Determination the Usefulness of AhHMA4p1::AhHMA4 Expression in Biofortification Strategies

Abstract

AhHMA4 from Arabidopsis thaliana encodes Zn/Cd export protein that controls Zn/Cd translocation to shoots. The focus of this manuscript is the evaluation of AhHMA4 expression in tomato for mineral biofortification (more Zn and less Cd in shoots and fruits). Hydroponic and soil-based experiments were performed. Transgenic and wild-type plants were grown on two dilution levels of Knop's medium (1/10, 1/2) with or without Cd, to determine if mineral composition affects the pattern of root/shoot partitioning of both metals due to AhHMA4 expression. Facilitation of Zn translocation to shoots of 19-day-old transgenic tomato was noted only when plants were grown in the more diluted medium. Moreover, the expression pattern of Zn-Cd-Fe cross-homeostasis genes (LeIRT1, LeChln, LeNRAMP1) was changed in transgenics in a medium composition-dependent fashion. In plants grown in soil (with/without Cd) up to maturity, expression of AhHMA4 resulted in more efficient translocation of Zn to shoots and restriction of Cd. These results indicate the usefulness of AhHMA4 expression to improve the growth of tomato on low-Zn soil, also contaminated with Cd.

Keywords: Biofortification; Cadmium; HMA4; Tomato; Transformation; Zinc.

Fig. 1

Zn concentration in tomato plants expressing AhHMA4 (lines 6 and 8) and wild type (WT). Zn concentration in the roots (a), leaves (b), and leaves/roots Zn concentration ratio (c) of 19-day-old plants grown hydroponically in 1/2 and 1/10 Knop’s medium without Cd or exposed to 0.25 μM Cd for 4 days. Values correspond to arithmetic means ± SD (n = 6); values for transgenic plants significantly different from WT at each experimental variant are highlighted by an asterisk (P ≤ 0.05). Different letters represent significantly different values at P ≤ 0.05 for wild-type plants grown upon different medium composition (evaluated by Student’s t test)

Fig. 2

Cd concentration in tomato plants expressing AhHMA4 (lines 6 and 8) and wild type (WT). Zn concentration in the roots (a), leaves (b), and leaves/roots Cd concentration ratio (c) of 19-day-old plants grown hydroponically in 1/2 and 1/10 Knop’s medium without Cd or exposed to 0.25 μM Cd for 4 days. Values correspond to arithmetic means ± SD (n = 6). Different letters represent significantly different values at P ≤ 0.05 for wild-type plants grown upon different medium composition (evaluated by Student’s t test)

Fig. 3

Fe concentration in tomato plants expressing AhHMA4 (lines 6 and 8) and wild type (WT). Fe concentration in the roots (a), leaves (b), and leaves/roots Fe concentration ratio (c) of 19-day-old plants grown hydroponically in 1/2 and 1/10 Knop’s medium without Cd or exposed to 0.25 μM Cd for 4 days. Values correspond to arithmetic means ± SD (n = 6); values for transgenic plants significantly different from WT is highlighted by an asterisk (P ≤ 0.05). Different letters represent significantly different values at P ≤ 0.05 for wild-type plants grown upon different medium composition (evaluated by Student’s t test)

Fig. 4

Expression analysis of LeIRT1 (a), LeChln (b), LeNRAMP1 (c), LeZIP4 (d), and AhHMA4 (e) genes by quantitative real-time RT-PCR (qPCR), in the roots and leaves of 19-day-old AhHMA4-transformed tomato plants (lines 6 and 8) and wild type (WT), grown hydroponically in 1/2 and 1/10 Knop’s medium without Cd or exposed to 0.25 μM Cd for 4 days. Values correspond to arithmetic means ± SD (n = 6); values for transgenic plants significantly different from WT at each experimental variant are highlighted by an asterisk (P ≤ 0.05). Different letters represent significantly different values at P ≤ 0.05 for wild-type plants grown upon different medium composition; (evaluated by Student’s t test)

Fig. 5

Number of fruits (a), fresh weight of fruits (b), number of seeds (c), and fresh weight of seeds (d) collected from tomato plants transformed with AhHMA4 (lines 6 and 8) and from wild type (WT) grown for 101 days in soil spiked with 10 mg Cd/kg d.m. and without Cd (control conditions). Each value represents a total number of a given plant organ collected from five plants. Values are given for fruits and seeds collected from the whole plants, upper, and lower bunches

Fig. 6

Zn concentration in upper leaves (a), lower leaves (b), and roots (c) of tomato plants expressing AhHMA4 (lines 6 and 8) and wild type (WT), grown for 101 days in soil spiked with 10 mg Cd/kg d.m. and without Cd (control soil). Values correspond to arithmetic means ± SD (n = 6); values for transgenic plants significantly different from WT at each experimental variant are highlighted by an asterisk (P ≤ 0.05). Different letters represent significantly different values at P ≤ 0.05 for wild-type plants grown upon different medium composition (evaluated by Student’s t test). Zn concentration from fruits collected from upper and lower bunches, and from seeds collected from these fruits, were not significantly different between transgenic and WT plants. Zn concentrations [mg Zn/kg d.m. of soil] are as follow: fruits from upper bunches: (−Cd) - Line 6: 19.05 ± 3.34; Line 8: 18.45 ± 0.82; WT: 20.51 ± 2.42, (+ Cd) - Line 6: 17.79 ± 9.67; Line 8: 18.49 ± 8.04; WT: 18.85 ± 1.32; fruits from lower bunches: (−Cd) - Line 6: 18.96 ± 3.30; Line 8: 19.16 ± 0.91; WT: 20.46 ± 2.02; (+Cd) - Line 6: 15.79 ± 1.27; Line 8: 18.18 ± 1.38; WT: 18.42 ± 0.97; seeds from upper bunches: (−Cd) - Line 6: 60.71 ± 5.87; Line 8: 64.94 ± 5.76; WT: 61.43 ± 6.29; (+Cd) - Line 6: 63.48 ± 5.72; Line 8: 67.78 ± 2.66; WT: 55.48 ± 3.69, seeds from lower bunches: (−Cd) - Line 6: 59.59 ± 7.50; Line 8: 61.20 ± 8.25; WT: 58.12 ± 5.03; (+Cd) - Line 6: 61.39 ± 7.25; Line 8: 61.98 ± 3.67; WT: 60.65 ± 7.24

Fig. 7

Cd concentration in upper leaves (a), lower leaves (b), roots (c) of tomato plants expressing AhHMA4 (lines 6 and 8) and wild type (WT), grown for 101 days in soil spiked with 10 mg Cd/kg d.m. and without Cd (control medium). Values correspond to means ± SD (n = 3); values for transgenic plants significantly different from the WT at each experimental variant are indicated by an asterisk (P ≤ 0.05) (evaluated by Student’s t test). Cd concentration from fruits collected from upper and lower bunches, and from seeds collected from these fruits, were not significantly different between transgenic and WT plants. Cd concentrations [mg Cd/kg d.m. of soil] are as follow: fruits form upper bunches - Line 6: 0.14 ± 0.07; Line 8 0.24 ± 0.08; WT: 0.23 ± 0.05, fruits from lower bunches: − Line 6: 0.11 ± 0.10; Line 8: 0.20 ± 0.07; WT: 0.32 ± 0.12, seeds form upper bunches: − Line 6: 0.77 ± 0.15; Line 8: 1.30 ± 0.54; WT: 1.01 ± 0.15, seeds from lower bunches: − Line 6: 0.73 ± 0.076; Line 8: 1.21 ± 0.55; WT: 0.86 ± 0.19