SHORT COMMUNICATION
Effect of Fe deficiency on mitochondrial alternative NAD(P)H dehydrogenases in cucumber roots

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Abstract

Iron deficiency affects the function of the respiratory chain, primarily at the complex I and complex II levels. Because plant mitochondria possess alternative NAD(P)H dehydrogenases located in the inner membrane, oxidizing NAD(P)H from both cytosol and matrix, we investigated these activities in mitochondria of Fe-deficient roots. External and internal NAD(P)H dehydrogenase activity increased in Fe-deficient mitochondria. Accordingly, NDB1 protein strongly accumulated, while NDA1 did not show differences in Fe-deficient roots. The data presented support, for the first time, the hypothesis that Fe deficiency induces the alternative NAD(P)H dehydrogenases, bypassing the impaired complex I.

Introduction

Strategy I plants (Römheld and Marschner, 1986) respond to Fe shortage in the soil by increasing three main types of activity at the root level: Fe(III)-chelate reductase (FC-R), the H+-ATPase and the iron-regulated transporters (IRT) (Curie and Briat, 2003). Their induction strongly increases the demand for NAD(P)H and ATP, leading to an overall adaptation of the metabolism. Indeed, in Fe-deficient plants, several metabolic changes occur (Zocchi, 2006 and references therein). Moreover, mitochondria are strongly affected by Fe deficiency, mainly at the respiratory chain level (Vigani et al., 2009). Notwithstanding these effects, they might still play a pivotal role in the metabolic changes that occur under these conditions (Vigani and Zocchi, 2009). In fact, plant mitochondria are characterized by the presence of several alternative pathways able to oxidize reducing equivalents, such as alternative NAD(P)H dehydrogenases (ND-DHs) (Møller and Lin, 1986; Rasmusson et al., 1998). Distinct external rotenone-insensitive enzymes oxidize cytoplasmic NADH and NADPH in a calcium-dependent manner (Roberts et al., 1995). Matrix NADH can be oxidized via two alternative enzymes. NADH oxidation, through the rotenone-sensitive complex I, mediates ATP production via proton pumping, whereas the activity of the rotenone-insensitive NADH dehydrogenases is not restricted by coupling to energy conservation (Rasmusson and Møller, 1991a). Additionally, a calcium-dependent rotenone-insensitive NADPH dehydrogenase is present at the matrix surface of the inner membrane (Rasmusson and Møller, 1991b; Melo et al., 1996).

As reported by Vigani et al. (2009), ND-DHs could be activated to bypass the dramatic loss of complex I and complex II activities occurring in Fe-deficient cucumber roots. The external and internal localization of these enzymes in the inner mitochondrial membrane allows them to oxidize the NAD(P)H from both the cytosolic and the matricial sides, but there is no evidence thus far to support their involvement in the response to Fe deficiency. In this work, we report results supporting this involvement.

Section snippets

Plant material and isolation of mitochondria

Seed germination and growth of cucumber (Cucumis sativus L. cv. Marketer) plants were performed according to Vigani et al. (2009). Iron-deficient plants showed the morphological responses typical of Fe deficiency (development of leaf Fe deficiency chlorosis, stunted growth and appearance of lateral roots) and the increases in specific activities (Fe3+-chelate reductase and H+-ATPase) typical of Strategy I plants (Supplementary Table 1). Mitochondria were isolated from Fe-sufficient and 10-day

Alternative NAD(P)H dehydrogenase activities in Fe-deficient mitochondria

In order to confirm the hypothesis postulating the induction of alternative NAD(P)H dehydrogenase activity to bypass the inhibition of complex I induced by Fe deficiency, mitochondria were purified from roots of cucumber plants grown in the presence or in the absence of Fe. Purified mitochondria from control and Fe-deficient roots showed high integrity (86% and 84%, respectively) (Table 1). It was possible to discriminate between external and internal NAD(P)H oxidation by an osmotic burst of

Discussion

In Fe-deficient roots, the relationships between reducing and oxidizing processes are quite complex, since the strong activation of FC-R and H+-ATPase activities leads to a strong need for energetic substrates. The lack of Fe strongly induces the NAD(P)H oxidizing activities over the NAD(P)+ reducing activities (Schmidt and Schuck, 1996; López-Millán et al., 2000, López-Millán et al., 2009).

We found (Vigani et al., 2009) that Fe deficiency strongly impaired the respiratory chain activity in

Acknowledgements

The authors wish to thank Prof. Allan Rasmusson for the kind gift of NDB1 and NDA1 antisera, and Dr. M. Dell'Orto and Dr. S. Morgutti for critical reading of the manuscript.

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