedlings treated with 40 µM DCMU in the presence (+) or absence (−) of 90 mM sucrose (Suc) and/or DCMU for six hours. (e,f) Effects of sugars on the expression of AtIPT3 (e) and CYP735A2 (f ) in Col-0 roots. Seedlings were incubated on plates with 90 mM sorbitol (Sorb), mannitol (Man), sucrose (Suc), glucose (Glc), with 45 mM sucrose (Suc45), or without sugar (-sugar) for six hours at 280 ppmv CO in the dark. (g,h) Changes in cytokinin levels in seedlings treated with sucrose. iP-type CK precursor levels (g) and tZ-type CK precursor levels (h) in shoots and roots are presented. Twelve-day-old seedlings grown on 1/2 MS agar plates at 280 ppmv were treated with 45 mM sucrose (+Suc) or without sucrose (−Suc) at 280 ppmv for 24 h. The concentrations of cytokinin molecular species are shown in Supplementary Table S3. Asterisks indicate statistically significant Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 6 www.nature.com/scientificreports www.nature.com/scientificreports/ differences (*p < 0.05; Student’s t-test). Error bars represent standard deviations of four biological replicates. Asterisks indicate statistically significant differences (*p < 0.05; Student’s t-test). Different lower-case letters indicate statistically significant differences as indicated by Tukey’s HSD test (p < 0.05). Expression levels were analysed by quantitative RT-PCR and normalized using At4g34270 as an internal control. t he ipt3 ipt5 ipt7 and cyp735a1 cyp735a2 mutants are impaired in elevated CO -triggered growth enhancement. Since the ipt3 cyp735a2 double mutant still accumulated CKs in response to high CO (Supplementary Fig. S4d,e), we employed higher order CK-biosynthetic mutants, ipt357 and cyp735a1 cyp735a2 (cypDM). The ipt357 mutant lacks three major IPT genes and, thus, has a dramatically reduced ability to de novo synthesize both iP- and tZ-type CKs. The cypDM mutant lacks all CYP735A genes and, thus, is expected to accumulate iP-type CKs but not tZ-type CKs under elevated CO . To verify that elevated CO -induced de novo CK biosynthesis is attenuated in ipt357 and cypDM, the CK concentrations in shoots and roots were measured. Seedlings were grown and treated as in Fig. 2 (24 h high CO treatment). In the ipt357 mutant, the accumulation level of all CKs was relatively low compared with the wild type. The iP-type CK precur - sor concentrations were unae ff cted in shoots and roots, but the levels of tZ-type precursor CKs increased slightly in shoots with a high CO treatment (Fig. 7a,b; Supplementary Table S8). In the cypDM mutant, iP-type precursor CKs accumulated but tZ-type precursor CKs levels were consistently low in shoots and roots under elevated CO (Fig. 7a,b; Supplementary Table S8). These observations confirmed the inability of the mutants to accumulate CKs of the expected types under elevated CO . These results showed that de novo CK biosynthesis, most likely medi- ated by IPT3, IPT5, IPT7, CYP735A1 and CYP735A2, plays an important role in CK accumulation in response to high CO . We then investigated whether these mutants are impaired in growth enhancement under elevated CO . Seedlings were grown and treated on agar plates as in Supplementary Fig. S4, and the FW was measured before and aer t ft reatment, and the RGRs were calculated. In Col-0, the FW and RGR of both the shoot and the root were dramatically increased in response to high CO (Fig. 7c,d; Supplementary Fig. S5). The ipt357 mutant also gained more FW both in shoots and roots in high CO compared with the low CO treatment, but the extent of the 2 2 increase was smaller compared with that of Col-0 (Fig. 7c). RGR analysis revealed that shoots and roots of ipt357 grew faster in high CO than in low CO but at a lower rate compared with those of Col-0, whereas the RGR in 2 2 low CO was similar among all genotypes in this growth system (Fig. 7d). Interestingly, the cypDM mutant dis- played essentially the same growth response defects to elevated CO as the ipt357 mutant (Fig. 7c,d), showing that accumulation of tZ-type CKs is critical for the response. We also analysed the growth response of soil-grown plants. The mutants were germinated and grown on soil together with Col-0 under low or high CO , and shoot growth was analysed at 17 and 31 days aer ft germination (DAG) by measuring dry weight (DW). Note that it was not possible to evaluate root growth in this system. Although Col-0 plants grown in high CO had significantly higher shoot biomass compared with those grown in low CO at the beginning of analysis (17 DAG), they gained more biomass by further growth in high CO (31 2 2 DAG, Supplementary Fig. S6). RGRs between 17 and 31 DAG were significantly higher in high CO -grown Col-0 plants than in the mutants (Fig. 7e). The number of rosette leaves counted on 31 DAG also significantly increased (Fig. 7f ). Although the cypDM mutant gained more biomass under high CO (Supplementary Fig. S6), no sig- nificant change in RGR in response to high CO treatment was observed (Fig. 7e). The RGR of the ipt357 mutant was slightly enhanced by high CO treatment (Fig. 7e). Rosette leaf numbers did not change in the cypDM mutant and were only marginally increased in the ipt357 mutant (5.4 more leaves in the wild type compared with 1.9 in ipt357) in response to high CO (Fig. 7f ). These results show that the ipt357 and cypDM mutants are impaired in the acceleration of shoot growth and development under elevated CO during the growth period examined and that the cypDM mutant, which cannot accumulate tZ-type CKs, is severely compromised. Together, these growth analyses suggest that CK accumulation, especially of the tZ-type, through de novo biosynthesis contributes to robust growth enhancement under elevated CO . photosynthetically generated sugars induce ABCG14 in roots. It has been reported that tZ-type 17,20,21,58 CKs are translocated from root to shoot by the ABCG14 protein to act as shoot growth signals . To get insight into whether root-to-shoot translocation of CKs is relevant to the observed CK accumulation in shoots, ABCG14 expression in roots was investigated (Fig. 8). Interestingly, ABCG14 expression responded to high CO and sugars in a similar manner to that of AtIPT3 and CYP735A2 (Fig. 8). Since ABCG14 has been reported to be CK-inducible , we tested whether the CKs that accumulate in response to elevated CO and sugars are rele- 51–53 vant to ABCG14 induction. The ipt357 and the cytokinin receptor mutants ahk2 ahk3 and ahk3 ahk4 were analysed as in Fig. 5a,b. ABCG14 induction in response to elevated CO and sugars was maintained in these mutants (Fig. 5c,d), indicating that sugars induce this gene independent of CK. Together, these results suggest that root-to-shoot translocation of CKs via ABCG14 might be involved in robust growth enhancement under elevated CO by mediating tZ-type CK accumulation in the shoot. Discussion 7,43,48,59,60 9,61–63 9,64 e a Th vailability of macronutrients such as nitrogen , phosphate and sulphate affects IPT expres- sion as well as CK levels. er Th efore, macronutrient availability has been proposed to regulate CK levels through 9,27,65 de novo biosynthesis to control plant growth and development . Our investigation has revealed another pathway in which photosynthesis-derived sugars regulate de novo CK biosynthesis to control plant growth and development. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 7 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 5. Expression of CYP735A2 and ABCG14 in cytokinin biosynthetic and signaling mutants treated with high CO or sugars. (a,c) Wild-type (Col-0), ipt3 ipt5 ipt7 (ipt357), ahk2 ahk3 (ahk23) and ahk3 ahk4 (ahk34) seedlings grown on 1/2MS agar plates at 280 ppmv CO for 12 days were exposed to 280 ppmv or 780 ppmv for six hours and then roots were harvested. (b,d) ipt357 seedlings were transferred to new plates containing 90 mM of sorbitol (Sorb), mannitol (Man), sucrose (Suc) or glucose (Glc), or without any sugar (-sugar). Roots were harvested aer six h ft ours. Expression levels were analysed by quantitative real-time PCR and normalized using At4g34270 as an internal control. Error bars represent standard deviation of three biological replicates. Different lower-case letters indicate statistically significant differences as indicated by Tukey’s HSD test (p < 0.01). Our study suggests that de novo CK biosynthesis is triggered by photosynthetically generated sugars (Figs 1–6). There are several other reports indicating that sugars induce the expression of genes involved in the de novo 66 67,68 synthesis of CKs. Transcriptome analyses show that glucose and sucrose treatments up-regulate AtIPT3 and CYP735A2. However, how sugars are perceived (as signalling molecules, energy sources or building blocks) to induce the expression of these genes is still not understood. u Th s, it is possible that sugars act indirectly through the signalling pathways of macronutrients because the metabolism of carbon and macronutrients are tightly inter- twined. Although our data suggests that sugars induce CK precursor accumulation independent of nitrate-related pathways (Fig. 6), Kamada-Nobusada et al. reported a pathway in which the internal nitrogen status regulates CK biosynthesis. Since the internal nitrogen status can also be modulated by carbon availability, we cannot rule out the possibility that sugars ae ff ct CK biosynthesis through this pathway. Further studies on sugar and internal nitrogen sensing and signalling mechanisms are required to resolve this problem. In any case, we propose that sugars generated by photosynthesis in shoots directly or indirectly promotes de novo CK biosynthesis. Under our experimental conditions, AtIPT3 was the only gene of the AtIPT family induced by elevated CO and sugars (Figs 3, 4). AtIPT3 expression is also regulated by various environmental signals to control CK levels; 9,43,48,63 increases in nitrogen, phosphate, and sulphate availability induce AtIPT3 expression , whereas drought and Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 8 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 6. Cytokinin levels in wild-type seedlings exposed to high CO or treated with sucrose under different nitrogen nutrient conditions. (a) Cytokinin (CK) levels in wild-type (Col-0) whole seedlings exposed to low or high CO under different nitrogen nutrient conditions. Seedlings were exposed to 280 ppmv (280) or 780 ppmv (780) CO for 24 h on modified 1/2 MS agar plates containing 10 mM KNO (NO ) or 10 mM NH Cl 2 3 3 4 (NH ), or without any nitrogen source (-N). The concentrations of cytokinin molecular species are shown in Supplementary Table S5. (b) Cytokinin levels in wild-type (Col-0) whole seedlings treated with (+Suc) or without (−Suc) 45 mM sucrose for 24 h on modified 1/2 MS agar plates containing 10 mM KNO (NO ) or 3 3 10 mM NH Cl (NH ), or without any nitrogen source (-N). The concentrations of cytokinin molecular species 4 4 are shown in Supplementary Table S6. Twelve-day-old seedlings grown at 280 ppmv were used. Error bars represent standard deviations of four biological replicates. Asterisks indicate statistically significant differences 6 2 (*p < 0.05; Student’s t-test). FW, fresh weight; tZ, trans-zeatin; iP, N -(∆ -isopentenyl)adenine. salt stress repress AtIPT3 expression . Although it remains unclear – with the exception of nitrate – whether these 70,71 signals regulate AtIPT3 expression directly , the available evidence suggests that AtIPT3 functions to integrate and translate various signals in the root into de novo CK biosynthetic activity. We also found that CYP735A2 and ABCG14 are high CO - and sugar-inducible (Figs 3, 4, 5, 8). Although CYP735A2 and ABCG14 are known to be 18,21 CK-inducible genes , we showed that sugars induce these genes independent of CK (Fig. 5). These results indi- cate that CYP735A2 and ABCG14 are controlled by two independent signals: shoot-derived signals (sugars) and root internal cues (root-synthesized CKs) in the response to elevated CO . Thus, CYP735A2 and ABCG14 might act to integrate signals from shoots and roots and translate these signals into tZ-type CKs translocated from root to shoot. However, it remains to be determined whether ABCG14-mediated root-to-shoot translocation activity is regulated at the level of expression or by some other means. In our expression analysis, AtIPT3 and CYP735A2 were the only genes induced under elevated CO among the de novo CK biosynthetic genes (Fig. 3). However, the ipt3 cyp735a2 double mutant still accumulated CKs, although at a lower level than WT (Supplementary Fig. S4d,e). The ipt357 and cypDM mutants were unable to accumulate iP-type and tZ-type CKs, respectively, in response to high CO suggesting that not only AtIPT3 and 2, CYP735A2 but also AtIPT5, AtIPT7, and CYP735A1 are involved in the accumulation. Since these genes were not found to be regulated at the level of transcript accumulation, post-transcriptional regulation might be involved. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 9 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 7. Cytokinin levels and growth of wild-type, ipt3 ipt5 ipt7 and cyp735a1 cyp735a2 seedlings exposed to high CO . (a,b) The concentration of iP-type cytokinin (CK) precursors (a) and tZ-type CK precursors (b) in shoots and roots of wild-type (Col-0), ipt3 ipt5 ipt7 (ipt357) and cyp735a1 cyp735a2 (cypDM) plants exposed to 280 ppmv (280) or 780 ppmv (780) CO for 24 h. Asterisks indicate statistically significant differences (**p < 0.01; *p < 0.05; Student’s t-test). The concentrations of cytokinin molecular species are shown in Supplementary Table S7. (c,d) Fresh-weight (c) and relative growth rate (RGR) (d) of 19-day-old wild type (Col-0), ipt3 ipt5 ipt7 (ipt357), and cyp735a1 cyp735a2 (cypDM) seedlings treated under 280 ppmv (280) or 780 ppmv (780) CO for seven days. (d) RGR was calculated using the fresh weight (FW) data obtained previously (Supplementary Fig. S5) and aer (c) lo ft w or high CO treatment. (e,f) Shoot growth of soil-grown wild-type, ipt3 ipt5 ipt7 and cyp735a1 cyp735a2 plants under low or high CO . (e) Relative growth rates (RGR) of shoots of Col-0, ipt357, and cypDM grown under 280 or 780 on soil. Dry weights of shoots shown in Supplementary Fig. 6b were used to calculate the RGR. Asterisks indicate statistically significant differences (**p < 0.001; Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 10 www.nature.com/scientificreports www.nature.com/scientificreports/ *p < 0.01; not significant (ns), p > 0.01; two-way ANOVA). (f) Rosette leaf number of Col-0, ipt357, and cypDM counted at 31 DAG. Error bars represent standard deviations (a, n = 3; b, n = 3; c, n = 9; f, n = 10) and standard error (d, n = 9; e, n = 9). Lower-case letters denote statistically significant classes (Tukey’s HSD test, p < 0.05). Consistently, it has been reported that AtIPT3 farnesylation modulates this protein’s subcellular localization and enzymatic properties . It should be noted that we cannot exclude that other genes involved in CK biosynthesis, modification, and/or degradation, and/or post-transcriptional regulation might be relevant to the accumulation of CKs. In this study, the role of CKs in growth enhancement under elevated CO was evaluated by analysing the growth of ipt357, a mutant deficient in iP- and tZ-type CKs, and cypDM , a mutant deficient in tZ-type CKs. Both mutants displayed similar growth response defects (Fig. 7), indicating that tZ-type CKs are required for robust growth enhancement of shoots and roots under elevated CO . A reduction in shoot growth acceleration in these mutants is consistent with previous reports that tZ-type CKs and their root-to-shoot translocation act to promote 17,20,21 shoot growth . However, a reduction in root growth acceleration cannot be explained by CK action because 73,74 CKs generally act to repress root growth . This result suggests that CK is not the major determinant of root growth rate. It is plausible that slowed root growth is a consequence of reduced photosynthesis (as sources of energy and building blocks) by smaller shoots, but it is also possible that complex crosstalk might exist between CK and sugars. Here, we revealed that sugar-induced de novo biosynthesis of CKs plays a role in the robust growth enhance- ment under elevated CO . This finding provides some insight into the mechanisms that plants employ to optimise growth in a fluctuating environment. Taking into account that AtIPT3, CYP735A2, and ABCG14 are induced in the root by photosynthetically generated sugars (Figs 3, 4, 5, 8), it is tempting to speculate that there is a systemic growth regulatory mechanism in which photosynthetically generated sugars induce de novo tZ-type CK biosyn- thesis in the root and root-to-shoot translocation of the CK via ABCG14 for growth regulation of the shoot. Materials and Methods Plant material and growth conditions. Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type. The cytokinin biosynthetic triple mutants ipt3 ipt5 ipt7 , the cytokinin receptor double mutants ahk2 53 17 ahk3 and ahk3 ank4 , and the cyp735a1-2 cyp735a2-2 double mutant were characterized previously. The ipt3 cyp735a2-1 and ipt3 cyp735a2-2 double mutants were generated by crosses between the ipt3 ipt5 ipt7 and the cyp735a1-2 cyp735a2-1 mutant, and cyp735a1-2 cyp735a2-2 mutants. For studies on soil-grown plants, strati- fied seeds were sown directly on nutrient-rich soil (Supermix A, Sakata, Japan), and grown in a CO -controlled growth chamber (LPH-0.5P-SH; Nippon Medical & Chemical Instrument) at 280 ppmv or 780 ppmv CO under −2 −1 150 µmol m s fluorescent light (12 h light/12 h dark) at 22 °C. For studies on seedlings, plants were grown on half-strength MS (1/2 MS) agar plates (pH 5.8; 1% agar) placed vertically at 22 °C in the CO -controlled growth −2 −1 chamber at 280 ppmv or 780 ppmv CO under continuous light (120 µ mol m s ) unless otherwise noted. To avoid any chamber effects, we used two growth chambers simultaneously with different CO concentrations and repeated each experiment at least twice with different chamber and CO concentration combinations. Although the data presented are from one representative experiment, similar results were obtained from different chamber and CO concentration combinations. Quantification of plant hormones. Cytokinin level was determined using an ultra-performance liquid chromatograph coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface as described previously . IAA and ABA levels were determined using an ultra-high-performance liquid chromatog- raphy (UHPLC)-electrospray interface (ESI) and a quadrupole-orbitrap mass spectrometer (UHPLC/Q-Exactive; er Th mo Scientific) as described previously . In the results reported, the category iP-type CK precursors comprise iPR and iPRPs; inactivated iP-type CK comprise iP7G and iP9G; tZ-type CK precursors comprise tZR and tZRPs; and inactivated tZ-type CK comprise tZ7G, tZ9G, tZOG, tZROG, and tZRPsOG. Gene expression analysis. Total RNA was extracted from root and shoot samples using the RNeasy Plant Mini kit (QIAGEN) in combination with the RNase-Free DNase set (QIAGEN). Total RNA was used for first strand cDNA synthesis by the SuperScript III First-Strand Synthesis System (Life Technologies) with oligo(dT) primers. Quantitative reverse transcription-PCR (RT-PCR) was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems) with the KAPA SYBR Fast qPCR kit (KAPA Biosystems). At4g34270 was used as an 77,78 internal control because this gene has been shown to be one of the most stably expressed genes in Arabidopsis . Similar results were obtained using other internal control genes (At1g13320 and At2g28390) as described by Czechowski et al. . Primer sets are listed in Supplementary Table S9. DCMU and sugar treatment. For 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) treatment, 8-day-old Col-0 seedlings grown on 1/2 MS agar plates (1% agar) placed vertically under continuous fluorescent light (120 −2 −1 µ mol m s ) at 22 °C in a CO -controlled growth chamber at 280 ppmv CO were sprayed with 40 µ M DCMU 2 2 −2 −1 or mock solution (0.05% ethanol) and exposed to 280 ppmv or 780 ppmv CO under 120 µ mol m s light or in the dark. The DCMU stock solution was 40 mM in 50% ethanol. For DCMU and sucrose co-treatment, seedlings were treated with 40 µ M DCMU or mock solution (0.05% ethanol) and then transferred to 1/2 MS agar plates (1% agar) containing 90 mM sucrose. For sugar treatment, seedlings were transferred to 1/2 MS agar plates (1% agar) containing 90 mM of sorbitol, mannitol, sucrose, glucose, or 45 mM sucrose. Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 11 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 8. Effects of high CO , photosynthesis and sugars on the expression of ABCG14 in roots. (a) Expression levels of ABCG14 in roots of Col-0 seedlings exposed to 280 ppmv (280) or 780 ppmv (780) CO for the indicated periods. Treatment was conducted as in Fig. 3. (b) Effects of dark and DCMU on ABCG14 in Col-0 roots. Treatments were conducted as in Fig. 4. (c,d) Effects of sugars on ABCG14 in Col-0 roots. Treatments were conducted as in Fig. 4. Expression levels were analysed by quantitative RT-PCR and normalized using At4g34270 as an internal control. Error bars represent standard deviations of four biological replicates. Asterisks indicate statistically significant differences (*p < 0.05; Student’s t-test). Different lower-case letters indicate statistically significant differences as indicated by Tukey’s HSD test (p < 0.05). High Co and sugar treatment under different nitrogen conditions. Wild-type seedlings were pre-grown for 11 days on modified 1/2 MS agar plates (1% agar) containing 10 mM KNO , 10 mM NH Cl or 3 4 5 mM NH NO as the sole nitrogen source in the CO -controlled growth chamber at 280 ppmv. Seedlings grown 4 3 2 with 10 mM KNO , 10 mM NH Cl or 5 mM NH NO were then transferred to new 1/2 MS agar plates (1% agar) 3 4 4 3 containing 10 mM KNO , 10 mM NH Cl or no nitrogen source, respectively. Aer 24 ft h incubation at 280 ppmv, 3 4 seedlings were subjected to high CO and sugar treatments under the same nitrogen conditions. Growth analysis under low or high CO . For growth analysis of soil-grown plants, stratified seeds were sown directly on nutrient-rich soil (Supermix A, Sakata, Japan), and grown in a CO -controlled growth chamber −2 −1 at 280 ppmv or 780 ppmv CO under 150 µ mol m s fluorescent light (12 h light/12 h dark) at 22 °C and 60% relative humidity. Shoots were harvested at 17 and 31 days aer g ft ermination (DAG) and their dry weights were determined ae ft r drying them in an oven set at 80 °C for three days. Rosette leaf number was counted on 31 DAG. For seedling growth analysis, surface sterilized seeds were sown on 1/2 MS agar plates (1% agar) containing 1% −2 −1 sucrose. Aer s ft tratification, plates were placed vertically in a CO -controlled growth chamber (120 µmol m s continuous fluorescent light, 22 °C) at 280 ppmv. Five-day-old seedlings were transferred to 1/2 MS agar plates (1% agar without sucrose) and grown vertically for another 7 days at 280 ppmv. Then, the 12-day-old seedlings were exposed to 280 ppmv or 780 ppmv CO for seven days. The shoots and roots were separated and their fresh Scientific Repo R ts | (2019) 9:7765 | https://doi.org/10.1038/s41598-019-44185-4 12 www.nature.com/scientificreports www.nature.com/scientificreports/ weights were measured before (Supplementary Figs S4a; S5) and after exposure (Fig.  7c). Relative growth rate (RGR) was calculated from the dry and fresh weights as described elsewhere . To avoid any chamber ee ff cts, we used two growth chambers simultaneously with die ff rent CO concentra- tions and repeated each experiment at least twice with different chamber and CO concentration combinations. Although the data presented are from one representative experiment, similar results were obtained from different chamber and CO concentration combinations. Statistical analysis. Data are given as means ± standard error (SE) or means ± standard deviation (SD) of one representative experiment. In order to examine whether hormone concentration, gene expression, or shoot growth were significantly different between treatments, Student’s t-test, two-way ANOVA, and Tukey’s honest significant difference (HSD) test were performed using KaleidaGraph ver. 4.1 software (Synergy Software). Accession numbers. Sequence data for the genes described in this article can be found in The Arabidopsis Information Resource database (see http://www.arabidopsis.org) under the following accession numbers: CYP735A1 (At5g38450), CYP735A2 (At1g67110), AtIPT1 (At1g68460), AtIPT3 (At3g63110), AtIPT4 (At4g24650) AtIPT5 (At5g19040), AtIPT6 (Ay1g25410), AtIPT7 (At3g23630), AtIPT8 (At3g19160), CKX1 (At2g41510), AtCKX2 (At2g19500), CKX3 (At5g56970), CKX4 (At4g29740), CKX5 (At1g75450), CKX6 (At3g63440), CKX7 (At5g21482), ARR4 (At1g10470), ARR6 (At5g62920), ARR15 (At1g74890), ABCG14 (At1g31770). 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This work was, in part, supported by the Grant- in-Aid for Scientific Research on Innovative Areas (No. 21114005, JP16H01477, JP17H06473, JP18H04793) from the Ministry of Education, Culture, Sports, Science & Technology of Japan. Author Contributions T.K. and H.S. conceived the research. T.K., Y.T. and M.K. conducted the experiments. T.K., Y.T. and M.K. and H.S. analysed and discussed the data. T.K. and H.S. wrote the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44185-4. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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