Function of Rhizodermal Transfer Cells in the Fe Stress Response Mechanism of Capsicum annuum L

Abstract

A variety of red pepper (Capsicum annuum L., cv Yaglik) responds to Fe deficiency stress with simultaneously enhanced H+ extrusion, reduction of ferric ions and synthesis of malic and citric acid in a swollen subapical root zone densely covered with root hairs. It is demonstrated that these stress responses temporally coincide with the development of rhizodermal and hypodermal transfer cells in this root zone. During stress response the transfer cells show a marked autofluorescence which could arise from endogenous iron chelators of the phenolic acid type. The presence of organelle-rich cytoplasm which often exhibits rotational cytoplasmic streaming points to high physiological activity and makes these cells, with their increased plasmalemma surface, particularly well suited for the entire stress response mechanism. Since Fe stress-induced acidification is diminished by vanadate and erythrosin B, both specific inhibitors of plasmalemma ATPases, it seems reasonable to suppose that Ho pumping from transfer cells is activated by an ATPase located in their plasmamembrane. H' extrusion is also shown to be inhibited by abscisic acid. Raised phosphoenolpyruvate carboxylase activity and simultaneous accumulation of malate in the swollen root zone point to the action of a pH stat preventing a detrimental rise in cytoplasmic pH of transfer cells during enhanced H' extrusion. The simultaneous increase in citric acid concentration favors chelation of iron at the site of its uptake and thus ensures long distance transport to the areas of metabolic demand. A direct link between citrate accumulation and ferric ion reduction as proposed in recent literature further supports the crucial role of transfer cells in the response to Fe deficiency stress. Rhizodermal transfer cells induced by mineral stress were first reported for the halophyte Atriplex hastata treated with high NaCl concentrations (13). The striking similarity of the accompanying morphological symptoms such as root tip swelling and root hair formation to analogous symptoms induced in Fe deficient sunflower plants (16) led to the discovery of transfer cells in Fe-starved roots (15). Even under salt stress conditions, the important role of iron nutritional status for the development of transfer cells is emphasized by the fact that their formation can be hindered by increasing Fe supply (14). This suggests that a NaCl-induced Fe shortage was the immediate trigger of transfer cell differentiation in that experiment. In a first attempt to characterize the physiological function of transfer cells under Fe deficiency, it was proposed (18) that their development is related to enhanced Fe"' reduction and H+ efflux, both of which are well known key reactions of the so-called Fe stress response mechanism (for review see Ref. 5). On the other hand, Romheld and Kramer (27) stated in a comparative study of various plant species that transfer cell formation is directly related only to increased proton secretion. However, this generalization seems problematic. Their measurements of Fe"' reduction were expressed for the fresh weight ofexcised roots in general and not for the actual transfer cell zone of only a few mm that varies substantially in length from species to species. The purpose of the present paper is to provide a more detailed elucidation of the functional role of rhizodermal transfer cells under Fe deficiency stress. The results demonstrate that, at least for red pepper, these specialized cells are involved not only in proton secretion but are also related to enhanced Fe"' reduction, organic acid accumulation and possibly the production of phe-nolic iron chelators. MATERIALS AND METHODS Growth of Plants. Seeds of red pepper (Capsicum annuum L. cv Yaglik) were germinated and grown to the seedling stage in quartz sand moistened with CaSO4 solution (0.2 mM). The seedlings were then transferred for 2 weeks to a continuously aerated nutrient solution that contained, in mM: Ca(NO3)2, 2.0; K2SO4, 0.75; KH2PO4, 0.50; MgSO4, 0.65; H3BO3, 1.0 x 10-2; MnSO4, 1.0 x I0-; CuSO4, 5.0 x 10-4; ZnSO4, 5.0 x 10-4; (NH4)6Mo7024, 5.0 x l0-5; Fe as Fe"'EDTA, 2.5 x 10-2. All plants were grown in a controlled growth chamber with a day/night regime of 16/8 h and light intensity near 28 W/m2 (fluorescent tubes, Osram-L 40 W/25 white and 40/77 Fluora, ratio 4:1). The temperature was 24°C (light and dark), the RH approximately 70%. pH Measurements. To induce Fe deficiency plants were transferred to plastic jars (one plant per jar), containing 600 ml nutrient solution without FeEDTA. The pH of the solution was recorded continuously. After 3 to 4 d plants were selected which showed a similar degree of chlorosis and for which the pH decrease of the ambient medium started at nearly the same time. Patterns of H+ efflux from intact Fe-stressed roots were visualized as previously shown (18). Selected single roots were rinsed with distilled H20 and (remaining attached to the plant) placed into darkened Petri dishes filled with 0.6% agar medium containing Fe-free nutrient solution and bromocresol purple (50 mg L-') with a pH being adjusted to 5.8. The assembly was kept under the lights in the growth chamber. For inhibitor studies orthovanadate solutions were prepared according to O'Neill and Spanswick (25). ABA stock solution was prepared by dissolving ABA in a minimal volume of KOH and neutralizing with HCI. Organic Acids. Hot water extracts containing glutaric acid as internal standard were cleared by passage through cation and anion exchange resins (Dowex 50-X8, 200 to 400 mesh, H+ form, and Dowex I-X8, 200 to 400 mesh, formate form). After elution with 16 M HCOOH the anionic fraction was brought to dryness under vacuum at 40C and taken up in l-ml Reacti-511