Arabidopsis thaliana zinc accumulation in leaf trichomes is correlated with zinc concentration in leaves

Zinc (Zn) is a key micronutrient for plants and animals, and understanding Zn homeostasis in plants can improve both agriculture and human health. While root Zn transporters in plant model species have been characterized in detail, comparatively little is known about shoot processes controlling Zn concentrations and spatial distribution. Previous work showed that Zn hyperaccumulator species such as Arabidopsis halleri accumulate Zn and other metals in leaf trichomes. To date there is no systematic study regarding Zn accumulation in the trichomes of the non-accumulating, genetic model species A. thaliana. Here, we used Synchrotron X-Ray Fluorescence mapping to show that Zn accumulates at the base of trichomes of A. thaliana. Using transgenic and natural accessions of A thaliana that vary in bulk leaf Zn concentration, we demonstrate that higher leaf Zn increases total Zn found at the base of trichome cells. Our data indicates that Zn accumulation in trichomes is a function of the Zn status of the plant, and provides the basis for future studies on a genetically tractable plant species to understand the molecular steps involved in Zn spatial distribution in leaves.


Scientific Reports
| (2021) 11:5278 | https://doi.org/10.1038/s41598-021-84508-y www.nature.com/scientificreports/ Metal-Associated transporters) perform Zn loading from the root symplast into the xylem for long distance transport 24 , and are also involved in Zn loading in seeds 25 . Zn accumulation and storage in shoots, on the other hand, is not well understood. Previous work in Zn hyperaccumulator species A. halleri and Nocceae caerulescens [26][27][28] which are close relatives of A. thaliana, show how critical Zn homeostasis genes are in establishing their remarkable tolerance to Zn levels that are lethal to other species. Both A. halleri and N. caerulescens rely on multiple copies of genes such as MTP1 and HMA4 for their Zn hyperaccumulation phenotypes [29][30][31] . Previous work has shown that Zn accumulation might be related to herbivory deterrence 32,33 . A. halleri accumulates high concentration of Zn in its trichomes. This accumulation occurs specifically at the base of tricomes as a narrow ring [34][35][36] Elements such as cadmium (Cd), for which A. halleri is also hypertolerant, also accumulate in trichomes 37 . Because the hyperaccumulator/hypertolerant species A. halleri and the non-hyperaccumulator A. lyrata both accumulate Zn and Cd in trichomes 34,35,37 in a similar pattern, it seems unlikely that this is a hyperaccumulation mechanism.
A. thaliana non-glandular tricomes are derived from epidermal cells that undergo endoreduplication, which consists of replication of the genome without mitosis, and results in increased cell size 38 . These trichomes differ from the secretory trichomes, such as those found in tobacco, which secrete Cd and Zn 39,40 . Despite not having a secretory function, non-glandular trichomes are likely hotspots for metal accumulation 41,42 . Cd has been detected in A. thaliana non-glandular trichomes 43,44 . Non-glandular trichomes of sunflower (Helianthus annuus) accumulate Mn when the metal is in excess in the growth medium 41 , while Zn accumulates rapidly at the trichome base when Zn is sprayed on leaves 45 , a phenomenon also observed in soybean 46 , suggesting that trichomes are a sink for metals both from foliar application or the transpiration stream. Despite the work on A. lyrata and A. halleri, little is known about metal accumulation in the trichomes of the model species A. thaliana.
Our aim in this study was to understand how Zn availability affects A. thaliana Zn accumulation in trichomes and in leaves. To do this we conducted elemental mapping using synchrotron X-ray fluorescence (SXRF) of both A. thaliana natural variants and transgenic plants provided with a variety of Zn concentrations in the growth medium. Non-glandular trichomes accumulated more Zn as leaf Zn concentrations increased and Zn accumulation at the base of the trichome was observed. Our results suggest that plants may actively change the partitioning of Zn to trichomes in response to leaf Zn supply.

Results
OsZIP7 constitutive expression leads to altered Zn distribution in leaves. We previously showed that OsZIP7 expression under the control of 35S promoter in A. thaliana leads to increased Zn concentration in leaves 16 . Here, to investigate alterations in Zn localization, we used two-dimensional SXRF mapping of leaves of wild type (WT) and OsZIP7-expressing plants (hereafter OsZIP7-FOX) to identify possible changes in Zn distribution. This technique provides information about all Zn regardless of chemical speciation. Leaves of plants grown under 50 nM Zn (our control condition) showed Zn evenly distributed throughout the leaf, with higher concentrations in hydathodes and closer to the petiole detachment site (Fig. 1A). In leaves of OsZIP7-FOX, Zn was found highly concentrated in small, punctuated areas at the leaf surface (Fig. 1A). When Zn localization was overlaid with localization of Ca, which is known to accumulate in leaf trichome papillae 47 , it was clear that Zn was accumulating at the base of trichomes in leaves of OsZIP7-FOX plants, a distribution that was not observed in WT (Fig. 1B). Leaves from glabra-1 mutant plants 48 , which lack trichomes, showed a uniform Zn distribution across the leaf surface similar to WT when plants are grown under 50 nM Zn (Fig. 1A,B). However, when plants were grown on media supplemented with 50 µM or 100 µM Zn, the punctate pattern of Zn distribution associated with the base of trichomes was observed in both WT and OsZIP7-FOX leaves ( Fig. 1C-F) whereas leaves of the trichome-less glabra-1 mutant showed evenly-distributed Zn and Ca. Such high Zn concentrations, although not typically seen in the environment, were used to maximize Zn availability to plants and therefore increase the likelihood of Zn accumulation in trichomes. These results confirm that Zn is accumulating at the base of trichomes, as observed for A. lyrata and A. halleri 34,36 .
User-defined region of interest (ROI) analysis allowed us to determine total Zn per trichome from mapping data collected via SXRF. The OsZIP7-FOX leaf grown under 50 nM Zn had the lowest total Zn per trichome, considering the maps in which Zn in trichomes was observed (i.e., excludes WT Col-0 leaves under 50 nM Zn conditions; Fig. 2). In leaves of plants growing at 50 µM Zn, total Zn per trichome was higher in both WT and OsZIP7-FOX compared to OsZIP7-FOX under 50 nM Zn. The total Zn per trichome in WT and OsZIP7-FOX grown with 50 µM Zn were not significantly different (Fig. 2). However, comparing WT and OsZIP7-FOX leaves from plants grown with 100 µM Zn, OsZIP7-FOX trichomes had clearly higher total Zn per trichome (Fig. 2). We showed previously that OsZIP7-FOX lines accumulate higher Zn concentration in their leaves compared to WT under these conditions 16 . Our data indicate that OsZIP7 expression in A. thaliana, which leads to increased accumulation of Zn in leaves, also leads to accumulation of Zn at the base of trichomes.
Trichomes accumulate Zn in a ring around the base in Arabidopsis thaliana. To gain more information on the nature of the characteristic pattern of Zn accumulation at the base of trichomes, we used higherresolution SXRF mapping of fresh trichomes. Plants grown at 50 µM Zn were chosen because both WT and OsZIP7-FOX leaves accumulate Zn in trichomes under these conditions (Fig. 1C,D), and this concentration was not toxic to A. thaliana plants under our experimental conditions 16 . Zn localization maps of WT leaves showed a ring-shaped pattern around the base of the trichome (Fig. 3A,B). In the mapped trichome of the OsZIP7-FOX plant, the Zn ring was thicker than in the mapped trichome of the WT plant, appearing to increase its domain farther away from the trichome base and up into the stalk (Fig. 3C,D). Because these are individual trichomes, and the median total Zn per trichome from plants cultivated under these conditions are not statistically different (Fig. 2), the distinct Zn distribution pattern observed is likely found in trichomes of both WT and OsZIP7 Scientific Reports | (2021) 11:5278 | https://doi.org/10.1038/s41598-021-84508-y www.nature.com/scientificreports/  www.nature.com/scientificreports/ plants. Therefore, our data show that Zn accumulation at the base of the trichome occurs in a ring shape, which can vary in thickness depending on Zn concentration.    www.nature.com/scientificreports/ to leaves 50 . Col-0 was included as the reference accession. Fab-2 leaves had several trichomes without Zn, and others with very low Zn fluorescence (Fig. 4C, D). Conversely, Kn-0 showed higher Zn fluorescence in its trichomes compared to Col-0 ( Fig. 4A,B,E,F). ROI analyses of these maps showed that Kn-0 had higher total Zn per trichome, whereas Fab-2 trichomes showed lower total Zn per trichome, compared to the reference accession Col-0 (Fig. 4G). These data suggest that increased levels of Zn in leaves are correlated with increased Zn accumulation at the base of trichomes.

Variations in leaf Zn concentration influence accumulation at the base of trichomes in
In addition to Col-0, Kn-0 and Fab-2, we mapped other 13 accessions with contrasting leaf Zn concentrations for a total of 16 accessions (Table 1), also cultivated under 50 µM Zn. These accessions span the high and low ranges of Zn distribution in the iHUB, available at www.ionom icshu b.org, allowing us to explore wide natural variation in leaf Zn concentration and its relationship to trichome Zn accumulation. Using ROI analyses, we determined the (1) mean count per pixel (a proxy for Zn concentration) per leaf; (2) percentage of total Zn sequestered within trichomes; and (3) percentage of Zn not associated with trichomes (Fig. 5).
Next, we compared the mean fluorescence counts per pixel with the percentage of total Zn found in trichomes, for each accession (Fig. 5). We found a positive correlation (R = 0.6871, p = 0.003) between the two variables, and consequently a negative, inverse correlation of Zn abundance not in trichomes (i.e., elsewhere in the leaf surface) and mean count per pixel. These data support the observations that A. thaliana accessions with higher leaf Zn concentrations accumulate a higher percentage of Zn at the base of trichomes, with a lower percentage elsewhere in the leaf. We therefore propose that Zn accumulation at the base of trichomes increases with increased Zn accumulation in the whole leaf, indicating trichomes may be an important Zn allocation site in leaves with high Zn concentrations.

Discussion
Zn accumulates at the base of trichomes in A. thaliana. Here we have demonstrated that A. thaliana accumulates Zn at the base of the trichome. In several plant species, trichomes are common sites of excess metal accumulation, including Pb, Zn and Cd in Nicotiana tabacum 39,40 , Cd in Brassica juncea 51 , Mn in Helianthus annuus 41 and Ni in the hyperaccumulator species Alyssum lesbiacum 52 . In the Zn hyperaccumulator/hypertolerant A. halleri, the base of trichomes accumulates the highest concentrations of Zn in leaves 34,36 . Despite being regions of high accumulation, trichomes do not account for the majority of Zn in leaves: a comparison with the non-hyperaccumulator A. lyrata showed that Zn in trichomes of A. halleri accounts for 10% of the total, while A. lyrata trichomes account for 20% of the total 35 . The data for A. lyrata agrees with our findings in A. thaliana, another non-hyperaccumulator, with Zn accumulation in trichomes ranging from 4 to 23% (Fig. 5).
Our data show clear localization of Zn in trichome cells. Zn localization in the trichome cell itself is quite obvious in A. halleri, as the narrow Zn ring is localized more distal to the base, on the trichome stalk [34][35][36] . In A. lesbiacum, Ni has a similar distribution along the stalk, and the authors suggested that Ni could be stored inside vacuoles 52 . However, our mapping data from intact trichomes of WT and OsZIP7-FOX leaves showed Zn in a ring shape at the base and continuing up into the trichome stalk (Fig. 3), observations which are more consistent with an extracellular localization, because a vacuolar localization would presumably fill the trichome cell. In A. halleri trichomes, Zn accumulates in a small compartment at its base. Although in roots of A. halleri Zn precipitates in the apoplast, in trichomes both the chemical form and subcellular localization of Zn remain to be determined. Zn might be in a soluble form with O-donors such as citrate, or found in a precipitated solid form as Zn oxides given the high Zn concentration (> 1 M when plants are treated with excess Zn) 34 .
Previous work showed that Cd associated with trichomes was predominantly bound to oxygen (O) and nitrogen ligands in non-hyperaccumulators A. thaliana and A. lyrata, and in the hyperaccumulator A. halleri 37,53,54 . Cd may be associated with the cell wall in trichomes, bound to O ligands 37,54 . Similarly, in A. halleri, Zn also binds mainly to carboxyl and/or hydroxyl groups 55 . Phosphate, thiol and silanol groups were excluded as potential Zn ligands 35 . Interestingly, the non-hyperaccumulator A. lyrata showed more Zn bound to cell wall (40% of the Zn in trichomes) compared to hyperaccumulator A. halleri (20%) 35 . Recently a new structure, the Ortmannian ring, www.nature.com/scientificreports/ was described in A. thaliana trichomes 56 . Ortmannian ring formation is dependent on the EXO70H4 exocyst subunit and is a callose-rich secondary cell wall layer, localized between the basal and apical regions of the trichome stalk. Loss-of-function exo70h4 plants showed no callose ring accumulation. Strikingly, WT and exo70h4 differed in their ability to accumulate Cu in trichomes, with WT plants showing Cu accumulation at the base of the trichome, while exo70h4 plants, which lack the Ortmannian ring, contain no Cu in the same region 56 . The data strongly suggest that the Ortmannian ring is involved in metal localization in trichomes. Therefore, Zn may co-localize with the Ortmannian ring, or its distribution is being limited by it. Further investigations are needed to clarify this. Moreover, experiments to fully characterize Zn subcellular localization in A. thaliana trichomes and its speciation and coordination environment should be performed in the future.
Physiological significance of Zn accumulation in trichomes. We found that natural accessions of A. thaliana with higher whole leaf Zn have increased amounts of Zn in their trichomes (Figs. 4, 5). When comparing the percentage of Zn in trichomes with whole leaf total Zn, we found that accessions with higher Zn concentrations in whole leaves have an increased percentage of Zn in trichomes (Fig. 5). This leads us to hypothesize that trichome Zn accumulation might have a role in metal detoxification by providing a location for metal sequestration in A. thaliana. Metal accumulation in trichomes has been shown before. In a previous study, four crop species were analyzed for Mn tolerance: sunflower (Helianthus annuus), white lupin (Lupinus albus), narrow-leafed lupin (Lupin angustifolius) and soybean (Glycine max). All but soybean could tolerate 100 µM Mn without showing toxicity symptoms 41 . Differently from the other Mn tolerant species, sunflower non-glandular trichomes accumulated Mn at the base, while Ca was distributed along the trichome length, resembling the pattern we observed in A. thaliana non-glandular trichomes with Zn and Ca. Fluorescence-XANES indicated that 66% of the Mn present at the base of the trichome is in the form of manganite [Mn(III)] 41 . The authors suggest that Mn is translocated from the apoplast to trichomes and then oxidized to manganite, thus preventing Mn accumulation in the cytoplasm and cell wall in leaf cells 41 . In the Ni-hyperaccumulator Alyssum murale, excess Mn accumulated in trichomes, where it was associated with phosphorous (P) 57 . It is possible that Zn in A. thaliana trichomes is being transported to the trichome apoplast directly via the transpiration stream, as has been proposed for Mn 41 .
A. halleri and A. lyrata (a non-hyperaccumulator) were found to accumulate Zn at the tricome base [34][35][36][37] . Zn accumulates to a higher extent in the mesophyll in A. halleri compared to veins, whereas the opposite is found in A. lyrata 35 . Another study observed that Zn concentration in mesophyll cells of A. halleri increased 30-fold upon exposure to high Zn, whereas Zn concentration in trichomes increased only 3-fold 34 . These results indicate that, despite their high Zn accumulation, trichomes are not important for Zn hyperaccumulation/hypertolerance in A. halleri. In tobacco (Nicotiana tabacum), the role of trichomes in heavy metal excretion is well documented: Zn and Cd are secreted from trichome tips as crystals 39,40,58 . However, these glandular, multicellular trichomes are very different from the unicellular, non-glandular trichomes found in A. thaliana 59 .
Cd and Mn accumulated at the base of A. thaliana trichomes 43,54 . Interestingly, A. thaliana transgenic lines that accumulate varying concentrations of Cd showed trichome metal accumulation varying in a similar way: more Cd in leaves resulted in more Cd in trichomes 43 . This is consistent with what we observed here for Zn in trichomes using 16 different accessions (Figs. 4,5). Upon exposure to Cd, A. halleri accumulated Cd first in trichomes, and only later in other leaf tissues 44 , which suggests that trichome cells might be relevant for short-term response to metal excess, whereas leaves hyperaccumulation/hypertolerance becomes necessary in prolonged exposure. Likewise, A. lyrata, a non-hyperaccumulator, has more Zn in trichomes (20%) than in the hyperaccumulator A. halleri (10%) 35 . Thus, trichome metal sequestration and possibly detoxification might be more important in non-tolerant and non-accumulators A. thaliana and A. lyrata than in a metal hyperaccumulator, hypertolerant species.
Transgenic plants or natural accessions with increased Zn accumulation in leaves showed a higher percentage of Zn in trichomes. Therefore, our findings support the idea that Zn found in trichomes increases linearly with higher Zn leaf concentrations (Fig. 5). In B. juncea, experiments where mass flow is decreased by application of abscisic acid (ABA) to induce stomata closure, Cd accumulation in leaves is dependent upon transpiration, although root uptake is not affected 51 . This would indicate that Cd accumulation in trichomes is dependent on transpiration rate. However, our data shows that the amount of Zn found in trichomes change depending on how much Zn a leaf accumulates, which suggests an active mechanism for Zn accumulation in trichomes. Whether this mechanism involves cell wall modifications or symplast transport remains to be answered. Moreover, experiments addressing possible changes in trichome density and/or development under varying Zn (and other metal) concentrations should be key to unravel a possible function of these cells in tolerance or accumulation of Zn and other metals.

Conclusion
Our work provides evidence for Zn accumulation in trichomes of A. thaliana, a genetically tractable model species, allowing exploration of the functional role of Zn distribution in trichomes and its relevance to leaf function. We also demonstrate that Zn accumulation in trichomes changes depending on Zn concentration in leaves, suggesting that plants might actively control this process to some extent. Future work should focus on the importance of such distribution in leaves and how it is molecularly controlled.

Materials and methods
Plant materials and growth conditions. All seeds from accessions used in this work were requested from the Arabidopsis Biological Resource Center (ABRC; https ://abrc.osu.edu/). OsZIP7-OE lines used were previously described 16  www.nature.com/scientificreports/ For growth in axenic conditions, we performed experiments as described 16 , with minimal changes. Briefly, seeds were sterilized for 15 min in 25% NaOH and 0.05% SDS, washed 5 times in sterile H 2 O and stratified at 4 °C for three days. Sterile 0.1% agar was used to suspend seeds, which were sown using a pipette onto plates made with full strength Gamborg's B5 media plus vitamins, 1 mM MES (2-(N-morpholino)ethanesulfonic acid), 2% sucrose and 0.6% agar. After five days, seedlings were transferred to minimal media containing 2 mM MES, 2 mM Ca(NO 3  Preparation of samples and mapping by two-dimensional XRF. In all experiments, we compared the detached 7th true leaf of each plant cultivated under the axenic conditions described above. 2D elemental mapping (between 7 and 2 µm steps) was conducted on fresh, unfixed leaf samples. Dehydration was minimized by sealing the leaf sample between two layers of Kapton tape, and the duration of mapping was restricted to less than 2 h. Samples did not show clear signs of dehydration, such as change in size or dried margins. Elemental maps were collected at beamline BL2-3 of the Stanford Synchrotron Radiation Lightsource (SSRL) as described 60 . Beam line 2-3 uses a water-cooled double crystal monochromator with either a Si(220) or Si(111) crystal and a Vortex single element detector. The beam was focused using a Pt-coated Kirkpatrick-Baez mirror pair (Xradia Inc.) and tuned to 11 keV. All maps were collected from the 7th true leaf. Elemental mapping was performed in 7 μm steps with a 50 ms dwell time for whole leaf maps, and in 2 µm steps and 50 ms dwell time for trichome 2D mapping. We used only one leaf per genotype for this analysis, so we confirmed our observations with separate pilot experiments conducted at NSLS beamline X26A, in which we observed the same trichome Zn distribution pattern (Supplemental Fig. 1).
The XRF maps were analyzed using Sam's Microanalysis Toolkit 61 (https ://www.sams-xrays .com/smak). To determine total abundance of elements in whole leaf maps and in individual trichomes, user-defined region of interest (ROI) analyses was performed. First, we defined a region at each trichome base, from which Zn counts were summed. The total number of pixels defined in each ROI was also obtained. The number generated contains all Zn within trichomes plus the Zn in the underlying leaf tissue. To subtract the background counts in each trichome ROI, we selected most leaf areas without trichomes and obtained the mean count per pixel in the leaf. The mean count per pixel in leaf was multiplied by the number of pixels in each ROI and subtracted from the total Zn per trichome value. The percentage of elements in trichomes was calculated by the sum of total abundance in each trichome divided by total abundance in whole leaf map.