Potassium Chloroaurate-Mediated In Vitro Synthesis of Gold Nanoparticles Improved Root Growth by Crosstalk with Sucrose and Nutrient-Dependent Auxin Homeostasis in Arabidopsis thaliana

Abstract

In a hydroponic system, potassium chloroaurate (KAuCl₄) triggers the in vitro sucrose (Suc)-dependent formation of gold nanoparticles (AuNPs). AuNPs stimulate the growth of the root system, but their molecular mechanism has not been deciphered. The root system of Arabidopsis (Arabidopsis thaliana) exhibits developmental plasticity in response to the availability of various nutrients, Suc, and auxin. Here, we showed the roles of Suc, phosphorus (P), and nitrogen (N) in facilitating a AuNPs-mediated increase in root growth. Furthermore, the recuperating effects of KAuCl₄ on the natural (IAA) auxin-mediated perturbation of the root system were demonstrated. Arabidopsis seedlings harboring the cell division marker CycB1;1::CDB-GUS provided evidence of the restoration efficacy of KAuCl₄ on the IAA-mediated inhibitory effect on meristematic cell proliferation of the primary and lateral roots. Arabidopsis harboring synthetic auxin DR5rev::GFP exhibited a reinstating effect of KAuCl₄ on IAA-mediated aberration in auxin subcellular localization in the root. KAuCl₄ also exerted significant and differential recuperating effects on the IAA-mediated altered expression of the genes involved in auxin signaling and biosynthetic pathways in roots. Our results highlight the crosstalk between KAuCl₄-mediated improved root growth and Suc and nutrient-dependent auxin homeostasis in Arabidopsis.

Keywords: Arabidopsis; KAuCl4; auxin; gold nanoparticles; hydroponic system; nutrients; sucrose.

Figure 1

The solution color, UV–Vis spectrum, and TEM images of KAuCl₄-mediated synthesis of AuNPs in different media. The effects of (a) deionized H₂O, (b) one-half-strength MS medium, (c) NR comprising one-half-strength MS medium supplemented with 1.5% (w/v) Suc, and (d) wild-type Arabidopsis seedlings grown in NR medium supplemented with 1.5% (w/v) Suc and different concentrations (0–100 ppm) of KAuCl₄ for 14 d on (A) color and (B) UV–Vis spectrum. A shift from colorless to different shades of bluish to bluish–purple and an increase in the absorbance at 530 nm, corresponding to the plasmon absorbance of AuNPs, suggested its formation in the medium in KAuCl₄ concentration-dependent manner. (C) TEM images of AuNPs in different media (a–d) supplemented with KAuCl₄ (100 ppm).

Figure 2

A low dosage of KAuCl₄ triggers augmented the growth of Arabidopsis seedlings. Wild-type Arabidopsis seedlings were hydroponically grown in a nutrient-rich (NR) medium for 7 d and then transferred to an NR medium supplemented with 0, 1, 10, 25, 50, and 100 ppm KAuCl₄ and grown for a further 7 d. Dosage-dependent effects of KAuCl₄ on the development responses of the root and shoot were documented.

Figure 3

A low dosage of KAuCl₄ triggered augmented developmental responses of the shoot and root. Wild-type Arabidopsis seedlings were initially grown hydroponically in the NR medium for 7 d and then transferred to the NR medium (control) and NR medium supplemented with 10 ppm KAuCl₄ (NR.KAuCl₄) and grown for a further 7 d. The seedlings were removed from the hydroponic system, and then (A) shoots and (B) roots were separated under the stereomicroscope and spread on an agar plate (1.0%; w/v) to document their phenotype and quantification of different traits. (C–G) Data are presented for (C) total shoot area, (D) primary root length, (E) the number of first- and higher-order lateral roots, (F) total length of first- and higher-order lateral roots, and (G) total root length. Values (C–G) are means ± SE (n = 12) and different letters on the histograms indicate significant differences (p < 0.05).

Figure 4

The deficiency of Suc and different essential nutrient elements affected KAuCl₄-mediated augmented developmental responses of the shoots and roots. Wild-type Arabidopsis seedlings were initially grown hydroponically in NR medium for 7 d and then transferred to an NR medium deprived of Suc (Suc-), Pi (P-), N (N-), Fe (Fe-), and Zn (Zn-), and these media were supplemented with 10 ppm KAuCl₄ (Suc-.KAuCl₄, P-.KAuCl₄, N-.KAuCl₄, Fe-.KAuCl₄, and Zn-.KAuCl₄) for further 7 d. (A) The seedlings were removed from the hydroponic system, shoots and roots separated, and spread on an agar plate (1.0%; w/v) to document their phenotypes. (B–F) Data are presented for (B) total shoot area, (C) primary root length, (D) the number of first- and higher-order lateral roots, (E) total length of first- and higher-order lateral roots, and (F) total root length. Values (B–F) are means ± SE (n = 12), and different letters on the histograms indicate significant differences (p < 0.05).

Figure 5

The differential recuperating effects of KAuCl₄ on the natural and synthetic auxin-mediated perturbation of the developmental responses of the root. Wild-type Arabidopsis seedlings were initially grown hydroponically in the NR medium for 7 d and then transferred to the NR medium supplemented with 0.1 µM each of natural (IAA) and synthetic (NAA and 2,4-D) auxins (NR.IAA, NR.NAA, and NR.2,4-D), and these media were supplemented with 10 ppm KAuCl₄ (NR.IAA.KAuCl₄, NR.NAA.KAuCl₄, and NR.2,4-D.KAuCl₄) for 7 d. (A) The seedlings were harvested, shoots and roots separated, and spread on an agar plate (1.0%; w/v) to document their phenotype. (B–F) Data are presented for (B) total shoot area, (C) primary root length, (D) the number of first- and higher-order lateral roots, (E) total length of first- and higher-order lateral roots, and (F) total root length. Values (B–F) are means ± SE (n = 12) and different letters on the histograms indicate significant differences (p < 0.05).

Figure 6

KAuCl₄ restored the inhibitory effect of IAA on the meristematic cell proliferation of the primary and lateral roots. The transgenic Arabidopsis seedlings harboring the cell division marker CycB1;1::CDB-uidA reporter gene were initially grown hydroponically in the NR medium for 7 d and then transferred to the NR, NR.KAuCl₄, NR.IAA, and NR.IAA.KAuCl₄ media for further 7 d, as described in the legend of Figure 5. Roots were harvested for the histochemical GUS expression analysis of CycB1;1::CDB-uidA in the primary and lateral roots. The red arrow indicates the effect of NR.IAA on perturbation in the meristematic cell proliferation in the primary root.

Figure 7

KAuCl₄ reinstated the IAA-mediated elevated expression of auxin-responsive genes and auxin subcellular localization in the root. Arabidopsis (wild-type and transgenic DR5rev:GFP) seedlings were initially grown hydroponically in the NR medium for 7 d and then transferred to NR, NR.KAuCl₄, NR.IAA, and NR.IAA.KAuCl₄ media for a further 7 d, as described in the legend to Figure 5. Root tissues of the wild-type and transgenic DR5rev:GFP seedlings were harvested for qRT-PCR and fluorescence microscopy, respectively. (A) The relative expression levels of GH3.3 and IAA6 in the root were determined by qRT-PCR. ACT2 was used as an internal control. Values are means ± SE (n = 6) and different letters on the histograms indicate significant differences (p < 0.05). (B) Microscopic images of the primary roots showing the effect of NR, NR.KAuCl₄, NR.IAA, and NR.IAA.KAuCl₄ on the spatial expression pattern of the transgenic DR5rev:GFP. Asterisks (***) indicate the normal expression of DR5:GFP in the QC and columella cells. Red arrows indicate the surrounding region of the QC and columella cells where DR5:GFP expression was induced upon IAA treatment.

Figure 8

Differential recuperating effects of KAuCl₄ on IAA-mediated spatial expression patterning of the PIN genes in the primary root. The pPINs:GUS transgenic seedlings were grown hydroponically in the NR medium for 7 d and then transferred to NR, NR.KAuCl₄, NR.IAA, and NR.IAA.KAuCl₄ media for a further 7 d, as described in the legend of Figure 5. Histochemical GUS-stained primary root tip showing the expression of pPIN1:GUS, pPIN2:GUS, pPIN3:GUS, pPIN4:GUS, and pPIN7:GUS. Black arrows indicate the NR.IAA-mediated reduced expression of the PIN genes, and red arrows show the effects of NR.IAA.KAuCl₄ treatment upon the restoration of the spatial expression pattern of these genes.

Figure 9

Differential recuperating effects of KAuCl₄ on IAA-mediated effects on the genes involved in the auxin pathway in the root. Wild-type Arabidopsis seedlings were hydroponically grown in the NR medium for 7 d and then transferred to NR, NR.IAA, and NR.IAA.KAuCl₄ for a further 7 d, as described in the legend of Figure 5. Roots were harvested, and the relative expression levels of the genes involved in auxin biosynthesis, its influx, intracellular transporters, and signaling were assayed by qRT-PCR. ACT2 was used as an internal control. Values are means ± SE (n = 6) and different letters on the histograms indicate significant differences (p < 0.05). Blue and red dots on the histogram indicate the suppression and induction of the genes, respectively, in response to NR.IAA treatment and their subsequent recuperation upon treatment with NR.IAA.KAuCl₄.

Figure 10

A model depicting the differential effects of KAuCl₄ on the genes involved in the biosynthesis, transport, and signaling of auxin in the root. (A) A schematic diagram of the primary root tip with 11 specific cell types indicated with color codes. ASA1, ASB1, NIT1, AUX1, PILS2, PILS7, ARF6, and ARF8 are expressed in all the cell types. However, TAA1 (vascular initials, quiescent center, cortex/endodermal initials, lateral root cap/epidermal initials, and columella stem cells), YUC9 (vascular initials, quiescent center, cortex/endodermal initials, lateral root cap/epidermal initials, columella stem cells, columella, and lateral root cap), LAX1 (stele cells), and PILS5 (stele cells, endodermis, cortex, quiescent center, and epidermis) show expressions in only some of the specific cell types. (B) Blue and red dots on the genes indicate their suppression and induction, respectively, in response to NR.IAA treatment and their subsequent recuperation upon treatment with NR.IAA.KAuCl₄. Solid arrows indicate pathways in which the genes, enzymes, or intermediates are known, and dashed arrows indicate pathways that are not well-defined.