Na+ recirculation in alfalfa and phloem transfer cell structure. N?ziha Boughanmi 1, Philippe Michonneau 2 and Pierrette Fleurat-Lessard 2

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1 Na+ recirculation in alfalfa and phloem transfer cell structure N?ziha Boughanmi 1, Philippe Michonneau 2 and Pierrette Fleurat-Lessard 2 1 D?partement des Sciences de la Vie. Facult? des Sciences de Bizerte Zarzouna. Tunisie. nezighanem@yahoo.fr 2 Laboratoire de Physiologie et Biochimie V?g?tales. UMR CNRS 6161, Universit? de Poitiers. France Abstract The native tunisian cultivar Gab?s of Medicago sativa shows, in response to a long term (4 weeks) NaCl treatment (150 mm), a growth reduction due to a disturbance in mineral nutrition (increasing of Na + and decreasing of K + content). However, among source leaves that support plant growth, the upper ones kept a better ionic status (low Na + and high K + content) than lower ones. In the latter, Na + toxicity induces chloroplast alteration in mesophyll cells. By contrast, plastids in upper leaves are unalterated, as a possible consequence of Na + recirculation. To this point, in upper leaves of NaCl treated plants, the wall ingrowths area is increased in phloem transfer cells of minor veins. This increase in veins mainly implicated in nutrient collection, might also be related to transfer cell involvement in Na + recycling from upper leaves towards lower leaves and roots, so that Na + content decreased in the upper ones. Moreover, in main veins, implicated in long distance transport, wall ingrowth enlargement observed after NaCl treatment can be linked with a rise of assimilates flux from source leaf towards sinks. Therefore, a positive growth balance would be maintained in this perennial M. sativa cv Gab?s, that must cope with long term salinity. In addition, the NaCl treatment alters transfer cells in lower leaves only. This effect, observed both in minor and main veins, would result from recirculation Na + recycling from upper leaves towards lower leaves and roots, in association with assimilate unloading. Media summary In alfalfa, ionic and structural modifications induced by NaCl in blade and phloem complex of upper and lower exporting leaves have been related to Na + recirculation Key words NaCl, ion, ultrastructure, companion cell, leaves Introduction In plants exposed to a long term NaCl stress, tolerance was found to be correlated with high K + (Zhu et al 1998) and low Na + (Munns and Termaat 1986) content in assimilating sites. To conteract Na + accumulation in leaves by xylem import, this ion is reexported by phloem. In Arabidopis, this recirculation of Na + from shoots to roots is controlled by the gene AtHKT1, whose expression was shown to be restricted to the phloem tissues. The recessive sas2-1 mutation of this gene gives plants with high sensitivity to NaCl associated with a decrease of Na + concentration in phloem sap and an increase of its accumulation in leaves (Berthomieu et al. 2003). Retranslocation of mineral nutrients via the phloem from shoot is closely associated with transport of carbohydrates from source leaves to sink sites in shoot and roots (Marschner et al. 1997). In minor vein, that are sites of assimilates collection, the loading from mesophyll to sieve tube is realized through companion cells. When these cells present transfer cells features, as in Fabaceae, the loading pathway is apoplasmic (van Bel 1996). Moreover these cells confer a particular adaptation to intraveinal recirculation of solutes (Pate and Gunning 1972). Na + recycling by this mechanism was thought to protect Trifolium alexandrinum leaves from NaCl toxicity (Winter 1982). On the other hand, as fodder crops, Fabaceae are of large agro-economic interest because of high protein content and symbiotic nitrogen fixation. Salinity reduces their biomass productivity, perennial species as Medicago sativa cv Gab?s are particularly concerned because they must cope with this stress. In this context, in this plant exposed to a long term NaCl treatment, we have studied ionic and structural

2 aspects of blade in exporting leaves that support plant growth. We have especially focused our interest on the phloem complex involved in ionic recirculation in this cultivar known for its tolerance to NaCl. Methods The germination of seeds of Medicago sativa cultivar Gab?s was performed on distilled water. Plants were grown on hydroponics (Long Ashton solution) in a greenhouse (22-32?C, 68-82%). NaCl stress (150 mm) was applied when the first trifoliate leaf was developed and by daily step of 50 mm to avoid osmotic shock. Ionic measures and cytology concerned upper (9 th and 7 th from the base for respectively control and NaCl treated plants) and lower (3 rd from the base of control and NaCl treated plants) exporting leaves after a 4 week NaCl treatment. In blade leaves, Na + and K + were measured by flame photometry after extraction in 0.1N nitric acid. Ultrastructural studies were realized in mesophyll and blade veins in upper and lower exporting leaves prepared according to classical techniques. Results Effects of NaCl on growth and ionic content After a 4 week NaCl treatment (150 mm), a growth reduction was observed at the whole plant level. However stems and leaves were the most affected (Fig. 1 A). The NaCl stress induces a new biomass partitioning as shown by the ratio Roots/Aerial parts that reached 17 % in control and 26% in salt treated plants. These observations are correlated with a decrease in K + and an increase in Na + content, in aerial part (Fig 1 B and C). In addition, upper leaves with a high K + and a low Na + content, have kept a better ionic statuts than the lower ones (Table 1). Figure 1. Dry weight (A), K + (B) and Na + (C) contents in leaves, stems and roots of Medicago sativa cv Gab?s treated by NaCl for 4 weeks (0 and 150 mm) (Values are means with standard deviations from 10 samples). Table 1: Na + and K + contents of blades of upper and lower exporting leaves of Medicago sativa cv Gab?s treated by NaCl (0 and 150 mm) for 4 weeks (Values are means with standard deviations from 10 samples). NaCl treatment Ion content (meq/ g of dry weight) Upper leaves Lower leaves

3 K + Na + K + Na + Ions 0 mm 1.20? ? ? ? mm 0.87? ? ? ?0. 05 Ultrastructure changes induced by NaCl In mesophyll, salt induces chloroplast alterations (vesicule dilatation, numerous plastoglobuli, large starch grains) particulary in lower leaves (Fig. 2 C). In upper leaves chloroplast structure is relatively well preserved (Fig. 2 B) by comparison to controls (Fig. 2 A). In minor veins, the presence of large wall ingrowths in transfer cell gives support for the implication of these cells in assimilates collection (Fig. 3 A, C). Salt stress increased by six times the wall ingrowth area, but only in lower leaves whereas it is unmodified in the upper ones. In main veins involved in long distance transport of assimilates, under salt treatment the wall ingrowth area was increased by three times both in upper and lower leaves (Fig. 3 B, Table 2). Similar deep alterations induced by NaCl occured in transfer cells of both minor and main veins in lower exporting leaves (Figs. 3 E, F). Figures 2. Chloroplasts of control (A), treated upper (B) and lower (C) exporting leaves of Medicago sativa cv Gab?s treated by NaCl for 4 weeks. G: grana, P: plastoglobuli, S: starch. bar: 0.1 μm Table 2. Wall ingrowth area in transfer cells of the blade of Medicago sativa cv Gab?s treated by NaCl (0 and 150 mm) for 4 weeks (Values are means with standard deviations from measurements on 20 cells per group of 10 minor and 10 main veins). NaCl treatment % Wall ingrowth area of transfer cell in leaves Upper leaves Lower leaves

4 VeinType Minor Main Minor Main 0 mm 14.5? ? ? ? mm 16.6? ? ? ?3.2 Figures 3. Ultrastructure of phloem complex in the exporting leaves in minor (A, C and E) and main veins (B, D and F) in control (A and B) and NaCl-treated Medicago sativa cv Gab?s (C and D: upper leaf, E and F: lower leaf). CC: companion cell, P: plastid, PP: phloem parenchyma,tc: transfer cell, SE: sieve element, arrow: walls ingrowths, star: nacreous wall, bar: 1μm Discussion A long term NaCl stress exposes Glycophytes to ionic specific stresses such as Na + toxicity and K + deficiency. Survival in such conditions is linked to the maintain a low level of the toxic Na + in assimilating sites, it might be achieved through recirculation by phloem. In Arabidopsis, this mechanism allows to efficiently protect aerial tissues from Na + invasion (Berthomieu et al. 2003). In Medicago sativa cv Gab?s, upper exporting leaves, with low Na + content have kept a preserved structure by comparison with the lower ones, where a high Na + content induces margin chlorosis. In Medicago sativa, Na + might recycle from upper leaves towards lower leaves and roots as in Trifolium alexandrinum (Winter 1982 b). The structural basis of such a mechanism is the presence of transfer cells in minor veins. These cells confer to plants a particular adaptation to intraveinous recycling (Pate et Gunning 1972). More recently, their presence in minor veins has been correlated with apoplasmic loading of assimilates (van Bel 1996). Moreover, these cells are also known to accumulate Na + under saline conditions (Flowers and L?uchli 1983). In Medicago sativa cv Gab?s and in lower leaves, the Na/K ratio measured using SIMS imaging, in phloem complex of minor and main veins is increased by salt stress in minor main veins (Boughanmi 2003) it might be the consequence of Na + storage in the enlarged apoplasm that will induce transfer cells necrosis. In these leaves, starch grains accumulate in mesophyll, in consequence, the development of phloem wall ingrowth transfer cells is not due to an increased collection assimilates but rather to a raise of Na + loading.

5 Na + may be then reexported through the main veins from upper towards lower exporting leaves, but would not leave the vein and reach the blade. Indeed, assimilates are always transported from source towards sinks (Eschrich 1980) and Na + is retranslocated from upper leaves towards roots. In the main veins, the alteration of transfer cell structure would result from Na + recirculation whereas the the increasing of wall ingrowth would be associated with a rise of assimilates unloading. Under salt stress this unloading assimilates would be done faster to meristems allowing a positive growth balance and towards roots, whose large development might insure a rapid growth of new stems after cutting in this perennial crop, well-known for its sturdiness. Conclusion The tolerance of the cultivar Gab?s of Medicago sativa exposed to a long term high NaCl stress is linked with the maintenance of biosynthetic activity of upper leaves that is related to the preservation of structure and ionic status. Our results support the hypothesis that transfer cell are involved in the stress response. Under NaCl treament, their increased apoplasm and plasmalemme surface would favor, in minor veins, the recycling of Na + towards the roots. In main vein, these structural features would intensify the transport of assimilates towards the sinks allowing therefore a positive growth balance. Acknowledgments This work was supported by the Service Interdisciplinaire de Microscopie et d Imagerie Scientifiques, University of Poitiers, France and the University of Bizerte, Tunisia. References Berthomieu P, Con?j?ro G, Nublat A, Nublat A, J.Brackenbury W, Lambert C, Savio C, Uozomi N, Oiki S, Yamada K, Cellier F, Gosti F, Simonneau T, A.Essah P, Tester M, V?ry A-A, Sentenac H and Casse F (2003). Functional analysis of AtHKt1 in Arabidopsis shows that Na + recirculation by the phloem is crucial for salt tolerance. EMBO Journal 22,

6 Boughanmi N, Michonneau P, Verdus M-C, Piton F, Ferjani E, Bizid E and Fleurat-Lessard P (2003). Structural changes induced by NaCl in companion and transfer cells of Medicago sativa blades. Protoplasma 220, Eschrich W (1980). Phloembeladung und verwandte Prozesse. Ber Deutsch Bot Ges 93, Flowers TJ and L?uchli A (1983). Sodium versus potassium: substitution and compartmentation. In: L?uchli A, Bieleski RL (eds) Inorganic plant nutrition. Berlin Heidelberg New York Tokyo, springer, pp Encyclopedia of Plant Physiology, vol 15). Marschner H, Kirkby EA and Engels C (1997). Importance of cycling and recycling of mineral nutrients within plants for growth and development. Bot Acta 110, Munns R and Termaat A (1986). Whole plant response to salinity. Aust. J. Plant Physiol. 13, Pate JS and Gunning BES (1972). Transfer cells. Annu. Rev. Plant Physiol. 23, van Bel AJE (1996). Interaction between sieve element and companion cell and the consequence for photoassimilate distribution: two structural hardware frames with associated physiological software packages in dicotyledons. J. Exp. Bot. 47, Winter E (1982 b). Salt tolerance of Trifolium alexandrinum L. III: effects of salt on ultrastructure of phloem and xylem transfer cells in petioles and leaves. Aust J Plant Physiol 9, Zhu JK, Liu J and Xiong L (1998). Genetic analysis of salt tolerance in Arabidopsis: evidence for a critical role of potassium nutrition. Plant Cell 10,

POTASSIUM IN PLANT GROWTH AND YIELD. by Ismail Cakmak Sabanci University Istanbul, Turkey

POTASSIUM IN PLANT GROWTH AND YIELD by Ismail Cakmak Sabanci University Istanbul, Turkey Low K High K High K Low K Low K High K Low K High K Control K Deficiency Cakmak et al., 1994, J. Experimental Bot.