Soil Zn concentration and deficiency
The soils used for rice cultivation in this work have widely variable compositions and textures (Table 2). Most relevant to this study are bioavailable Zn levels in each soil. Zinc deficiency in soil is a common problem in Cambodia (Blair and Blair 2014) for one of the four major reasons:
-
Low total and bioavailable Zn concentrations. Soil fertility is commonly assessed by measuring the concentration of Zn extracted from soil using DTPA, or somewhat less precisely, based on total Zn concentrations. Normally, DTPA-extractable Zn in soils < 0.5 mg kg-1 is associated with Zn deficiency in rice. DTPA extractions of the soils in this study are all uniformly low, ranging from < 0.1 mg/kg to about 0.3–0.4 mg/kg. Using this definition, Zn nutrient status varies widely but all the soils in this study are Zn deficient. Care must be used in evaluating DTPA extractions, however, as measurements performed in dry soils are often not representative for flooded soils because Zn availability can vary temporally (Johnson-Beebout et al. 2009). As such, we define Zn limitation based on total Zn levels in the soil. DTPA-extractable Zn typically is only 1–2% of the total Zn in tropical paddy soils in South Asia (Wisawapipat et al. 2017). This implies that soils are likely to be Zn deficient if they contain < 25–50 mg kg-1 Zn. Accordingly, we conservatively define paddy soils as highly deficient if they have Zn concentrations less than the limit of detection (LOD, about 4 mg kg-1) and potentially somewhat inadequate at levels up to 25 mg kg-1. This designation has also supported the fact that Zn levels in rice plants are correlated to soil Zn in this concentration range (Rutkowska et al. 2013).
-
Flooding-Induced Zn Mineral Precipitation. Flooding affects Zn availability by changing the chemical form of Zn. Flooding induces sulfate reduction to sulfide followed by the precipitation of insoluble sulfides, decreasing bioavailability (Du Laing et al. 2007). The amount of flooding varied considerably between sites and between growing seasons in this study, potentially modulating Zn limitation, but this effect could not be tracked in this research, and it is not used to distinguish between different rice samples. Sulfide mineral precipitation requires adequate sulfate to be present, and thus is associated with high soil sulfate (> 1000 mg kg-1). In these soils, all soils have sufficient S to induce precipitation during flooding.
-
Low Soil Fe Concentrations. Soil Fe oxides often are effective at buffering sulfide concentrations to levels too low for ZnS precipitation and thereby maintaining Zn solubility and bioavailability when soils are flooded (Bunquin et al. 2017). Iron oxides also adsorb Zn but release that Zn into solution during reductive dissolution (Du Laing et al. 2009). Zn limitation thus is often associated with low total soil Fe (< 1500 mg kg-1).
-
Other Factors. Soil texture affects Zn bioavailability and the retention of soluble Zn. Zn fertilizer is more available in sandy soils than clayey soils (Rutkowska et al. 2013), but soluble Zn also is easily leached from sandy soil. Here no Zn fertilizer is applied to any soils and all the sandy soils are extensively leached with Zn concentrations lower than clay soils and well below limiting thresholds. As such, most sandy soils are likely Zn limited. Other factors also could play secondary roles. For example, soil pH affects metal solubility by influencing the formation of metal complexes with dissolved organic carbon (DOC), precipitation of carbonate phases, and the adsorption of Zn to Fe and Mn (hydro)oxides (Kirk 2004). DOC also has a limited effect in this study because all the soils are pH 6–7.
Table 2
Soil Properties and Zn Limitation for Cambodian Paddy Soils
Sample ID
|
Sampling date
|
pH
|
Soil Texture
|
DTPA extractable (mg kg− 1)
|
Element Concentration in Soil (mg kg− 1)
|
|
Main Soil Zn Deficiency Factors
|
Soil bioavailable
Zn level e
|
Soil Zn deficiency
level f
|
Zn
|
Fe
|
Zn
|
S
|
|
Total Zn a
|
Soil Texture b
|
Total
Fe c
|
Total
S d
|
KCH119_102R
|
Nov. 2018
|
6–7
|
Clay
|
0.35
|
6445
|
11
|
833
|
|
-
|
+
|
-
|
-
|
Higher
|
Lower
|
KCH119_104R
|
Nov. 2018
|
6–7
|
Clay
|
0.34
|
11116
|
10
|
699
|
|
-
|
+
|
-
|
-
|
Higher
|
Lower
|
KPC119_8R
|
Nov. 2018
|
6–7
|
Clay
|
0.29
|
6100
|
10
|
778
|
|
-
|
+
|
-
|
-
|
Higher
|
Lower
|
PP119_22R
|
Nov. 2018
|
6–7
|
Sand
|
0.12
|
4468
|
< 4
|
644
|
|
++
|
-
|
-
|
-
|
Lower
|
Higher
|
PP119_2R
|
Nov. 2018
|
6–7
|
Sand
|
0.21
|
2613
|
9
|
912
|
|
-
|
-
|
+
|
-
|
Higher
|
Lower
|
PP119_3R
|
Nov. 2018
|
6–7
|
Silt
|
0.1
|
2613
|
9
|
912
|
|
-
|
-
|
+
|
-
|
Higher
|
Lower
|
PP119_103R
|
Nov. 2018
|
6–7
|
Sand
|
< 0.1
|
878
|
< 4
|
996
|
|
++
|
-
|
++
|
-
|
Lower
|
Higher
|
PP119_13R
|
Nov. 2018
|
6–7
|
Sand
|
< 0.1
|
638
|
< 4
|
932
|
|
++
|
-
|
++
|
-
|
Lower
|
Higher
|
PP3_Rice1
|
Jan. 2019
|
6–7
|
Sand
|
< 0.1
|
2400
|
< 4
|
816
|
|
++
|
-
|
+
|
-
|
Lower
|
Higher
|
PP3_Rice2
|
Jan. 2019
|
6–7
|
Sand
|
< 0.1
|
2400
|
< 4
|
816
|
|
++
|
-
|
+
|
-
|
Lower
|
Higher
|
PP5_Rice1
|
Jan. 2019
|
6–7
|
Sand
|
< 0.1
|
2400
|
< 4
|
816
|
|
++
|
-
|
+
|
-
|
Lower
|
Higher
|
PP6_Rice1
|
Jan. 2019
|
6–7
|
Sand
|
< 0.1
|
2400
|
< 4
|
816
|
|
++
|
-
|
+
|
-
|
Lower
|
Higher
|
PP7_Rice1
|
Jan. 2019
|
6–7
|
Sand
|
< 0.1
|
1400
|
< 4
|
848
|
|
++
|
-
|
++
|
-
|
Lower
|
Higher
|
PT3_Rice1
|
Jan. 2019
|
6–7
|
Clay
|
0.31
|
34500
|
70
|
845
|
|
-
|
+
|
-
|
-
|
Higher
|
Lower
|
PT5_Rice1
|
Jan. 2019
|
6–7
|
Clay
|
0.29
|
2800
|
< 4
|
850
|
|
++
|
+
|
+
|
-
|
Lower
|
Higher
|
PT8_Rice1
|
Jan. 2019
|
6–7
|
Clay
|
0.34
|
2800
|
< 4
|
850
|
|
++
|
+
|
+
|
-
|
Lower
|
Higher
|
Notes:
( a ): Primary deficiency factor, Zn deficiency is presumed when soil Zn is low (< 4 mg kg − 1 (limit of detection, LOD)), while Zn is considered replete (sufficient) when soil Zn is 25–50 mg/kg;
( b ): Zn deficiency related to soil texture, all the sandy soil are considered less at risk of Zn limitation than clay soils if Zn concentrations are similar;
( c ): Zn deficiency is associated with low (< 2000 mgFe/kg) levels of Fe in paddy soil, concentrations of 2000–3000 mg/kg are considered potentially ow enough to induce limitation;
( d ): Zn deficiency increased when soil S concentrations are high (> 1000 mg/kg), S levels are all near levels sufficient to induce sulfide precipitation
( e ): Identification of soil bioavailable Zn based on the combination of soil composition and texture;
( f ): Identification of rice growing Zn deficiency possibility based on the combination of soil composition and texture;
(++): Indicative of more extensive Zn deficiency (relative to typical rice-growing soils);
(+): Indicative of potential Zn deficiency;
(-): Indictive to no Zn deficiency, or minimal deficiency.
Based on these factors, levels of Zn deficiency for each rice-soil pair were classified as either low-Zn and likely Zn-deficient, or moderate to high-Zn and indicative of moderate Zn availability and less Zn-limited (Table 2). This classification is conservative in that it probably underestimates Zn limitation, and it does not account for the potential effects of rice variety (Alloway 2009; Wissuwa et al. 2008).
Rice Composition
In contrast to Zn concentrations in paddy soils varying over nearly 2 orders of magnitude, the Zn level of rice grown was consistently 15–30 mg/kg and was not correlated to soil total Zn levels (Table 3). Pooled analysis that grouped rice samples based on their Zn fertility status, however, does reveal some difference in grain composition (Fig. 1). Rice grown in soils with lower soil Zn levels have variable but somewhat higher grain Zn concentrations than rice samples grown in soils with higher Zn levels, and similar to market rice samples. Most rice samples in both groups clustered between the interquartile range of 18–25 mg/kg Zn. This likely reflects the homeostasis in Zn concentrations in rice grains. Rather than producing rice grains that have too little Zn to be viable, the rice plant either grows less or produces fewer grains under limiting conditions. Unfortunately, this effect is difficult to evaluate without yield information from each site. These Zn concentrations that are observed are consistent with Zn concentrations typical of rice grown under Zn deficient (< 25 mg kg− 1) or borderline sufficient (18–35 mg kg− 1) soil conditions (Wissuwa et al. 2008), and our conservative estimates of Zn limitation based on soil composition (Table 2).
Table 3
Concentrations of Zn and selected elements in Cambodian rice grain
Sample ID
|
Sampling date
|
Rice Variety
|
Element Concentration (mg kg− 1)
|
Zn
|
P
|
S
|
Fe
|
Cu
|
Mn
|
KCH119_102R(a)
|
Nov. 2018
|
Rice 85
|
15.7
|
3417
|
1304
|
7.46
|
0.98
|
16.3
|
KCH119_104R(a)
|
Nov. 2018
|
Rice 85
|
17.7
|
852
|
1105
|
0.86
|
2.49
|
10.2
|
KPC119_8R(a)
|
Nov. 2018
|
Tgon, Rice 85, Pka Mleas
|
20.1
|
2415
|
1023
|
4.65
|
2.36
|
28.3
|
PP119_22R(b)
|
Nov. 2018
|
Unknown
|
24.2
|
2868
|
872
|
3.29
|
1.37
|
16.0
|
PP119_2R(a)
|
Nov. 2018
|
Chhmar Prom
|
19.9
|
1851
|
985
|
4.70
|
1.63
|
19.1
|
PP119_3R(a)
|
Nov. 2018
|
Chhmar Prom
|
22.1
|
2866
|
1051
|
7.70
|
2.06
|
23.4
|
PP119_103R(b)
|
Nov. 2018
|
Srov Khmao
|
18.1
|
2391
|
880
|
3.07
|
1.83
|
21.8
|
PP119_13R(b)
|
Nov. 2018
|
Chhmar Laet
|
20.8
|
2371
|
1159
|
4.07
|
3.27
|
43.1
|
PP3_Rice1(b)
|
Jan. 2019
|
Romdol
|
27.3
|
3773
|
902
|
14.10
|
2.48
|
20.7
|
PP3_Rice2(b)
|
Jan. 2019
|
Romdol
|
26.1
|
3724
|
876
|
9.00
|
1.80
|
20.3
|
PP5_Rice1(b)
|
Jan. 2019
|
Romdol
|
24.9
|
3610
|
869
|
10.96
|
1.00
|
27.2
|
PP6_Rice1(b)
|
Jan. 2019
|
Unknown
|
29.7
|
3593
|
900
|
15.06
|
1.85
|
21.4
|
PP7_Rice1(b)
|
Jan. 2019
|
Chonarlar
|
23.6
|
3119
|
985
|
11.25
|
3.88
|
42.0
|
PT3_Rice1(a)
|
Jan. 2019
|
Unknown
|
15.3
|
2479
|
756
|
9.78
|
2.73
|
24.4
|
PT5_Rice1(b)
|
Jan. 2019
|
Unknown
|
27.8
|
3352
|
1047
|
11.33
|
2.76
|
29.3
|
PT8_Rice1(b)
|
Jan. 2019
|
Unknown
|
16.4
|
2876
|
883
|
10.57
|
2.76
|
24.8
|
Market2(c)
|
-
|
China (black rice)
|
13.4
|
2449
|
891
|
5.25
|
1.43
|
26.5
|
Market3(c)
|
-
|
Thai (sweet rice)
|
18.3
|
511
|
939
|
2.09
|
1.50
|
7.4
|
Market9(c)
|
-
|
Thai (Red cargo rice)
|
19.7
|
2471
|
916
|
3.52
|
1.96
|
23.6
|
Notes:
(a): Rice grains are grown in soil with less Zn deficiency (high soil bioavailable Zn);
(b): Rice grains are grown in soil with more Zn deficiency (low soil bioavailable Zn);
(c): Market rice grains are grown under unknown levels of Zn deficiency;
(-): Not available.
Genotype or rice variety also can affect nutrient uptake, nutrient storage, and overall susceptibility to nutrient limitation (Alloway 2009; Wissuwa et al. 2008). It is important to note that this study does not control for rice variety. In fact, within our sampled sites, nearly half of all rice samples were a unique variety, and only a few varieties were observed more than a few times. In contrast, more developed agricultural systems like the US or China typically grow only a few varieties of rice. The genetic diversity of commonly-cultivated Cambodian rice is much higher; there are over 2500 rice varieties grown in Cambodia, most isolated to a few growing areas or growth conditions (Sar et al. 2012). These Cambodian rice varieties are not well characterized but chosen based on local experience, in many cases probably favoring the ability to grow under more variable nutrient availability and water stress conditions common to under-fertilized crops and irregular rainfed irrigation. In most cases, the net result of using these unimproved/native varieties of rice under a severe nutrient limitation is the slow maturation of rice (one crop per year), and low overall yields, often < 3 tons ha− 1 (Blair and Blair 2014).
Zn localization in rice embryo, aleurone, and endosperm
Zinc in grains facilitates protein synthesis, cell elongation, membrane function, and resistance to abiotic stresses during germination (Cakmak 2000; Farooq et al. 2012). The embryo is the nascent growing plant, and it is often enriched in metals including Zn because protein synthesis and cellular functions are localized there. The endosperm is the most crucial storage tissue that supplies Zn to the developing embryo, but the concentration of most elements are significantly lower in the endosperm relative to the developing embryo and aleurone layer (Bewley et al. 2013). The aleurone layer is a proteinaceous layer on the outer part of the endosperm that is responsible for nutrient uptake during grain filling that also can store nutrients.
XRF-measured elemental distributions indicate that most elements are heterogeneously distributed within the rice grain (sample KPC-119-3, Fig. 2). Zn levels are much higher in the embryo and aleurone than the endosperm, so are Fe, Zn, S, Mn, and P. Within the embryo, Zn concentrations are also somewhat heterogeneously distributed, with higher Zn levels associated with the radicle than the plumule. Elements such as Ti, which are primarily from inorganic (soil) sources external to the grain, are very low (near the detection limit) but concentrated on grain surfaces. Importantly, the aleurone is preserved in our analysis, indicating that the polishing method was sufficient to remove the rice husk without also removing the aleurone and embryo.
The total quantity of each element within the embryo, aleurone, and endosperm (the major divisions of the grain) is estimated by sectioning the image into their corresponding areas or regions using masking tools (Figure S2). Using these, we can calculate the mean and variance of each element concentration relative to the endosperm within each masked area (Fig. 3a). By summing the counts or relative element concentrations within each region (Webb 2011), we can estimate the percentage of a given element in different tissues corresponding to these masks (Fig. 3b). This analysis indicates that most elements are most concentrated in the embryo, including Zn, but more abundant overall in the endosperm because of its higher volume. Iron and Si are notable exceptions that are more concentrated in the aleurone, but also are largely in the endosperm. For Fe, this may reflect the accumulation of Fe in storage compounds within the aleurone. The reason for Si appearing to be enriched in the aleurone is unknown but could result from instrumental factors. Silicon is a light element and its X-rays are sufficiently low energy (1740 eV) that they are absorbed within the material, and thus are preferentially observed at surfaces and edges such as the aleurone (Fig. 2).
The concentration ratios for Zn in embryo, aleurone and endosperm are 5.7: 1.3: 1. For comparison, Zn concentrations embryo and endosperm in barley are 164 mg kg− 1 and 14 mg kg− 1 (Persson et al. 2009), corresponding to a ratio of 11.7 (Table S2). Although the embryo contains > 5 times higher Zn concentrations than that in the endosperm, it contains around 30% of the total Zn because it is small relative to the endosperm and aleurone. The aleurone and endosperm have similar Zn concentrations, but endosperm contains 60% of the total Zn because of its large size (Fig. 3b). This differs from other studies that suggest Zn is localized primarily in the aleurone as phytate complexes stored in protein storage vacuoles (Raboy 2009; Bohn et al. 2008). In this case, it is likely that this difference stems from the fact that this study directly measures the locations of Zn and P localization, while most other work either infers it based on biological function, or operationally defines differences using ex situ approaches like extractions.
Element concentration maps of P and S are particularly relevant in this study because phosphorus and sulfur (as thiol) potentially complex Zn. Most P in grains is usually present as phytate, while much of S is associated with proteins. The distribution of P (and thereby phytate) is similar to that of Zn, while S is more evenly distributed (Fig. 2). In this study, the relative concentration ratios of P and S in embryo, aleurone and endosperm are around 4.1:3.0:1 and 2.6: 2.1:1, respectively (Fig. 3a). Our results, however, suggest that much more P and S are stored in the endosperm (80% for P) than in the aleurone. Our higher estimates of endosperm-associated-Zn and P than previous estimates (Raboy 2009; Bohn et al. 2008) may reflect differences in analysis methods, rice variety or growth conditions, or an underestimation of endosperm P in the previous studies.
The spatial correlations of Zn:P and Zn:S count ratios also can be used to better understand how Zn in retained in these phases. The relationship between Zn and S or P depends on which region of the grain is analyzed, suggesting it might be bound in different environments in each (See supporting information part 2, Figure S3 and Figure S4). Zn counts correlate with P counts within the embryo, suggesting that Zn may be associated with P, or enriched in parallel to P, in that tissue. There is a much weaker or absent correlation between Zn and P in endosperm and aleurone, suggesting that the Zn is also associated with other elements in these regions (Figure S3). The slope of the Zn:P ratio also provides potential information about the relative importance of P to Zn complexation in specific tissues. The magnitude of the Zn:P ratios/slopes follow the order of aleurone < embryo < endosperm. Given that the aleurone has much higher P levels (and presumably also higher phytate concentrations) than other regions (Raboy 2009; Bohn et al. 2008; Iwai et al. 2012), the low Zn:P ratio suggests that Zn only complexes a small fraction of the total P/phytate in the aleurone, and that much of the aleurone’s phytate is stored in vacuoles with other metals. In contrast, the endosperm contains less P and phytate, but Zn is a more significant portion of metals bound in those regions. Minimal correlations between Zn and S are found in any of these three tissues (Figure S4). This correlation might be observable if much of the Zn was bound to protein, which commonly binds Zn through thiol groups in cysteine. Thus, it appears that protein or other thiol-bound Zn is an insufficient portion of total Zn to produce a trend with S levels, and minor relative to its complexation in more abundant storage compounds.
Zinc Local Structure and Speciation in Rice Grain
Bulk EXAFS spectra of each rice sample contained a combination of tetrahedral Zn-O, octahedral Zn-O and tetrahedral Zn-S indicative of the primary Zn species present: Zn-Phytate, Zn-NA (or mineral Zn containing octahedral Zn-O) and Zn-protein/thiol (Table 4, example fits are shown in Figure S5). Also present in each sample is a distinct Zn-P shell at 3.1 Å consistent with Zn in phytate. In most cases, tetrahedral Zn-O coordination predominates and is accompanied by the presence of two Zn-P second-shells, indicating that Zn-phytate is the most abundant form of Zn in the grain.
Table 4
Structural parameters determined by simulating extended X-ray absorption fine structure (EXAFS) spectra for Zn in rice grain. The tetrahedral Zn-O likely represents Zn phytate complexes, the octahedral Zn-O is likely representative of Zn nicotinamide(NA) complexes or Zn mineral, and a tetrahedral Zn-S represents Zn thiol complexes similar to those found for Zn bound to thiols in proteins.
Sample ID
|
Coordination Number ± Uncertainty
|
χ2
|
Reduced χ2
|
R factor
|
Zn-O tet
[Zn Phytate]
(1.95 Å)
|
Zn-O oct
[Zn-NA/mineral]
(2.11Å)
|
Zn-S tet
[Zn Thiol/Protein]
(2.34 Å)
|
Zn-P (*)
[Zn Phytate]
(3.1 Å)
|
Zn-P (*)
[Zn Phytate]
(3.6 Å)
|
KCH119_102R(a)
|
3.0 ± 0.6
|
0.7 ± 0.8
|
0.4 ± 0.5
|
1.0 ± 0.7
|
3.9 ± 1.0
|
150.26
|
23.25
|
0.0175
|
KCH119_104R(a)
|
2.9 ± 0.7
|
1.0 ± 0.9
|
0.5 ± 0.5
|
2.0 ± 0.6
|
4.9 ± 1.0
|
62.37
|
9.65
|
0.0165
|
KPC119_8R(a)
|
2.5 ± 0.4
|
1.0 ± 0.5
|
0.2 ± 0.3
|
1.1 ± 0.4
|
3.0 ± 0.6
|
96.28
|
14.89
|
0.0088
|
PP119_22R(b)
|
2.6 ± 0.4
|
0.9 ± 0.5
|
0.1 ± 0.3
|
1.1 ± 0.4
|
2.9 ± 0.6
|
79.09
|
12.23
|
0.0102
|
PP119_2R(a)
|
2.9 ± 0.5
|
1.3 ± 0.5
|
0.2 ± 0.4
|
1.1 ± 0.6
|
3.2 ± 1.0
|
58.55
|
9.06
|
0.0171
|
PP119_3R(a)
|
2.5 ± 0.4
|
1.0 ± 0.5
|
0.3 ± 0.3
|
1.1 ± 0.4
|
2.9 ± 0.6
|
154.16
|
23.85
|
0.0077
|
PP119_103R(b)
|
3.4 ± 0.5
|
0.6 ± 0.7
|
0.2 ± 0.4
|
2.1 ± 0.4
|
4.1 ± 0.7
|
19.48
|
3.01
|
0.0076
|
PP119_13R(b)
|
3.3 ± 0.5
|
0.8 ± 0.6
|
0.4 ± 0.4
|
1.0 ± 0.7
|
2.8 ± 1.0
|
41.57
|
6.43
|
0.0152
|
PP3_Rice1(b)
|
3.3 ± 0.5
|
0.6 ± 0.7
|
0.3 ± 0.4
|
1.4 ± 0.6
|
3.7 ± 0.9
|
58.65
|
9.07
|
0.0121
|
PP3_Rice2(b)
|
3.1 ± 0.5
|
1.0 ± 0.6
|
0.3 ± 0.4
|
1.1 ± 0.5
|
3.2 ± 0.8
|
32.96
|
5.10
|
0.0104
|
PP5_Rice1(b)
|
3.1 ± 0.5
|
1.1 ± 0.5
|
0.3 ± 0.4
|
1.1 ± 0.5
|
3.2 ± 0.8
|
315.01
|
48.74
|
0.0098
|
PP6_Rice1(b)
|
3.2 ± 0.5
|
0.8 ± 0.6
|
0.2 ± 0.4
|
1.2 ± 0.7
|
3.3 ± 1.1
|
82.70
|
12.79
|
0.0197
|
PP7_Rice1(b)
|
3.4 ± 0.5
|
0.5 ± 0.6
|
0.1 ± 0.4
|
1.2 ± 0.5
|
3.6 ± 0.8
|
28.37
|
4.39
|
0.0097
|
PT3_Rice1(a)
|
2.9 ± 0.7
|
1.1 ± 0.8
|
0.3 ± 0.5
|
1.8 ± 0.4
|
4.5 ± 0.6
|
22.45
|
3.47
|
0.0072
|
PT5_Rice1(b)
|
3.4 ± 0.5
|
0.6 ± 0.7
|
0.2 ± 0.4
|
1.5 ± 0.5
|
3.9 ± 0.8
|
44.19
|
6.83
|
0.0096
|
PT8_Rice1(b)
|
3.5 ± 0.5
|
0.4 ± 0.7
|
0.1 ± 0.4
|
1.2 ± 0.6
|
3.4 ± 0.9
|
53.76
|
8.32
|
0.0127
|
Market2(c)
|
3.0 ± 0.6
|
1.0 ± 0.8
|
0.5 ± 0.4
|
1.5 ± 0.6
|
3.8 ± 0.9
|
36.31
|
5.62
|
0.0135
|
Market3(c)
|
3.0 ± 0.7
|
0.8 ± 0.9
|
0.5 ± 0.5
|
2.2 ± 0.6
|
4.9 ± 1.0
|
75.21
|
11.63
|
0.0155
|
Market9(c)
|
3.1 ± 0.5
|
1.0 ± 0.6
|
0.2 ± 0.4
|
1.3 ± 0.5
|
3.4 ± 0.8
|
31.11
|
4.81
|
0.0102
|
Notes:
(*): The structural parameters from the Zn-P shell are determined in independent fits with fixed Zn-O/S coordination numbers to reduce the number of fit variables;
(a): Rice grains are grown in soil with less Zn deficiency (high soil bioavailable Zn);
(b): Rice grains are grown in soil with more Zn deficiency (low soil bioavailable Zn);
(c): Market rice grains are grown under unknown levels of Zn deficiency.
The relative fraction of each Zn species in 19 rice samples is quantified based on their coordination numbers (Table 5). Zn-phytate was most abundant, with 66–88% of Zn identified as combined with phytate Zn, and around 10–29% of Zn is combined with NA or as Zn minerals. The proportion of Zn combined with protein or other thiols is very limited (2%-11%) and insignificant (with estimated errors over the fractional abundance). Consequently, we also report quantitative results omitting the Zn-S shell entirely, which increases the relative proportion of the other Zn phases slightly (Table S3). Thus, EXAFS indicates that most Zn is bound principally in phytate complexes, which are mostly not bioavailable, with smaller fractions of more available organic complexes or mineral phases.
Table 5
The normalized component fraction of different Zn components presents in rice grain based on coordination numbers obtained by extended X-ray absorption fine structure (EXAFS) shell fitting and presented in Table 4. The tetrahedral Zn-O likely represents Zn phytate complexes, an octahedral ZnO is likely representative of Zn nicotinamide (NA) complexes or Zn mineral, and a tetrahedral ZnS represents Zn binding with thiol complexes similar to those found for Zn bound to thiols in proteins. Normalization factor accounts for a total of unnormalized ZnO tet, ZnO oct and ZnS tet fractions, representing the disorders of the first shell coordination (see EXAFS analysis and model compounds for EXAFS fitting in experimental section). Reported uncertainties reflect variable correlation, fit and data quality.
Sample ID
|
Normalized Component Fraction ± Uncertainty
|
Normalization Factor
|
Zn-O tet
[Zn Phytate]
(1.95 Å)
|
Zn-O oct
[Zn-NA/mineral]
(2.11Å)
|
Zn-S tet
[Zn Thiol/Protein]
(2.34 Å)
|
KCH119_102R(a)
|
0.77 ± 0.16
|
0.12 ± 0.14
|
0.11 ± 0.12
|
0.96
|
KCH119_104R(a)
|
0.72 ± 0.17
|
0.17 ± 0.15
|
0.12 ± 0.13
|
1.02
|
KPC119_8R(a)
|
0.75 ± 0.13
|
0.20 ± 0.09
|
0.05 ± 0.10
|
0.83
|
PP119_22R(b)
|
0.85 ± 0.14
|
0.09 ± 0.12
|
0.05 ± 0.11
|
1.01
|
PP119_2R(a)
|
0.79 ± 0.11
|
0.12 ± 0.10
|
0.09 ± 0.09
|
1.04
|
PP119_3R(a)
|
0.78 ± 0.12
|
0.18 ± 0.09
|
0.04 ± 0.09
|
0.82
|
PP119_103R(b)
|
0.75 ± 0.13
|
0.21 ± 0.09
|
0.04 ± 0.10
|
0.98
|
PP119_13R(b)
|
0.73 ± 0.12
|
0.19 ± 0.09
|
0.08 ± 0.09
|
0.84
|
PP3_Rice1(b)
|
0.82 ± 0.13
|
0.10 ± 0.12
|
0.08 ± 0.10
|
1.01
|
PP3_Rice2(b)
|
0.76 ± 0.12
|
0.16 ± 0.09
|
0.08 ± 0.09
|
1.01
|
PP5_Rice1(b)
|
0.75 ± 0.12
|
0.19 ± 0.09
|
0.07 ± 0.09
|
1.03
|
PP6_Rice1(b)
|
0.81 ± 0.13
|
0.14 ± 0.11
|
0.05 ± 0.10
|
0.99
|
PP7_Rice1(b)
|
0.89 ± 0.13
|
0.09 ± 0.11
|
0.02 ± 0.10
|
0.97
|
PT3_Rice1(a)
|
0.73 ± 0.17
|
0.19 ± 0.13
|
0.08 ± 0.13
|
0.99
|
PT5_Rice1(b)
|
0.86 ± 0.14
|
0.10 ± 0.12
|
0.04 ± 0.11
|
1.00
|
PT8_Rice1(b)
|
0.90 ± 0.13
|
0.07 ± 0.11
|
0.03 ± 0.10
|
0.97
|
Market2(c)
|
0.73 ± 0.14
|
0.16 ± 0.12
|
0.11 ± 0.11
|
1.03
|
Market3(c)
|
0.75 ± 0.17
|
0.13 ± 0.15
|
0.11 ± 0.13
|
1.00
|
Market9(c)
|
0.79 ± 0.13
|
0.16 ± 0.10
|
0.05 ± 0.10
|
1.00
|
Notes:
(a): Rice grains are grown in soil with less Zn deficiency (high soil bioavailable Zn);
(b): Rice grains are grown in soil with more Zn deficiency (low soil bioavailable Zn);
(c): Market rice grains are grown under unknown levels of Zn deficiency.
Our XRF results help constrain where this Zn-phytate is localized in the grain. The µ-XRF results indicate that more than 60% of Zn in rice grain is stored in the endosperm, similar to the fractions of Zn-phytates calculated by EXAFS fitting. This combination of findings could result if all of the Zn bound in phytate is present in the endosperm; however, µ-XRF results (Fig. 2) also indicate that much of the rice P (and thus most phytate) is localized in the aleurone. We thus conclude that Zn phytate is distributed in all tissues. For example, if Zn phytate were abundant in aleurone and embryo, at least (15–35%) of Zn would also need to be present as Zn-phytate complexes within the endosperm to explain its overall prevalence in the grain. There is limited information to confirm these associations in rice, but it appears that Zn is more likely complexed with phytate and other unknown species in endosperm than other portions of the grain (Iwai et al. 2012). NA or mineral Zn also seems to be somewhat more abundant and correlated to Zn concentrations in embryos (r = 0.647, significant at P < 0.05) (Díaz-Benito et al. 2018). If the Zn is bound within mineral forms, this could reflect the precipitation of mineral grains potentially as storage compounds within this layer, similar to phytate crystals stored there. Alternatively, Zn-NA/mineral may be stored in the aleurone or embryo. Given these constraints, we conclude that the Zn-phytate and NA/mineral complexes are found in all tissues, but that Zn-NA/mineral complexes are somewhat more abundant in aleurone and embryo, while Zn-phytate is slightly more abundant in the endosperm.
Linking Soil Zn Deficiency to Rice Zn Speciation and Bioaccessibility
The chemical form of Zn in the studied rice samples is related to the total Zn concentration in those grains (Figure S6). In general, phytate Zn and mineral Zn content are somewhat correlated with total Zn in the grain (in mg kg− 1 of each species, calculated with the fraction of each species and the XRF-measured Zn concentration in the grain). This correlation across all samples implies that there is a systematic relationship between total Zn and the speciation of that Zn.
Because this study examines both the rice and the soil from which the rice was grown, it is possible to qualitatively attribute variation in growing conditions and Zn availability to structure of Zn bound in rice, and thus to the chemical form and concentration of Zn in rice. There is a significant (P < 0.05) increase in the coordination number of tetrahedral Zn-O (2.7 to 3.2) that accompanies a decrease in the coordination number of octahedral Zn-O (1.0 to 0.7) in more Zn-limited rice samples, while coordination of Zn to S is low (< 0.4) but not affected (Fig. 4). This change in coordination indicate that Zn-phytate becomes more abundant under Zn limitation, while Zn bound to nicotianamine or other octahedral forms decreases.
The level of Zn deficiency significantly (P < 0.05) affects concentrations of Zn phytate and Zn-NA/mineral (Fig. 5). Zn-phytate concentrations increase about 10% (almost 5 mg kg− 1 in concentration) under higher levels of limitation. This increase in phytate is matched by a corresponding 7% (~ 3 mg kg− 1) decrease in the concentration of Zn-NA/mineral, and to some extent by a statistically insignificant increase in the total concentration of Zn. In contrast, Zn bound to thiols in proteins are small but unaffected by nutrient status.
Paradoxically, total Zn levels appear to increase slightly in response to lower Zn fertility, in parallel with increases in Zn-phytate concentrations (Fig. 5). We can probe this effect by examining how grain Zn and P concentrations vary as a function of Zn concentration in soil (Fig. 6). Zn concentrations in grain are generally higher in the grain and they increase with grain P content (which is primarily present in phytate). This is unexpected given that higher Zn limitation indicates low Zn availability, and which presumably would reduce its bioaccessible concentration. However, the concentration of Zn in the grain is not in itself a measure of total Zn uptake because it does not account for variation in grain yield, nor does it account for the physiological response of the rice plant to nutrient limitation. The most pronounced effect of nutrient limitation is diminished growth and lower crop yields. Thus, the nutrient limitation that decreases the amount of grain produced can lead to overall less Zn uptake, even if the concentration of Zn in that grain stays constant or potentially increases. The physiological response to Zn limitation is the induction of Zn uptake, the increased production of phytate in the grain, leading to an increase in phytate that can increase Zn concentrations. Overall, this increased uptake is modest does not counter the effect of loss of yield due to nutrient limitation. Our data suggest that homeostasis effectively regulates Zn levels in the grain to ensure that grain produced is viable. Future studies should measure grain yields, and control for rice variety differences to better understand the connection between Zn phytoaccesibility in soils, and Zn grain concentrations. Measuring gene expression of relevant genes, or protein activities relevant to Zn acquisition and transport also would provide additional insight into this process.
It is useful to contrast the composition and speciation of the Cambodian rice samples with rice from other sources, presumably that are well fertilized. The Chinese and Thai rice grains included in this study also have high levels of Zn phytate, and concentrations of Zn and P are similar to the concentrations observed in Cambodian rice samples. Specifically, Market rice 2 (from China), Market 3 (Thailand), Market 9 (Thailand) have 73%, 75% and 79% Zn-phytate based on EXAFS spectra, respectively. These changes are minor and probably insignificant but in combination with changes in the chemical composition of those rice samples, these data suggest that Market rice 9 (which has the highest Zn-phytate content), could be produced under more Zn limitation, while Market rice 2 is potentially grown in replete Zn conditions, or is a variety that is more effectively able to access soil Zn.