Review

CALCIUM AND ITS MANY ROLES IN PLANT RELATIONS

By Charles H. Walker, II

1) CALCIUM IN SUPPORT TISSUE

Calcium is a macronutrient that is sequestered in vacuoles and the endoplasmic reticulum, and it is associated with the middle lamella of cell walls playing a role in support and growth (Wu et al 2002). After methyl groups have been cleaved from what is believed to be a highly methyl-esterified polymer by the enzyme pectin methylesterase (PME), the resultant carboxylate ions are bound to Ca²⁺ (Buchanan et al 2001).

The outer fibrillar layer of the pollen tube wall is largely composed of pectin that is cross-linked by Ca²⁺ that results in a supportive framework for the tube (Taylor and Hepler 1997). In addition, a calcium gradient is present at the tip of the pollen tube and this dictates the direction that the pollen tube will grow (Buchanan et al 2001). Nakata and McConn (2000), in their greenhouse experiment with Medicago truncatula, call into question the hypothetical role of calcium oxalate in support tissue as the growth and development of the Cod 5 mutant exhibited normal growth even though it did not sequester calcium in the crystalline form. Over 73% of the 295 families of gymnosperms and angiosperms produce oxalate (McNair 1932).

2) Calcium uptake and root hair development

Ca²⁺ is taken up by plants via the apoplastic pathway. It must be maintained within the cytosol in the 0.1–0.6 µM range because it precipitates phosphate, the energy currency of cells (Sze 2000; Buchanan et al 2001). Salinity stress inhibits the uptake of calcium via the symplastic pathway more than the apoplastic pathway and leads to calcium deficiency in shoots Halperin et al 1997). Halperin et al (1997) concluded that the endodermis of the older portions of the root are more suberised and utilize the symplastic pathway and in his experiment were more sensitive to elevated levels of salinity than were the younger portions of the root that utilized the apoplastic pathway.

The major nutrient-uptake sites are the root hairs. Root hairs do not develop from all root epidermal cells, only those known as trichoblasts. Atrichoblasts do not develop root hairs and can be distinguished from trichoblasts by their larger vacuoles. Trichoblast development varies from species to species. In Phleum and Hydrocharis trichoblasts result from an asymmetrical division of a protodermal cell, while in Arabidopsis epidermal cells that bridge underlying cortical cells develop into trichoblasts (Gilroy and Jones 2000). In Vicia all epidermal cells develop into trichoblasts (Miller et al 1999). In a study of 77 eudicotyledonous species from 43 families, most species had epidermal cells that could all potentially develop into trichoblasts, 10 families were similar to Arabidopsis in trichoblast development (Brassicaceae, Limnanthaceae, Resedaceae, Amaranthaceae, Basellaceae, Caryophyllaceae, Plumbaginaceae, Polygonaceae, Portulacaceae, and Boraginaceae), but none of the families had trichoblasts resulting from the asymmetrical division of a protodermal cell (Pemberton et al 2001).

The [Ca²⁺]_cyt gradient prevents actin filaments (cytoskeleton) from extending all the way to the vesicle-rich apex of the root hairs. Growth at the root hair apex is dependent upon the exocytosis of Golgi vesicles that are brought there by the actin filaments where they fuse with the cell membrane (Miller et al 1999).

WEREWOLF (WER) is an MYB-class protein that is expressed in epidermal cells of the root and hypocotyl where it suppresses root hairs and stomatal cells respectively, while Glabra1 (GL1) is expressed in the epidermal cells of the shoot producing trichomes on leaves (Kellogg 2001). In Arabidopsis, a protein called Transparent Testa Glabra (TTG) controls both the production of trichomes in the leaf epidermis as well as root hairs in the root epidermis and the origin of the genes GL1 and WER may be tied to either the origin of roots or trichoblasts (Kellogg 2001).

3) The [Ca²⁺]_cyt gradient

There are numerous ionic gradients operating at the same time and the [Ca²⁺]_cyt gradient is important in many signaling pathways. Ca²⁺ enters the cytosine through channels in the plasma membrane and/or is discharged from internal stores (Sze et al 2000). Cation channels that are specialized transport proteins catalyze Ca²⁺ influx through the plasma membrane of root cells and only recently have the genes that encode putative Ca²⁺-permeable channels been identified (White et al 2002).

The Ca²⁺ channel is primarily involved in cell signaling through the rapid transport of Ca²⁺ across the plasma membrane, tonoplast, endoplasmic reticulum, chloroplast or nuclear membrane (White 2000). Calcium channels open when the cell has been signaled, transmitting up to 10⁶ atoms of Ca per second that enters the cytoplasm down its electrochemical gradient from intracellular stores and the cell wall (Trewavas 1999). [Ca²⁺]_cyt can rapidly increase by tenfold and this brings about changes in Ca²⁺ modulated proteins and their targets that bring about response (Sze et al 2000). Calcium-pumping ATPases are found in the chloroplast envelope, endoplasmic reticulum, plasma membrane, and vacuolar membranes. These enzymes pump Ca²⁺ back out of the cytosol after a signaling event (Buchanan et al 2001).

Signal mechanisms of hormonal or environmental origin are used to regulate various physiological processes through signal transduction pathways that includes essential components called second messengers, e.g. Ca²⁺-calmodulin, cGMP, and phosphoinositide (Volotovski et al 1998). In addition to cyclic GMP (cGMP), lipids and pH have been shown to be messengers involved in signaling pathways, but none of these have been shown to respond to more stimuli than cytosolic free Ca²⁺ ([Ca²⁺]_cyt (Sanders et al 1999, Table 1).

The elevated levels of Ca in the cytoplasm, sometimes referred to as a spike, may last from a few seconds to several minutes and is dependent on the specific signal (Trewavas 1999). It is in the cell's nuclear region that the calcium spike originates and it is spread distally to the cell apex (Shaw and Long 2003). The diffusion of Ca²⁺ in the cytosol is slow and is impeded by its uptake into chloroplasts, endoplasmic reticulum, mitochondria, and vacuoles as well as by its binding to protein (Buchanan et al 2001). Buchanan et al (2001) asserts that a slow dispersal rate leads to standing gradients of Ca²⁺ that is important in pollen tube growth, vesicle fusion and continued growth, and signaling.

The opening of Ca₂₊ channels is brought about by the pre-depolarization of the plasma membrane and channel recruitment is dependent upon the intensity and length of time that the membrane was depolarized (Thuleau et al 1994). The plasma membrane-bound proton pump (H⁺-ATPase) electrically counterbalances the depolarizing Ca⁺² current and the Ca²⁺ channels close with Ca moving back into the cell wall and the cell vacuole (Trewavas 1999, Miedema et al 2001). Miedema et al (2001) concluded that the movement of a type of ion across the plasma membrane does not occur alone, but in concert with all the other types of ions interacting with each other by means of membrane voltage (V_m). Arabidopsis has been shown to have 14 Ca²⁺ pumps and ECA1 can transport Mn²⁺and Zn²⁺ as well as Ca² (Wu et al 2002). Wu et al (2002) also found genetic evidence supporting the critical role that ER-located pump plays in growth and tolerance to Mn²⁺ toxicity.

Deposition of plasma membranes and cell walls at the tip of the expanding root tip is linked to [Ca²⁺]_cyt. The [Ca²⁺]_cyt gradient shows a higher influx at the root hair tip, K⁺ influx at the root hair tip is the same as at the sides, and H⁺ influx is highest at the base and sides of the root hair (Gilroy and Jones 2000). The major anion (Cl^-) is outwardly directed. A similar [Ca²⁺]_cyt gradient has been observed in pollen tubes with values above 3 µM at the apex and dropping quickly to 0.2 µM within 20 µm of the tip (Taylor and Hepler 1997). Stimulus-induced oscillations of [Ca²⁺]_cyt are thought to encode information specifying the outcome of the final response of calcium-based signal pathways (McAinsh et al 1997, Evans et al 2002, White et al 2002). The different amplitudes and frequencies of oscillations in Ca²⁺ may encode different information and lead to different physiological responses downstream pathways (McAinsh et al 1997). This may also explain how different stimuli that increase guard cell [Ca²⁺]_cyt can both initiate closing and opening stomata (Table 2). Circadian peaks of [Ca²⁺]_cyt were observed in 3 seedlings of transgenic tobacco (Nicotiana plumbaginifolia) with the peaks occurring at different times, indicating that this may show differing cellular control mechanisms associated with Ca²⁺ mediated signal transduction in plant cells (Wood et al 2001).

4) STOMATAL CLOSURE

In order for the stomata to close there must be a large efflux of K⁺ and anions accompanied by the conversion of malate to starch and this leads to a reduction in the turgor of guard cells (McAinsh et at 1997). Stomates close 5 to 10 minutes after the increase of [Ca²⁺]_cyt (McAinsh et al 1990). The inhibiting of K⁺ influx by [Ca²⁺]_cyt is of secondary importance to the K⁺ efflux from guard cells that is unaffected by increases in [Ca²⁺]_cyt (McAinsh et at 1997). Outward rectifying K⁺ channels are probably activated by membrane depolarization during salt loss (Buchanan et al 2001). Opening of the rapidly activating R-type and the slowly activating S-type Ca²⁺ anion channels results in the loss of Cl^- and the depolarization of the plasma membrane (Buchanan et al 2001).

Ca²⁺ has been shown to be an intracellular second messenger in the closing of stomata with abscisic acid (ABA) inducing an increase in [Ca²⁺]_cyt in guard cells (McAinsh et al 1990). Abscisic acid is a phytohormone that is produced in fruit, older leaves, and in the root cap of plants, initiating the closing of stomata due to drought conditions. Nitric oxide is generated by guard cells in response to ABA by a NOS-like enzyme and is required for the full closure of stomates (Neill et al 2002). Neill et al (2002) also found that cGMP and cADP-R were required for NO- and ABA-induced stomatal closure. The second messenger cADP-R is synthesized from NAD⁺ and can mobilize Ca²⁺ from intracellular stores (Buchanan et al 2001). McAinsh et al (1991) determined that the increase in [Ca²⁺]_cyt came at least partially from intracellular stores of Ca²⁺, but contributions from the apoplast could not be ruled out. Ca²⁺ may also act as a second messenger in the carbon dioxide signal transduction pathway, triggering the closing of stomata due to elevated CO₂ (Webb et al 1996). Guard cells were shown to have an ABA perception site on the exterior of the plasma membrane, as viable ABA injected cells and uninjected cells failed to close, while those with ABA external to the plasma membrane did close (Anderson et al 1994). Other studies (MacRobbie 1995) have demonstrated that both external and internal vacuolar sites of ABA perception are involved.

Several Ca²⁺ channels have been identified in the plasma membrane, rough endoplasmic reticulum, tonoplast, and possibly even in mitochondria (Buchanan et al 2001). There are three families of Ca²⁺ channels that have been discovered in plants by researchers using patch clamp technology (Buchanan et al 2001). One group of Ca²⁺ channels, stretch-activated channels, monitor tension in the plasma membrane and the membrane of the vacuole, opening when there is an alteration in tension (Buchanan et al 2001). Stretch-activated channels mediate the transport of Ca²⁺ across the plasma membrane and may also influence the other Ca²⁺ -permeable sites (Cosgrove and Hedrich 1991). In addition stretch-activated channels appear to regulate Ca²⁺ influx in a small region at the tip of the growing pollen tube where growth occurs and the deformation of the membrane reaches its maximum (Taylor and Hepler 1997).

A second group is comprised of the receptor- and second messenger-regulated Ca²⁺ channels (Buchanan et al 2002). Inwardly rectifying K⁺ channels in guard cells of the fava bean were inhibited by activation of G-protein by Ca²⁺ (Fairley-Grenot and Assmann 1992). G-proteins are a subset of GTPase that act as molecular switches, but their primary function may be to increase accuracy of recognition during signaling, secretion, and the synthesis of protein (Buchanan et al 2001).

Lastly, the voltage-dependent Ca²⁺ channels at the membrane of the vacuole open when negative conditions exist in the cytosol, or with increased Ca²⁺ levels and/or pH in the vacuole (Allen and Sanders 1994). Non-selective voltage-gated Ca²⁺ -permeable channels at the plasma membrane allow Ca²⁺ to pass into the cell when ABA levels are high and these channels also allow the passage of K⁺ ions (Schroeder and Hagiwara 1990). Slow vacuolar cation channels (SV-type) allow Ca²⁺ and K⁺ ions in a 3:1 ratio to pass from the vacuole into the cytosol, suggesting a pivotal role for these channels during stomatal closure (Ward and Schroeder 1994). Membrane depolarization over a period of several hundred milliseconds slowly activates SV channels, as does Ca²⁺-calmodulin (Buchanan et al 2001). Ca²⁺ activates vacuolar K⁺ (VK) channels that results in a shift in the vacuolar membrane potential to more positive potentials outside the vacuole and this activates the SV channels causing the efflux of Ca²⁺ from the vacuole into the cytosol (McAinsh et al 1997).

5) Calcifuges and calcicoles

The calcifuges ("chalk-escaping"), Veronica officinalis and Carex pilulifera, developed chlorosis when grown in calcareous soils as they were unable to translocate sufficient amounts of iron or they accumulated iron in their leaves that was not of an active form that could be extractable by iron complexing agents (Zohlen and Tyler 1997). However, calcifuge species of the limestone heaths in southwest Britain have persisted in that area despite soil erosion and soil pH that has been on the rise (Etherington 1981). It may not be the pH that is critical as clover and other plants can be grown on acidic soils with the addition of limestone, even though the pH remains acidic (Albrecht 1941). In addition the growth rate of 9 out of 10 calcifuge plants doubled or tripled with the addition of 5 mol m^-3 of CaHPO_4, indicating the inability to utilize the form of phosphate prevalent in the Ordovician limestone soils of pH 8 (Tyler 1994).

The concentration of calcium in both the roots and shoots of Agrostis capillaris (Tyler and Olsson 2001) and A. canina, A. setacea, A. stolonifera, and A. tenuis (Clarkson 1965) is lower at low soil solution pH. However at higher Ca levels, the amount of Ca sequestered in the shoots and roots of the calcifuge A. setacea was less than half as much as was sequestered in the calcicole A. stolonifera (Clarkson 1965).

Calcicoles ("chalk-loving") grow in soils with high pH and growth is stimulated by high calcium concentrations (Lambers et al 1998). Calcifuges have been shown to invade calcareous soils in areas of high rainfall and soils with low aeration (Grime 1963). Alfalfa can be grown in states like Kansas and Colorado without the addition of calcium to the soil, because annual rainfall is lower and calcium has not been lost due to leaching as it has in the southeast U.S (Albrecht 1941). The calcicoles Veronica spicata and Phleum phleoides are better able to uptake P, K, and Mn under increased levels of soil moisture, while Ca, Mg, and Zn uptake diminishes (Misra and Tyler 1999). Calcicoles exudate more dicarboxylic oxalate and tricarboxylic citrate, low-molecular organic acids (LOAs), than do calcifuge species (Ström 1998). Ström (1998) proposes that dicarboxylic oxalate and tricarboxylic citrate solubilize soil phosphate and iron, and this is what enables calicoles to grow in soils that are nutrient limited. The increased availability of phosphate and iron in the rhizosphere enhances the concentration of calcium in the soil and consequently in the xylem sap of calcicole species (Lambers et al 1998). As soil pH increases so does the total LOAs concentration and as the percent soil organic matter increases, the total LOAs concentration decreases and (Shen et al 1996). Shen et al (1996) found that the dicarboxylic and tricarboxylic acids were readily immobilized in the soil, but not most of the monocarboxylic acids. Micronutrients that are less available (Fe and Mn) or are not readily solubilized (Ca) have higher seed:leaf concentrations in calcicoles and calcifuges respectively, indicating a strategy for satisfying the need by seedlings for nutrients that are not readily available in the soil (Tyler and Zohlen 1998).

6) Bacteria and Oomycetes

Root cells produce exudates, usually flavonoids, that induce gene expression in bacterium in the rhizosphere, e.g. Rhizobium, Azorhizobium, Bradyrhizobium, and Sinorhizobium (Long 1996). In Rhizobium, flavonoids are first bound with a bacteroid gene product and then they interact with a promoter from the genetic complement of the bacteria (Lambers et al 1998). Only members of the Fabaceae family and the genus Parasponia in Ulmaceae have the ability to form nitrogen-fixing symbiosis with Rhizobium and other bacteria genera that are gram-negative Szczyglowski and Amyot 2003). Evans et al (2002) described how Nod factors (NFs) or lipochitooligosaccharide signals are synthesized by bacteria and are in response to the mixture of flavonoids and other inducers secreted from root cells. Gene expression in bacterium varies due to the wide range in specific mixtures that could be received (Long 1996). Nod factors are involved in the signal transduction pathway leading to root nodule formation. There are 2 responses to Nod factors that indicate the role of Ca²⁺ as a second messenger. Low concentrations of NF (< 1 nM) elicit an initial calcium spike near the tip of the hair cell, while a high NF concentration (10 nM) evokes a separate and independent calcium flux that occurs near the nuclear region (Shaw and Long 2003). One of the earliest responses by root hair cells to Nod factors with the subsequent infection by Rhizobium is calcium spiking (Szczyglowski and Amyot 2003). Spikes propagate along the root hair and may carry information from the nucleus to other parts of the cell (Felle et al. 1999). The nodulation mutants sym8, sym10, and sym19 did not exhibit chitin-oligomer-induced calcium spiking that is necessary for mycorrhizal infection, so the Fabaceae nodulation genes may be involved in both nodulation and mycorrhizal signaling (Walker et al 2000).

Another response in root hairs to NFs is a temporary and periodic increase in cytosolic calcium levels known as calcium flux (Engstrom 2002). The increase in cytosolic Ca²⁺ leads to a cascade of events at the plasma membrane including a rapid efflux of Cl^-, the alkalinization of the root hair zone, depolarization of the cell, and the delayed efflux of K⁺ (Felle et al 1998, Felle et al 1999). The efflux of Cl^- brings about the depolarization of the cell, and the efflux of K⁺ quickly repolarizes it again Felle et al 1998). Calcium involved in spikes originates from intracellular stores close to the nucleus, but the Ca that is involved in fluxes originates from outside the plasma membrane (Felle et al 1999). Initially, both potential symbiotic partners and microbial intruders invoke a Ca²⁺ flux. However, defense-related reactions by alfalfa root hairs to (GlcNAc)₈ included a slow persistent acidification of the cytosol and the root hair space, rather than a rapid and persistent alkalinization of the cytosol as induced by Nod factors (Felle et al 2000). Felle et al (2000) describes plant defense as occurring in two phases. Phase 1 entails an immediate reaction involving an oxidative burst and/or ion fluxes that creates conditions that are unfavorable to the attacking organism e.g. acidification of the cytosol and root hair space. Phase 2 is a long-term reaction involving an array of defensive measures to actively ward off an intrusion. Within 18 to 30 hours of an intrusion by Rhizobium cell division is initiated towards the outside of xylem cells within the cortex leading to the formation of nodule tissue (Buchanan et al 2001). There may be a signal emanating from within the stele that is responsible for this pattern of cell division and the products of nodulin genes, the plant hormones auxin, cytokinin, and especially ethylene have been linked to nodule formation in Fabaceae (Buchanan et al 2001).

Many oomycetes, e.g., Phytophthora, Pythium, etc., are phytopathogens that infest plant roots through chemoattractants, calcium, or electric fields (Tyler 2002). In many cases there is a sensitive attraction of a specific oomycete to a specific species, e.g., Phytophthora sojae to the isoflavones secreted by soybean roots (Morris and Ward 1992). When soil nutrient and Ca²⁺ are low, zoospores develop a sporangium that releases a single new spore termed a secondary zoospore and through repeated incarnations can progress a considerable distance through the rhizospere (Tyler 2002). Conversely, high concentrations of Ca²⁺ can trigger circular swimming and encystment by Pythium zoospores (Donaldson and Deacon 1993). Calcium could be responsible for signaling how close the swimming zoospore is to the root surface and bring about the change from a searching mode to a docking mode (Tyler 2002).

BIBLIOGRAPHY

Stimulus Red light Abscissic acid Gibberellin Salinity/drought Hypoosmotic stress Touch Fungal elicitors Cold KIN1 Heat shock Oxidative stress NOD factors

Example of Response Photomorphogenesis Stomatal closure -Amylase secretion Proline synthesis Osmoadaptation Growth retardation Phytoalexin synthesis gene expression Thermotolerance Free radical scavenger induction Root hair curling

Reference Shacklock et al. 1992 McAinsh et al. 1990 Bush and Jones 1988 Knight et al. 1997 Taylor et al. 1996 Knight et al. 1991 Knight et al. 1991 Knight et al. 1996 Gong et al. 1998 Price et al. 1994 Ehrhardt et al. 1996

Table 1. Some Physiological Stimuli That Elevate [Ca²⁺]_c in Plant Cells (Sanders et al 1999)

 

Table 2. Ca²⁺ response in guard cells to various stimuli (McAinsh et al 1997).

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