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A model system of 10–12 day-old, two-branched (2-B) pea (Pisum sativum L. cv. Adagumsky) seedlings was used to study the roles of endogenous auxin indole-3-acetic acid (IAA) and cytokinins (CKs) in the interaction between the shoots. The IAA export activity (IEA) from shoots was 2-fold higher in one-branched (1-B) plants with one shoot removed than in the 2-B plants, while tZ-type cytokinin contents in xylem sap were 4-fold greater in the 1-B plants than in 2-B plants. Exogenous 6-benzylaminopurine introduced into the vascular stream of one shoot enhanced its IEA. Therefore, xylem cytokinin appears to control both growth and IEA in branches. In the hypocotyls of 1-B and 2-B plants, IAA contents were equal in both cases, while the levels of tZ-type cytokinins were different. These data do not agree with the well-supported role of auxin in down-regulating CK content. The observed paradox may be explained by assuming that a steady-state IAA level in the hypocotyls is feedback regulated via xylem cytokinin, which controls the delivery of IAA from the shoots. As a result, the level of IAA in the hypocotyl is most likely maintained at a threshold below which a decrease in auxin content can switch on CK synthesis that will increase xylem cytokinin levels, thereby stabilizing the level of IAA in the hypocotyl. Therefore, our results suggest that correlative inhibition in the 2-B pea system is a function of an IAA/CK feedback loop, in which cytokinin essentially acts as a second messenger for IAA. Keywords: Correlative inhibition, cytokinins, indole-3-acetic acid, Pisum sativum, transport, two-branched pea seedlings, xylem sap. Introduction Plants can be considered to be ‘competing populations of transport from shoots is both an essential attribute of shoot redundant organs’ (Sachs, 2002). Early experiments on a model growth and an important means to affect the growth of other system of two-branched (2-B) pea seedlings, in which one shoot shoots. Accordingly, it was found that the correlatively inhib- was correlatively inhibited by the other, revealed a positive cor- ited branch in 2-B pea seedlings had active IAA efflux carriers relation between shoot growth and export from the shoot of but was unable to directionally transport IAA, while both auxin the auxin indole-3-acetic acid (IAA) (Morris, 1977; Morris and transport and branch growth could be restored by removal Johnson, 1990; Li and Bangerth, 1999). Removal of the domi- of the dominant branch (Morris, 1977; Morris and Johnson, nant shoot leads to increased IAA export from the repressed 1990). By analogy, following decapitation in pea, the axillary shoot; this observation led to the conclusion that active auxin buds are released from auxin-mediated apical dominance, which Abbreviations: ATA, auxin transport autoinhibition; 0-B, branchless plants; 1-B, one-branched plants; 2-B, two-branched plants; BA, 6-benzylaminopurine; CI, cor- relative inhibition; CK, cytokinin; IAA, indole-3-acetic acid; IEA, IAA export activity; iPR, isopentenyladenosine; iP-type CK, isopentenyladenine-type cytokinin; PBS, phosphate-buffered saline; PVPP, polyvinylpolypyrrolidone; RGR, relative growth rate; tZ, trans-zeatin; ZR, zeatin riboside. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which per- mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 2968 | Kotov and Kotova is correlated with the restoration of directional auxin export by an auxin-regulated process. This follows from the fact that subcellular polarization of PIN efflux carriers (Kalousek et al., in Arabidopsis seedlings the expression of the tZ-type CK 2010; Balla et al., 2011; Balla et al., 2016). To explain how auxin biosynthesis gene CYP735A significantly decreased 1 h after transport can be involved in correlative inhibition (CI) among treatment with auxin (Takei et al., 2004). Li and Bangerth the shoots, two possible models were proposed. (1992; 2003) showed that CKs can activate auxin export from According to the auxin transport model, CI between the shoot apex. In Arabidopsis, CK appears to promote the shoots (e.g. two branches) results from competition between expression of IAA17/AXR3 and SHY2, but the roles of these their auxin sources (shoot apices) when auxin transport from signaling genes, especially SHY2, in auxin biosynthesis is a dominant shoot can somehow inhibit auxin transport from not clear (Jones et al., 2010). On the other hand, it has been subordinate shoot(s) without the involvement of any sec- reported that treatment of axillary buds in intact pea plants ond messenger. Initially, it was suggested that such auxin with a synthetic CK, 6-benzylaminopurine (BA), increases transport competition occurs by some kind of auxin trans- the expression of genes encoding auxin influx (PsAUX1) and port autoinhibition (ATA) at the junction of two branches. efflux (PsPIN1) carriers, along with polarization of PsPIN1 (Li and Bangerth, 1999; Bangerth et al., 2000). Applying the protein in the buds (Kalousek et al., 2010). Interestingly, dir- competitive principle in auxin canalization previously pre- ect application of IAA to these buds led to higher PsAUX1 sented by Sachs (1969), Bennett et al. (2006) proposed that and PsPIN1 expression, but did not cause the repolarization the earlier-occurring IAA export from the dominant organ of PsPIN1 proteins (Kalousek et al., 2010), thus suggesting a inhibits later IAA export from subordinate organs due to specific role for CK in polar auxin transport regulation. the saturation of auxin transporters in the polar auxin trans- Many authors have argued that both models are consistent port stream of the main stem. However, auxin transport at with the concept of a hybrid model of branching in which physiological concentrations was not found to be limited by both second messenger and auxin transport effects occur the availability of auxin carriers (Brewer et al., 2009; Renton (Dun et al., 2013; Rameau et al., 2015; Seale et al., 2017). et al., 2012). Later, Prusinkiewicz et al. (2009) showed that the Results obtained in pea seedlings with partially reduced shoot assumption of saturation, while intuitive, is not required, and tips indicated that the growth rates of axillary buds are closely presented a computation model in which competition can correlated with the CK/IAA ratio in internode tissues (Kotov emerge from positive feedback between auxin flux and polar - and Kotova, 2000), data that might support the hybrid model ization of active auxin transport according to Mitchison’s of apical dominance. In the present study, using 2-B pea seed- equations (Kramer, 2009). This model successfully explained lings as a model system, we provide evidence that CI between CI specificity in various branched Arabidopsis mutants on shoots is a direct consequence of the interactions between the basis of specificity in their PIN-dependent IAA trans- IAA and CK and, hence, xylem CK appears to control both port, and accounted for ‘the apparent paradox that increased growth and IAA export from branches. branching can be achieved either by decreasing accumulation of active PINs on the membrane, as in tir3, or by increasing accumulation, as in the max mutants’ (Prusinkiewicz et al., Materials and methods 2009). Despite the above arguments in favor of an auxin Production of two-branched, one-branched, and branchless transport hypothesis, at the molecular level, flux-based prin- pea plants ciples of auxin efflux carrier polarization have still not been Seeds of Pisum sativum L. cv. Adagumsky were soaked and ger- elucidated (Kramer, 2009; Tanaka et al., 2013), and it is not minated between two layers of filter paper moistened with distilled known how auxin in the polar auxin transport stream is able water for 3 days at 25 °С in the dark (Kotov and Kotova, 2015). to inhibit auxin export from branches (Bennett et al., 2014). After 4 days, the epicotyl was removed from the cotyledonary node The second messenger model proposed that basipetal IAA to induce the growth of two axillary buds at this node. Forty seed- transport from one branch can inhibit IAA export from the lings were grown in tap water in a 0.5 l vessel at 20 ± 1°С under –2 –1 fluorescent light providing an intensity of 135 μmol m s , with a other, acting through cytokinins (CKs). CK synthesis or 14 h day/10 h night photoperiod. Four days later, the seedlings with xylem content can be regulated by negative feedback from two equal cotyledonary shoots (2-B) were selected and transferred IAA exported from the shoots (Bangerth, 1994; Li et al., to 0.4 l trays (6 × 16 × 6 cm) with tap water, with nine plants per tray, 1995; Kotova et al., 2004; Nordstrom et al., 2004), and in under the same conditions. To obtain one-branched (1-B) or branch- this case CKs function as intermediary signals by regulat- less (0-B) plants, one or two shoots were removed from 8-, 10-, or 14-day-old 2-B plants. Two days later, in the 10-, 12-n or 16-day-old ing auxin export from the branches (Li and Bangerth, 1992; plants, samples of hypocotyl tissues, xylem sap, and shoot diffusates 2003). Consistent with this model, the expression of two CK were taken depending on the experiment. biosynthesis genes, PsIPT1 and PsIPT2, in pea internodes was shown to be activated by decapitation and suppressed Shoot growth measurements by apical application of exogenous auxin (Tanaka et al., 2006; Dun et al., 2012). The precise mechanism of an auxin- For shoot growth analysis, shoot lengths of 2-B and 1-B plants produced on day 8 were measured daily during days 8–11 using a dependent repression of IPT genes remains unclear, and horizontal calibrated stereomicroscope. The shoot relative growth auxin-responsive factors such as SHY2/IAA3 and Aux/IAA9 rate (RGR ) at each day i was calculated as RGR =[(L – (i) (i) (i+1) (Wang et al., 2005; Weijers et al., 2005; Chapman and Estelle, L )/L ]/24 h, where L and L were shoot lengths on day i and (i) (i) (i) (i+1) 2009; Sakakibara, 2010) may be involved in this process. In 24 h later, respectively. The mean RGR value and standard error turn, the synthesis of trans-zeatin (tZ)-type CKs is most likely were determined from nine biological replicates for each treatment. Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 Auxin–cytokinin control of correlative inhibition | 2969 Hormonal treatments calculated by linear regression analysis (SigmaPlot 11 for Windows 7) conducted on the 5–10 data points for each treatment. The shoot For vascular supply, in 10-day-old 2-B plants a thread submerged IEA was expressed as the amount of diffusible IAA in the shoot tip in 1.5 ml of 1 µM or 10 µM BA (Serva, Heidelberg, Germany) or having a standard weight of 45 mg, and calculated using the lin- control solutions (all containing 0.1 % DMSO) was extended with ear regression equations in SigmaPlot 11 with standard error of the a needle through the stem base of one cotyledonary shoot (Fig. 3) estimate. according to Gomez-Roldan et al. (2008). In some experiments, 2 h diffusates were collected from whole 10-day-old, 2-B and 0-B seedlings that were previously de-rooted Estimation of IAA export activity in the cotyledonary shoots 1.5 cm below the cotyledonary node (Fig. 2D, right panel) by immersing the basal end in 150 µl of distilled water or in 0.5 mM Export of IAA from shoot apices (diffusible IAA) was measured by EDTA (pH 7.05) to measure possible phloem IAA transport (Kotov a diffusate method. Unless otherwise stated, each shoot tip (shoot and Kotova, 2000; Jager et al., 2007). apical bud with uppermost leaf) (Fig. 1A, B) was cut off and its basal end was placed individually into 120–150 µl of distilled water. The samples were incubated in a humid box for 2 h at 20 ± 1 °С Collection of xylem sap and estimation of acropetal CK in darkness, and then IAA-containing diffusates from each separ- transport ate shoot tip were stored at –20 °C before analysis. The shoot tips were cut off from the underlying internode and weighed. The dif- Xylem sap was collected in 10-day-old 2-B, 1-B, and 0-B plants by a fusates from 5–6 shoot tips of similar weight (not exceeding ±5 mg) vacuum-suction technique (Kotov and Kotova, 2015). In short, root were pooled and subjected to ELISA for measuring diffusible IAA. plus hypocotyl or root alone was cut off immediately below or 1.5 cm The correlation of IAA amount with average shoot tip weight was below the cotyledonary node, respectively, and a flexible silicone Fig. 1. (A, B) Influence of the medium on the time course of IAA diffusion out of excised shoot tips (diffusible IAA) from 10-day-old two-branched (2-B) and one-branched (1-B) pea seedlings. (C) Effect of defoliation on IAA export from shoots of 12-day-old 1-B plans. Schematic views of the experimental set-up for IAA export measurements is shown on the left. The 2-B plants were prepared by removing the epicotyl from 4-day-old seedlings to induce the growth of two axillary buds at the cotyledonary node. 1-B plants were then produced by removing one branch from 2-B plants at day 8 or 10. After 2 days, diffusible IAA from excised shoot tips of 10- or 12-day-old 2-B and 1-B plants was collected into 120–150 µl of aqueous medium stepwise during an incubation period of 2 h (A) or four 1 h periods (B, C), and IAA was measured by ELISA. Data are expressed as the mean ±SE of three samples pooled from six shoot diffusates. BA, 6-benzylaminopurine; K-Pi, potassium phosphate buffer. (This figure is available in colour at JXB online.) Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 2970 | Kotov and Kotova of –0.6 to –0.8 MPa. Xylem sap samples collected from each plant were transferred into pre-weighed 0.5 ml eppendorf vials, weighed, and stored at –20°С. For CK analysis, xylem sap collected from the 2–4 plants exud- ing similar amounts of sap were pooled, diluted with distilled water (1:2), and subjected to ELISA with anti-zeatin riboside (ZR) or anti- isopentenyladenosine (iPR) antibodies. Results were expressed in ZR or iPR equivalents. For each treatment, the amount of tZ-type CK measured in 12–18 samples was correlated with the correspond- ing sap flow rate using linear regression (SigmaPlot 11 for Windows –1 7). As transpiration rates (<0.01 µl s ) are very low in 10-day-old pea plants (Kotov and Kotova, 2015), the real concentration of tZ- type CK in xylem sap in vivo (the acropetal transport of CK) could be approximated as the concentration of CK in xylem sap at near- zero levels of sap flow rate. The values presented in the summary diagram of the xylem CK levels in Fig. 4C were extrapolated to null sap flow rate by linear regression and calculated with standard error of the estimate. For column chromatography of tZ-type CK, xylem sap was buff- ered with phosphate-buffered saline (PBS; 0.01 M sodium phosphate buffer with 0.15 M NaCl, pH 7.4). A sample aliquot of 500 μl was applied to a 1.5 ml Toyopearl HW 40F column (Toso, Japan), which was then eluted with PBS (Kotov and Kotova, 2000; 2015), and 0.5 ml fractions were analyzed by ELISA with anti-ZR antibodies. Collection and preparation of hypocotyl samples for IAA and CK analyses Hypocotyl segments (2–3 mm long) from 10-day-old 2-B, 1-B, or 0-B pea seedlings were cut 2–3 mm below the cotyledons, frozen, and stored in liquid nitrogen. Using four replicates per treatment (10 segments per replicate, one segment per plant), samples were extracted and purified for ELISA (Kotov and Kotova, 2000). Briefly, each frozen tissue sample (150–250 mg) was homogenized and extracted overnight at 4 °С with 5 ml of 80% (v/v) methanol con- –1 –1 taining 200 mg l butylated hydroxytoluene and 100 mg l ascorbic acid, with shaking. The extract was filtered and passed through a 50 mg Porolas-TM cartridge (hydrophobic sorbent with an average pore size of 4.24 nm, 50–250 μm particle size, OmskKhimProm, Russia). The filter and cartridge were washed with 0.5 ml or 1 ml of 80 % methanol, respectively, and the washings were combined with the corresponding eluents, giving a final volume of 6.5 ml per sample. These extracts were evaporated down to the aqueous resi- due (~0.5 ml per sample) at 40 °C under a stream of nitrogen. The residue was diluted to a final volume of 2 ml of 50 mM potassium phosphate buffer (pH 3.0) and this aqueous sample was purified on Fig. 2. IAA export from excised shoot tips as a function of the shoot tip a column (internal diameter 8 mm) containing 1 ml of pre-swollen weight for (A) 10-day-old, (B) 12-day-old, and (C) 16-day-old 2-B and 1-B polyvinylpolypyrrolidone (PVPP, Sigma, USA) to remove poly- pea seedlings. A schematic of the experimental set-up is shown on the phenolic compounds. The PVPP column was washed with 6 ml of left. (D, left side) Age dependence of IAA export activity from 2-B and 1-B the same buffer. The washes were combined with the column eluent shoot tips with a standard weight of 45 mg. (D, right side) IAA transport to obtain the total CK fraction (8 ml). After washing the PVPP col- from intact 2-B and 0-B de-rooted seedlings collected for 2 h into distilled umn with 1 ml H O and 0.5 ml PBS (pH 7.4), IAA was eluted with water or 0.5 mM EDTA, pH 7.05 (150 µl per plant). Plants were prepared 3 ml PBS and the obtained IAA fractions were subjected to ELISA as in Fig. 1 and diffusible IAA from each excised shoot tip was collected with anti-IAA antibodies. into 120–150 µl of distilled water for 2 h. Diffusates from six shoot tips with A 1 ml aliquot of the total CK fraction was neutralized to pH similar weight (≤5 mg) were combined for ELISA. In (A–C), the data were 7.4 with 200 μl of 200 mM K PO , and subjected to ELISA with 3 4 analyzed using linear regression (SigmaPlot 11 for Windows 7); in (D, left anti-iPR antibodies. The remaining 7 ml was used to isolate the frac- side), values were interpolated by linear regression of existing data points tion containing both zeatin and ZR (Z/ZR) from the total CKs on in (A–C) ±SE. In (D, right side), data are expressed as the mean ±SE of a C18 column (300 mg sorbent was combined from three Amprep three samples pooled from five shoot diffusates. In (D), asterisks indicate С18 100 mg columns (# RPR 1900, Amersham, UK) into one col- statistically significant differences compared with 2-B plants: *P<0.05; umn). For this, the sample was loaded on to the C18 column, and **P<0.01, ***P<0.001. (This figure is available in colour at JXB online.) after column washing with 2 ml 50 mM potassium phosphate buffer (pH 3.0) and 2 ml H O, Z/ZR was eluted with 5.5 ml 0.2 N acetic acid in 20% methanol into a Porolas-TM cartridge (0.8 ml). From tube (length 17 mm, internal diameter 2–2.5 mm) was stretched over this cartridge Z/ZR was collected with 4 ml methanol, evaporated the stump of the hypocotyl or root. The other end of the tube was to dryness at 40 °C under a stream of nitrogen, redissolved in 1 ml attached to a xylem sap receiver (1 ml pipette tip) connected to an PBS, and stored at –20°С prior to ELISA. Aliquots of the obtained air duct system in which negative pressure was generated by a water- Z/ZR fractions were chromatographed on a 1.5 ml Toyopearl HW suction pump controlled by a manometer and manually adjusted 40F column with PBS (pH 7.4) as an eluent (as described above). using an inlet valve. Suction was applied for 30 min at a pressure Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 Auxin–cytokinin control of correlative inhibition | 2971 The recoveries of IAA, ZR, and iPR standards after the purification plants (Fig. 1A). Diffusion from both 1-B and 2-B shoots was procedure were 88%, 85%, and 84%, respectively. No compensation stable only throughout the first 2 h of the incubation (Fig. 1B) was made for their losses in our experiments. and decreased with time, coming to a stop after 6 h (Fig. 1A). Diffusion into 60 mM potassium phosphate buffer (pH 6.8) ELISA of IAA, Z-type, and isopentenyladenine-type cytokinins (Johnson and Morris, 1989; Prusinkiewicz et al., 2009; Balla Antiserum against Z/ZR, iP/iPR, and IAA was produced by immu- et al., 2011) resulted in a similar decrease in IAA diffusion nizing rabbits with bovine serum albumin conjugated with ZR, iPA, (Fig. 1B). The use of phosphate buffer resulted in a slightly or IAA, respectively (Kotov and Kotova, 2000). Antigens for coat- suppressed IAA diffusion for the first hour, relative to the use ing plates used ovalbumin as the carrier protein, and dihydrozea- of distilled water (Fig. 1B). This effect might be explained by tin riboside was conjugated with ovalbumin for heterologous assay initial neutralization of the apoplastic solution changing the of Z/ZR (Kotov and Kotova, 2015). Competitive indirect ELISA was performed on 96-well microtiter plates (Costar # 9018, USA) diffusion of IAA according to the chemiosmotic model for (Kotov and Kotova, 2000; 2015). For the detection of primary anti- auxin transport (Friml, 2010). bodies, horseradish peroxidase-conjugated sheep anti-rabbit IgG To check whether the observed decline in IAA diffusion was used (Medgamal, Moscow, Russia). Non-specific interference was not caused by damage to the stem tissue during the long was tested by adding known amounts of a hormone standard into period of contact with aqueous media, we recut the basal end the sample (Neuman and Smit, 1990). The added/found ratio was within 100 ± 15% for all types of sample (diffusates, xylem sap, and of the shoot tip every 2 hours. This procedure did not affect hypocotyl tissue). IAA export (Fig. 1A). The addition of 1% sucrose to the dif- fusion media transiently promoted IAA diffusion (Fig. 1B). The addition of 10 µM BA, which activates IAA export/syn- Statistical analysis thesis in shoot apices (Li and Bangerth, 1992; 2003), increased Regression analysis (Figs 2 and 4) was performed with SigmaPlot 11 the initial IAA diffusion rate from the shoot tips, although for Windows 7. Where comparisons were made between two treat- ments (Figs 2–5), data were subjected to one-way ANOVA using all rates declined over time (Fig. 1A). It can be concluded SigmaPlot 11 for Windows 7. The variance was analyzed by Tukey’s that the decline of IAA export from excised shoots does not significant difference, with the level of significance set to P<0.05. appear to be affected by the medium or caused by assimilate or CK deficiency. We can speculate that the pool of transportable IAA con- Results tained in shoot cells, at a constant rate of replenishment, Two-branched seedlings as a model system for could be quickly exhausted by a greatly enhanced capacity investigating correlative inhibition to transport IAA, thus leading to the observed drop in IAA diffusion from the shoot tips. Therefore, the IAA export These experiments were mostly conducted using 10- to measured in diffusates may most likely be related to the IAA 12-day-old pea seedlings bearing two equal growing cotyle- that was previously accumulated in auxin-transporting cells donary shoots (2-B). In 1-B plants one shoot was removed rather than to the real values of IAA export occurring in on day 8. Shoot RGR (over a period of 24 h) of 2-B plants vivo. Presumably, IAA concentrated in the transport pool can compared with 1-B plants on day 9 was 0.55 ± 0.03 versus determine the rate of auxin transport, which, in turn, must 0.67 ± 0.04 (n=9; P<0.05), while the shoot tip weights (shoot be equivalent to the rate of refilling of this pool by IAA syn- apical bud with uppermost leaf, see Fig. 1) on day 12 were thesis; hence, auxin synthesis, the transport pool, and export/ 63.4 ± 4.0 mg versus 98.3 ± 4.2 mg (n=10; P<0.001). These transport should be dynamically interrelated. data show that CI in our system was not strong; the removal Compared with the IAA diffusion from shoots of 10-day- of one branch resulted in a 22% increase in RGR in the old plants, the diffusion from excised 12-day-old shoots con- remaining branch. An explanation for this is that 2-B plants taining a mature leaf and shoot tip was stable during the first have synchronously growing branches, in contrast to previous 3 h (Fig. 1C). The removal of the leaf did not affect the initial experiments on plants with two unequal shoot systems (dom- IAA diffusion rate but reduced the period of stable IAA dif- inant/inhibited branches) (Morris, 1977; Li and Bangerth, fusion to 2 h (Fig. 1C), as was observed for 10-day-old shoot 1999). However, as will be seen, the differences observed in tips (Fig. 1B, open squares). Mature leaves do not appear to IAA and CK transport and content between 2-B and 1-B be involved. Therefore, the diffusable IAA collected into dis- plants were much greater, and the explanation for this became tilled water from the shoot tip during the initial 2 h stable the main objective of our study. period of diffusion can objectively be characterized as active IAA export from the whole shoot. In the current study, this Estimation of IAA export from shoots 2 h testing procedure was used as the basis for estimation of shoot IEA. IAA export from shoots was determined by a diffusate method, and we specify this export as IEA (Li and Bangerth, 1999). To optimize the sampling of diffusible IAA, we tested Mutual inhibition of IAA export from shoot between two the relevance of some factors in the course of diffusible IAA branches of pea collection. As mentioned above, the removal of one branch in 10- and Shoot diffusates into water at pH 6 (Kotov and Kotova, 12-day-old 2-B plants (to produce 1-B plants) led to approxi- 2000; Kojima et al., 2002) revealed a 2-fold difference in the mately double the IAA export from the remaining branch, amount of diffusible IAA between 1-B and 2-B 10-day-old Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 2972 | Kotov and Kotova thereby compensating for the loss of one IAA source their growth was close to the maximum limit. Hence, even (Fig. 2A, B, D). This indicates CI of IEA, and of growth, a small increase in the shoot growth activity would require between the two branches in our pea plant model. In 2-B a substantial increase in CK supply, and possibly the satur- plants with unequal shoots, the IEA of the dominant shoot ation in the growth response occurs before the saturation of was of an intermediate level between that of the shoots of the IEA response. Nevertheless, in the experiments with 0 or 2-B plants with equal growing shoots and 1-B plants (Fig. 2B, 1 µM BA treatments, the shoots differing significantly in IEA D). The IEAs of shoots from 16-day-old 2-B and 1-B plants had similar fresh weights. Such results may be a consequence were approximately the same (Fig. 2C, D), with values close of the low sensitivity of the method for assaying growth in to those of the shoots of 10- and 12-day-old 2-B plants. These this case, in comparison with the early estimations of shoot data suggest an age-dependent loss of CI in two-shoot plants. RGR or measurements made 4 days after treatment instead It appears that in 10- and 12-day-old plants, irrespective of the 2 days used here. Previous work has shown the activa- of shoot number, the total export of IAA from shoots was tion of shoot IEAs by exogenous CK (Li and Bangerth, 1992; equal (Fig. 2A, B, D). To examine whether the shoot IEA 2003), and so we further examined in detail the involvement value is the result of shoot IAA transported from the shoots, of endogenous CKs in the establishment of CI in two-shoot rather than just a response to loss of a shoot, the IAA export plants. from one shoot of 12-day-old 2-B plants was stimulated by It is well established that the roots play a major role in reg- BA introduced to the vascular stream. The increase in IEA ulating shoot branching, and we tested the possibility of root of the BA-treated shoot was accompanied by a decrease in CK being regulated by shoot-derived auxin. Unexpectedly, IEA from the untreated shoot (Fig. 3). Interestingly, short- the levels of diffusible IAA obtained from intact 2-B and term incubation of cut shoots in 10 µM BA solution was less branchless (0-B) seedlings previously de-rooted 1.5 cm below effective in increasing IEA (increase of ~25%; see Fig. 1A) the cotyledons were low and similar (Fig. 2D, right, in H O). than the long-term experiments where the vascular supply of Thus, IAA transport from the shoots did not follow into the 10 µM BA resulted in a 4-fold increase in IEA (Fig. 3), sug- root. The use of 0.5 mM EDTA to estimate IAA translocated gesting that the CK effect needs time to develop. along the phloem (Kotov and Kotova, 2000; Jager et al., 2007) A 10-fold increase in BA concentration resulted in only a gave similar results (Fig. 2D, right, in EDTA). This is consist- 27% increase in shoot apex weight, even though it induced ent with observations that decapitation did not change the a 2-fold increase in IAA export (Fig. 3). Similarly, the IAA content (Kotova et al., 2004) in pea roots, and that the removal of one branch led to a 2-fold increase in the IEA expression of the IAA-regulated genes PsIPT1 and PsIPT2 of the remaining branch (Fig. 2A, B, D), but only slightly was not detected in the roots after decapitation (Tanaka et al., activated its growth (see above). It is possible that the shoots 2006). It was previously shown in 2-B pea plants that radi- in our 2-B model plants were not deficient in CK supply and olabeled IAA applied on the shoot apex can be transported Fig. 3. Effects of 6-benzylaminopurine (BA) treatment on the IAA export activity of shoots of 12-day-old, 2-B pea seedlings. BA was supplied into the vascular stream of one shoot (treated shoot; T). NT, non-treated shoot. A schematic of the experimental set-up is shown on the left. 2-B plants were prepared as for Fig. 1. For vascular supply, a thread submerged in 1.5 ml of 0, 1, or 10 µM BA solution (all containing 0.1% DMSO) was passed with a needle through the stem of one shoot at its base. Two days later, the IEA of T and NT shoots was determined by collecting 2 h diffusates from excised shoot tips into distilled water and subjecting the diffusates to ELISA. For each treatment, the amount of IAA measured in 5–6 diffusate samples pooled from five shoot tips was correlated with the average weight of corresponding shoot tips using linear regression (SigmaPlot 11 for Windows 7). IEA values estimated as diffusible IAA in the shoot tips with a standard weight of 45 mg were calculated using the linear regression equations (±SE). Asterisks indicate statistically significant differences compared with control (0 µM BA): *P<0.05, **P<0.01. The fresh weights of shoot apical buds were measured at 12 days (2 days after treatments) and are shown above the points as mg per bud ±SE (n=30); **P<0.01. (This figure is available in colour at JXB online.) Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 Auxin–cytokinin control of correlative inhibition | 2973 and accumulated into roots (Morris, 1977; Li and Bangerth, agreement with other literature data for pea plants (Beveridge 1999). However, in these studies, root was defined as all plant et al., 1997; Foo et al., 2007; Kotov and Kotova, 2015). parts below the cotyledons, which included the hypocotyl as Gradual removal of shoots from 2-B pea plants resulted in well as the root itself. pronounced increases in tZ-type CKs in xylem sap obtained from the root plus hypocotyl compared with sap obtained from the root itself (Fig. 4A–С). There was a small but significant Number of branches in pea model plants determines effect of shoot-derived auxin on root CK synthesis (Fig. 4C); the xylem CK levels due to activity of the hypocotyl however, this was much smaller than the effect in the hypocotyl. ELISA of xylem sap from 10-day-old 2-B and 0-B pea plants Therefore, hypocotyl, rather than root, is the major determin- showed low relative contents of isopentenyladenine (iP)- ant involved in the response to branch reduction through the type CKs, comprising 3–10% of tZ-type CKs, and showing regulation of xylem CK levels. This conclusion is consistent ZR to be a dominant tZ-type CK (Fig. 4D). Our data are in with the observations showing that shoot IAA is apparently Fig. 4. Concentrations of tZ-type CKs in the xylem sap from root plus hypocotyl (A) or from root alone (B) as a function of sap flow rate in 10-day-old 2-B, 1-B, and 0-B pea seedlings. (C) Summary diagram of xylem CK levels extrapolated to null sap-flow rate from data in (A) and (B) with the ratios of 2-B/1-B/0-B values shown above the bars. (D) Immunohistogram of the reactivity of anti-ZR antibodies against the Toyopearl HW 40F column-separated fractions of xylem sap. Plants were prepared as in Fig. 1 at day 8, and two days later xylem sap samples were taken by cutting off the roots, with hypocotyl (A) or without (B). Xylem sap was collected for 30 min under reduced pressure. Sap samples from 2–4 plants displaying similar sap flow rates were pooled and subjected to ELISA. Values are expressed in ZR equivalents. For each treatment (A, B), the relationship was fitted with linear regression giving P-values of ≥0.998 at α=0.05 for all variants, except for 0-B plants in (B), where P=0.739. The values in (C) extrapolated to null sap-flow rate are 3 –1 closely related to xylem CK levels in vivo given near-zero transpiration rates (<0.01 mm s ) previously observed in 10-day-old pea seedlings by Kotov and Kotova (2015). Asterisks indicate statistically significant differences compared with 2-B plants: *P<0.05, **P<0.01, ***P<0.001. In (D), the column chromatography of xylem sap was performed using PBS (pH 7.4) as eluent buffer. Fractions were analyzed by ELISA; the elution of CK standards is indicated by horizontal bars: Z, t-zeatin; ZN, zeatin nucleotide; ZR, zeatin riboside. Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 2974 | Kotov and Kotova not transported to the root (Fig. 2D, right; Tanaka et al., 2006). The levels of tZ-type CKs in 2-B plants were similar in xylem sap collected from root with or without hypocotyl, and differed only after shoot excision (Fig. 4C). These data suggest that the roots most likely play a role in supplying CK to the upper plant parts, independent of shoot IAA export. Although our results show that shoot-derived auxin does not have a major role in regulating root CK synthesis, the up-regula- tion of root CK synthesis by nitrate can play a key role in regu- lating branching in Arabidopsis and tomato (Takei et al., 2001; Rahayu et al., 2005); however, in rice, nitrogen enhanced the amount of CK by promoting the expression levels of OsIPTs in stem nodes rather than in roots (Xu et al., 2015). Root CK may provide a necessary minimum CK supply for the shoot. This is consistent with our data in which a high IAA export from one branch was not able to completely suppress the IEA of another branch (Fig. 3), suggesting that this residual export of IAA might be supported by the root CK. Furthermore, in experiments with strigolactone-deficient pea mutants, apical auxin applications reduced bud growth in de-rooted nodal explants, but had a smaller effect in rooted decapitated plants (Young et al., 2014), in agreement with a growth-promoting role Fig. 5. IAA and CK contents in the hypocotyl tissues of 10-day-old 2-B, 1-B, and 0-B pea seedlings. The ratio of 2-B/1-B/0-B plant CK levels is for root CK which may be largely independent of shoot auxin. shown above the bars. 2-B, 1-B, and 0-B plants were prepared as for Our results indicated that the levels of the hypocotyl- Fig. 4, and hypocotyl samples from all treatments of 10-day-old plants derived tZ-type CKs in ascending xylem sap entering shoots were collected for analysis. IAA and tZ+iP-type CKs were purified and were four times higher in 1-B than in 2-B plants (Fig. 4A, C). fractionated on PVPP columns, whereas Z/ZR were separated from the Together with the stimulation of shoot IEA by exogenous total CK fractions on Amprep С18 columns. IAA and CKs were measured by ELISA and expressed as ZR or iPR equivalents per g fresh weight. Data CK (Fig. 3), the data provide strong evidence that xylem CK are the means of four replicates (10 plants in each) ±SE. Asterisks indicate controls shoot IEA and possibly shoot growth as well. The statistically significant differences compared with 2-B plants: *P<0.05, results also show that the number of branches in our model **P<0.01, ***P<0.001. plants determined xylem CK levels, and that this effect is con- trolled by the CK-synthetic activity of the hypocotyl. observed in xylem sap (Fig. 4A, C). This observation is difficult to reconcile with the potential for IAA to regulate CK levels, as shown above for 0-B plants. To explain this paradox we pro- A dynamic model for auxin–cytokinin inter-regulation in pose a dynamic interaction model that includes the transport two-branched pea plants of IAA and CK along with the IAA–CK interactions (Fig. 6). In order to account for how shoots can regulate the levels of We assume that the IAA homeostasis observed in the hypo- xylem CK in our model plants, we analyzed the contents of cotyl is being maintained by shoot IAA, which is positively con- IAA and CKs in the hypocotyl. As expected, in 0-B plants the trolled by xylem CK synthesized mainly in the hypocotyl, under IAA level in the hypocotyl was rather low (Fig. 5), indicating negative auxin control. In this model, the hypocotyl IAA con- that previously accumulated auxin could not be transported tent can be sustained at a constant level, below which auxin-reg- to the roots (Fig. 2D) and was most likely degraded. The dra- ulated CK synthesis will be switched on. The resulting increase matically increased concentrations of tZ-type and iP-type in xylem CK levels returns IAA content to the previous level CKs in the hypocotyls of 0-B plants (Fig. 5) were consistent through CK-activated IAA export from shoots. Accordingly, the with published accounts of the role of auxin in the negative removal of one shoot from a 2-B plant will decrease IAA con- regulation of CK levels (Li et al., 1995; Kotova et al., 2004; tent in the hypocotyl and so triggers CK synthesis, which con- Takei et al., 2004; Nordstrom et al., 2004; Tanaka et al., 2006; tinues until CK levels in xylem sap are enough to induce IAA Dun et al., 2012; Kotov and Kotova, 2015). In 0-B plants, the export from the remaining shoot. This export restores the IAA levels of tZ-type CKs were highly increased compared with level in the hypocotyl and stops further CK synthesis (Fig. 6). 2-B plants both in xylem sap obtained from root plus hypo- cotyl (Fig. 4A, C) and in hypocotyl tissue (Fig. 5). However, Discussion this increase was 3-fold less for xylem sap from roots (Fig. 4A, C), thus implying the hypocotyl origin of xylem CK. Correlative inhibition in the two-branched pea model In hypocotyls of 1-B and 2-B plants, IAA levels were equal system is age dependent (Fig. 5). This can be explained if the total IEA from two shoots in 2-B plants equals that from one shoot in 1-B plants (Fig. 2A, When studying 2-B plants at various ages, we found that dif- D). At the same time, the concentrations of tZ-type CKs were ferences in shoot IEA between 2-B and 1-B plants, and hence dissimilar in 1-B and 2-B plants (Fig. 5), similar to the findings CI in this system, disappeared in 16-day-old plants bearing Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 Auxin–cytokinin control of correlative inhibition | 2975 Fig. 6. Schematic representation of the IAA–CK balance in the model system of pea seedlings. Roots were shown not to be involved in the hormonal interactions (Fig. 2D, right side; Fig. 4A–C). The interaction between IAA and CKs occurs by transport of these hormones. Xylem CK (dotted lines) acts positively on the production/export of shoot auxin (dashed lines) (Fig. 3; Li and Bangerth, 1992, 2003), while IAA acts negatively on the synthesis/content of CKs in hypocotyl (0-B variant in Fig. 5; Li et al., 1995; Tanaka et al., 2006; Kotov and Kotova, 2015). Our data suppose that IAA homeostasis in the hypocotyl is key to the formation of the IAA–CK balance; a steady-state tissue IAA level is sustained near a threshold level, below which CK synthesis is switched on, activating the export of IAA from shoot(s) through ascending xylem flow of CKs. The removal of one shoot from 2-B plants results in an initial decrease of hypocotyl IAA content that triggers CK synthesis, which continues until the concentration of CKs in hypocotyl (Fig. 5) and, as a consequence, in xylem sap (Fig. 4A, C) becomes sufficient to increase IAA export from the remaining shoot (Fig. 2A, B, D). This compensates for the loss of a shoot, restoring the IAA concentration in the hypocotyl, and stopping further CK synthesis and elevation of xylem CK levels. two mature leaves (Fig. 2A–D). Similar loss of CI has also (Fig. 2A, D). The correlation obtained between IEA (Fig. 2A, been observed in two-shoot plants bearing equal shoots D) and xylem CK (Fig. 4C) in 1-B and 2-B plants on the one with three mature leaves (Li and Bangerth, 1999. Thus, it is hand, and the ability of ВА supplied to the vascular stream evident that shoots develop independence. Similarly, as the to affect shoot IEA (Fig. 3) on the other hand, together argue branches grow, they eventually became resistant to strigol- an important role for endogenous CK signaling in the shoot/ actones, which inhibit the outgrowth of axillary buds (Dun branch competitive interaction, at least in our model 2-B et al., 2013). However, in 2-B pea plants with unequal shoots, system. This possibility is supported by the observations of removal of the dominant shoot results in growth and IEA Li and Bangerth (2003) and of CK-driven polarization of activation in the subordinate shoot (Li and Bangerth, 1999) PsPIN1 proteins in pea axillary buds (Kalousek et al., 2010). which suggests that the ability of branches to compete does Localizing the hypocotyl as the site of control of acropetally not disappear completely with increasing age. It appears that transported CKs (Fig. 4), which in turn regulate the shoot if a shoot/branch lags behind others and thus remains physi- IEA (Fig. 3), has suggested a model in which CI is a function ologically young it can remain responsive to the CI stimuli. of an IAA–CK feedback loop, where CK essentially acts as a second messenger for IAA (Fig. 6). The phenomenon of CI has led to alternative auxin trans- Mutual inhibition between two branches of pea occurs port models (Li and Bangerth, 1999; Bangerth et al., 2000). according to the second messenger model Li and Bangerth (1999) proposed that IAA transported from Our studies, as in the experiments of Li and Bangerth (1999), a dominant shoot can somehow competitively impede IAA lead us to similar basic conclusions, namely, that competition outflow from subordinate shoots [auxin-transport autoinhibi- between branches on the same plant occurs at the level of tion (ATA) at junctions]. Li and Bangerth (1999) did not find their IEA. A clear understanding of this variable and the rea- significant differences in auxin transport capacity between sons leading to its regulation is the key to elucidating the CI subordinate and dominant shoots, reporting only 20% ele- phenomenon. IEA reflects IAA synthesis, yet is also depend- vation in internodes of the dominant shoot. Therefore, they ent on quantitative regulation of IAA transport. The latter considered ATA only as a result of a possible competition at follows from the results showing that IAA contents in the the level of auxin transport and not a change in auxin trans- hypocotyl (Fig. 5) were directly correlated with the shoot IEA port capacity. However, we showed a substantial increase in Downloaded from https://academic.oup.com/jxb/article/69/12/2967/4953358 by DeepDyve user on 12 July 2022 2976 | Kotov and Kotova the xylem CK level after shoot removal in 2-B pea plants, and dependent on the bud size, decreasing with an increase in size that could enhance auxin transport from a residual shoot. (Chatfield et al., 2000; Kotov and Kotova, 2010), which has Although it is possible to explain ATA using a second mes- periodically raised the question of whether auxin is the hor- senger model, other direct action models have been proposed. mone of apical dominance (Cline and Sadeski, 2002; Mason The auxin transport experiments with Y-shaped pea explants et al., 2014). Moreover, the bud inhibition by exogenous auxin demonstrated the autoinhibition of H-IAA transport in in decapitated pea plants was often associated with bud swell- one arm by the simultaneous transport at various unlabeled ing that did not occur in the dormant buds in intact wild-type IAA concentrations in the other arm, but high (unphysiologi- plants (Beveridge et al., 2000). The possible reason for these cal) concentrations of IAA (up to 50 mM IAA in lanolin) observed anomalies could be connected to increased cellu- were needed to achieve reasonable autoinhibition (Li and lar osmotic pressure, which has proved to be up to 0.15 MPa Bangerth, 1999). Next, in the more sensitive ‘endogenous ATA’ higher in the buds of decapitated plants treated with auxin system, it was shown that the transport of H-IAA through than that of intact plants (Kotov and Kotova, 2010), with pedicels of young tomato fruits decreased with increases in the difference obtained being equivalent to 50 mM (1.8 %) the concentration of unlabeled IAA from 1 to 50 µM in the sucrose solution. These data suggest that the suppression of agar receiver (Bangerth et al., 2000). In our study, the IAA bud growth by exogenous auxin most likely occurs against a contents in the hypocotyl were similar in 1-B and 2-B plants background of an increased amount of assimilates/sugars, the (Fig. 5), whereas the shoot IEAs were different (Fig. 2A, D), a transport of which to the buds was shown to be activated fol- fact that is difficult to reconcile with the ATA effect. Further, lowing the removal of a dominant sink organ, and which has our data showed that high IAA export from one branch does the potential to promote bud outgrowth (Mason et al., 2014; not completely inhibit export from another branch (Fig. 3); Barbier et al., 2015). Gene expression profiling in Arabidopsis this observation does not agree with the model outlined by suggests that the sugar-repressive element (SRE) is one of Prusinkiewicz et al. (2009), which predicts that high auxin the potential regulatory elements involved in the down-reg- flux from one source should fully shut down auxin flux from ulation of gene expression after decapitation, and SRE may another source. We believe that our data from 2-B pea are contribute to the nutritional regulation of gene expression in consistent with the second messenger model for auxin action, Arabidopsis axillary buds (Tatematsu et al., 2005). Most likely, rather than the auxin transport model. However, data from the independence of axillary bud growth in nodal Arabidopsis other systems, discussed below, could be consistent with the explants from СKs (Müller et al., 2015) could be accounted auxin transport model. for by the availability of sugars (Barbier et al., 2015). Auxin-induced bud inhibition mechanisms in other Acknowledgements model systems We would like to thank to Dr NV Kataeva (Timiryazev Institute of Plant Müller et al. (2015) reported that in Arabidopsis plants CK Physiology, RAS, Moscow) for the kind gift of rabbit antiserum against is not necessarily required to promote bud outgrowth. This iP/iPR, and Prof. Richard Napier for thorough correction of the English language. conclusion followed from experiments indicating that the axillary buds in nodal explants of CK mutants defective in CK synthesis (ipt 3, 5, 7) and signaling (arr3, 4, 5, 6, 7, 15), References grew equivalent to wild-type buds. 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Journal of Experimental Botany – Oxford University Press
Published: May 25, 2018
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