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Journal of Plant Physiology 165 (2008) 1274—1279 n of nutrient-limited plants: Its impact on Lidiya B. Vysotskaya, Institute of Biology, Ufa Research Received 15 June 2007; received Water relations of growth in favour of roots under limited nutrient supply. The ABA content was mineral nutrients appears to be modulated by accumulation of ABA in roots. This ABA may originate in the shoots, where its synthesis is stimulated by the loss of leaf plant shoot growth, while root growth is often less the importance of this reaction for acclimation to water and ion shortage is widely recognized. Mechanisms enabling this growth allocation in favour of roots have been assessed by Chapin ARTICLE IN PRESS �Corresponding author. Tel.: +7 3472355362; fax: +7 3472356247. (1990) and by Frensch (1997). These analyses 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2007.08.014 E-mail address: guzel@anrb.ru (G.R. Kudoyarova). turgor. & 2007 Elsevier GmbH. All rights reserved. Introduction Shortage of mineral nutrients in soil rapidly slows inhibited, thereby increasing the relative surface area available for ion absorption (Chapin, 1990). Plants also respond in this way to soil drying, and greater in shoots and growing apical root parts of starved plants than in nutrient sufficient plants. Accumulation of ABA in shoots of nutrient deficient plants was linked to a decrease in leaf turgor. Increased flow of ABA in the phloem apparently contributed to the accumulation of ABA in the apical part of the roots. Thus, partitioning of growth between roots and shoots of wheat plants limited in KEYWORDS ABA; Deficit in mineral nutrition; Durum wheat; Root and shoot growth; Alla V. Korobova, Guzel R. Kudoyarova� Centre, Russian Academy of Sciences, 450054 Ufa, Russian Federation in revised form 23 August 2007; accepted 24 August 2007 Summary We describe the involvement of abscisic acid (ABA) in the control of differential growth of roots and shoots of nutrient limited durum wheat plants. A ten-fold dilution of the optimal concentration of nutrient solution inhibited shoot growth, while root growth remained unchanged, resulting in a decreased shoot/root ratio. Addition of fluridone (inhibitor of ABA synthesis) prevented growth allocation in favour of the roots. This suggests the involvement of ABA in the redirecting the differential growth of roots and shoots Abscisic acid accumulatio in the roots www.elsevier.de/jplph ARTICLE IN PRESS Abscisic acid accumulation in roots of nutrient-limited plants 1275 implicated accumulations of abscisic acid (ABA) in bringing about the larger root/shoot ratio of nutrient- and water-deficient plants (Chapin, 1990; Saab et al., 1990). It is therefore not surprising that the involvement of ABA in the control of root growth in droughted plants has been intensively studied (e.g. LeNoble et al., 2004). However, the likely involvement of ABA in growth responses to deficits in major mineral nutrients has received much less attention. Nitro- gen and potassium deficiencies are known to promote accumulation of ABA in root tissues of maize (Schraut et al., 2005). However, in castor bean, ABA was found to accumulate in shoots rather than in roots under phosphate deficiency (Jeschke et al., 1997). Moreover, the differences in ABA content in nutrient-deficient plants have mainly been discussed in relation to shoot rather than to root growth (Dodd et al., 2002). Another feature of this complex subject is the search for the actual signal that induces ABA accumulation in nutrient- deficient plants. Water shortage is well known to induce accumulation of ABA in plants (Davies et al., 2005). Because deficiency in mineral nutrients is frequently accompanied by water deficit (Dodd et al., 2002), accumulation of ABA in nutrient- deprived plants may be due to water deficit. The site of ABA synthesis in water-deficient plants has been discussed in detail by Hartung et al. (2002). ABA produced in roots of such plants was suggested to serve as a root-derived signal to the shoot that closes stomata and slows leaf expansion (Zhang and Davies, 1989). Supporting evidence for such root to shoot signalling in mineral-deficient plants revolves around finding increased concentrations of ABA in xylem sap of pepper and castor bean plants following nitrate and phosphate deprivation (Jeschke et al., 1997; Dodd et al., 2002). ABA may also be produced in shoots and exported to roots through the phloem (Jiang et al., 2004), its appearance in the xylem sap resulting from recirculation. Thus, the source of ABA in nutrient- deprived plants remains unclear and is the topic of the present paper, which examines the effect of dilution of the nutrient solution on root growth, ABA content and transport in wheat. We deter- mined whether dilution of nutrient solution led to accumulation of ABA in the roots and examined the possibility that changes in leaf hydration and osmotic potentials instigate ABA accumulation. Experiments included treating plants with an inhibitor of ABA synthesis (fluridone). We also examined the effect of nutrient shortage on root weight and branching and on indole acetic acid (IAA) levels, since this phytohormone is important for root growth (Reed et al., 1998). Materials and methods Plant material Seedlings of durum wheat (Triticum durum Desf., cv. Bezenchukskaya 139) were grown in containers filled with 0.1 strength (10%) Hoagland–Arnon nutrient solution under illumination of 90Wm�2 PAR from ZN and DNAT- 400 fluorescent lamps, in a 14-h photoperiod and at 22 1C. In preliminary experiments, a 10% solution (0.5mM KNO3, 0.5mM Ca(N03)2, 0.1mM KH2PO4, 0.1mM MgSO4, 0.5mM CaSO4) was shown to support a maximum growth rate of wheat seedlings. Increasing the concentration of nutrients 10-fold reduced growth in shoot dry mass by 20% over 7 d, while a 10-fold dilution to give 1% of full strength Hoagland–Arnon solution halved growth in shoot dry mass. Since the differences in shoot mass were observed within 2 d, short-term changes in growth rate and hormones were studied under limited nutrient supply in the present experiments to reveal the primary effects. In this work, 7-d-old plants bearing one true leaf that was partly expanded were exposed to nutrient solution diluted to yield 1% of full-strength Hoagland–Arnon solution. In some experiments, fluridone was added to the diluted nutrient solution to give a final concentration of 5mg/L. At this stage, the shoot of plants comprised only the first leaf. Accordingly, the terms leaf and shoot are used interchangeably throughout the text. Water relations and photosynthesis measurement The leaf water potential was measured using a Scholander-type pressure chamber. Osmotic potential of leaves was measured by means of a freezing point depression micro-osmometer (Camlab Limited, UK). Turgor pressure was calculated as the difference between water and osmotic potential. Changes in photosynthetic CO2 assimilation and stomatal conductance were mea- sured with a portable open system gas analyser (CIRAS-1, PP-Systems, Hitchin, Hertfordshire, UK). Phytohormone extraction and immunoassay For phytohormone extraction, shoots, whole roots and their distal growing parts (3.5mm long) were homoge- nized in 80% ethanol and incubated overnight at +4 1C. After filtration and vacuum evaporation of extracts to remove all traces of ethanol, the aqueous residue was acidified with HCl to pH 2.5 and partitioned twice with peroxide-free diethyl ether (ratio of organic to aqueous phases 1:3). The extracted hormones were subsequently transferred from the organic phase into 1% sodium hydrocarbonate (pH 7–8, ratio of organic to aqueous phases was 3:1), re-extracted with diethyl ether, methylated with diazomethane and immunoassayed using polyclonal antibodies against ABA and IAA as described by Vysotskaya et al. (2003) and Veselova et al. (2005). Antibodies against IAA and ABA had high immunoreactiv- ity to the corresponding hormones and low cross- reactivity to substances structurally related to them. Thus, in the case of immunoassay for ABA, cross- reactivity to ABA, phaseic acid and xanthoxin was 100%, 0.1% and 0.001%, respectively, while in the case of immunoassay for IAA, cross-reactivity to IAA, indole-3- acetamide and indole-3-acetaldehyde was 100%, 1% and 0.15%, respectively. Reliability of immunoassay for ABA was enabled by both the specificity of the antibodies and purification of the phytohormones according to a modified scheme of solvent partitioning (Veselov et al., 1992). Measurement of ABA flow from shoots to roots To evaluate ABA transport from the shoot to the root, 1.5mL of 5mM Na2EDTA was applied to the base of excised shoots to prevent plug formation in phloem sieve root/shoot ratio similar to well-fed plants. Photo- synthesis was inhibited by limited nutrition, while addition of fluridone to diluted nutrient solution did not significantly change photosynthesis and the extent of its inhibition remained similar compared to well-fed plants. Stomatal conductance was not influenced by either limited supply of nutrient or fluridone treatment, and was similar in all variants tested. The number of lateral roots on the main (longest seminal) root was similar in both well-fed and starved plants. However, nutrient deficiency decreased the number of root primordia (Figure 1). Decreasing the availability of mineral nutrients did not change the leaf water potentials (Table 2). However, the osmotic pressure was lowered ap- proximately 17% by nutrient deficit, and the turgor pressure was higher in well-fed plants. Root osmotic pressure was similar in well-fed and starved wheat plants. Dilution of nutrients did not change ABA content in the roots as a whole, but in the apical parts (3–4mm) levels were increased. Concentration of ABA in the leaf was also increased two-fold by ARTICLE IN PRESS oto don tica L.B. Vysotskaya et al.1276 elements and kept in darkness at 24 1C for 3 h (Caputo and Barneix, 1999). Measurement of lateral root and primordia number Two days after dilution of the nutrient solution, lateral roots and their primordia were counted under the microscope in roots fixed in a mixture of ethanol and glacial acetic acid (3:1) and stained with acetocarmine (Vysotskaya et al., 2007). Results Measurement of shoot and root weight of wheat plants 2 d after dilution of the nutrient solution showed that a deficit in mineral nutrients inhibited shoot growth while that of the roots remained similar to that of well-fed plants (Table 1). Maintaining root growth while shoot growth was inhibited resulted in lower shoot/root ratios in nutrient-deficient plants compared to well-fed plants. When fluridone (an inhibitor of ABA synth- esis) was added to the nutrient solution simulta- neously with the dilution of nutrients, root growth was inhibited by the nutrient deficit to the same extent as that of the shoot, resulting in a Table 1. Shoot and root fresh weight and their ratio, ph 2 d after dilution of nutrient solution and addition of fluri Plant treatment Control (well-fed plants) Shoot weight, mg 22575 Root weight, mg 13275 Shoot/root ratio 1.7 CO2 assimilation, mmolm �2 s�1 1171 Stomatal conductance, mmolm�2 s�1 8674 Means of 10 replicates and their SE are presented. Means statis (n ¼ 10). synthesis rate and stomatal conductance of wheat plants e Diluted nutrient solution Diluted nutrient solution+fluridone 20276* 19377* 13176 11174* 1.5* 1.7 671* 571* 9575 7974 lly different from control (t-test) are indicated by * (Po0.05) Figure 1. Number of primordia (Pr) and lateral roots (LR) per tap root of wheat plants continuously grown on 10% (well-fed) and 2 d after their transfer to 1% (nutrient- limited plants) Hoagland–Arnon solution (H–A). ARTICLE IN PRESS ave r to osm ure 0. 0. ally (10% H–A) H–A) Abscisic acid accumulation in roots of nutrient-limited plants 1277 ABA content in leaves 87711 178719* ABA content in whole roots 4574 3874 ABA content in apical 3–4mm of roots 98711 151714* Table 2. Characteristics of water relations (MPa) in le Hoagland–Arnon (H–A) solution and 1 d after their transfe Nutrition Leaf water potential Leaf press Well fed (10% H–A) 0.6570.04 1.247 Nutrient limited (1% H–A) 0.6170.05 1.057 Means of 5 replicates and their SE are presented. Means statistic Table 3. Phytohormone content in shoots and roots (pmol g�1 fresh weight), hormone flow in xylem and phloem (pmol plant�1 in h) of wheat plants grown continuously on 10% Hoagland–Arnon (H–A) solution and 1 d after their transfer to 1% H–A Well fed Limited (1% mineral shortage (Table 3). Phloem flow of ABA to the roots was higher in starved than in well-fed plants, while the flow of ABA from root to shoot in xylem sap did not change. The IAA content in the roots was changed little by mineral deficiency. Discussion In our 7-d-old nutrient wheat plants, mineral deficiency induced by diluting the nutrient solution inhibited shoot growth over 2 d while that of the root was kept constant. This resulted in a decrea- sed shoot/root ratio. A similar effect has been noted previously in tomato (Chapin, 1990) and many other species (Wilson, 1988). Decreased shoot growth in our nutrient-limited wheat plants may be explained by the inhibition of photosynthesis observed in our experiments. Reduced photosynth- esis occurred despite no marked closing of stomata, thus suggesting damage to the photosynthetic apparatus itself, perhaps due to shortages of nitrogen, potassium and other elements necessary Xylem flow of ABA 4.570.4 5.770.6 Phloem flow of ABA 9.871 1672* IAA content in roots 68711 5176 IAA content in apical 3–4mm of roots 251722 205730 Means of 5 replicates and their SE are presented. Means statistically different from well-fed (t-test) are indicated by * (Po0.05). for photosynthesis. It is of interest that ABA accumulation in leaves of our nutrient-limited plants was not accompanied by a decline in stomatal conductance, which is in apparent contra- diction to the well-known effect of ABA inducing the closure of stomata. However, the bulk amount of the leaf ABA cannot be related to stomatal conductance, which is reported to be predomi- nantly controlled by ABA in the xylem sap (Zhang and Davies, 1989; Hartung et al., 2002). No increase in ABA concentration in xylem sap was observed in our experiments, and thus the absence of stomatal response to the limited nutrient supply may be related to unchanged concentration of free ABA in xylem sap of the plants. Although dilution of nutrient solution inhibited shoot growth, root growth remained unchanged, resulting in a decreased shoot/root ratio. Addition of fluridone inhibited ABA synthesis and prevented growth allocation in favour of the roots. This suggests the involvement of ABA in redirecting growth in favour of roots when plants are mineral- deficient. Fluridone may have effects in addition to suppressing ABA production. It may therefore influence photosynthesis. However, addition of fluridone to diluted nutrient solution did not change photosynthesis significantly and the extent of its inhibition remained similar to that of well-fed plants. When fluridone was used in experiments with water-deficient plants as an inhibitor of ABA synthesis by Saab et al. (1990), they too observed that the root growth in the stressed plants decreased, implying a positive role for ABA in root growth. Although our measurements of the ABA in whole roots did not reveal any changes in concen- s and roots of wheat plants grown continuously on 10% 1% H–A otic Turgor pressure Root osmotic pressure 06 0.5870.03 0.4670.03 05* 0.4370.03* 0.4270.04 different from well-fed (t-test) are indicated by * (Po0.05). tration in comparison with well-fed plants, a marked accumulation was observed in the apical 3–4mm of the main roots. We conclude that this ABA that accumulated in the zone of apical cell division and expansion was sufficient to support root growth. Saab et al. (1990) also observed an accumulation of ABA in apical parts of roots of droughted plants. They considered the effect being responsible for continued elongation of roots at low water potentials. ARTICLE IN PRESS L.B. Vysotskaya et al.1278 Another striking feature of the roots under nutrient limitation was a decreased number of root primordia formed on primary roots of our plants. Current concepts of lateral root regulation focus on the role of auxin (Reed et al., 1998). However, the IAA content was unaffected by mineral shortage in whole roots or in their apical 3–4mm. Inhibition of lateral root growth in osmotically stressed plants was attributed by Deak and Malamy (2005) to ‘‘interplay’’ between promotive auxin and repres- sive ABA signalling. The initiation of root primordia takes place in those parts of roots where we observed accumulation of ABA in plants under nutrient deficit. Both ABA and auxins were also reported to be involved in the control of lateral root growth in droughted Arabidopsis plants (Vartanian et al., 1994). ABA stimulated the synthesis of auxins in maize plants (Ludwig-Muller et al., 1995). However, in our nutrient-deficient plants the observed increase in ABA content in root tips was not accompanied by IAA accumula- tion, and we conclude that the unchanged level of auxin was unable to counter the repressive effect of ABA on lateral root formation. Thus, the increased content of ABA in the apical 3–4mm of nutrient-deficient roots is probably responsible for the suppression of auxin-induced initiation of lateral roots. ABA accumulation in the root apices of nutrient limited plants and its apparent involvement in maintaining root growth raises the question of its origin. Root-derived ABA has been reported to be important in long-distance signalling in drought- treated plants (Zhang and Davies, 1989; Hartung et al., 2002; Davies et al., 2005). In the present experiments the flow of ABA in xylem sap, the most likely pathway for hormonal signalling from roots to shoots, was independent of the concentration of mineral nutrients. However, ABA is also synthesized in shoots of plants (Creelman and Mullet, 1991) and in our wheat plants the ABA content in leaves and its phloem flow to the roots was higher in nutrient- deficient plants, suggesting increased ABA synthesis in the shoots. It has been suggested that, in droughted plants, ABA accumulation is an outcome of decreased shoot water potential (Zabadal, 1974). However, in the hydroponically grown mineral-deficient wheat of the present experi- ments, no water deficits were detected. This is in contrast to other experiments with nutrient-defi- cient plants grown in soil by Dodd et al. (2002). Other studies have suggested that accumulation of ABA in shoots may be a function of the loss of leaf turgor (Creelman and Mullet, 1991). In the present experiments, calculation of leaf turgor in wheat plants revealed that it decreased in leaves of Caputo C, Barneix AJ. The relationship between sugar and amino acid export to the phloem in young wheat plants. Ann Bot 1999;84:33–8. Chapin III FS. Effects of nutrient deficiency on plant growth: evidence for a centralised stress-response system. In: Davies WJ, Jeffcoat B, editors. Importance of root to shoot communication in the responses to environmental stress. Bristol: British Society for Plant Growth Regulation; 1990. p. 135–48. Creelman RA, Mullet JE. Abscisic acid accumulates at positive turgor potential in excised soybean seedling growing zones. Plant Physiol 1991;95:1209–13. Davies WJ, Kudoyarova G, Hartung W. Long-distance ABA signaling and its relation to other signaling pathways in the detection of soil drying and the mediation of the plant’ response to drought. J Plant Growth Regul 2005;24:285–95. Deak KI, Malamy J. Osmotic regulation of root system architecture. Plant J 2005;43:17–28. Dodd I, Munns R, Passioura J. Dose shoot water status limit leaf expansion of nitrogen deprived barley. J Exp Bot 2002;