Apoplastic pH and Fe3+ Reduction in Intact Sunflower Leaves

Abstract

It has been hypothesized that under NO 3؊ nutrition a high apoplastic pH in leaves depresses Fe 3؉ reductase activity and thus the subsequent Fe 2؉ transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO 3 ؊ nutrition significantly increased apoplastic pH at distinct interveinal sites (pH > 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO 3 ؊ /proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH 4 ؉ or NH 4 NO 3 nutrition. Complementary to pH measurements, the formation of Fe 2؉ -ferrozine from Fe 3؉ -citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe 3؉ reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5.0. In analogy to the monitoring of Fe 3؉ reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe 3؉ reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.

Nitrate Does Not Result in Iron Inactivation in the Apoplast of Sunflower Leaves

“…The pH of apoplastic fluid from leaves of NO 3 Ϫ -fed plants collected 3 h after light onset was always higher by about 0.1 to 0.3 units than in NH 4 ϩ -or NH 4 NO 4 -fed plants (Table III); however, these differences were not statistically significant. These findings are in accord with the results of Kosegarten et al (1999) showing mean data of the apoplastic pH determined by the fluorescence microscope imaging technique. However, a clear increase in apoplastic pH above 6.5 found in their study was only in the small intervenial area of young leaves (about 10% of the total apoplastic space), …”

“…It has been argued that NO 3 Ϫ nutrition induces an alkalization of leaf apoplast Kosegarten et al, 1999Kosegarten et al, , 2001), although there is no clear evidence that this occurs to any great extent (Mü hling and Sattelmacher, 1995;Sattelmacher et al, 1998;Mü hling and Läuchli, 2001). Also, there are other factors, including NO 3 Ϫ reductase activity in the roots (Andrews, 1986) and light-induced pH changes in the leaf apoplast (Mü hling and Läuchli, 2001), which may modify any possible potential effect of NO 3 Ϫ supply in the nutrient solution on leaf apoplastic pH.…”

“…Kosegarten, B. Hoffmann, K. Mengel [1999] Ϫ supply (up to 40 mm) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted 59 Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO 3 Ϫ (6 mm) showed no effect on 59 Fe uptake by the symplast of leaf cells.…”

“…The presence of high HCO 3 Ϫ in the nutrient solution was shown to affect neither the bulk pH of apoplastic fluid obtained by centrifugation of intact leaves Rö mheld, 1999, 2002) nor the apoplastic pH measured by using a pH-sensitive fluorescent dye (fluorescein isothiocyanate-dextran) after infiltration of xylem sap into excised leaves (Kosegarten et al, 1999). Furthermore, HCO 3 Ϫ does not appear as a cause of physiological Fe inactivation in the leaf apoplast nor does it cause an inhibition of Fe uptake into the leaf symplast (Nikolic and Rö mheld, 2002), which is also in agreement with the recent conclusions of Kosegarten et al (1999Kosegarten et al ( , 2001.…”

“…In this study, we tested Mengel's hypothesis of Fe inactivation in leaves caused by NO 3 Ϫ nutrition using the same model plant, sunflower, as has commonly been used in many experiments performed by Mengel et al (Mengel et al, 1994;Hoffmann and Kosegarten, 1995;Kosegarten et al, 1998aKosegarten et al, , 1998bKosegarten et al, , 1999Kosegarten et al, , 2001. The objectives of the work presented here were: (a) to clarify the effect of NO 3 Ϫ supply to roots on postulated Fe inactivation in the leaf apoplast as a primary cause of NO 3 Ϫ -induced Fe deficiency chlorosis; (b) to investigate the influence of form of N supply and pH of nutrient solution stabilized by pH buffers on concentration of Fe in the two-leaf compartments, apoplast, and symplast; and c) to characterize the leaf apoplast for various binding forms of Fe.…”