Protoplasma

, Volume 247, Issue 3, pp 215–231

Calcium storage in plants and the implications for calcium biofortification

Authors

  • Maclin Dayod
    • Waite Research Institute, School of Agriculture, Food and WineUniversity of Adelaide
  • Stephen Donald Tyerman
    • Waite Research Institute, School of Agriculture, Food and WineUniversity of Adelaide
  • Roger Allen Leigh
    • Waite Research Institute, School of Agriculture, Food and WineUniversity of Adelaide
    • Waite Research Institute, School of Agriculture, Food and WineUniversity of Adelaide
Review Article

DOI: 10.1007/s00709-010-0182-0

Cite this article as:
Dayod, M., Tyerman, S.D., Leigh, R.A. et al. Protoplasma (2010) 247: 215. doi:10.1007/s00709-010-0182-0

Abstract

Calcium (Ca) is an essential nutrient for plants and animals, with key structural and signalling roles, and its deficiency in plants can result in poor biotic and abiotic stress tolerance, reduced crop quality and yield. Likewise, low Ca intake in humans has been linked to various diseases (e.g. rickets, osteoporosis, hypertension and colorectal cancer) which can threaten quality of life and have major economic costs. Biofortification of various food crops with Ca has been suggested as a good method to enhance human intake of Ca and is advocated as an economically and environmentally advantageous strategy. Efforts to enhance Ca content of crops via transgenic means have had promising results. Overall Ca content of transgenic plants has been increased but in some cases adverse affects on plant function have been observed. This suggests that a better understanding of how Ca ions (Ca2+) are stored and transported through plants is required to maximise the effectiveness of future approaches.

Keywords

ApoplasmApoplastBiofortificationBioavailabilityCalciumCAXOsteoporosis

Abbreviations

At

Arabidopsis thaliana

Arabidopsis

Arabidopsis thaliana

Ca

Calcium

Ca2+

Calcium ion(s)

[x]

Concentration of x

CDC2a

Cell division cycle 2a gene promoter

CMV

Cauliflower mosaic virus

EDTA

Ethylenediaminetetraacetic acid

ER

Endoplasmic reticulum

PM

Plasma membrane

RDI

Recommended daily intake

Introduction

Calcium (Ca) is an essential macronutrient for plants and animals where it plays indispensable structural and signalling roles. Soluble calcium ions (Ca2+) are extracted from the soil solution by plants to form the primary source of Ca in the food chain. In recent years, our understanding of the signalling role of Ca2+ in organisms has developed significantly (e.g. Dodd et al. 2010; Hogan et al. 2010). However, fundamental information regarding the mechanisms that regulate Ca2+ transport and storage in plants is still lacking. The human dietary intake of Ca is, in the majority of cases, below the recommended daily intake (RDI) of 800–1,300 mg, and this is believed to result in widespread health and economic costs (Milott et al. 2000; Kranz et al. 2007; Lanham-New 2008; Vatanparast et al. 2010). Plants potentially represent a cheap and convenient source of dietary Ca, however, attempts to biofortify crops with greater amounts of Ca have had mixed results (Hirschi 2009). Therefore, further exploring how plants transport and store Ca2+ may have far-reaching benefits.

The role of Ca in plants has been the topic of numerous wide-ranging reviews in the context of: Ca as a plant nutrient (White and Broadley 2003); a signalling element (McAinsh and Pittman 2009; Dodd et al. 2010); or a dietary source for animals and humans (Hirschi 2009). A recent review also focused on how discrete expression patterns of Ca2+-transporters may influence Ca2+ storage in plants (Conn and Gilliham 2010). Here, we highlight how an understanding of Ca2+ transport and storage in plants may inform research efforts aimed at producing plants with increased Ca content for improved shelf life, nutrition and stress tolerance.

Plants as a source of dietary Ca for humans

Low dietary Ca intake has been linked to diseases such as osteoporosis (Heaney 1993a; Chan et al. 2007) and rickets (Bhatia 2008; Pettifor 2008); both are conditions of low bone density and are underlying cause of bone fragility in humans. There is debate on whether an excess of fluoride in diets acts synergistically with Ca deficiency to cause rickets (Pettifor et al. 2008; Teotia and Teotia 2008a, b). However, severe infantile Ca deficiency leading to rickets (synonymous with bone deformities and increased infant mortality) is rare and mostly confined to extreme conditions e.g. children previously described in regions of South Africa, Nigeria and Bangladesh with near zero Ca intake (Pettifor et al. 1978; Oramasionwu et al. 2008; Pettifor 2008). Low Ca diets have also been linked to hypertension, and with colorectal and other cancers (Heaney and Bargerlux 1994; Centeno et al. 2009). Calcium intake in pregnant woman is particularly important for foetal skeleton development, increasing birth weight and avoidance of pre-eclampsia and prenatal hypertension (Chan et al. 2006). Conversely, osteoporosis is a condition that mainly affects older people and is linked to enhanced decalcification and dimineralisation of bones (Michaelsson et al. 2005). Most, but not all, studies show that increasing Ca intake in later life decreases the occurrence of osteoporotic fractures and it is universally accepted that sustaining the RDI of Ca throughout life is beneficial for health later in years (Heaney 1993b; Michaelsson 2009).

Bone demineralisation usually occurs after the age of 35 unless associated with other conditions such as alcoholism or eating disorders (Prentice 2004; Binkley 2009; Setnick 2010). Although heritable genetic factors play a significant role, in almost a third of cases dominant factors are nutrition, hormone levels, exercise and body mass (Kelly et al. 1990; Michaelsson et al. 2005). In Australia, 50% of females and 33% of males over the age of 60 years will have an osteoporotic bone fracture, with on average, one person admitted to hospital every 6 min; the total direct costs are AU $1.9 bn per annum, with further associated indirect costs in excess of AU $8 bn per annum (OA 2010; AIHW 2010). The problem is clearly not unique to Australia, for instance in the USA direct costs are an estimated US $13.8 bn each year and in the EU osteoporosis-related hospital admission occurs every 30 s (Bachrach 2001; OA 2010). Clearly, Ca nutrition in humans is something that needs to be addressed to improve quality of life and reduce health care costs. However, Ca intake in 90% of adolescent girls and 50% of adolescent boys in the U.S.A. is sub-optimal; adolescence being the stage when bone mass increase, and the body’s requirement for Ca is at its highest and most important (Bachrach 2001; Vatanparast et al. 2010).

The largest potential source of dietary Ca is found in dairy products but due to dietary preferences or dairy-product intolerances, consumption may be minimal in some individuals. After dairy products, plant products make the largest potential contributor to Ca intake (Weaver and Plawecki 1994; Weaver et al. 1999; Lanham-New 2006). However, most staple cereal foods (e.g. rice, maize and wheat) have low Ca content (Jeong and Guerinot 2008). For instance, the average Ca content of rice or wheat is 12–15 mg.g−1 DW (Table 1) (Weaver et al. 1991; Jiang et al. 2007), therefore to receive the full recommended adult intake of Ca, if only consuming cereals, at least 10 kg must be consumed each day—this is clearly not acceptable or likely. Furthermore, the total concentration of Ca ([Ca]) present in a food source does not necessarily reflect the amount of Ca that will be absorbed from that food (Table 1) (Hirschi 2009). Therefore, human feeding trials to test absorbability of nutrients from food sources, especially to test claims that certain biofortified foods will improve the diet, are advocated as a necessity (Hirschi 2008; Hirschi 2009).
Table 1

Calcium content in selected vegetables and its absorbability in premenopausal women

Calcium source

 

Ca content (mg per 100 g)

Calcium content (mg per serving)

Fraction of Ca absorbed (±SD)

References

Broccoli (Brassica oleracea)

51

83

0.478 ± 0.089

Heaney et al. 1993

Bok Choy leaves (Brassica rapa chinensis)

97

83

0.520 ± 0.074

Heaney et al. 1993

Bok Choy stems (Brassica rapa chinensis)

84

83

0.519 ± 0.089

Heaney et al. 1993

Carrot (Daucus carota)

Wildtype transgenic (sCAX1)

32

38

0.488 ± 0.048

Morris et al. 2008

58

38

0.421 ± 0.043a

Chinese cabbage flower leaves (Brassica rapa pekinensis)

306

200

0.402 ± 0.017a

Weaver et al. 1997

Chinese mustard greens (Brassica juncea)

265

200

0.379 ± 0.026a

Weaver et al. 1997

Chinese spinach (Amaranthus spp.)

460

200

0.093 ± 0.007a

Weaver et al. 1997

Common beans (Phaseolus vulagaris)

Red

179

71.6

0.193 ± 0.050

Weaver et al. 1993

White

138

71.6

0.225 ± 0.061

Pinto

158

71.6

0.231 ± 0.053

Pintoc

158

71.6

0.318 ± 0.071

Ivy gourd (Coccinia grandis)

100

100

0.476 ± 0.109

Charoenkiatkul et al. 2008

Kale (Brassica oleracea)

127

83

0.527 ± 0.091

Heaney et al. 1993

Milk

110

110

0.377 ± 0.056

Heaney et al. 1991

110

82

0.463 ± 0.095

Weaver et al. 1993

110

200

0.451 ± 0.088

Heaney et al. 1993

110

200

0.379 ± 0.026a

Weaver et al. 1997

120

250

0.217 ± 0.040b

Zhao et al. 2005

110

220

0.552 ± 0.119

Charoenkiatkul et al. 2008

Rhubarb (Rheum rhabarbarum)

237

120

0.092 ± 0.008a

Weaver et al. 1997

Soy bean milk (Glycine max)

H. phytate

0.13

110

0.310 ± 0.070

Heaney et al. 1991

L. phytate

0.13

111

0.414 ± 0.074

Heaney et al. 1991

Fortified

100

250

0.211 ± 0.057b

Zhao et al. 2005

Sweet potato (Ipomoea batatas)

54

63

0.228 ± 0.021a

Weaver et al. 1997

Wheat flour, milled (Triticum arvense)

19

15

0.817 ± 0.124

Weaver et al. 1991

Winged bean (Psophocarpus tetragonolobus)

65

52

0.391 ± 0.128

Charoenkiatkul et al. 2008

a±SEM

bNot significantly different

cPhytate treated

Many nuts, fruits and vegetables contain significantly more Ca than cereals. Although total [Ca] may be higher, the fraction of bioavailable Ca in these foods may be much lower when compared with dairy products. This is due to a significant presence of ‘anti-nutrients’ such as phytate and oxalate that reduce the absorbability of Ca by humans (Table 1) (Hirschi 2009). When [Ca] and/or [phytate] or [oxalate] are high, this decrease in absorbability can be particularly strong. For instance, Amaranthus spp. (Chinese spinach) and Rheum rhabarbarum (rhubarb) have very high Ca content but less than 10% of it can be absorbed from the food. Whereas, some foods such as Coccinia grandis (ivy gourd), Brassica oleracea (kale) and Brassica juncea (Chinese mustard greens) have similar Ca content and absorbability to dairy products (Table 1) due to low ‘anti-nutrient’ content, and as such are suitable alternatives to increase Ca nutrition of humans.

Dietary fortification and supplements, such as Ca addition or phytase treatment, are also an effective solution to increase plant Ca content and bioavailablilty. Phytase treatment of beans increased absorbable Ca by a third but Ca content was still low when compared with cow’s milk (Table 1) (Weaver et al. 1993). Soybean milk supplemented with Ca to levels equivalent of cow’s milk also had equivalent absorbability but this was dependent on the form of Ca added (Table 1) (Weaver et al. 1993; Zhao et al. 2005), similar results have been shown in orange juice and soft-drinks (Schroder et al. 2005). Costly extraction processes may also be used to increase available Ca (Zhao et al. 2005). However, all such manipulations are expensive and therefore not viable for increasing nutrition in low income groups, particularly those in developing countries (Table 1) (Heaney et al. 1993; Hirschi 2009; Gomez-Galera et al. 2010). For this reason, strategies that enhance Ca content or bioavailability of plants without significant additional inputs or processing are being explored (White and Broadley 2005).

Improving plants as a source of Ca for humans

Calcium content of plants generally increases with an increase in external Ca2+ supply (White 2001) so Ca fertilisation of food crops may be an option to increase bioavailable Ca, but again, such an approach may have significant economic and environmental costs (Gomez-Galera et al. 2010). Biofortification of major food crops through transgenic or breeding may provide an alternative approach to enhance the [Ca] and/or the concentration of other essential nutrients (White and Broadley 2005). It is hoped that food may be obtained in this way to provide an array of nutrients in a form that the body is evolved to process, this is in contrast to the way nutrients are often supplied by dietary supplements (Genc et al. 2005; White and Broadley 2005; Graham et al. 2007). Furthermore, in addition to enriched nutrient profiles it is hoped that crops produced in this way will not have diminished productivity or inferior taste, or require additional inputs, although this would obviously need testing. An added complication may be on the horizon due to climate change; elevated CO2 has been reported to decrease [Ca] in leaves and grain of wheat (Loladze 2002; see ‘The transport of Ca in plants’). This could compound the already limited Ca intake of those dependent on plant-derived Ca sources and is further incentive to study the processes of Ca accumulation in plants.

It is an aim of plant breeding or genetic modification to increase productivity and stress tolerance of plants, and hence increase food production in general. Such approaches may inadvertently improve nutritional values of diets by increasing availability of food and/or lowering prices (Gomez-Galera et al. 2010). However, specific genetic modification or breeding of plants to enhance [Ca] or availability is a desirable parallel strategy to improve dietary Ca. The removal of oxalate or the breeding of low phytate crops may be an important step in increasing Ca absorbability and has met with some success (Morris et al. 2007; Hirschi 2009). For instance, low phytate varieties of soybean have Ca absorbability equal to cow’s milk but Ca content was still orders of magnitude less, as such, soymilk products are still commonly fortified with additional Ca if they are to be used as a dairy substitute (Table 1) (Heaney et al. 1993; Heaney et al. 2000). Transgenic approaches for directly increasing Ca content of food crops, have so far produced promising but mixed results. Hirschi and co-workers have increased the Ca content of some economically important crops (i.e. tomato, potato, lettuce, carrot, watermelon and tobacco) by constitutively increasing expression of a number of tonoplast localised Ca2+-transporters (Table 2) (Hirschi 1999; Park et al. 2004; Kim et al. 2005; Park et al. 2005a; Park et al. 2005b; Park et al. 2009; Han et al. 2009). In the case of carrot, Ca content was doubled and, despite a 10% decrease in the absorbable Ca, there was effectively a 41% increase in the Ca assimilated in humans fed the same mass of transgenic carrot over those fed control carrots (Table 1) (Morris et al. 2008). The reasons for the decrease in absorbable Ca in the carrots is unknown but it is likely to have its origin in how the Ca is stored within the tissue, and linked to the ‘anti-nutrient’ content of the plant (see ‘Future prospects for biofortification of plants with additional Ca’).
Table 2

Transgenic approaches to increasing Ca content and/or availability in plants

Species

Approach

Target

Ca (and other element) content

Other phenotypes

References

Medicago trunculata

EMS mutagenesis of population to find mutants lacking Ca oxalate crystals

Mutants with no (cod5) or reduced (cod6) calcium oxalate crystals identified—no gene identified yet

∼10% reduction in Ca content of leaves but 23% increase in Ca absorbed by mice in feeding trial

Increased palatability of cod5 and cod6 mutants in insect feeding studies

Korth et al. 2006; Morris et al. 2007 (and references within)

Tomato (Lycopersicon esculentum Mill.)

Vacuolar Ca2+-transporter overexpression

AtsCAX1 under control of AtCDC2a promoter (less than half activity of 35S)

20% increase in leaves, 100% increase in fruit (increase from ∼2.5 to ∼5 mg.g−1 DW); also increases in Cu, Fe, Mg, Mn and Zn

More compact growth, 40% increase in root mass, thicker leaves, delayed fruit-set (∼5, increase in shelf-life (∼40 day) rescued seed size, day) reduced fertility, necrotic lesions on primary transformants, required additional exogenous Ca to alleviate deficiency symptoms (blossom end rot)

Park et al. 2005a

AtCAX4 driven by 35S promoter

50% increase in fruit (no change in other elements)

Increase in fruit hardness/shelf life (∼5 day) Otherwise indistinguishable from vector control plants

Park et al. 2005a

AtsCAX2A driven by 35S promoter

100% increase in fruit (from 0.3 to 0.6 mg.g−1 DW)

Similar to wildtype

Chung et al. 2010

Potato (Solanum tuberosum)

AtsCAX1 driven by CDC2a or 35S promoter

300% increase in tubers (from ∼0.5 to ∼1.5 mg.g−1 DW) in 1st and 2nd generation, 200% increase in 3rd; 200% increase in leaves (from ∼5 to ∼10 mg.g−1 DW); no other elements mentioned

Indistinguishable from wildtype, although lesions in leaves and requirement for exogenous Ca reported in Kim et al. 2006

Park et al. 2005b

AtsCAX2B driven by 35S promoter

∼50–65% increase in Ca in tubers (no Mn increase) from ∼1.5 to ∼2.5 mg.g−1 DW; no Ca increase in leaves

Indistinguishable from wildtype

Kim et al. 2006

Carrot taproot (Daucus carota)

Vacuolar Ca2+-transporter overexpression

AtsCAX1 driven by CDC2a or 35S promoter

160% increase in Ca from ∼3 to 5 mg.g−1 DW

Indistinguishable from wildtype

Park et al. 2004; Morris et al. 2008

Rice (Oryza sativa)

AtsCAX1 under control of 35S promoter

None reported

None reported

Kim et al. 2005

Tobacco (Nicotiana tobacum)

AtsCAX1 using 35S promoter

100% increase in roots from ∼15 to ∼35 mg.g−1 DW; 30% increase in shoots from ∼15 to ∼22 mg.g−1 DW

Necrotic lesions, reduced root mass, ameliorated by supplemental Ca

Hirschi 1999

AtsCAX1 using AtCDC2a promoter

15% increase in shoots

Not reported

Reported in Park et al. 2009

Lettuce (Latuca sativa)

AtsCAX1 using 35S promoter

19 mg.g−1 DW compared, 25–32% more Ca than control; no increase in other elements

Indistinguishable from control, also in sensory characteristics

Park et al. 2009

AtsCAX1 using AtCDC2a promoter

19 mg.g−1 DW compared, 25–32% more Ca than control; no increase in other elements

Indistinguishable from control, also in sensory characteristics

Park et al. 2009

Bottle Gourd (Lagenaria siceraria Standl.)

Transgenic bottle gourd used as a rootstock to watermelons to overexpress vacuolar Ca2+-transporter

AtsCAX2B using 35S promoter

9% increase in root; no increase in shoot of rootstock; 8% increase in K in root; 45% increase in Na in 27% shoot and in root

46% increase in biomass in rootstock. No change in grafted watermelon fruit weight or size but increase in Brix, osmotic pressure and soluble solids

Han et al. 2009

Despite an increase in total Ca content of plants constitutively overexpressing tonoplast Ca2+-transporters, many of the transgenic plants showed signs of increased incidence of Ca2+-deficiency in the plant tissues that traditionally show symptoms, i.e. leaf tip necrosis or blossom end rot of fruit (Table 2) (Hirschi 1999; Park et al. 2005a; Park et al. 2005b). Such an approach may therefore be detrimental to plant function (Hirschi 1999; Cheng et al. 2003) (see ‘Calcium is differentially distributed in plant tissues’ and ‘Future prospects for biofortification of plants with additional Ca’). How this may occur is outlined in the following sections which describe how Ca is distributed, transported and compartmentalised within leaves.

Calcium as a plant nutrient

Calcium is an essential plant macronutrient. Calcium ions are required for plant membrane stability, cell wall stabilisation and cell integrity, in some cells they are a major osmoticum and in all cells they are fundamental to multiple intracellular signalling events (Sanders et al. 1999; Knight and Knight 2001; White and Broadley 2003; McAinsh and Pittman 2009; Conn and Gilliham 2010; Dodd et al. 2010). Once Ca2+ have been transported into cells they are relatively immobile and are not readily redistributed from the mature to the actively growing parts of plants; only under very special conditions are they redistributed, i.e. when damage occurs to tissues (Malone et al. 2002).

The relatively immobile nature of Ca within plants appears contrary to the multitude of roles and functions it has in plant physiology. Furthermore, the lack of redistribution, combined with limitations on transport pathways (see ‘The transport of Ca in plants’) can lead to local deficiencies despite abundance in supply. Local Ca deficiency in plant tissues affects the development of the cell wall and causes local cell necrosis (White and Broadley 2003). When present in crop plants, Ca deficiency can be a major horticultural problem leading to decreased quality and yield (White and Broadley 2003). Treatment of plants with supplemental soil or foliar Ca2+ sources can help to alleviate many of these symptoms, and may also help to prevent many parasitic diseases presumably through strengthening of the cell wall or priming plant defences against pathogen attack by some unknown mechanism (Table 3) (White and Broadley 2003). However, exogenous application of Ca is not universally successful in improving quality of crops. In particular, Ca deficiency in internal meristems, which results for instance in tipburn of leaves, is not ameliorated by simply supplying the plant with additional Ca (Leclerc et al. 1992).
Table 3

The external application of calcium salts can alleviate crop diseases

Host plant

Disease

Pathogen

Calcium source

References

Apple (Malus domestica Biorkh.)

Bitter rot

Colletotrichum gloeosporioides and Colletotrichum acutatum

CaCl2

Biggs 1999

Ca propionate

Citrus (Citrus aurantium L.)

Phytophthora root rot

Phytophthora nicotianae

CaO

Campanella et al. 2002

Dry bean (Phaseolus vulgaris)

White mold

Sclerotinia sclerotiorum

CaCl2

Paula et al. 2009

Ca3SiO5

Peach (Prunus persica)

Brown rot

Monilinia fructicola

CaCl2

Elmer et al. 2007

Potato (Solanum tuberosum)

Soft rot

Erwinia carotovora

CaSO4

McGuire and Kelman 1986

Pink rot

Phytophthora erythroseptica

Ca(NO3)2

Benson et al. 2009

Rice (Oryza sativa)

Sheath rot

Sarocladium oryzae

CaSO4

Narasimhan et al. 1994

Soybean (Glycine max)

Phytophthora stem rot

Phytophthora sojae

CaCl2

Sugimoto et al. 2008

Ca–formate

Ca(NO3)2

CaSO4

Strawberry (Fragaria x naassa)

Grey mould

Botrytis cinerea

CaSO4

Naradisorn et al. 2006; Singh et al. 2007

CaCl2

Tomato (Lycopersicon esculentum Mill.)

Powdery mildew

Erysiphe orontii

CaCl2

Ehret et al. 2002

Ca–EDTA

Ca(NO3)2

CaCl2 Ca chloride, EDTA CaNa2C10H16N2O8, formate (CaHCOO)2, Ca(NO3)2 nitrate, CaO oxide, propionate (CaC2H5COO)2), Ca3SiO5 silicate, CaSO4 sulphate

Shelf life and storage potential of vegetables are of great importance to the horticultural industry and consumers alike. Table 4 outlines selected studies that show improvements in various physiological traits, crop quality or shelf life by supplying additional Ca. Exogenous Ca application increases shelf life of some crops by delaying ripening and softening of fruit by: decreasing fruit respiration rate and ethylene production by an unknown mechanism (Gerasopoulos et al. 1996); increasing cell wall integrity (by generating cross-links with non-esterified pectins in the primary cell wall and middle lamella (Cosgrove 2005) and reducing transpiration (Saladié et al. 2007) which will lead to slower turgor loss and fruit softening. But fertilisation of crops with additional Ca, or exogenous application directly to fruits, obviously has a cost, so if improvements in shelf life or fruit quality can be made by breeding or transgenic means this will be advantageous. In the case of tomato, watermelon and potato this has already been observed (Table 2; see ‘Future prospects for biofortification of plants with additional Ca’) but the mechanisms by which this occur are still unknown.
Table 4

Beneficial effects on the physiological and quality parameters in economically important crops

Crop

Parameter improved

Calcium source

References

Apple (Malus domestica Biorkh.)

Increased fruit Ca content and firmness, simultaneous application with B increased these parameters further

CaCl2

Sen et al. 2010

Cherry (Prunus avium L., ‘Vogue’)

Greater fruit firmness, lower soluble pectin content, more resistance to stem removal and less stem browning

CaCl2

Tsantili et al. 2007

Cucumber (Cucumis sativus)

Increased photosynthesis under low light intensity and sub-optimal temperature

CaCl2

Liang et al. 2009

Table grape (Vitis Vinifera)

Berries were larger, more turgid, with lower dry matter and larger cells—although more likely associated with increase in berry chloride

CaCl2

Bonomelli and Ruiz 2010

Kiwifruit (Actinidia deliciosa)

Increased fruit pericarp, core and skin Ca, fruit firmness and storage life

CaCl2

Gerasopoulos et al. 1996

Mango (Mangifera indica)

Higher Ca content in the peel and flesh, a lower cumulative physiological loss in weight and a reduced respiration rate

CaCl2

Singh et al. 1993

Ca(NO3)2

Peach (Prunus persica)

Enhanced Ca content in fruits

CaCl2

Elmer et al. 2007

Prolong fruit firmness and shelf life

CaCl2

Singh and Sharma 2009

Potato (Solanum tuberosum)

Enhanced Ca content in tubers

CaSO4

McGuire and Kelman 1986

Strawberry (Fragaria x anaassa)

Firmer and brighter fruits with higher ascorbic acid content and acidity

CaCl2

Singh et al. 2007

Addition of: CaCl2 Ca chloride, Ca(NO3)2 nitrate or CaSO4 sulphate

Calcium also plays a role in tolerance to some abiotic stresses such as cold and salt (NaCl). Sodium ions (Na+) can reduce the yield of plants and these effects can be exacerbated when external [Ca] is low; apoplasmically1 supplied Ca has dual roles in improving Na+ tolerance by reducing toxic Na+ influx into cells and maintaining cell wall integrity (Munns and Tester 2008). Sodium stress also induces Ca-deficiency symptoms within the cell wall, presumably by competing for cell wall binding sites but not providing strong cross-linking of pectins. Therefore, the role of apoplastic1 Ca is seen as critical in maintaining cell wall integrity under saline conditions (Reid and Smith 2000). Many abiotic stresses also have calcium dependent signalling pathways that confer some degree of cold acclimation and salt tolerance, and have been outlined in detail elsewhere (Knight and Knight 2001; Munns and Tester 2008; McAinsh and Pittman 2009; Dodd et al. 2010). Influx of apoplasmic Ca into the cytosol, and cytosolic [Ca] transients, are required for many responses involved with salt tolerance or cold acclimation (Knight and Knight 2000; Tracy et al. 2008). If Ca homeostasis is perturbed the responses to certain stresses will be altered and tolerances affected (see ‘Future prospects for biofortification of plants with additional Ca’). Therefore, when considering strategies to manipulate transport and storage of Ca, particularly by the manipulation of Ca2+-transport proteins, the tolerance of transgenic crops to stresses must be evaluated.

Unfortunately, the exact mechanisms that lead to cell necrosis due to Ca2+-deficiency are not known, but it appears to be particularly prevalent in organs with low transpiration (Olle and Bender 2009a, b). It is not known whether necrosis is initiated within the cell wall or within cells, or both, and it has been speculated to be co-associated with Ca-deficiency and/or stress signalling or giberrelic acid synthesis (Saure 1998; Olle and Bender 2009a, b). The fact that Ca fertilisation, despite increasing Ca content of plants, is not always effective in ameliorating localised deficiency symptoms suggests that it is the way that Ca2+ is transported in the plant, and not the supply, that is the dominant factor influencing the severity of deficiencies (White 2001; Hartz et al. 2007; Johnstone et al. 2008).

The transport of Ca in plants

Calcium is normally acquired from the soil solution in the form of Ca2+. Transport of Ca2+ toward the root is linked to mass flow of water, more so than most other nutrient ions (Barber 1995) which accounts for accumulation of Ca2+ at the root surface (Barber and Ozanne 1970). Once in the root apoplasm (essentially the cell wall space), Ca2+ bind to negatively charged residues within the Donnan free space or on membranes, are taken up by cells down the electrochemical gradient for Ca2+, or pass through the water-free space of the cell wall to the xylem where they are transferred to the shoot (White and Broadley 2003). The presence of a suberised Casparian band in the radial and transverse walls of the endodermis can form a partial barrier to the radial movement of Ca2+ (and water) to the xylem via the apoplasm (Clarkson 1984; Schreiber et al. 2005; Baxter et al. 2009). Where this barrier is effective, Ca2+ must cross cell membranes and be taken up into the symplasm at least until the Casparian band has been passed. However, there appear to be large differences between plant species in the degree to which water and Ca2+ can bypass the endodermis and flow continually across the root to the xylem exclusively via the apoplasmic pathway, and thus the extent to which the symplasmic pathway (cell-to-cell, across membranes or plasmodesmata) is used (White 2001; Cholewa and Peterson 2004; Bramley et al. 2009, 2010).

Experimental evidence, using radioactive tracers and transport inhibitors, implicates a substantial role for Ca2+-ATPases in transport from the soil to the root xylem. However, considering the known density of proteins in the inner portion of the root, and the required amount of ATP, the overall Ca2+ flux into the xylem appears to be much greater than possible if purely protein mediated (Clarkson 1984; White 2001; Cholewa and Peterson 2004). Root exodermis bypass flow to the xylem (via the apoplasmic discontinuities in the exodermis in lateral roots) can allow Na+ to enter the shoot and cultivar differences have been observed in salt tolerance related to the degree of bypass flow in rice (Faiyue et al. 2010a, b). It is possible that this may also affect Ca2+ transfer to the shoot in rice, although this appears not to have been investigated (Faiyue et al. 2010a, b). In this context, it would be interesting to examine the impact of silicate on Ca2+ transport to the shoot as it dramatically reduces bypass flow and shoot accumulation of Na+ in rice (Gong et al. 2006).

Interestingly, in Arabidopsis, an increase in suberisation of the endodermis, and hypothetically a reduction in endodermal bypass flow, leads to a decrease in Ca2+ content of the shoot but an increase in Na+ (Baxter et al. 2009); this indicates that the routes that Na+ and Ca2+ take across the endodermis differ. It is likely that the proportion of Ca2+ transported through the symplasm increases as total flux of Ca2+ to the shoot decreases and this may occur following conditions that have increased suberisation of the endodermis, or under a low Ca supply, or at a low rate of transpiration (Clarkson 1984; Baxter et al. 2009). An appropriate strategy for increasing total Ca2+ accumulation in shoot tissue may be to decrease the resistance to apoplasmic flow of Ca2+ across roots into the xylem and reduce the need for the symplasmic pathway, but this may well have implications for Na+ tolerance of plants (Faiyue et al. 2010a,b), at least in soils where this ion is prevalent.

Organs that are predominantly xylem fed and have high rates of transpiration have high [Ca], conversely those that are predominantly phloem fed, and have low rates of transpiration, have a low [Ca] (White and Broadley 2003; Conn and Gilliham 2010). Therefore, it is not surprising that the tissues most susceptible to Ca deficiencies are those that have low relative rates of transpiration (White and Broadley 2003). Experiments to demonstrate the requirement for transpiration to deliver nutrients to the shoot show that root pressure and recycled phloem water (Munch water) is sufficient to deliver nutrients to the shoot, with the exception of Ca (Tanner and Beevers 2001). This link between transpiration and transport of Ca (which is almost certainly through the extracellular pathway) highlights the low rate of symplasmic transport of Ca2+ within most tissues.

It is predicted that climate change will decrease plant transpiration as rising atmospheric CO2 concentration, drought stress and salinity stress all reduce transpiration depending on cultivar and plant organ (Martinez-Ballesta et al. 2010). A meta-analysis of nutrient concentrations, including Ca for herbaceous and woody species under high CO2 (double ambient) showed a general decline in nutrient concentration, with Ca declining by about 7.5% in leaves and by about 15% in wheat grain (Loladze 2002). However, in other plants, leaf Ca supply can be inversely related to Ca accumulation in low transpiring organs such as tomato fruit (Adams and Ho 1993). As such, Ca deficiencies in low transpiring organs can be more prevalent when leaf transpiration is high (Olle and Bender 2009a, b). Hypothetically, if leaf transpiration was decreased, the transpirational draw of Ca away from other tissues would not be as significant and more of the Ca pool would be available to tissues which have the majority of Ca supplied symplasmically. When the transpiration rate of these regions, such as the meristem, is increased then the severity of deficiencies such as tipburn is reduced (Frantz et al. 2004; Chang and Miller 2004). It would be expected that Ca deficiencies would be less prevalent if symplasmic flows were ordinarily greater and were able to fulfil the Ca requirement for tissues, this may occur with a general decline in transpiration which may be of initial benefit to the horticultural industry in terms of crop appearance. However, the reduction in total Ca accumulation associated with a reduction in transpiration is a problem that must be overcome as this may impact both Ca bioavailability and shelf life (see ‘Future prospects for biofortification of plants with additional Ca’).

Calcium is differentially distributed in plant tissues

It is well known that Ca is differentially distributed intracellularly and the concentration of free Ca ([Ca2+]) is tightly controlled (White and Broadley 2003). Calcium compartmentation in different subcellular organelles appears important in preventing (1) precipitation reactions of Ca2+ with inorganic phosphorous species (Pi), ATP and other organic phosphates, (2) competition for enzyme binding sites preferably reserved for Mg2+ and (3) allowing the effective use of Ca2+ as a 2nd messenger within the cytosol during signalling (Marschner 1995). For instance, cytosolic [Ca2+] is usually nearer 100 nM when the cell is ‘resting’ but can be above 1 μM during signalling events. However, elevations in cytosolic [Ca2+] are generally not prolonged and instead oscillate in order to maintain cell viability (McAinsh and Pittman 2009). In contrast, vacuolar [Ca2+] is usually in excess of 1 mM (Fricke et al. 1994) and other intracellular compartments such as the endoplasmic reticulum (ER), mitochondrion and chloroplast are also proposed to have elevated [Ca2+] compared to the cytosol, although few reliable measurements in the literature exist (Subbaiah et al. 1988; Sai and Johnson 2002; Logan and Knight 2003; McAinsh and Pittman 2009).

Measured values of leaf apoplasmic [Ca2+] vary considerably but the total [Ca2+] is often high due to binding sites of molecules such as pectates within the Donnan free space (Sattelmacher 2001). The cation exchange capacity of the cell wall for Ca2+ varies greatly with tissue type and environmental conditions ∼0 ≥ 1,000 mM, but within the water-free space [Ca2+] is generally in the micromolar range (Sattelmacher 2001; Fritz 2007) and must be maintained below about 750 μM in order to prevent stomatal closure (DeSilva et al. 1996a). Elevations in apoplasmic [Ca2+] have been used in experiments to manipulate cytoplasmic [Ca2+] and stomatal movements (Allen et al. 2001) but the extent to which apoplasmic [Ca2+] is regulated or even sensed is not known.

After Ca2+ enters cells it is actively transported into organelles (predominantly the vacuole but also other compartments—see above) or back into the apoplasm to maintain a low cytosolic [Ca2+]. Despite the constant and significant electrochemical gradient for the movement of Ca2+ into the cytoplasm, plant cells appear able to regulate how apoplasmic [Ca2+] affects cytoplasmic [Ca2+]. For instance, mutant guard cells devoid of the chloroplast-localised ‘extracellular’ Ca2+ sensor do not, unlike wildtype stomata, raise cytoplasmic [Ca2+] or close the stomatal pore with an elevation in extracellular [Ca2+] to 5 mM (Weinl et al. 2008). Examples of apoplasmic localised Ca2+-‘sensors’ or modulators include pectin, calmodulin, annexins and ATP (Hepler and Winship 2010). Therefore, it is possible that different cells respond differently to extracellular calcium dependent upon its intra- and extracellular sensing complement.

It is widely known that as Ca cannot be readily remobilised once unloaded from the xylem, and in particular, to points downstream of transpirational flow (White and Broadley 2003; Conn and Gilliham 2010). This results in highly transpiring organs such as leaves with high [Ca] and those with low transpiration (such as fruit) with low [Ca] (White and Broadley 2003). It is less well-appreciated that Ca2+ is also differentially stored between different cell types. Point source stores of Ca within the form of oxalate are frequently observed in crystal containing idoblasts within the roots and shoots of many ‘oxalate plants’ (Franceschi and Nakata 2005) but soluble Ca is also differentially abundant within plants (Conn and Gilliham 2010). However, it has also been observed that soluble Ca appears to be located preferentially in specific cell types of the shoot (e.g. Karley et al. 2000a). For instance, in cereals total [Ca] is highest within the epidermal leaf vacuoles (up to 300 mM) but lowest in the mesophyll (<10 mM), whereas in most eudicots studied [Ca] is <10 mM within the epidermis and bundle sheath and an order of magnitude higher in the mesophyll (Karley et al. 2000a, b; Storey and Leigh 2004; Conn and Gilliham 2010). In root cells [Ca] has not been reported above 10 mM unless precipitated with oxalate (Storey et al. 2003).

Compartmentation of Ca2+ into vacuoles of specific leaf cells may occur for similar reasons to compartmentation away from the cytosol; to prevent precipitation with phosphates, which are usually compartmentalised into different cell types, but Ca2+ will also be a significant osmoticum when at high concentrations in certain cells (Fricke et al. 1994; Conn and Gilliham 2010). Differential compartmentation has been hypothesised to be the result of the distinct capacity of different cell types to accumulate Ca2+ through the expression of particular Ca2+-transporters (reviewed in Conn and Gilliham 2010). The transport characteristics of the tonoplast, in particular, appear to play a key role in storage capacity and flux of Ca2+ and water across the plasma membrane (PM) (Karley et al. 2000a; MacRobbie 2006a, b; Conn and Gilliham 2010). In addition, the role of Ca2+-supply and water movement through the apoplasm is also likely to impinge upon Ca2+-distribution (Karley et al. 2000b; Kerton et al. 2009) and warrants further investigation.

The impact of calcium on plant water flow and physiology

A constant influx of Ca2+ from the roots to the shoots in the transpiration stream may lead to a very high total [Ca] in leaves (White and Broadley 2003). High [Ca2+] especially in the cytosol of the guard cells has been linked to closure of stomata (DeSilva et al. 1996a). Closure of stomata for an extended period may lead to limited leaf internal CO2 concentrations which consequently affect photosynthesis and eventually plant productivity. Calcicoles are able to tolerate high rhizosphere [Ca2+] despite similar sensitivity of guard cells to high apoplasmic [Ca2+] (DeSilva and Mansfield 1994). It has been proposed that stomatal function is maintained in these plants by a high capacity of mesophyll and, in particular, trichomes to store Ca2+ (DeSilva et al. 1996b). Trichomes secrete Ca away from epidermal cells in the form of Ca oxalate in Centuarea scabiosa and Leontodon hispidus (DeSilva et al. 1996b). The ability of trichomes to secrete Ca was reduced by ozone treatment and this resulted in a reduced stomatal conductance presumably from a higher apoplasmic Ca load, although this was not measured (DeSilva et al. 2001).

It would also be important to limit [Ca] in the apoplasm because of consequent elevation of cytosolic Ca (Allen et al. 2001). This will affect an array of signalling processes including stomatal regulation as alluded to above, but Ca also regulates aquaporins of the PM intrinsic protein (PIP) family. A direct effect may occur on the cytosolic facing residues of PIPs (Hedfalk et al. 2006), where Ca in low and high ranges of concentration have been shown to increase and decrease aquaporin activity respectively (Alleva et al. 2006). This opens the possibility that [Ca] may regulate the pathway of water flow through leaves after it exists the xylem, and that this effect on aquaporins may in turn redistribute apoplasmic Ca. Furthermore, the potential role of Ca2+ in regulation of mesophyll conductance to CO2, where aquaporins appear to be involved in facilitating CO2 diffusion (Uehlein et al. 2008), and interaction with water flow, has yet to be investigated. These will be elaborated upon further in a forthcoming review.

The flow of water to fruit during development is not constant. As fruits mature xylem functionality can be minimal which results in low Ca content in most organs that are fed predominantly through the phloem for greater periods of time (Greenspan et al. 1994). In such fruits, the majority of Ca present is loaded through the xylem in the early phases of fruit development (Rogiers et al. 2006). For instance, in grapevine berries, xylem flow to the berry reduces during ripening, though there are large differences between cultivars (Tilbrook and Tyerman 2009) and this corresponds with a cessation of Ca inflow to the berry (Rogiers et al. 2006). Co-incident with these changes is the onset of loss of vitality in mesocarp cells and increased berry deformation (Tilbrook and Tyerman 2008). Interestingly parallels have been seen in tomato where fruit softening has been correlated with an increase in fruit water loss and reduction in turgor rather than breakdown of the primary cell wall (Saladié et al. 2007). The role of [Ca] in apoplasm in this phenomenon is yet to be explored.

Future prospects for biofortification of plants with additional Ca

The aim of Ca biofortification strategies is to increase Ca content of harvested organs of crops without adversely affecting plant growth or increasing the plant requirement for additional inputs i.e. Ca fertilisation or increased water use. Strategies to increase Ca content of plants could conceivably include: (1) increasing Ca supply to cells; (2) increasing Ca uptake into cells; and/or (3) increasing Ca retention within cells and/or tissues. Additional free [Ca2+] in the apoplasm, without an increased capacity to isolate the excess Ca2+ away from sites of action or signalling cascades, may reduce leaf hydraulic conductivity and close stomata - which would ultimately reduce the potential for Ca deposition in leaves by reducing transpiration (Tanner and Beevers 2001; DeSilva et al. 1996b). At the other extreme, if the Ca2+ secretion/binding capacity of plant tissues was increased too much, insufficient Ca2+ would be available for normal cell, or cell wall, function which would presumably result in symptoms of Ca deficiency and susceptibility to biotic and abiotic stresses (see Table 3 and 4; ‘Calcium as a plant nutrient’). Therefore, any approach aimed at increasing Ca content of plants needs to be carefully considered and offer a compromise between Ca supply and storage and the requirements for water use.

An obvious way to increase Ca2+ supply to plants would be to increase mass flow of water (and consequently Ca2+) to the roots (see ‘The transport of Ca in plants’). This could be achieved by increasing water demand from the plant by modifying stomatal pore regulation or tissue hydraulic conductivity; however this would not be a favoured strategy if water is limiting, so in the context of decreased water security due to climate change, this will not be explored further in this review. Alternative options to increase Ca2+ entry into plant tissues would be to increase the demand for Ca, this could be in the form of increasing cell wall binding (see below) or entry of Ca2+ into cells. To increase Ca2+ entry into cells the expression or activity of Ca2+-permeable transporters on the PM or tonoplast would need to be modified (Conn and Gilliham 2010). Calcium influx across the PM is passive (i.e. down the Ca2+ electrochemical gradient) and mediated by Ca2+ channels (White and Broadley 2003). Plasma membrane Ca2+ channels have been reported in all known cell types (Very and Sentenac 2002) but there are few reports of the genes that encode these proteins. Candidates include Glutamate Receptor Like Proteins (Gilliham et al. 2006a) and cyclic nucleotide gated channels (Demidchik and Maathuis 2007) although there is still some uncertainity as to whether these genes encode transport proteins or sensors related to transport processes (Roy et al. 2008; Conn and Gilliham 2010). MCA1 and MCA2 from Arabidopsis, are reported to encode PM mechanosensitive Ca2+ permeable channels but knockout of these genes did not reduce plant Ca content, although an Arabidopsis mca2 mutant did have a reduced rate of Ca influx into roots (Yamanaka et al. 2010). Finally, TPC1, the slow-vacuolar channel, is likely to encode a Ca2+ and K+ release pathway into the cytosol and therefore is not be related to Ca2+ accumulation (Peiter et al. 2005; Ranf et al. 2008). However, as overexpression and knockout of most of the above genes has not been reported to increase whole plant Ca accumulation, although many have Ca-sensitive phenotypes (Conn and Gilliham 2010), it appears unlikely that a constitutive approach to their misexpression is an appropriate strategy for Ca biofortifcation. Furthermore, it appears likely that Ca accumulation is ultimately controlled by active tonoplast processes (Karley et al. 2000a, b; MacRobbie 2006a,b; Conn and Gilliham 2010).

For Ca2+ to be accumulated into the vacuole, active transport of Ca2+ is required and both Ca2+-ATPases (ACA) or Ca2+/H+ antiporters (CAX; using the proton motive force) perform this role (Rea et al. 1992; Conn and Gilliham 2010). The vacuolar lumen constitutes the largest volume within plants and is consequently the largest potential storage compartment (Leigh 1997) and therefore is the natural choice for increasing plant Ca storage capacity. Both CAX and ACA proteins have N-terminal autoinhibitory domains, CAX may be activated by phosphorylation or CAX interacting proteins (CXIP) (Pittman et al. 2002; Cheng and Hirschi 2003) and ACA by Ca2+/Calmodulin (CaM) (Baxter et al. 2003). ACAs and CAXs have quite distinct expression patterns but it is not known which transcription (or epigenetic) factors regulate this expression, for instance AtCAX1 is highly expressed in shoots but not in roots (Cheng et al. 2005). No reports to date have confirmed which are more important out of CAX or ACA proteins for Ca2+-accumulation into the vacuole but the likely lower energy demands of transport through CAX proteins make them a good candidate for initial studies (Conn and Gilliham 2010). An approach to investigate this further may be to test the effect of overexpression of vacuolar Ca2+-transporters, or potential regulators of transport, on the magnitude of Ca2+ fluxes across the PM. This may identify key components in Ca accumulation and test the hypothesis that vacuolar transport controls Ca2+ flux across the PM (Gilliham et al. 2006b; MacRobbie 2006a, b; Conn and Gilliham 2010).

Hirschi and co-workers have successfully increased the Ca content of a number of crop species (see ‘Improving plants as a source of Ca for humans’) primarily by driving expression of an Arabidopsis thaliana modified Ca2+/H+ antiporter AtsCAX1, and also AtCAX4 and AtsCAX2A/B, using the 35S CMV promoter or AtCDC2a promoter (Table 2) (Park et al. 2005a; Kim et al. 2006; Han et al. 2009; Chung et al. 2010). To form constitutively active CAX protein, as denoted by an s prefix (e.g. AtsCAX1), the first 36 amino acid were removed from the N-terminal autoinhibitory domain of AtCAX1 (Pittman and Hirschi 2001). Plants expressing AtsCAX1 in tissues show 2- or 3-fold increases in Ca (Table 2). However, in tissues that readily show signs, Ca-deficiency symptoms have been observed. Interestingly, fewer deficiency symptoms were reported in carrot and potato than tomato, but overexpression of AtCAX4 or a modified AtsCAX2B, both being weaker Ca transporter alleles than AtsCAX1, also increased Ca content but do not result in Ca-deficiency symptoms (Park et al. 2005a; Chung et al. 2010). The decrease in Ca deficiency using weaker transport alleles suggests that when AtsCAX1 is expressed it is too successful at locking Ca away from the cytosol and makes it unavailable to the plant for cellular or cell wall processes. Whereas AtCAX2 ordinarily has a significant transport capacity for manganese or cadmium ions (Mn2+ or Cd2+), mutant AtsCAX2A/B were constructed to contain different regions of CAX1 and do not transport Mn2+, although they retain Cd2+ transport ability (Shigaki et al. 2003). There is no information on Cd accumulation in the above studies overexpressing any CAX in plants, and it would seem necessary to examine, as CAX2 and 4 overexpression in tobacco roots has been used to reduce Cd accumulation in shoots by increased accumulation in roots Korenkov et al. (2009). It is likely that even if Cd accumulation is increased in plants, a similar strategy to the modification of CAX2 to reduced Mn2+ transport could be performed (Shigaki et al. 2003). CAX transporters are clearly excellent candidates for increasing bioavailable Ca in plants but future approaches need to avoid inducing deficiency symptoms in susceptible tissues, this coupled with significant gains in total Ca content should result in plants that could become major Ca sources for humans.

There are initial reports that the use of calcium binding proteins may be a way to decrease the negative effects of large Ca increases in tissues without having to sacrifice the large gains in Ca content that can be accrued by overexpressing AtsCAX1. Tobacco plants overexpressing AtsCAX1 usually have leaf tip necrosis and significant growth retardation (Hirschi 1999). However, calreticulin (an ER-localised calcium binding protein) overexpressed in tobacco plants already overexpressing AtsCAX1 no longer have symptoms of Ca deficiency despite an increased [Ca], even over plants overexpressing AtsCAX1 (without calreticulin) (Wu et al. 2009). Furthermore, the overexpression of calreticulin in Arabidopsis resulted in an increase in ‘bioavailable’ Ca for the plant (Wyatt et al. 2002), which when taken together with observations in tobacco could be interpreted as the diversion of Ca to the ER and away from the vacuole, and may be a strategy to avoid deficiencies.

The Ca storage capacity of phloem fed organs; carrots, potatoes and tomatoes has been doubled by constitutive overexpression of AtCAX (Table 2). However, as all are predominantly phloem fed the total amount of Ca in the organ was still low. In reality increasing the Ca content of phloem fed tissues may be more useful in improving the post-harvest storage properties and disease resistance of these organs than Ca biofortification of diets. Although no change in susceptibility to pathogens has been reported there have been reports of increased shelf life of a number of crops, this is not surprising as pathogen defence and ripening involve very distinct processes, although they both share calcium regulated and cell wall-localised phenomena (Moscatiello et al. 2006; Saladié et al. 2007; Cantu et al. 2008). Transgenic approaches to alter the ripening of fruit to increase shelf life of crops has focused mainly on the development of flavour and aroma compounds elicited by ethylene and a complex network of transcription factors (Matas et al. 2009). However, little progress has been made on the processes that control the softening of fruit, also integral to ripening, although it is known to be multifactorial and include the disassembly of polysaccharide networks in the primary cell wall, increases in water loss and reductions in turgor (Saladié et al. 2007). Interestingly, many of the these processes are under the control of Ca2+ to some degree so it may be of no surprise that alterations in supply and storage of Ca by transgenic means may alter the ripening process (e.g. Park et al. 2005b). Manipulations of vacuolar storage capacity and apoplasmic Ca will have implications for ion accumulation in tissues, water loss and turgor (e.g. Han et al. 2009). Interestingly, parallels in grapevine berries have been seen when Ca supply is reduced (see ‘The impact of calcium on plant water flow and physiology’). Apoplastic Ca is required for cross-linking pectins in the cell wall and maintaining cell wall integrity and also controls transpiration through regulation of stomatal aperture (see ‘The impact of calcium on plant water flow and physiology’). It has also been long-known that cell wall strength can also impinge upon stomatal function directly (and hence transpiration) (DeMichele and Sharpe 1973). A priority will be to examine the role of Ca2+ in these processes further by exploring the mechanisms in which it can increase shelf life of crops (and/or pathogen defence).

Furthermore, AtCAX1 and AtCAX3 (a protein that is 77% identical at the amino acid level) have been shown to have roles in tolerance to abiotic stresses or certain physiological phenomenon. Knockout mutant plants of AtCAX1 and AtCAX3 genes have numerous phenotypes including delayed germination on sucrose, increased sensitivity of germination to ABA, tolerance to ethylene with respect to germination and inhibition of hypocotyl elongation, decreased salt tolerance (Atcax3) and increased freezing tolerance after cold acclimation (Atcax1) (Cheng et al. 2003; Catala et al. 2003; Cheng et al. 2005; Zhao et al. 2008). Whether these phenotypes highlight specific roles of these transporters or reflect a general perturbation to Ca homeostasis is not known. However, different AtCAX genes have tissue type-specific expression which may confer some of the specific phenotypes of these mutants (Cheng et al. 2005). Pleiotropic affects of AtCAX misexpression have also been observed at the whole plant level. For instance, when AtCAX1 expression is knocked-out, AtCAX3 and AtCAX4 expression increases, similarly other transport processes are also affected such as V-ATPase or Ca2+-ATPase activity, and consequently protein gradients across the tonoplast, and this has knock-on effects on the storage of other ions (Cheng et al. 2005). Therefore, it would seem necessary to evaluate not only the total elemental content of tissues (as has often been performed) but also the intracellular pool sizes of Ca (and other ions) in response to overexpression of Ca2+-transporters. Also an evaluation of the abiotic stress tolerance of plants overexpressing Ca2+-transporters should be examined.

An alternative approach to increasing Ca bioavaiblility is to reduce Ca ‘anti-nutrients’ such as oxalate or phytate in tissues (Heaney et al. 1991; Morris et al. 2007). The Medicago trunculata (medic) mutant cod5 is deficient in oxalate synthesis but does not have reduced Ca content and no Ca-sensitive phenotype. Medic may be a useful model for other high oxalate plants such as spinach which have reduced bioavailable Ca. Interestingly, there has been a recent report of low oxalate spinach so a priority will be to test the relative absorbability of Ca for humans from this plant (Murakami et al. 2009). The Ca absorbability of soybean milk has been measured in low phytate varieties and shown to be equivalent of that in cow’s milk and is 10% greater than in higher phytate varieties (Table 1). A potential issue with this approach is the removal of potential herbivory defence compounds such as oxalate (Table 2) (Korth et al. 2006) which may increase the palatability to such plants to pests and therefore increase the need for pesticide applications to crops. But, if it is possible to increase Ca bioavailability in a way that does not affect plant production then it is potentially a good strategy. It may also be combined with alternative strategies if deleterious phenotypes do occur such as overexpression of vacuolar Ca transporters or calcium binding proteins.

The cation-binding capacity (CEC) of the cell wall also represents a potential target for increasing Ca content of plant tissues (Marschner 1995). Increasing the Ca content of cell wall tissues may also help remedy some symptoms of Ca deficiency or increase biotic (i.e. pathogens) and abiotic (i.e. excess Na+) stress tolerances by improving relevant cell wall properties. The CEC of different tissues and plant species clearly varies (Fritz 2007) so there may be some considerable scope for this approach. Furthermore, the CEC of cereals is generally low and this has been correlated with the low Ca content in shoots (Marschner 1995). So it is possible that increasing the CEC of roots may be a strategy to get more Ca into plants. However, processes that regulate Ca signalling in the apoplast are poorly understood as are the mechanisms by which Ca can affect cell wall processes such as cellulose deposition or extensibility (Cosgrove 2005). Therefore, any such approach to modify cell wall Ca binding capacity would need to be monitored closely.

All approaches that modify Ca content of plant tissues, be they specific to the cell wall, PM or vacuole may benefit from taking cell-specific requirements into consideration. The CEC and properties of cell walls surrounding particular tissues varies (Fritz 2007; Thompson 2008) and this is likely to have biological relevance so constitutive modification may have detrimental pleiotropic effects. It is clear that some nutrient elements are not usually stored in the same cell types for instance, Ca is usually in a different cell type to phosphorus (P) (Conn and Gilliham 2010). If Ca and P were forced to accumulate within the same vacuole it is likely to decrease the dietary availability of both nutrients and possibly lead to deficiencies. It would be interesting to see if an organ-specific approach, similar to that taken with sodium tolerance studies (Møller et al. 2009), would eliminate the deficiency symptoms that were detected in potatoes and tomatoes. The use of developmentally regulated promoters to maximise Ca entry into organs (e.g. grapevine berry or tomato) during life-stages that rely upon xylem-fed Ca may also be beneficial to overall plant health. The use of developmentally regulated promoters may also be of use in attempts to eliminate Ca deficiencies in organs with low transpiration by directing Ca into these tissues to increase symplasmic flows (perhaps using PM targeted channels or Ca2+-ATPases) (see ‘Calcium as a plant nutrient’), which may be of use to the horticultural industry to reduce spoilage. Many different approaches could be used to fine tune Ca delivery to organs by using native or synthetic promoters, for instance, transcript abundance of transporters could be up-regulated under conditions of adequate Ca supply or downregulated by RNAi technologies during conditions of Ca deficiency. Proteins that modify activity of transporters could also be regulated in a similar fashion i.e. CaM (for Ca2+-ATPases) or CXIP (CAX) (Baxter et al. 2003; Cheng and Hirschi 2003). Galon et al. (2010) describe suites of Ca-regulated genes that could also be used directly or indirectly by replicating appropriate elements (e.g. ABRE, CMATA, E-Box) within their promoters to form chimeric synthetic constructs in combination with other developmental-stage or cell-specific elements to manipulate Ca-transport activity across the appropriate cell membranes at the appropriate time (Peremarti et al. 2010).

Conclusions

The advent of molecular breeding tools provides opportunities to enhance Ca2+ accumulation in plants cells, in particular harvested organs, for the biofortification of food crops for animal and human consumption and the improvement of diets. Enhancement of Ca levels in tissues could also increase stress tolerance of plants and enhance post-harvest storage quality. We are only beginning to understand the complexity, and links between, Ca transport and storage in plants. It is apparent that more fundamental studies are needed before we will be able to manipulate greater Ca content of plants without adversely affecting plant function. However, it is likely that manipulation of the expression or activity of specific transporters on specific membranes at specific times will be a beneficial approach to advances in Ca biofortification of crops. Through such techniques it is expected that viable value-added crops with enhanced Ca content will provide a cheaper alternative for dietary calcium for both humans and animals.

Footnotes
1

In this article, the term apoplasmic is used to refer to transport through the apoplasm, and symplasmic through the symplasm. The term apoplast and symplast are used only when referring to growth deformation of plant tissue (following Erickson 1986).

 

Acknowledgements

Work in our laboratory is supported by the Australian Research Council and the Faculty of Sciences, University of Adelaide. MD is supported by a doctoral scholarship from the Sarawak Government, Malaysia.

Conflict of interest

The authors declare that they have no conflict of interest.

Copyright information

© Springer-Verlag 2010