Introduction

In vegetables and fruits, calcium (Ca2+) deficiency is a critical factor reducing their quality and yield due to Ca2+-related physiological disorders, such as blossom-end rot (BER) in tomato, pepper, eggplant and melon, tipburn in lettuce, celery and cabbage, and bitter pit in apple fruit (White and Broadley 2003; Dayod et al. 2010; de Freitas et al. 2011). Moreover, low human dietary intake of Ca2+ has been associated with a disease, osteoporosis, which may lead to a bone fracture (Bachrach 2001). Plant-based foods are good sources of dietary Ca2+; however, increased amounts in particular foods may help ameliorate the incidence of osteoporosis caused by consumption of inadequate dietary Ca2+ (Hirschi 2009; Park et al. 2009). Therefore, a better understanding of Ca2+ improvement in plant cells is required in order to positively impact human nutrition and improve fruit and vegetable production.

Calcium is unique amongst the elements in plants and animals because it plays both a pivotal structural and, an essential, signaling role (White and Broadley 2003; Hirschi 2004). Consequently steep gradients for Ca2+ exist across cell membranes and cell endomembranes: the plasma membrane (PM), tonoplast (TN), and the endoplasmic reticulum (ER). Gradients across these organelles are important for normal cellular function and for the regulation of metabolic processes which requires punctilious regulation of cytosolic Ca2+. These gradients are established by a dynamic balance between influx and efflux of Ca2+ across each of the cellular membranes.

The concentration gradient of Ca2+ across the TN is established partially by high-capacity H+/Ca2+ antiporters (Zhao et al. 2009). Among them, CAXs (Cation/H+ exchangers), a group of high-capacity, low-affinity transporters that export cations out of the cytosol to maintain ion homeostasis across biological membranes (Pittman and Hirschi 2003), have been physiologically characterized from a variety of plants. The first Arabidopsis CAX gene, CAX1 was identified by its ability to suppress the Ca2+ sensitivity of a yeast mutant deleted in vacuolar Ca2+ transport (Hirschi et al. 1996). CAX1 contains an additional 36 amino acid at its N-terminus that reduces the transport activity in both yeast and plant expression assays (Pittman and Hirschi 2001; Mei et al. 2007). When the N-terminal truncated version (sCAX1) is ectopically expressed in potato, carrot and lettuce, Ca2+ content in their edible tissues increases (Park et al. 2005b; Park et al. 2009). However, in some cases, these changes also produce deleterious phenotypes that impact yield (Hirschi 1999; Park et al. 2005a). Tempering expression of sCAX1 driven by a different promoter results in healthier plants but they often accumulate less Ca2+ (Park et al. 2005a).

Tobacco lines expressing sCAX1 increase Ca2+ content in their tissues, but also display severe Ca2+ deficiency-like symptoms, such as apical leaf tip burning and sensitivity to ion imbalances (Hirschi 1999). In addition, while the fruits of sCAX1-expressing tomato plants accumulate higher total Ca2+ than vector control plants, the sCAX1-expressing tomatoes show increased incidence of distinct necrotic lesions in the distal portion of fruits, termed BER, which is presumed to be caused by aberrant Ca2+ homeostasis in fruit cells (Park et al. 2005a). These phenomena are an obstacle for the development of Ca2+-biofortified crops.

Our working hypothesis is that the increased expression of sCAX1 in conjunction with Ca2+ binding proteins on another endomembrane may reduce these deleterious phenotypes. Calreticulin (CRT), a Ca2+-binding protein mainly resident in the ER, has been known as an effective Ca2+ buffer protein that may allow the transient storage of Ca2+ and play a role in stress responses (Jia et al. 2009). Over-expression of a maize CRT cDNA in tobacco suspension cells results in a two-fold increase in Ca2+ accumulation in the ER-enriched fraction in vitro (Persson et al. 2001) and could improve growth of tobacco cell suspensions in high-Ca2+ medium (Akesson et al. 2005).

Here, we express a maize CRT in sCAX1-expressing tobacco and tomato plants to test our hypothesis if the expression of CRT gene can mitigate Ca2+-related cellular dysfunction resulted from expressing of sCAX1 in tobacco and tomato plants while maintaining enhanced Ca2+ content. Our findings suggest that co-expressing transporters and binding-proteins may be a means of boosting plant nutrient content without adversely affecting yield. To our knowledge, this study represents the first attempts to increase the Ca2+ content of plants using co-expression of two genes which play important roles in the regulation of Ca2+.

Materials and methods

Bacterial strain and plasmid

The pCaMV::sCAX1 [sCAX1 driven by the cauliflower mosaic virus (CaMV) 35S promoter] expression vector was previously constructed and described (Park et al. 2005b) (Fig. 1a). The maize CRT (NCBI accession number: AF190454) open reading frame was cloned into the SacI site of pE1775 binary vector (Lee et al. 2007) (Fig. 1a), and the pE1775::CRT and pCaMV::sCAX1 were introduced into Agrobacterium tumefaciens strain LBA 4404 (Hoekema et al. 1983) using the freeze–thaw method (Holsters et al. 1978). The pE1775 expression vector contains a superpromoter, which consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2′ (mas2′) transcriptional activating element plus minimal promoter, and has been proved to be a strong promoter when being expressed in tobacco and maize (Lee et al. 2007). 35SCaMV promoter was intentionally avoided to drive CRT gene because previous studies suggest that two transgenes driven by the same promoter might cause silencing of one or both genes (Park et al. 1996).

Fig. 1
figure 1

Molecular analyses of sCAX1-, CRT- and sCAX1 + CRT-expressing tobacco and tomato plants. a T-DNA regions of pCaMV35S::sCAX1 and pE1775::CRT. RB right border, LB left border, Nos-pro nopaline synthase promoter, Kan R the gene conferring resistance to kanamycin, neomycin phosphotransferase (NPTII), Nos-ter nopaline synthase terminator. 35S pro CaMV 35S promoter, sCAX1 short cut cation exchanger 1 coding region, Aos octopine synthase transcriptional activating element, AmasPmas mannopine synthase 2′ activating and promoter elements, CRT maize calreticulin coding region, ags-ter polyA addition signal from the agropine synthase gene, hptII gene conferring resistance to hygromycin, Pnos mopaline synthase promoter, tAg7 poly A addition signal for T-DNA gene 7. b Southern-blot analysis of transgenic tobacco plants. Ten micrograms of tobacco genomic DNA were digested with SacI, and hybridized with the sCAX1 probe. c Northern-blot analysis of transgenic tobacco plants. Ten micrograms of total RNA from leaves were hybridized with sCAX1 and CRT probe, respectively. Ethidium bromide-strained rRNA (bottom) is shown as a loading control. d Southern-blot analysis of transgenic tomato plants with CRT probe. Ten micrograms of tomato genomic DNA were digested with XbaI, and hybridized with CRT probe. The arrow indicates the endogenous tomato CRT gene that was detected by maize CRT probe. e PCR detection of sCAX1 in genomic level. f RT-PCR detection of the expression of sCAX1. g RT-PCR detection of the expression of CRT. SlPP2Acs was used as tomato housekeeping gene

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Plant material, transformation, and growth conditions

Tobacco (Nicotiana tabacum L.) cultivar KY14 was used in this study. Tobacco transformation was performed via Agrobacterium-mediated leaf disk transformation method as previously described (Horsch et al. 1985). Seeds were surface-sterilized and germinated on MS inorganic salt medium (Murashige and Skoog 1962) with 30 g l−1 sucrose, pH 5.7, and solidified using 8 g l−1 agar (PhytoTechnology, Shawnee Mission, KS, USA). Transformants were selected on standard medium containing 100 μg ml−1 kanamycin for sCAX1-, 50 μg ml−1 hygromycin for CRT-, and 100 μg ml−1 kanamycin plus 50 μg ml−1 hygromycin for sCAX1- and CRT-co-expressing tobacco. Tobacco plants were grown in a greenhouse as previously described (Hirschi 1999). For ion sensitivity analysis, surface-sterilized seeds were germinated in MS media. Ten days after plating, the seedlings were transferred to MS media supplemented with the appropriate ion. To make media deficient in Ca2+, we removed the CaCl2 from the nutrient solution. The T1 and T2 tobacco plants were grown in the greenhouse under a 16-h photoperiod within a temperature range of 25–30 °C. Leaves from 2-month-old T2 generation tobacco plants were sampled for Ca2+ concentration analysis.

Tomato (Solanum lycopersicum ‘Rubion’) transformation was performed via Agrobacterium-mediated transformation method using cotyledon and hypocotyls explants as previously described (Park et al. 2003). A. tumefaciens LBA 4404 was used for generating stable transgenic plants. After inoculation with A. tumefaciens, the plant cultures were maintained at 25 °C under a 16-h photoperiod. After 6–8 weeks, regenerated shoots were transferred to rooting medium for additional 6 weeks. The temperature of the greenhouse was maintained within a range of 25–28 °C.

T2 generation of tomato plants were grown in the greenhouse with the same conditions described above. We manually pollinated the flowers and marked the date of pollination. The number of healthy and BER fruits was counted and the BER ratio was examined. The fruits of 40-day after pollination (40 DAP) were harvested for Ca2+ content determination.

DNA isolation and DNA gel blot analysis

Genomic DNA of tobacco and tomato was isolated from 100 mg of fresh leaves using the DNeasy Plant Mini-Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. DNA gel analysis was carried out as described previously (Park et al. 2009). Genomic DNA (5–10 μg) was digested with XbaI, separated in a 0.9 % (w/v) agarose gel by electrophoresis and blotted on to a nylon membrane (Zeta-Probe GT membrane, BioRad Laboratories, Hercules, CA, USA). The probe for the sCAX1 gene was isolated by digesting pBluscript::sCAX1 (Park et al. 2009). The membranes were pre-hybridized at 65 °C in 7 % sodium dodecylsulphate (SDS) and 0.25 M Na2HPO4 for 3 h, and then hybridized overnight at 65 °C in the same solution containing the probe labeled by NEBlot Phototope Kit (New England Biolabs, Beverly, MA, USA). Membranes were washed twice for 40 min each with 20 mM Na2HPO4 and 5 % SDS at 65 °C and then washed twice again for 30 min each with 20 mM Na2HPO4 and 1 % SDS at 65 °C. The signal was detected using the Phototope-Star Detection Kit (New England Biolabs).

RNA isolation, RT-PCR, and RNA gel blot analysis

Total RNA of tobacco and tomato was extracted from leaves using RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA for RT-PCR was treated with RNase-free DNase prior to the synthesis of first-strand cDNA by oligo (dT) priming using moloney murine leukaemia virus-reverse transciptase (BD Biosciences Clontech, Palo Alto, CA, USA). One microliter of the reverse transcription reaction solution was used as a template in a 25 μl PCR solution. Total RNA (7 μg) was separated on a 1.2 % agarose gel containing 1.5 % formaldehyde, and blotted on to a Zeta-Probe GT membrane according to the manufacturer’s instructions. Hybridization and washing were performed as described previously in DNA gel blot analyses (Park et al. 2009).

Ca2+ and other mineral analysis

The tobacco leaves and tomato fruits were dried at 70 °C for 4 days. A total of 0.5 g (dry weight) of fruits was digested for analysis as described (Park et al. 2005a). Calcium content per gram of dry weight was determined by inductively coupled plasma emission spectrophotometry (Spectro, Kleve, Germany).

Results

Generation of sCAX1-, CRT-, and sCAX1- and CRT-co-expressing tobacco and tomato plants

The temporal and spatial regulation of sCAX1 is crucial for proper modulation of Ca2+ with plant cells (Park et al. 2005a). The 35S promoter confers strong constitutive expression in plants, and is often used to give high level expression of a given gene (Benfey et al. 1990). In previous studies, various sCAX1-expressing lines under the control of the 35S promoter showed symptoms similar to Ca2+ deficiency (Hirschi 1999; Park et al. 2005a), and this promoter may therefore be used effectively to identify the capacity to regulate Ca2+-related cellular dysfunction in sCAX1-expressing plants through manipulation of CRT. Initially 18 sCAX1- and 20 CRT-expressing lines were generated, respectively, and then we co-transformed CRT into two independent sCAX1-expressing T2 homozygous tobacco lines (sCAX1-1 and sCAX1-2). The stable integration of the 35S::sCAX1 chimeric construct in the genome of tobacco plants that were used for CRT co-transformation was confirmed by Southern-blot analysis (Fig. 1b). The line we termed sCAX1-2 appeared to contain a single-copy insertion, while line sCAX1-1 and sCAX1-5 had more than one integration event (Fig. 1b). Twenty independent sCAX1- and CRT-co-expressing tobacco lines (hereafter as sCAX1 + CRT) were generated by CRT co-transformation. Expression of sCAX1 and CRT transcripts were measured in T1 transgenic lines by RNA gel blot analysis. Two sCAX1- and CRT-co-expressing lines sCAX1 + CRT-2 and -3 appeared to show stronger bands compared to other lines sCAX1 + CRT-13, sCAX1-1, or CRT-1 (Fig. 1c). The intensity of the signal in sCAX1 + CRT-2 and -3 may result from high-level of expression in those particular lines by transformation variability, various technical issues such as an excess of loaded total RNAs, or the possible co-transformation effect of two different genes. Regardless, the results suggest that sCAX1 and CRT transcripts were expressed only in the sCAX1 and CRT transgenic lines, respectively; while both sCAX1 and CRT transcripts accumulated in the sCAX1 + CRT-2, -3, and -13 transgenic lines (Fig. 1c).

Previous tomato studies demonstrate that sCAX1 expression also causes apical burning and the development of distinct necrotic lesions in the distal portion of fruits (BER). Thus, we were interested in determining whether co-expression of CRT in sCAX1-expressing tomato plants would alleviate the symptoms. Initially 24 sCAX1- and 15 CRT-expressing lines were generated, respectively, and then we co-transformed CRT into a sCAX1-expressing-13 (a single-copy insertion) T2 homozygous tomato line that showed severe Ca2+ deficiency-like symptoms including BER (data not shown). Twelve independent sCAX1 + CRT-expressing tomato lines were generated. Two of each sCAX1-2 and 13, CRT-9 and 21, and sCAX1 + CRT-4 and 5 expressing transgenic lines were randomly selected and confirmed by Southern-blot and PCR analysis (Fig. 1d, e).

The stable integration of the CRT in the genome was confirmed by Southern-blot (Fig. 1d). We found a background band in every line, including wild-type, which might be caused by the endogenous CRT in the tomato genome. The Southern-blot result suggests that the CRT-21, sCAX1 + CRT-4, and sCAX1 + CRT-5 lines contained a single-copy of CRT, while CRT-9 line contained 3 copies of CRT. The integration of sCAX1 in the genome was confirmed by PCR using sCAX1 primers (Fig. 1e, Supplementary Table 1). The expression of CRT and sCAX1 was confirmed by RT-PCR using CRT and sCAX1 primers, respectively (Fig. 1f, g, Supplementary Table 1). All the molecular works were conducted using the T2 generation plants.

CRT suppresses sCAX1-induced Ca2+ deficiency-like symptoms of tobacco and tomato plants

As shown previously (Hirschi 1999), sCAX1-expressing tobacco lines including two independent sCAX1-expressing T2 homozygous tobacco lines (sCAX1-1 and sCAX1-2, Fig. 2a, b) that were used for CRT co-transformation have altered morphology and growth characteristics. All the sCAX1-expressing lines displayed necrosis on the tips of the new leaves from a young stage, which is a Ca2+ deficiency-like symptom (Fig. 2c). In addition to the necrosis, all the sCAX1-expressing tobacco plants showed severe stunting (Fig. 3a, bottom). In contrast, after introducing the CRT into sCAX1-expressing tobacco plants, the symptoms were alleviated (Figs. 2d, 3a, top).

Fig. 2
figure 2

Morphology of sCAX1-, and sCAX1 + CRT-expressing tobacco plants at young stage. a, b The sCAX1-expressing tobacco plants used for CRT transformation. c The morphology of sCAX1-expressing tobacco seedlings. d The morphology of sCAX1 + CRT-expressing tobacco seedlings

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Fig. 3
figure 3

Segregation of the Ca2+ deficiency-like symptoms. a Morphology of T1 generation of sCAX1-, and sCAX1 + CRT-expressing tobacco plants. b Segregation of the morphology in T2 generation of sCAX1 + CRT-expressing plants. Some of the plants maintained the normal morphology, but some returned to the Ca2+ deficiency-like symptoms. c Detection of the expression of sCAX1 and CRT in T2 generation sCAX1 + CRT-expressing plants by RT-PCR. d Ca2+ concentration of T2 generation tobacco leaves of different lines. All results shown here are the means of 3 biological replicates, and the error bars indicate the standard deviations (SD n = 3) (Student t test, *p < 0.05; **p < 0.01)

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To establish that the growth phenotypes were due to co-expression of the CRT, 40–45 each of sCAX1 + CRT-expressing T2 generation plants from 5 independent lines (sCAX1 + CRT-2, -3, -6, -13, and -27) were analyzed to determine if CRT segregated with the robust growth phenotype. As shown in Fig. 3b (right) and 3c, 4 of 5 lines showed a segregation pattern of 3:1 for the robust growth phenotype (Supplementary Table 2), and all the CRT-co-expressing lines were healthy while the absence of CRT caused the reappearance of the symptoms associated with sCAX1-expression (Fig. 3b (left) and 3c). This result suggests that CRT contributes to the recovering of sCAX1-expressing tobacco plants with Ca2+ deficiency-like symptoms.

To determine how the expression of sCAX1, CRT and sCAX1 + CRT alters Ca2+ concentration in the cells, we measured the total accumulation of Ca2+ in the tobacco leaves in T2 generation transgenic plants. As shown in Fig. 3d, sCAX1- and sCAX1 + CRT-expressing tobacco plants accumulated significantly more (up to 25 %) Ca2+ than wild-type plants; however, CRT-expressing tobacco plants did not significantly enhance Ca2+ accumulation as compared with wild-type plants. In addition, expression of sCAX1, CRT or sCAX1 + CRT did not affect the accumulation of other minerals (Cu2+, Fe2+, Mg2+, Mn2+, and Zn2+, Supplementary Fig. 1).

In order to ascertain whether CRT can suppress sCAX1-induced adverse symptoms in tomato plants, we introduced CRT into sCAX1-expressing tomato plants. As shown in Fig. 4a and Supplementary Fig. 2, the necrosis in leaf tips caused by sCAX1-expressing was alleviated by the co-expression of CRT. Furthermore, when we counted the number of the BER and healthy fruits of wild-type, sCAX1-, CRT-, and sCAX1 + CRT-expressing T2 generation transgenic plants, respectively, the results showed that the BER ratio could be reduced by introducing CRT to the sCAX1-expressing plants. Although the ratio of BER in sCAX1 + CRT-expressing plants was not statistically different from that of sCAX1-expressing plants, because the BER ratio shows a large variation among different plants even in the same line, the BER symptom in sCAX1 + CRT-expressing plants was indeed less severe than that in sCAX1-expressing plants according to our day-to-day observation (Fig. 4b, c, and data not shown).

Fig. 4
figure 4

Expression of CRT mitigated the Ca2+ deficiency-like symptoms of sCAX1-expressing tomato plants. a Expression of CRT mitigated the leaf tip burning of sCAX1-expressing tomato plants. b Expression of CRT reduced the BER incidence of sCAX1-expressing tomato plants. Left panel sCAX1-expressing tomato plants; right panel, sCAX1 + CRT-expressing tomato plants. c BER ratio of wild-type, sCAX1-, CRT-, and sCAX1 + CRT-expressing tomato plants. d Concentrations of Ca2+ in fruits of wild type, sCAX1-, and sCAX1 + CRT-expressing tomato plants. All results shown here are the means of 3 biological replicates, and the error bars indicate the standard deviations (SD n = 3). Means accompanied by the same letter are not significantly different using ANOVA analysis (p < 0.05)

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To determine how the co-expression of CRT in sCAX1-expressing tomato alters Ca2+ concentration in the fruit cells, the total accumulation of Ca2+ in the tomato fruits of wild-type, sCAX1-, CRT-, and sCAX1 + CRT-expressing T2 generation plants was analyzed. All the sCAX1- and sCAX1 + CRT-expressing tomatoes showed significantly higher Ca2+ content than wild-type tomatoes (Fig. 4d). However, among 15 CRT- expressing tomato lines, the majority of these lines did not significantly enhance Ca2+ content as compared to wild-type tomatoes while the fruits of line #9 and #21 increased ~9 and ~40 % more Ca2+ than wild-type fruits, respectively (Fig. 4d).

CRT suppresses sCAX1-induced ion sensitivity in tobacco lines under ion imbalance growth condition

We further tested whether introducing CRT could mitigate the ion sensitivity caused by sCAX1. After in vitro growing lines on standard MS media for 14 days, wild-type and transgenic seedlings (sCAX1-1 and -2; CRT-1; sCAX1 + CRT-2, -3, -6, -13, and -27) were transferred to media containing various concentrations of Mg2+ or K+, or reduced Ca2+. All the sCAX1-expressing seedlings were sensitive to the ion imbalance that failed to perturb the growth of wild-type and sCAX1 + CRT-expressing plants. For example, after being transferred in the Ca2+-depleted media, the sCAX1-expressing seedlings could not grow and develop leaves (Fig. 5a). In contrast, the sCAX1 + CRT-expressing seedlings grew vigorously without any abnormal morphological developments (Fig. 5a). In the medium containing 50 mM MgCl2, the sCAX1-expressing seedlings also showed hypersensitivity to the stress, such as necrotic lesions in the young leaves and stunted growth (Fig. 5b); however, the sCAX1 + CRT-expressing seedlings did not display any adverse growth (Fig. 5b). The sensitivity of sCAX1-expressing tobacco to K+ salt stress was not as severe as the Ca2+ or Mg2+ growth phenotypes. However, after transferring the seedlings to the media containing 100 mM KCl for 60 days, the necrotic lesions displayed on the sCAX1-expressing leaf tips, but not on the leaves of sCAX1 + CRT-expressing plants (Fig. 5c).

Fig. 5
figure 5

CRT suppresses sCAX1-induced ion sensitivity in tobacco plants. a Tobacco seedlings grown in medium with low Ca2+ for 30 days. b Tobacco seedlings grown in medium with 100 mM MgCl2 for 30 days. c Tobacco seedlings grown in medium with 100 mM KCl for 60 days. Upper panel overview of the plates; lower panel close up pictures of sCAX1 + CRT- (lower left) and sCAX1-expressing (lower right) seedlings. Four biological replicates were performed. Scale bar 1 cm

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Discussion

Conventional breeding strategies for mineral biofortification of crops rely on germplasm with limited genetic variation for many traits (White and Broadley 2009). In some cases, genetic diversity can be increased by crossing to distant related species and movement of the traits slowly into the agronomically useful cultivars. However, the variation in a trait, in particular Ca2+ concentration, may not cover the range desired for agronomic value. Thus, breeders may not have the appropriate level of genetic variation in Ca2+ concentration among varieties. Our genetic engineering approach allows over-expression of Ca2+ transporter genes and expression to a level not present in germplasm. However, a major impediment for the development of Ca2+-biofortified crops using Ca2+ transporters is that the transgenic lines expressing sCAX1 dramatically increase Ca2+ content in their tissues, but also display severe Ca2+ deficiency-like symptoms, leading to significant yield losses (Hirschi 1999; Park et al. 2005a). Previous studies in Arabidopsis suggest that CRT plays a key role in the regulation of Ca2+ status of the plant ER and that the ER, in addition to the vacuole, is an important Ca2+ store in plant cells (Persson et al. 2001). In fact, Arabidopsis plants over-expressing a version of CRT contained up to 35 % more total Ca2+, and the increased Ca2+ sequestered by the CRT appeared to benefit plants when grown in a Ca2+ deficient situation (Wyatt et al. 2002). Results from these studies also suggest that the CRT-mediated alteration of the ER Ca2+ pool could potentially make Ca2+ more readily accessible for release into the cytosol and further strengthens the notion that the increased Ca2+-buffering capacity generated by overproduction of CRT helps maintain Ca2+ homeostasis.

There are at least two different groups of CRT isoforms, CRT1/CRT2 and CRT3, in higher plants (Persson et al. 2003). Different isoforms of CRT exhibit differences in the tissue-specific and stress-dependent expression patterns, indicating that they are involved in different pathways for their functions in plants (Jia et al. 2009). Among different CRT isoforms, CRT1 can substitute for animal CRTs in terms of modulation of Ca2+ homeostasis (Christensen et al. 2008). In addition, the role of maize CRT1 in plant responses to stress has been previously studied (Wyatt et al. 2002; Akesson et al. 2005). Thus, a maize CRT1 was chosen in this study to further investigate whether co-expression of the CRT1 may mitigate the Ca2+ deficiency-like symptoms caused by expression of sCAX1. Indeed, co-expression of a maize CRT mitigates the Ca2+ deficiency-like symptoms including tip burning and BER (Figs. 2, 3, and 4) and the hypersensitivity to ion imbalance (Fig. 5) caused by expression of sCAX1 in tobacco and tomato plants. Although CRT expression alone was not sufficient to dramatically alter the Ca2+ content and incidence of BER in this study, our results here suggest that combining expression of transporters and binding proteins may be a strategy to alter the concentration of Ca2+ without negatively impacting plant growth and development.

CRT is also known to harbor chaperone-like functions that may influence protein folding by interacting with unfolded proteins (Crofts and Denecke 1998). Indeed, recent studies indicate that AtCRT1a (also known as AtCRT1) and CRT1b family members are components of a general ER chaperone network and AtCRT1a restores putative folding deficiencies (Christensen et al. 2008, 2010). Furthermore, CRT expression is induced by biotic and abiotic stresses and may ensure plants adapt to various stresses (Jia et al. 2009). Therefore, it cannot be ruled out that co-expression of CRT in sCAX1-expressing lines could mitigate adverse effects by working as a stress-inducible chaperone and/or a positive regulator in stress responses.

Most mature plant cells have a central vacuole, which often takes up more than 80 % of the cell volume (Martinoia et al. 2000). The vacuole is considered to be the largest intracellular storage compartment for Ca2+ (Gelli and Blumwald 1993), and fluxes of Ca2+ across the vacuole are similar in magnitude to those occurring across the PM (Bush 1995). The plant ER, like the vacuole, is thought to function as a substantial Ca2+ storage compartment (Iwano et al. 2009). In animals, total Ca2+ concentration can approach micromolar concentrations in the mammalian sarcoplasmic reticulum (SR) (Zucchi and RoncaTestoni 1997). Measurements of Ca2+ efflux from plant ER vesicles indicate that there is rapid exchange of Ca2+ across the ER (White and Broadley 2003). Our data suggest that increased expression of Ca2+ binding proteins on the ER can ameliorate the adverse effects caused by increasing sequestration of Ca2+ into the vacuoles. Recent technological advances should enable future studies to make a detailed analysis of Ca2+ dynamics in different cellular compartments to decipher the temporal and spatial characteristics of Ca2+ signatures caused by altered sCAX1 and CRT expression (Krebs et al. 2012).

In Arabidopsis mutants where CAX activity is greatly reduced, the lines show threefold more apoplastic Ca2+ (Conn et al. 2011). On the other hand, when sCAX1 expression is increased in tomato plants, apoplastic concentration of Ca2+ are reduced (de Freitas et al. 2011). Depleting the apoplastic Ca2+ pool by expression of sCAX1 may cause the Ca2+ deficiency-like symptoms. One of the important functions of apoplastic Ca2+ is cross-linking the homogalacturonans for the biosynthesis of cell wall (Cosgrove 2005). Thus, reducing the apoplastic Ca2+ concentration in sCAX1-expressing plants could disrupt the cell wall biosynthesis and further results in growth stunting, tip burning and BER, especially in the tissues that the cell division and wall formation are most rapid (Figs. 2, 3 and 4). Furthermore, recent studies show that suppressing expression of pectin methylesterases (PMEs) in tomato fruit reduces the amount of Ca2+ bound to the cell wall, subsequently increasing Ca2+ available for other cellular functions and, thereby, reducing fruit susceptibility to BER(de Freitas et al. 2012). Therefore, future research may focus on elucidating the effects of co-expression of CRT and sCAX1 on the distribution/partitioning of symplastic and apoplastic Ca2+.

Ca2+ disorders, likely involving altered CAX activity, may be responsible for losses in crop production (Ho and White 2005). These putative Ca2+ disorders have been thought to develop similarly (White and Broadley 2003) and to be associated with a Ca2+ deficiency within the cells (Saure 2001). BER in tomato and bitter pit in apples may also be linked to changes in CAX activity (Park et al. 2005a; de Freitas et al. 2010). To explain the primary causes of BER, two hypotheses have been considered, (1) Ca2+ deficiency and (2) aberrant Ca2+ homeostasis. The majority of studies on BER in recent years have proposed that Ca2+ imbalance events at the cellular level, triggered by environmental stresses, may result in aberrant intracellular Ca2+ signals, ultimately leading to BER. It is suggested that this phenomenon might be a consequence of aberrant cytosolic Ca2+ regulation, and therefore spatial and temporal control of cellular Ca2+ concentration is a key factor determining incidence of Ca2+-related physiological disorders (Hirschi 2004; Ho and White 2005; Park et al. 2005a; Karley and White 2009; White and Broadley 2009; Dayod et al. 2010; de Freitas et al. 2011). Regardless of mechanisms, our work here shows that elevated expression of CRT can reduce the severity of growth abnormalities caused by increased CAX activity.

Utilization of the sCAX1 for Ca2+ biofortification have been extensively investigated in various horticultural crop species (carrot, potato, tomato, lettuce) since the expression of sCAX1 can dramatically improve the Ca2+ accumulation in their edible tissues (Hirschi 1999; Park et al. 2004, 2005a, b, 2008, 2009). Interestingly, not all the increased Ca2+ in the transporter-modified carrots was bioavailable (Morris et al. 2008). This may be due to a fraction of the extra Ca2+ being bound to antinutrients within the carrot (Hirschi 2009). This serves as a cautionary example for scientists that assume that all increases in nutrient content directly equate to increased bioavailability. However, the modified carrots are a better source of Ca2+ because total Ca2+ absorbed was higher. Although we postulate that the Ca2+ content has increased within the vacuoles of the modified carrots, we have not yet addressed the intracellular Ca2+ redistribution in these plants experimentally. We postulate that co-expressing various transporters and CRTs will differentially increase total Ca2+ content and the fractional absorption of Ca2+ in animals. However, feeding studies must be conducted to address the bioavailability issues in the double transformants, including the CRT + sCAX1 transformed crops.

Our working hypothesis is that the Ca2+ content within these double transgenic plants is more evenly distributed throughout the plant cells. However, in order to decode the relationship between expression of transporters and binding proteins and location of Ca2+ within the cell, we must determine the spatial resolution of Ca2+ within the plant (Punshon et al. 2009, 2012; Conn et al. 2011). Various techniques exist to visualize the distribution and abundance of elements within plants. These techniques are useful because, in contrast with bulk or volume-averaged measures (such as inductively coupled plasma mass spectroscopy, ICP-MS) where the sample is homogenized, the confinement of elements within specific plant organs, tissues, cells and even organelles can be seen (Punshon et al. 2012). The potential of synchrotron x-rays in spatially resolved elemental imaging in plants has begun to be realized (Punshon et al. 2009). In fact, this work has recently been done to demonstrate the alterations of Ca2+ partitioning in seeds of Arabidopsis lines altered in CAX expression (Punshon et al. 2012), it will certainly be interesting to apply this technology to the edible portions of crops co-expressing both sCAX1 and CRT.

In conclusion, while genetic engineering strategies to increase Ca2+ content by expression of a single gene (either sCAX1 or CRT) alone have provided promising results, co-expressing of CRT and sCAX1 enhances the Ca2+ content of plants without any apparent detrimental effects potentially caused by sCAX1 expression. Manipulation of the partitioning of nutrients across various endomembranes may be a means to increase plant nutrient content while maintaining crop productivity.