Agronomic biofortification of zinc in rice: Influence of cultivars and zinc application methods on grain yield and zinc bioavailability

Field Crops Research 210 (2017) 52–60

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Agronomic biofortification of zinc in rice: Influence of cultivars and zinc application methods on grain yield and zinc bioavailability

MARK

⁎

Susmit Sahaa, , Mahasweta Chakrabortyb, Dhaneshwar Padhanb, Bholanath Sahac, Sidhu Murmub, Kaushik Batabyalb, Anindita Sethd, G.C. Hazrab, Biswapati Mandalb, R.W. Belle a

College of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Burdwan Sadar 713101, West Bengal, India Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, Kalyani 741235, West Bengal, India c Dr. Kalam Agricultural College, Bihar Agricultural University, Bihar 855107, India d Hebrew University of Jerusalem, Rehovot 760100, Israel e School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia b

A R T I C L E I N F O

A B S T R A C T

Keywords: Fe Loss mechanism Optimization Phytic acid Zn bioavailability

Zinc biofortification in rice can be improved by altering Zn application timing and placement and cultivar choice. We made a comprehensive assessment on this, analysing Zn, Fe and phytic acid in whole grains and processed brown, white and cooked rice obtained from six cultivars raised with Zn applied through soil and/or foliar supply at different phenological stages of the crop and measuring Zn bioavailability in cooked rice. Pathways for Zn enrichment (27.4–92.6% over control) by Zn fertilization with different application protocols and cultivars were elucidated. Such enrichment of Zn was associated with depletion in Fe (6.5–29.4%) and phytic acid (14.8–30.4%). However, the loss of Zn on processing of rice grains increased on Zn fertilization (12.6–28.7 mg kg−1) because of a preferential allocation of applied Zn into bran and aleurone of the grains. Despite such loss, application of Zn caused a net increase in Zn bioavailability (52.2% over control) in the cooked product. Using the ranksum scoring technique, we found cultivar GB 1 and Zn supply through soil (basal) + 2 foliar applications achieved the most effective biofortfication of Zn in rice by optimizing grain yield, and enriching Zn and its bioavailability in cooked grain with least antagonism of Fe availability.

1. Introduction Dietary deficiencies of Zn and Fe are a serious global public health problem affecting over two billion people and causing a loss of 63 million life-years annually (Myers et al., 2014). These cases of malnutrition are more acute in populations of Africa, South and South East Asia where cereals, the major staple foods, are low in dietary Zn and Fe. Rice is of major importance particularly in South and South East Asia because it contributes more than two thirds of the energy intake of its population (Timsina et al., 2010). Zinc concentration in rice grains can be enriched by: i) biofortification with popular Zn fertilizers (Cakmak, 2009), ii) manipulating Zn transporters and ligands in rice plants (Palmgren et al., 2008; Borrill et al., 2014) and iii) efficient germplasm screening for higher bioavailable Zn (Blair 2014; Trijatmiko et al., 2016). All these methods depend on fertilizer or the soil or both as the source of Zn to produce Zn enriched grains. Soil supplied Zn is, however, limited depending upon soil properties such as pH and redox potential, contents of CO32− and HCO3−, oxides of Fe and Al, and organic matter (Mandal and Mandal, 1990) and inherent Zn status in the ⁎

upper soil layer (Tuyogon et al., 2016). The problem of low Zn availability to plants is exacerbated when rice is grown in submerged soils (Meng et al., 2014). Application of Zn fertilizer is the most common option to overcome such problems. But recovery of applied Zn by rice hardly exceeds 2% of the applied amount (Alloway, 2008). Tailoring Zn application protocols may help to enhance transport of applied Zn to the edible parts in plants and thus its use-efficiency. We, therefore, designed Zn application protocols using key principles of Zn nutrition of rice namely: (i) soil applied Zn after undergoing reactions with soil components, is absorbed by roots, travels through xylem to storage tissues, leaves and subsequently to grains via phloem (Pottier et al., 2014) despite a number of impediments like high pH of phloem sap, chelation processes etc. (Impa and Johnson-Beebout, 2012); (ii) on the contrary, foliar applied Zn moves faster within plants but retranslocation is dependent on plant nutritional status, germplasm and plant phenological stage (Sperotto, 2013). Immature leaves are physiologically incapable of exporting nutrients until they mature, while mature leaves export nutrient directly via phloem to developing grains and other organs but are incapable of importing (Fernandez and Brown,

Corresponding author at: College of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, 741235, West Bengal, India. E-mail address: susmit_saha1984@rediffmail.com (S. Saha).

http://dx.doi.org/10.1016/j.fcr.2017.05.023 Received 28 January 2017; Received in revised form 20 May 2017; Accepted 28 May 2017 0378-4290/ © 2017 Elsevier B.V. All rights reserved.

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(Stomph et al., 2014) and iii) grain processing involves three steps viz., i.e. hulling, milling and cooking to make consumable products. All these may profoundly influence biofortification of Zn in rice compared to that in wheat (Stomph et al., 2009; Mabesa et al., 2013). Keeping the above in view, a series of experiments were conducted to test the hypotheses formulated to ascertain i) a suitable Zn fertilization protocol and cultivar for maximizing Zn loading in grains, ii) the loci (hull, bran or endosperm) of such enrichment of Zn in the grains and the extent of its loss during processing, iii) the amount of Zn bioavailability in cooked rice with compositional changes on Zn application, and iv) the best cultivar and Zn application protocol optimizing Zn loading with increased grain yield, reduced Zn-Fe antagonism, favourable phytic acid:Zn molar ratio and enhanced Zn bioavailability in ultimate food product (cooked rice) for human consumption.

2013). Leaf maturity determines whether a leaf competes with grain as a sink for Zn or whether it can act as a source for Zn translocation to grains. Hence an application of Zn both through soil and foliar methods may help to overcome the above difficulties and improve the efficiency of loading of Zn into grains from applied Zn. Zinc application either in soils or onto leaves at different phenological stages or both, may influence Zn enrichment in grains. However, cultivars are known to respond differently to Zn application depending upon their inherent grain Zn density (Hegelund et al., 2012; Saha et al., 2015a). Cultivars with high Zn density in grain are resistant to further enrichment; while those with low density are more responsive to Zn enrichment (Saha et al., 2015a). To capture the actual differences among the Zn application protocols, the tested cultivars thus need to be relatively similar in their native Zn density. Efficient Zn application protocols and cultivars may enrich grains with Zn but cannot ensure higher Zn bioavailability in the ultimate food products prepared from them. This is particularly true for rice, since it undergoes three steps of processing viz., hulling involving removal of the husk or the seed coat from the paddy grains yielding the brown rice, milling/polishing that removes the bran containing the embryo and aleurone from the brown rice producing white rice and cooking which involves removal of the boiled water soluble Zn from the white rice through gruel to yield low Zn cooked rice for consumption. A substantial loss of Zn may occur in each of the steps from the grains. Spatial distribution of Zn in those components of the grain ultimately determines the magnitude of loss of Zn during the processing. The enrichment of Zn in grains needs to be assessed in relation to changes in other important nutritional traits of grain viz., concentrations of Fe (Giordano and Mortvedt, 1972) and phytic acid, the phytic acid:Zn molar ratio (Cakmak et al., 2010; Hussain et al., 2012), protein (Cakmak, 2008) etc. Such changes in composition of grains or cooked rice may influence their Zn bioavailability, since it is widely understood that the two primary factors affecting dietary Zn absorption in adults are the quantities of Zn and phytate in the diet (Miller et al., 2007). Capturing all the above changes in nutritional traits in grains in response to Zn enrichment is thus necessary for a comprehensive evaluation of the net gains from Zn biofortification in crops. In our present study, we have used a model proposed by Miller et al. (2007) to estimate Zn bioavailability from cooked products for more accuracy rather than phytic acid:Zn molar ratio. Estimation of Zn bioavailability through Caco-2 cell model is another option in this regard (Jou et al., 2012). In our previous study on agronomic biofortification of Zn in wheat, we have shown i) the best timing for application of Zn along with suitable cultivars, ii) the magnitude of Zn induced Fe and phytic acid depletion with different treatments and cultivars, iii) the extent of loss of Zn on processing (milling and cooking i.e. two step processing) of wheat grains and iv) the net magnitude of bioavailability of Zn from flat breads made from Zn-loaded wheat grains (Saha et al., 2017). Rice differs from wheat in many ways particularly its i) growing environments (submerged, low redox potential soil causing significant variations in nutrient availability), ii) native tendency for enriching seed endosperm with Zn by lowering Zn concentration in vascular tissues

2. Materials and methods 2.1. Experiment 1 2.1.1. Experimental sites The experiment was conducted in the University Research Farm located (22°60′N, 88°23′E) under hot and humid climate with annual average rainfall of about 1480 mm, and maximum and minimum monthly temperature of 36.2 ± 2.0 °C and 12.5 ± 1.0 °C, respectively. After primary land preparation, soil samples were collected (0–0.2 m layer) from the experimental fields (two adjacent Zn-deficient fields were chosen for consecutive years to avoid residual effect of applied Zn) for analysis of pH (soil: water: 1:2.5), oxidizable organic carbon (Walkley and Black, 1934), 0.32% KMnO4 extractable N, 0.5 M NaHCO3 (pH 8.5) extractable P, 1.0 M NH4CH3CO2 (pH 7.0) extractable K (Jackson, 1973) and 0.005 M DTPA extractable Zn and Fe (Lindsay and Norvell, 1978) following standard methods. The soil was an Aeric Endoaquept with loamy texture (sand, silt and clay = 35–38, 43–45 and 23–25%, respectively), neutral reaction (pH 6.7–7.5), medium organic C (5.0–6.0 g kg−1) and extractable N, P and K (340–360, 20–24 and 180–200 kg ha−1 respectively), low DTPA extractable Zn (0.5–0.6 mg kg−1) but adequate Fe (30–40 mg kg−1). 2.1.2. Cultivars used The major characteristics of the six selected rice cultivars viz., Gobindobhog, GB 1, MTU 7029, KRH 2, Satabdi and Lalat used for the study (Table 1) were determined before the commencement of the present experiment. The seeds, raised with same management practices (without Zn application), were taken from a gene bank of the university and considered to be true to type with their native characteristics including similarity in Zn density. The cultivars are widely grown and cover almost the entire rice-area of this region of the world. 2.1.3. Management practices Twenty one day-old seedlings of the six cultivars were transplanted with 6 treatments of Zn (Table 1) in the fields both in 2011–12 and 2012–13 during 10 to 15th July at 25.0 cm × 25.0 cm (row to row and

Table 1 Characteristics of the rice cultivars tested before the commencement of the experiment. Cultivars

Parentage

Duration (days)

Average yield (t ha−1)

Zn in grains (mg kg−1)

Zn Harvest Index*

Fe in grains (mg kg−1)

Phytic acid in grains (g kg−1)

Gobindobhog GB 1 MTU 7029 KRH 2 Satabdi (IET 4786) Lalat

Folk Rice Selection from Assam Rice Collection Vasistha × Masuri IR 58025A × KMR 3R CR-10–114 × CR-10-115 Obs 677 × IR–207 × Vikram

130–135 110–115 140–145 130–135 120–125 125–130

2.5c 5.0a 5.2a 5.5a 4.5b 4.5b

18.6b 20.6b 21.0b 19.8b 20.8b 28.1a

0.09c 0.19a 0.17a 0.19a 0.13b 0.18a

49.8ab 43.6b 44.6b 38.8b 59.0a 56.7a

17.5c 27.0a 21.9b 27.4a 26.3a 26.6a

Different superscripted letters denote significant differences at P < 0.05. * Zn Harvest Index = [Zn in grains (mg kg−1) × grain yield (t ha−1)]/[{Zn in grains (mg kg−1) × grain yield (t ha−1)} + {Zn in straw (mg kg−1) × straw yield (t ha−1)}].

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plant to plant) spacing in plots of 30 m2 (6.0 × 5.0 m) with three replications. Fertilizers were applied (N-P-K: 80–17.5–31.0) on the basis of soil test values. Half of the nitrogen and the entire amounts of P and K were applied at the time of transplanting and the other half of N at maximum tillering stage (∼20–25 days after transplanting) of the crop. Standing water up to 5.0 cm depth (700 l water/m2/growing seasons) was maintained in all the rice plots throughout from transplanting to grain filling stages. All other recommended practices, as advocated by the University for the region, were followed for raising the crop. Zinc treatments were designed such that there was a boost in Zn supply at critical phenological stages of rice (sowing, maximum tillering and flowering) and combinations of stages to study the effects of root uptake and remobilization of Zn from source tissues to grains for Zn enrichment. Basal (soil) application of Zn was made to avoid Zn deficiency at initial growth stage, since the experimental soils contained low Zn (0.5–0.6 mg kg−1) and it affected the yield of rice; while foliar Zn application(s) was done for loading Zn in grains and straw. Zinc was applied through soil [basal at 5.0 or 4.5 kg Zn ha−1 (to equalize the amount of total Zn applied when foliar application was made except treatment Ftf) in the form of ZnSO4, 7H2O] by broadcasting at the time of final land preparation and by foliar spray (0.50/0.25% Zn in solution using a Knapsack sprayer, at 660–1320 l ha−1) at maximum tillering and/or flowering stages. We have applied 0.50% ZnSO4. 7H2O twice – one at maximum tillering and another at flowering stage for the treatment Ftf. All these treatments have been designed according to our previous study on agronomic biofortification of Zn in wheat (Saha et al., 2015a,b, 2017). The details of six Zn treatments imposed are described in Table 2. Two levels of farmyard manure (FYM) i.e. no FYM (OM0) and FYM at 5.0 t ha−1 (OM1) were also applied 14 days before the transplanting of rice seedlings. The experiment was conducted in a strip-strip design for the two seasons mentioned. For brevity and due to non-significant effects of year and FYM (Table S1, Supporting information), values are averaged over years and FYM levels.

produce brown rice and husk. The entire amount of the husk was collected, weighed and stored for analysis. The whole quantity of the brown rice thus produced was divided into two parts. One part was milled to white rice and brans; while the other part was kept as such for cooking to prepare cooked rice. On milling, both the white rice and brans were collected, weighed and stored for analysis. The hulling and milling operations were performed using a compact mill running for 30 s for each step yielding husk, bran and white rice. These, along with the brown rice, were subsequently analysed for Zn, Fe and phytic acid content following standard procedures described above.

2.3. Experiment 3 2.3.1. Cooking of the processed grains Both the brown and white (milled) rice thus produced from each of the six cultivars raised with the six Zn treatments were cooked separately in stainless steel pressure-cookers after addition of double distilled water (450 ml for 250 g of rice). The cooking was continued for 20 min and left as such with pressure inside the oven for another 15 min when the whole brown and white rice were transformed into cooked (boiled) rice. After decantation of the gruel (traditional way of cooking rice in South Asia) and on cooling, representative samples of the cooked rice were taken (50.0 g) in glass petri-dishes and dried in a hot-air oven (45 °C), processed and subsequently analysed for Zn, Fe and phytic acid following the standard methods mentioned. To evaluate the accuracy of estimation of Zn and Fe content in grains and cooked rice, recovery tests were performed with five levels (0, 0.5, 1.0, 2.0 and 2.5 μg g−1) of standard additions of the elements. The recovery varied between 92 and 97 and 94–98% for Zn and Fe, respectively.

2.3.2. Estimation of Zn and Fe bioavailability We estimated Zn bioavailability in grains and cooked rice by two methods: i) using a model factoring in Zn and phytate concentration (Miller et al., 2007), and ii) using phytic acid:Zn (PA:Zn) molar ratio as its indicator (Morris and Ellis, 1989); since it is widely understood that the two primary factors affecting dietary Zn absorption in adults are the quantities of Zn and phytate in the diet. The model (Miller et al., 2007) used for calculating the bioavailability was as follows:

2.1.4. Collection and analysis of rice grains At maturity, the cultivars with different treatments were harvested individually whole plot-wise, threshed and weighed separately for grains and straw and their representative samples were collected. The samples were thoroughly cleaned by winnowing through hot air and oven-dried (45 °C), processed and dry-ashed (0.5 g at 550 °C in a muffle furnace for four and half hours) and dissolved in 2.0 M HCl solution for analysis of Zn and Fe using atomic absorption spectrophotometer (GBC Avanta, model no. 912). Phytic acid content of the grains was also estimated by extracting phytins (Ca or Mg phytates) with trichloroacetic acid (TCA) with subsequent precipitation of Fe-phytate on addition of FeCl3 and measuring Fe concentration in the form of Fe(NO3)3 using atomic absorption spectrophotometer assuming a Fe:P ratio of 4:6 (Wheeler and Ferrel, 1971).

TAZ = 0.5 [Amax + TDZ + KR (1 + TDP/KP)2 − {(Amax + TDZ + KR (1 + TDP/KP))2 − 4 Amax + TDZ}1/2] Where, TAZ = total daily absorbed Zn (m mol d−1), TDP = total daily dietary phytate (m mol d−1), TDZ = total daily dietary Zn (m mol d−1), Amax = maximum Zn absorption (0.091), KR = equilibrium dissociation constant of Zn-receptor binding reaction (0.033), KP = equilibrium dissociation constant of Zn-phytate binding reaction (0.680) developed through isotope studies in gastrointestinal tract of humans with diet containing Zn and phytic acid concentration similar to ours (Leland V. Miller, personal communication). The PA:Zn and PA:Fe molar ratios in grains and cooked rice were calculated as follows:

2.2. Experiment 2 2.2.1. Processing of the harvested grains As mentioned, the harvested grains were thoroughly cleaned by winnowing through hot air and oven-dried (45 °C). A representative sample (2000 g) of the grains thus obtained from different Zn application protocols and cultivars was then subjected to de-hulling to

PA: Zn molar ratio =

(Phytic acid content in mg kg−1)/660 (Zn content in mg kg−1)/65

Table 2 Zinc application protocols tested in the experiment . C S SFt SFf Ftf SFtf

No application (control) Basal application @ 5 kg Zn ha−1 through ZnSO4·7H2O Basal application @ 4.5 kg Zn ha−1 + one foliar application at maximum tillering @ 0.5 kg Zn ha−1 supplied as 0.5% aq. solution of ZnSO4·7H2O (660 l/ha) Basal application @ 4.5 kg Zn ha−1 + one foliar application at flowering @ 0.5 kg Zn ha−1 supplied as 0.5% aq. solution of ZnSO4·7H2O (660 l/ha) Foliar application twice at maximum tillering and flowering @ 0.5% aq. solution each of ZnSO4·7H2O (1320 l/ha) Basal application @ 4.5 kg Zn ha−1 + foliar application twice at maximum tillering and flowering @ 0.25% aq. solution each of ZnSO4·7H2O (1320 l/ha)

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PA: Fe molar ratio =

(Phytic acid content in mg kg−1)/660 (Fe content in mg kg−1)/56

(25.0%) but lowest with GB 1 (17.6%) and MTU 7029 (20.8%)(Table S2, Supporting information).

(Using 660, 65 and 56 Da as molecular weight of Phytic acid, Zn and Fe respectively). The Fe bioavailability was estimated using a factor 17.2%, a fraction of total Fe in diet (Beard et al., 2007) for rice considering its Fe and phytic acid concentration.

3.2. Loss of Fe and phytic acid in grains on Zn application An antagonism was observed between Zn and Fe loading in grains (r = − 0.50, p < 0.05) (Fig. 1). The magnitude of such effect, as calculated in loss of Fe per unit gain in Zn (ΔFe/ΔZn) in grains, was higher with Lalat (−1.29) followed by Satabdi (−0.80) and MTU 7029 (−0.71), moderate with Gobindobhog (−0.51) and KRH 2 (−0.52), but lower with GB 1 (−0.29) (Table S3, Supporting information). Among the treatments, the antagonism was more acute when Zn was applied through soil (S) (−0.79), followed by soil + foliar at maximum tillering (SFt) (−0.75), but less with only two foliar applications of Zn (Ftf) (−0.51) and with SFtf (−0.60). Zinc enrichment also reduced the phytic acid (PA) level in grains (r = − 0.33, p < 0.05) (Fig. 1). Unlike Fe, such reduction was highest (30.4%) when Zn was applied through SFtf but lowest (14.8%) with its application through soil (S) only. Of the cultivars, reduction in PA was more in Lalat (32.3%) followed by MTU 7029 (28.6%) and KRH 2 (26.9%), but less in Satabdi (12.8%).

2.3.3. Evaluation of Zn application protocols and cultivars We screened out the Zn application protocols and cultivars tested as to their effectiveness for effecting Zn biofortification using six criteria viz., i) total Zn content of cooked rice, ii) total Fe content of cooked rice, iii) total phytate content of cooked rice, iv) amount of bioavailable Zn, v) amount of bioavailable Fe, and vi) increased grain yield on Zn application through the rank-sum scoring technique. In this technique, the mean values of all the above parameters for the 5 different Zn application protocols and 6 cultivars were ranked (scored) from 1 to 5 and 1 to 6 respectively based on their performance with the most suitable choice as 1. The minimum ranksum score of these six criteria for each of the Zn application protocols and cultivars indicates the best option(s) for adoption.

3.3. Loss of Zn, Fe and phytic acid on processing

2.4. Statistical analysis

There was substantial loss of Zn, Fe and PA during processing of harvested paddy grains to cooked rice. The magnitude of such losses was directly proportional to the native contents of Zn (r = 0.99, p < 0.01), Fe (r = 0.99, p < 0.01) and PA (r = 0.73, p < 0.01) of the tested cultivars. Out of a total loss of 12.6–28.7 mg kg−1of Zn, hulling, milling and cooking accounted for about 31.0–51.0 (40.5), 24.6–41.5 (33.0) and 23.8–27.5 (25.6)%, respectively (Fig. 2A); for Fe, out of a total loss of 26.4–35.3 mg kg−1, a major part (19.0–25.7 mg kg−1, 69.0–72.9%) was lost by milling followed by cooking (6.1–8.3 mg kg−1, 21.7–25.3%) and hulling (1.3–2.4 mg kg−1, 4.8–7.4%) (Fig. 2B). However, for phytic acid, the loss was maximum for hulling (11.2–15.9 g kg−1, 66.8–72.5%) followed by milling (3.1–3.5 g kg−1, 14.9–19.4%) and cooking (2.2–2.8 g kg−1, 12.6–14.3%) (Fig. 2C). Such loss of Zn was maximum when Zn was applied through soil + 2 foliar (SFtf) (28.7 mg kg−1) followed by 2 foliar (Ftf) (26.8 mg kg−1) and soil + foliar at flowering (SFf) (23.3 mg kg−1) but minimum with the control (C) (12.6 mg kg−1); while it did not change much for Fe and PA over the six tested regimes of Zn. Among the cultivars, the maximum loss of Zn and Fe occurred with Lalat (29.4 and 38.4 mg kg−1) followed by Satabdi (27.3 and 37.5 mg kg−1), but minimum with Gobindobhog for Zn (21.9 mg kg−1), and KRH 2 for Fe (21.0 mg kg−1); while for PA it was maximum with GB 1 (22.5 g kg−1) but minimum in MTU 7029 (17.4 g kg−1). We also estimated the% loss of applied Zn (30–80%) due to these processing operations, wherein hulling, milling and cooking caused about 5–10, 20–50 and 5–25% loss respectively (results not shown).

The results obtained were analysed following strip-strip design with cultivars in the main plot, Zn treatments in sub plots and organic matter (FYM) level in sub plots with the help of SPSS software (SPSS 14.0). Regression analyses between Zn and Fe/phytic acid were done to evaluate the effect of Zn loading on these traits in grains. The significance of the effects of treatments and cultivars was evaluated by two-way ANOVA since year and FYM effects were not significant, as mentioned earlier (Table S1, Supporting information). The mean effects of 3 replicates were further subjected to Post-Hoc test (Duncan Multiple Range Test) to identify the homogenous means at P < 0.05. 3. Results 3.1. Grain yield and its Zn enrichment Zinc application through soil (basal) + foliar supply at maximum tillering and flowering stages (SFtf) caused the largest increase in Zn concentration in grains (40.8 vs 21.5 mg kg−1 in control) (Table 3). Its application through foliar supply at late growth stage (flowering) was more effective (36.2 mg kg−1) than that at early stage (maximum tillering) (31.2 mg kg−1) in producing Zn dense grains. The effect differed significantly among the tested cultivars and was highest with Satabdi (45.3% increase) followed by MTU 7029 (36.1%) but lowest with Lalat (23.4%). Zinc application through SFtf again caused maximum increase in grain yield (17.6–33.3% over control) closely followed by soil + foliar supply at flowering (SFf) (Table 4). Such response was highest in Gobindobhog (33.3%) followed by Satabdi (31.3%) and Lalat

3.4. Bioavailability of Zn and Fe in cooked rice

Table 3 Effect of Zn application protocols on Zn concentration (mg kg−1) in grains of the tested rice cultivars. Treatments/Cultivars

C

S

SFt

SFf

Ftf

SFtf

Mean

Gobindobhog GB 1 MTU 7029 KRH 2 Satabdi Lalat

18.6 20.6 21.0 19.8 20.8 28.1 Mean

23.9 29.7 26.1 25.0 27.1 31.1 21.5b

29.7 32.4 31.3 32.1 29.9 31.3 27.2b

33.2 38.0 36.4 34.8 40.1 34.8 31.2ab

34.8 40.0 38.8 36.5 42.9 38.8 36.2a

37.9 40.0 42.0 41.7 41.2 41.9 38.6a

29.7a 33.5a 32.6a 31.7a 33.7a 34.3a 40.8a

Assuming that an adult consumes 300 g of cooked rice (dried) in their daily diet, the amount of bioavailable Zn in cooked rice prepared from Zn loaded grains varied from 1.5 to 1.9 and 1.4 to 2.1 mg d−1, respectively, among the cultivars and Zn application protocols tested (Table 4). There was insignificant variation in the amount of bioavailable Zn (1.5-1.9 mg d−1) among different cultivars, excepting for the lower amount in cooked grain of Gobindobhog (1.5 mg d−1); while among the Zn application protocols, cooked rice prepared from grains raised with SFtf (2.1 mg Zn d−1) and Ftf (2.0 mg Zn d−1) provided higher amounts of bioavailable Zn. Iron bioavailability, however, varied from 0.41-0.57 mg d−1 and 0.40-0.50 mg d−1 among the cultivars and Zn application protocols, respectively (Table 4). Cultivar GB 1 (0.57 mg Fe d−1) and KRH 2 (0.49) and protocols S (0.50) and SFt

Interaction was not significant. Means in a row or column with different letters are statistically significant at P < 0.05.

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Table 4 Ranking of rice cultivars and Zn application protocols based on different tested parameters for inclusion in an effective Zn biofortification program (Values of different parameters are either the mean of all the Zn treatments or the cultivars tested). Values in the first bracket denote the ranking of the specific cultivar or the treatment depending upon its performance for the particular parameter. Bioavailable Zn (mg) in 300 g cooked rice

Parameters/ Cultivars

Zn in cooked rice (mg kg−1)

GobindoBhog GB 1 MTU 7029 KRH 2 Satabdi Lalat

11.2 15.4 15.5 14.1 15.6 15.1

(6)b (3)a (2)a (5)a (1)a (4)a

1.5 1.7 1.9 1.7 1.8 1.7

Treatments S SFt SFf Ftf SFtf

12.0 13.2 16.3 17.2 18.2

(5)b (4)b (3)ab (2)a (1)a

1.4 1.6 1.9 2.0 2.1

Phytate in cooked rice (mg kg−1)

Grain Yield (t ha−1)

(4)b (1)a (5)b (2)ab (3)ab (6)b

1400(1)a 2600(5)e 1500(2)b 2200(3)c 2400(4)d 2400(4)d

2.4 5.6 5.4 4.6 3.9 3.7

(6)c (1)a (2)a (3)b (4)bc (5)bc

25 13 17 18 17 28

(1)a (2)a (4)ab (3)ab (5)b

2200(5)e 2100(4)d 2000(3)c 1900(2)b 1800(1)a

4.2 4.4 4.5 4.2 4.6

(4)a (3)a (2)a (4)a (1)a

21 19 19 16 14

Fe in cooked rice (mg kg−1)

Bioavailable Fe (mg) in 300 g cooked rice

(4)b (3)ab (1)a (3)ab (2)a (3)ab

10.5 (4)b 13.2 (1)a 10.2 (5)b 11.3 (2)ab 11.2 (3)ab 9.6 (6)b

0.45 0.57 0.44 0.49 0.48 0.41

(5)b (4)b (3)a (2)a (1)a

11.6 (1)a 11.2 (2)a 10.3 (4)ab 10.6 (3)ab 9.2 (5)b

0.50 0.48 0.44 0.46 0.40

Ranksum score

Different letters are statistically significant at P < 0.05. Fig. 1. Relationship between Zn with Fe (mg kg−1) and Phytic acid concentration (g kg−1) in the harvested paddy grains.

developing grains in plants especially after the onset of senescence (Fernandez and Brown, 2013). In contrast, application of Zn early through soil and/or foliar at maximum tillering is transported only by xylem to the leaves (sink tissues) where it remains until leaf senescence begins. After senescence, a meagre amount of such applied Zn is expected to flow into the developing grains. Stomph et al. (2014) suggested that rice plants can sequester ∼40% of the total Zn in matured grains from post flowering uptake, while a meagre amount (12% of the total grain Zn) is reallocated into the grains from its uptake between transplanting and panicle initiation. Rescheduling Zn application from early to late growth stage thus significantly enhanced Zn sequestration in grains. This was more with cultivars Satabdi (45.3%) and MTU 7029 (36.1%) but less in Lalat (23.4%) and GB 1 (26.7%). Besides Zn, a significant amount of Cadmium uptake by rice can be expected owing to its morpho-physiological features e.g. fibrous root system exploring more surface area for metal absorption (Sebastian and Prasad, 2014). The soil of our experimental fields show a trace amount of Cd (Hazra et al., 2012) and the area did not receive industrial effluents or city wastes in the past. Moreover, incorporation of organic matter under submergence (low redox potential) accentuates the possibility of binding Cd to organic matter and thus reduces available Cd for plants (Sauve et al., 2003). For brevity, we, therefore, exclude Cd analysis in grains from our experiment.

(0.48) excelled over the others for supplying bioavailable Fe. 3.5. Screening of cultivars and Zn application protocols On the basis of the six criteria used for assessing the effectiveness of Zn biofortification, the ranking of the five Zn application protocols and six cultivars tested showed that Zn application through SFtf (ranksum score 14) with cultivar GB 1 (ranksum score 13) was the most effective closely followed by Zn application through Ftf (ranksum score 16) with Satabdi and MTU 7029 (ranksum score 17) for a successful Zn biofortification in rice (Table 4). 4. Discussion 4.1. Efficient Zn application protocols and cultivars for Zn enrichment Both cultivars and changes in the form and timing of Zn fertilizer application altered the Zn concentration in grains of rice. However, all the tested application protocols and cultivars maintained a Zn concentration in grains higher than the critical value of 28.0 mg kg−1 to achieve an effective Zn biofortification (Trijatmiko et al., 2016). Zinc applied late, at flowering, is known to retranslocate readily from leaves into the developing grains via the phloem (Olsen and Palmgren, 2014). As leaves develop and mature, they transform from sink organs to source organs that export nutrients to other organs particularly 56

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Fig. 2. Loss of A) Zn (mg kg−1) B) Fe (mg kg−1) and C) phytic acid (g kg−1) from harvested paddy grains on different processing techniques. Values are means of three replicates. Vertical bars denote the standard errors.

in the vegetative stage may create an acute competition between these two elements for an efficient xylem translocation. Conversely we postulate that as the plant matures, synthesis of abundant transporters may reduce such competition (Saha et al., 2017). Earlier, we observed that the transfer coefficients of Fe from wheat shoot to grains remained mostly unaffected by the application of Zn; but Zn supply caused a significant depletion in Fe in grains and straw indicating that the antagonism occurred mainly in the soil and roots rather than during remobilization from source to sink tissues (Saha et al., 2015a). The extent of Zn-Fe antagonism was greatest in the cultivar Lalat (−1.29) followed by Satabdi (−0.80) and least with Gobindobhog (−0.51) and KRH 2 (−0.52). Hence the latter group of cultivars when used for Zn biofortification represent least compromise to Fe nutrition.

4.2. Reducing Fe and phytic acid depletion on Zn application Zinc enrichment caused a net depletion in Fe in grains (r = −0.50, p < 0.05). This can be attributed to a known competition between Zn and Fe for i) absorption by roots in soils (Dutta et al., 1989), ii) loading into the xylem (Alloway, 2008), iii) chelation for translocation (KabataPendias 2001), and iv) cross membrane transport by particular carrier proteins (ZIP family proteins) (Palmgren et al., 2008). The magnitude of the antagonism, expressed as the loss of Fe per unit gain in Zn (ΔFe/ ΔZn) in grains of the tested cultivars, was lower when Zn was applied through late foliar treatments (SFf, Ftf and SFtf). While Zn and Fe translocation in plants is facilitated by substrate-specific transporters (Ricachenevsky et al., 2015), a limited expression of such transporters 57

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amount of it (4.9 vs 2.6 mg kg−1) (Tables S4 and S5, Supporting information). During cooking, boiling water comes directly in physical contact with the embyo and aleurone as well as the endosperm and on the physical disruption, brings into solution whatever boiled water soluble Zn there was in the grains for subsequent loss during decantation in the form of gruel. Since the embryo and aleurone of the grains contained a good amount of Zn (∼23.0% of the total) and are susceptible to disintegration during cooking more than its endosperm, Zn associated with the former was not protected during cooking of brown rice causing a higher percent loss of Zn from the grains. Nevertheless, cooked rice prepared from the brown rice always had a net higher amount of Zn than those prepared from white rice (16.4 vs 14.5 mg kg−1, on an average of all the treatments). The net Fe content was also higher in cooked rice prepared from the brown rice; while the PA/Zn and PA/Fe molar ratios in cooked rice were almost similar for both the types prepared either from brown rice or white rice. All these results thus showed that the cooked rice prepared from brown rice had higher Zn and Fe retention and increased bioavailability of Zn and Fe.

Like Fe, Zn application also reduced the phytic acid content in grains (r = −0.33, p < 0.05). Existence of an antagonism between P and Zn in roots on Zn application (Bharati et al., 2013) and the attendant reduction in P translocation from roots (Mai et al., 2011) can explain the observed low phytic acid level in the grains (Imran et al., 2015). Further, a disruption in P-metabolism in plants with high Zn levels was also not unexpected. The antagonistic effect was greater, unlike Fe, when Zn was applied late through foliar supply, lowering significantly the phytic acid: Zn molar ratio below the critical value of 15.0 (Turnlund et al., 1984) for better bioavailability of Zn in humans. Significant variations were found in (PA/Zn) ratio among the cultivars tested and those (Gobindobhog, MTU 7029 and Lalat) with lower ratio in ultimate food product (cooked rice) may be preferred for Zn biofortification programmes. 4.3. Curbing Zn loss during processing of grains Zinc loading into the starchy endosperm of rice grains is the ultimate requirement for biofortification to improve Zn nutrition in humans. This is because paddy grains undergo three steps of processing, viz., hulling, milling/polishing and cooking, each of which causes a substantial loss of Zn from the grains yielding ultimately to a low Zn endosperm in cooked rice. Hulling removes the husk or the seed coat from the paddy grains, while milling/polishing removes the bran containing the embryo and aleurone, and together they removed 30–80% of Zn in grain. Further, while preparing cooked rice, the endosperm was boiled with water and physically disrupted which caused addition loss of endosperm Zn in the decanted gruel. Spatial distribution of Zn in tissues of the whole grains of rice and the methods used for its processing (Lu et al., 2013) determine how much of the element would be retained in the processed foods i.e. cooked rice. Estimation of loss of Zn during processing showed that the bulk of the whole grain Zn present in the seed coat (6.5–8.9 mg kg−1, 24.2%), and aleurone and embryo (3.1–11.9 mg kg−1, 23.2%) was lost during hulling and milling. A good part of Zn (3.0–7.9 mg kg−1, 15.6%) in the endosperm was then lost on cooking leaving a smaller amount (12.0–18.2 mg kg−1, 37.0%) in the cooked rice. The net amount of Zn left over in cooked rice was higher with grains raised through SFtf (18.2 mg kg−1, 78.4% increase over control), Ftf (17.2 mg kg−1, 68.6% increase over control) and SFf (16.3 mg kg−1, 59.8% increase over control) treatments over the control (10.2 mg kg−1) and the other three application methods tested (Table S4, Supporting information). Similarly, a higher amount of net Zn left over in cooked rice was found with those prepared from the grains of Satabdi (15.6 mg kg−1), MTU 7029 (15.5 mg kg−1) and GB 1 (15.4 mg kg−1) than those from Gobindobhog (11.2 mg kg−1), and KRH 2 (14.1 mg kg−1) (Table S5, Supporting information). Results thus indicated that Zn application through SFtf followed by Ftf and SFf with the cultivars Satabdi, MTU 7029 and GB 1 would cause an effective biofortification of Zn in rice. With increasing Zn load in grains following Zn application, loss of Zn on hulling, milling and cooking increased significantly (r = 0.99, p < 0.01). However, there was little variation among Zn treatments and cultivars in losses due to hulling over milling and cooking (5.5–12.5% vs 16.4–45.8 and 9.1–25.5% respectively). This suggests that loading of applied Zn occurred preferentially in the aleurone, embryo and endosperm, not even proportionally with its native content in the outer seed coat (hull). All these results are useful for identifying the suitable cultivar that retains more Zn in the final consumable products.

4.5. Improving Zn and Fe bioavailability The phytic acid:Zn (PA:Zn) and phytic acid:Fe (PA:Fe) molar ratios are considered to be the indicators for Zn and Fe bioavailability in food (Morris and Ellis, 1989; Hussain et al., 2012). We found a significant reduction in the values of PA:Zn molar ratio (from 112.3 in control to 43.7 in SFtf) on Zn application due to a decrease in phytic acid and an increase in Zn content in the grains. The beneficial effect was accentuated in the cooked rice (from PA:Zn 24.1 in control to 9.7 in SFtf) (Table S4, Supporting information). On the other hand, PA:Fe molar ratio did not show much variations in grains (39.1–45.2) or as in the cooked rice (15.2–16.6) on Zn application, possibly due to simultaneous depletion in both phytic acid and Fe (Tables S4 and S5, Supporting information). The values of PA:Zn and PA:Fe ratio, however, differed significantly among the cultivars tested both in their grains (from 79.3 in Satabdi to 53.8 in Gobindobhog for PA:Zn and from 51.1 in GB 1 to 29.8 in Gobindobhog for PA:Fe) and cooked rice (from 16.6 in GB 1 to 9.5 in MTU 7029 for PA:Zn and from 21.2 in Lalat to 11.3 in Gobindobhog for PA:Fe) (Table S5, Supporting information). Use of Zn application protocols and cultivars that maintain lower PA:Zn and PA:Fe molar ratios than the critical value of 15.0 (Turnlund et al., 1984) would enhance bioavailability of the elements in humans. The daily dietary requirement of Zn by an adult is about 10–11 mg (FAO/WHO, 2004). The two primary factors affecting dietary Zn absorption in adults are the quantities of Zn and phytate in the diet (Miller et al., 2007). The cooked rice we prepared from the Zn biofortified grains could supply about 5.2 mg Zn d−1 compared with 3.5 mg through normal grains, assuming that an adult consumes 300 g of cooked rice (dried) daily. The amount constituted about half (46.9–52.0%) and one-third (30.0–32.2%) of the daily dietary requirement of Zn supplied through biofortified and normal grains respectively, indicating a significant gain in Zn supply to human through Zn biofortification. Moreover, we observed a significant drop in phytic acid in grains/cooked rice on Zn biofortification. Using Miller’s model (Miller et al., 2007) we calculated that about 1.5 (normal) and 2.3 (biofortified) mg of Zn d−1 would be absorbed by an adult consuming 300 g of cooked rice. These indicated a 53% improvement in the net amount (0.8 mg d−1) of Zn absorption by humans when fed with Zn biofortified cooked rice compared with the normal one. Such enhancement in Zn bioavailability was accompanied by a significant loss in Fe content in cooked rice (Table S4, Supporting information). The daily dietary Fe requirement of an adult is about 8 (male)–18 mg (female) d−1 (Institute of Medicine, Food and Nutrition Board, 2001). The amount of Fe supplied through 300 g of cooked rice prepared from our normal grains (3.8 mg d−1) contributed about 21.1–47.5% of its total requirement. The above amount dropped down to 2.8 mg d−1 (contributing only 15.5–35.0%) when the cooked rice

4.4. Benefits of cooking brown vs white rice We prepared cooked rice both from brown and white rice to compare their effectiveness for Zn enrichment. The brown rice with embryo and aleurone contained a higher amount of Zn (25.3 mg kg−1) than the polished white rice (17.4 mg kg−1) but, on cooking, lost a greater 58

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was prepared from the Zn biofortified grains. We also calculated the amount of actual Fe absorption by an adult consuming the same amount of cooked rice using a factor proposed by Beard et al. (2007) and observed that about 0.66 (normal) and 0.48 (biofortified) mg of Fe d−1 respectively, were absorbed from the same amount of cooked rice prepared from normal and Zn biofortified grains. Hence to cause a net additional absorption of 0.8 mg d−1 of Zn in humans from cooked rice, a net loss in absorption of about 0.18 mg d−1 of Fe occurred due to Zn biofortification. These findings are critical for areas with both acute Fe and Zn deficiency in humans and to optimize the choice of cultivars and Zn application protocols for effecting a successful Zn biofortification. 4.6. Optimizing the biofortification process Since several properties need to be optimized for biofortification to be effective, six Zn application protocols and cultivars for effecting Zn biofortification were evaluated through ranksum scoring technique using six criteria described above. Out of the six treatments and cultivars tested, supply of Zn through soil + foliar applications at maximum tillering and flowering (SFtf) (ranksum score 14) followed by 2 foliar (Ftf) (ranksum score 16) with cultivar GB 1 (ranksum score 13) followed by Satabdi and MTU 7029 (ranksum score 17) (Table 4) were found to be the best combination for a successful Zn biofortification program in rice. 5. Conclusion Zinc supply through soil + foliar application at maximum tillering + flowering (SFtf) yielded the most Zn-dense grains and the greatest increase in bioavailability of Zn in the cooked rice. A favourable reduction in Zn-Fe antagonism and phytic acid content in the tested grains can also be successfully pursued by the mentioned Zn application protocol in rice. The resultant biofortification was maximized with the cultivars GB 1 followed by Satabdi and MTU 7029. Rice is the major staple diet of a large population in South, South East and East Asia. Low Zn in rice is responsible for widespread Zn deficiency in this population. Our evidence of improved Zn loading in rice grains by Zn application protocols, and of more Zn-dense cooked rice with higher Zn bioavailability can support programmes to mitigate Zn malnutrition of vulnerable populations. Acknowledgement We thank Department of Science and Technology, Govt. of India for providing the senior author (S S) an INSPIRE Fellowship during the study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fcr.2017.05.023. References Alloway, B.J., 2008. Zinc in Soils and Crop Nutrition. IZA and IFA Brussels Press, France, pp. 14–53. Beard, J.L., Murray-Kolb, L.E., Haas, J.D., Lawrence, F., 2007. Iron absorption: comparison of prediction equations and reality: results from a feeding trial in the Phillipines. Int. J. Vitam. Nutr. Res. 77, 199–204. Bharati, K., Pandey, N., Shankhdhar, D., Srivastava, P.C., Shankhdhar, S.C., 2013. Improving nutritional quality of wheat through soil and foliar zinc application. Plant Soil Environ. 59, 348–352. Blair, M.W., 2014. Mineral biofortification strategies for food staples: the example of common bean. J. Agric. Food Chem. 61, 8287–8294. Borrill, P., Connorton, J.M., Balk, J., Miller, A.J., Sanders, D., Uauy, C., 2014. Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops. Front. Plant Sci. 5, 1–8. Cakmak, I., Kalayci, M., Kaya, Y., Torun, A.A., Aydin, N., Wang, Y., Arisoy, Z., Erdem, H., Gokmen, O., Ozturk, L., Horst, W.J., 2010. Biofortification and localization of zinc in

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