Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings

Summary

Seedlings of sweet orange (Citrus sinensis) were fertilized for 14 weeks with boron (B)-free or B-sufficient (2.5 or 10 μM H₃BO₃) nutrient solution every other day. Boron deficiency resulted in an overall inhibition of plant growth, with a reduction in root, stem and leaf dry weight (DW). Boron-starved leaves showed decreased CO₂ assimilation and stomatal conductance, but increased intercellular CO₂ concentrations. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and stromal fructose-1,6-bisphosphatase (FBPase) were lower in B-deficient leaves than in controls. Contents of glucose, fructose and starch were increased in B-deficient leaves while sucrose was decreased. Boron-deficient leaves displayed higher or similar superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activities, while dehydroascorbate reductase (DHAR) and catalase (CAT) activities were lower. Expressed on a leaf area or protein basis, B-deficient leaves showed a higher ascorbate (AsA) concentration, but a similar AsA concentration on a DW basis. For reduced glutathione (GSH), we found a similar GSH concentration on a leaf area or protein basis and an even lower content on a DW basis. Superoxide anion (O₂⁻) generation, malondialdehyde (MDA) concentration and electrolyte leakage were higher in B-deficient than in control leaves. In conclusion, CO₂ assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. Although B-deficient leaves remain high in activity of antioxidant enzymes, their antioxidant system as a whole does not provide sufficient protection from oxidative damage.

Introduction

Boron (B) is an essential element required for the normal growth of higher plants. Boron deficiency is a widespread problem in many agricultural crops, including citrus (Shorrocks, 1997). The role of B in plant nutrition is little understood, which is surprising, since on a molar basis the requirement for B is, at least for dicotyledons, higher than that of any other micronutrient (Marschner, 1995; Alves et al., 2006).

Previous studies have shown that B deficiency decreases plant photosynthetic capacity (Kastori et al., 1995; Zhao and Oosterhuis, 2002, Zhao and Oosterhuis, 2003). In mustard (Brassica campestris), decreased rate of CO₂ assimilation appears to result from both decreased Hill reaction activity and low intercellular CO₂ concentration (Sharma and Ramchandra, 1990). Plesničar et al. (1997) suggested that the decreased rate of photosynthetic O₂ evolution in B-deficient sunflower (Helianthus annuus) leaves could be correlated with reduced chlorophyll (Chl) content, photosynthetic electron transport rate and photophosphorylation. El-Shintinawy (1999) investigated the structural and functional damage caused by B deficiency in sunflower leaves and suggested that B deficiency affected photosynthesis directly or indirectly. Accumulation of starch and hexoses occurs despite decreased photosynthesis (Kastori et al., 1995; Zhao and Oosterhuis, 2002; Camacho-Cristóbal et al., 2004) because growth is more affected than photosynthesis, and they are not used. This leads us to hypothesize that CO₂ assimilation is feedback-regulated by excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes.

A consequence of the imbalance between photosynthetic production of carbohydrates and their use in growth is that less of the absorbed photon-energy captured by the light harvesting system is used in CO₂ assimilation, so the photosystem electron transport chain becomes over-reduced. This leads to accelerated production of reactive oxygen species (ROS) such as single oxygen (¹O₂), superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH). To minimize cellular damage caused by ROS, plants have evolved a scavenging system composed of antioxidants such as ascorbate (AsA) and reduced glutathione (GSH) and antioxidant enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), monodehydroascorbate reductase (MDAR, EC 1.6.5.4), and dehydroascorbate reductase (DHAR, EC 1.8.5.1) involved in the water–water cycle, as well as catalase (CAT, EC 1.11.1.16) involved in scavenging the bulk H₂O₂ generated by photorespiration (Chen et al., 2005). Although there have been several reports investigating the antioxidant responses in leaves of plants treated with excess B (Keles et al., 2004; Molassiotis et al., 2006), data are limited on the effects of B deficiency. Liu and Yang (2000) reported that low B decreased SOD, APX, and CAT activities, and AsA content of soybean (Glycine max) leaves. In sunflower leaves, B deficiency increased APX activity, decreased GR and CAT activities, decreased AsA and non-protein SH-compound contents, but did not affect SOD activity (Cakmak and Römheld, 1997; Dube et al., 2000). Thus, it is not well known how B deficiency affects antioxidant system in plants.

In China, B deficiency is frequently observed in citrus orchards, and is responsible for considerable loss of productivity and poor fruit quality. To our knowledge, there is hardly any information on gas exchange, photosynthetic enzymes, carbohydrates, and antioxidant system of citrus leaves in response to B deficiency. The objectives of this study were to test the hypothesis that CO₂ assimilation is feedback-regulated by excessive accumulation of starch and hexoses in B-deficient leaves, and to determine how B deficiency affects antioxidant system in B-deficient leaves.