Calcium is an essential macronutrient for plant growth derived from the growing medium. Calcium ions are essential for cell wall strengthening and cell-cell adhesion (Marry et al., 2006; Marschner, 1995). Pectic polysaccharide rhamnogalacturonan portions of the middle lamella are cross linked by calcium ions (Matoh and Kobayashi, 1998) Calcium bound to the outer surface of the plasma membrane maintains membrane stability and cell integrity (Hanson, 1984; Hirschi, 2004; Palta, 1996). For example, cell death following freezing injury has been attributed to ion leakage caused by calcium loss from the plasma membrane (Arora and Palta, 1986). In addition, calcium is known to have a prominent role as a second messenger coupling stimuli such as environmental stress, and light and plant hormones to a response (Bush, 1995; Ng and McAinsh, 2003; Sanders et al., 1999).
Calcium is largely immobile in the phloem, and is distributed with water in the transpiration stream (White and Broadley, 2003). Thus, calcium deficiency symptoms are observed in tissues with a low transpiration rate, including young expanding leaves, enclosed shoot tissues, fruits, underground tubers, and in portions of the plant principally fed by phloem rather than xylem (White and Broadley, 2003). Shoot tip necrosis, a physiological disorder observed during S. tuberosum in vitro culture has been attributed to calcium deficiency (McCown and Sellmer, 1987; Sha et al., 1985). This condition is typified by the browning and death of the shoot tip, the loss of apical dominance, and axillary branch development in an in vitro shoot culture system (McCown and Sellmer, 1987; Sha et al., 1985). Transpiration is limited during in vitro culture by high humidity brought about with nearly closed culture vessels. Therefore, the uptake and transport of calcium ions, which is dependent on transpiration, may be limited during in vitro culture (Williams, 1993). In addition to in vitro shoot culture of S. tuberosum, shoot tip necrosis has been observed in shoot cultures of Pistacia vera L. in relation to calcium deficiency (Abousalim and Mantell, 1994). Results from these studies suggest that shoot tip necrosis, a symptom of calcium deficiency, can be precisely controlled and studied by regulating root zone calcium concentration using in vitro culture. Despite this fact, shoot culture systems have not been exploited to study growth and development regulation by nutrients in the root zone.
Our objective was to study shoot tip necrosis symptoms at the cellular level. We precisely defined the medium calcium concentration for the development of shoot tip necrosis symptoms. Sha et al. (1985) used media containing 0.3, 3.0, and 30.0 μm calcium and culture vessels containing multiple plants in each vessel. While the authors were able to show a relationship between shoot tip necrosis and the medium calcium concentration, they were not able to do precise experiments at the individual plant level owing to the interplant competition in the culture vessels. The exact medium calcium concentration for a given shoot could not be defined. The in vitro system we used allowed us to describe the structural basis of the symptoms at the tissue and cellular level. We further show that primary symptoms develop just below the shoot apical meristem where internode elongation occurs—the subapical region. We also demonstrate that this primary injury results in a collapse of tissue in the subapical region, causing the death of the shoot apical meristem and loss of apical dominance. Using 45Ca as a tracer, we tracked the distribution of calcium in the shoot under sufficient and deficient media calcium levels. Our results provide evidence for the involvement of calcium in subapical necrosis and we demonstrate that a shoot culture system can be used to further study the mechanism by which a lack of calcium causes loss of apical dominance.
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