Plant-Growth-Promoting Bacteria Can Impact Zinc Uptake in Zea mays: An Examination of the Mechanisms of Action Using Functional Mutants of Azospirillum brasilense

Abstract

Among the PGPB, the genus Azospirillum-with an emphasis on A. brasilense-is likely the most studied microorganism for mitigation of plant stress. Here, we report the investigation of functional mutants HM053, ipdC and FP10 of A. brasilense to understand how the biological functions of these microorganisms can affect host Zn uptake. HM053 is a Nif⁺ constitutively expressed strain that hyper-fixes N₂ and produces high levels of the plant's relevant hormone auxin. FP10 is a Nif^- strain deficient in N₂-fixation. ipdC is a strain that is deficient in auxin production but fixes N₂. Zn uptake was measured in laboratory-based studies of 3-week-old plants using radioactive ⁶⁵Zn²⁺ (t_½ 244 days). Principal Component Analysis was applied to draw out correlations between microbial functions and host ⁶⁵Zn²⁺ accumulation. Additionally, statistical correlations were made to our prior data on plant uptake of radioactive ⁵⁹Fe³⁺ and ⁵⁹Fe²⁺. These correlations showed that low microbial auxin-producing capacity resulted in the greatest accumulation of ⁶⁵Zn. Just the opposite effect was noted for ⁵⁹Fe where high microbial auxin-producing capacity resulted in the greatest accumulation of that tracer.

Keywords: 65Zn and 59Fe radiotracers; maize; plant-growth-promoting bacteria; zinc nutrient uptake.

Figure 1

Experimental setup used for plant ⁶⁵Zn uptake studies. During exposure to radiotracer, plants were maintained at a constant 500 μmol m⁻² s⁻¹ light intensity and 21 °C temperature.

Figure 2

Dynamic ⁶⁵Zn transport over ~3-hours of acquisition. The data reflect ⁶⁵Zn transport measured by a radiation detector located along the plant stem at approximately 8 cm from the base of the stem. Each data point represents a 5-minute average ± SD of 300 data points sampled at 1 Hz across 5-6 biological replicates. (Panel A): Transport of ⁶⁵Zn in HM053-inoculated plants is shown in red with the non-inoculated control data shown in black. (Panel B): Transport of ⁶⁵Zn in ipdC inoculated plants is shown in yellow, with the non-inoculated control data shown in black. (Panel C): Transport of ⁶⁵Zn in FP10 inoculated plants is shown in green, with the non-inoculated control data shown in black. In all cases, data were normalized to the same starting level of radioactivity for direct comparison of transport across the bacteria strains. Data were fitted to trendlines (depicted by the dashed lines).

Figure 3

(Panel A): ‘Cut and count’ measurements yielded information on plant uptake of ⁶⁵Zn presented as the percentage of tracer dose administered to the beaker that was assimilated by the plant over 3 h. (Panel B): Root-to-shoot allocation of ⁶⁵Zn is presented as the percentage of the administered dose of radiotracer. Data reflect means for N = 5-6 replicates (±SE). Statistical significance p < 0.05 was designated by ‘a’ in a comparison of treatment to control, ‘b’ in a comparison of ipdC or FP10 to HM053, and ‘c’ in a comparison of FP10 to ipdC treatment.

Figure 4

Principal Component Analysis correlates ⁶⁵Zn uptake and root–shoot allocation (Panel A) to the biological functions of the beneficial microbes. HM053 and FP10 mutant strains were most like non-inoculated plant behavior, while ipdC was dissimilar, exhibiting the highest levels of root uptake and transport. Statistical correlations were made to ⁵⁹Fe²⁺ (Panel B) and ⁵⁹Fe³⁺ data that we acquired in prior work [16].

Figure 5

Radiographic images of maize plants after exposure to ⁶⁵Zn as a function of inoculation with ipdC, FP10, and HM053 bacteria. For comparison, we also show radiographic images of maize plants after exposure to ⁵⁹Fe³⁺ and ⁵⁹Fe²⁺ radiotracers from our prior work [16]. These plants were also inoculated with HM053, which was shown to exert the greatest influence on host ⁵⁹Fe³⁺ and ⁵⁹Fe²⁺ uptake and allocation.

Figure 6

Kernel Zn content was measured using ion chromatography. Data reflect means for N = 12 replicates (±SE). Statistical significance p < 0.05 was designated by ‘a’ in the comparison of treatment type to untreated control, and ‘b’ in a comparison of ipdC or FP10 treatments to HM053 treatment.