Seasonal Zinc Storage and a Strategy for Its Use in Buds of Fruit Trees1[OPEN]

Ruohan Xie,a,b Jianqi Zhao,a,b Lingli Lu,a,b Patrick Brown,c Xianyong Lin,a,b Samuel M. Webb,d Jun Ge,a,b Olga Antipova,e Luxi Li,e and Shengke Tiana,b,2,3 Ministry of Education (MOE) Key Laboratory of Environment Remediation and Ecological Health, College of Environmental and Resource Science, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Subtropic Soil and Plant Nutrition, Zhejiang University, Hangzhou 310058, China Department of Plant Sciences, University of California, Davis, California 95616 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025 Advanced Photon Source, Argonne National Laboratory, Lemont, Illinois 60439

Bud dormancy allows deciduous perennial plants to rapidly grow following seasonal cold conditions. Although many studies have examined the hormonal regulation of bud growth, the role of nutrients remains unclear. Insufficient accumulation of the key micronutrient zinc (Zn) in dormant buds affects the vegetative and reproductive growth of perennial plants during the subsequent year, requiring the application of Zn fertilizers in orchard management to avoid growth defects in fruit trees. However, the mechanisms of seasonal Zn homeostasis in perennial plants remain poorly understood. Here, we provide new insights into Zn distribution and speciation within reproductive and vegetative buds of apple (Malus domestica) and four other deciduous fruit trees (peach [Amygdalus persica], grape [Vitis vinifera], pistachio [Pistacia vera], and blueberry [Vaccinium spp.]) using microscopic and spectroscopic characterization techniques comprising synchrotron-based x-ray fluorescence and x-ray absorption near-edge-structure analyses. By establishing a link between bud development and Zn distribution, we identified the following important steps of Zn storage and use in deciduous plants: Zn is preferentially deposited in the stem nodes subtending apical and axillary buds; Zn may then be sequestered as Zn-phytate prior to dormancy; in spring, Zn effectively releases for use during budbreak and subsequent meristematic growth. The mechanisms of Zn homeostasis during the seasonal cycles of plant growth and dormancy described here will contribute to improving orchard management, and to selection and breeding of deciduous perennial species.
Bud dormancy is one of the most important evolutionary strategies of the deciduous perennial growth habit to ensure survival of seasonal extreme-cold conditions. Budbreak and subsequent meristematic growth in spring reflects the availability of resources during growth-promoting conditions (Horvath et al., 2003;Paul et al., 2014;Considine and Considine, 2016;Martín-Fontecha et al., 2018). This process is generally thought to be controlled by a set of intricate interactions between plant hormones and environmental cues (Horvath et al., 2003;Leyser, 2003;Rohde and Bhalerao, 2007;Martin-Fontecha et al., 2018), whereas the role of nutrients in such physiological networks is regarded as secondary. However, since 1925, the nutritional hypothesis proposed that access to plant nutrients is a major factor regulating bud growth (Loeb, 1924;Gregory and Veale, 1957;Cline et al., 2009). Further, in recent years, a growing body of evidence indicates that budstored nutrients are strongly associated with budbreak and subsequent growth (Zhu and Kranz, 2012;Kebrom, 2017). However, direct evidence for the involvement of nutrients in budbreak and subsequent growth is still scarce at best.
In perennial deciduous trees, interruption or reduction of Zn availability during a given year will strongly affect vegetative growth and the success of new reproductive and vegetative development during the following year (Hu and Sparks, 1990;Swietlik, 2002). Thus, Zn deficiency in deciduous plants results in stunted growth, small leaves, and a rosette appearance at the shoot tip (Swietlik, 2002). This inhibition of shoot elongation and leaf development usually becomes visible in the early stages of spring growth after budbreak (Ojeda-Barrios et al., 2012;Xie et al., 2019), indicating that the emerging vegetative bud and early meristematic development have a very strong demand for Zn. Since spring budbreak occurs prior to active root growth and soil Zn uptake, deciduous plants must acquire and maintain an adequate Zn storage pool in buds for successful budbreak and initial growth (Maillard et al., 2015). Nevertheless, our understanding of the role of Zn bud storage in overwintering deciduous perennials remains limited.
The aforementioned significant gaps in our knowledge of plant biology are partly owing to a lack of information on the distribution of mineral nutrients within the bud. To date, research on the dynamic nutrient changes that occur during bud formation and budbreak has relied exclusively on whole bud analysis, which does not permit cellular localization of metals. To provide insight into the mechanisms whereby plants store and regulate Zn distribution during the seasonal cycles of growth and dormancy, it is essential to go beyond the quantification of total metal content in a bulk sample. Thus, here, we selected five species of deciduous fruit trees (apple [Malus domestica), peach (Amygdalus persica L.), grape (Vitis vinifera], pistachio [Pistacia vera], and blueberry [Vaccinium spp.]) to investigate the in vivo localization of Zn within terminal buds by application of microscopic and spectroscopic characterization techniques comprising synchrotronbased x-ray fluorescence (XRF) and x-ray absorption near-edge-structure (XANES) analyses. Most fruit crops are sensitive to Zn deficiency, and in many fruit orchards, especially in areas with calcareous and saline soils, Zn deficiency results in severe annual yield losses and deterioration of fruit quality (Swietlik, 2002). An examination of the spatial localization and the in vivo speciation of Zn in buds will provide a better understanding of the physiological processes responsible for Zn accumulation and the mechanisms of Zn supply during budbreak and early meristematic growth. This information will allow for improved management, selection, and breeding of deciduous perennial species. A, Microscope image of the cross section of an apple tree stem node. B, Micro-XRF images of stem nodes at upper, middle, and lower sections of apple tree stems, and fluorescence intensity values of Zn through the vascular bundles of stem nodes. The selected scanning sites from points (a) to (b) are marked by white dotted lines in m-XRF images. Fluorescence intensity values of elements were normalized, such that the brightest spots correspond to the highest concentrations. Images were digitally extracted and made into a composite for comparison. Xy, Xylem; Ph, phloem.

Localization of Zn in Stem Nodes
Here we applied micro XRF (m-XRF) analysis to investigate the localization of Zn in stem nodes in the upper, middle, and lower sections of apple-tree stems (Fig. 1). Our m-XRF scans clearly showed that Zn was preferentially present in the vascular bundles at the point of stem-petiole connection (stem node). To further examine nutrient distribution patterns, we conducted spatial imaging of stem nodes having an axillary bud ( Fig. 2A). In addition to Zn distribution in the vascular tissues of the stem node, axillary dormant buds also showed a remarkably pronounced localization of Zn, whose signal was detected in the axillary meristems protected by leaf primordia (Fig. 2B). In contrast, Zn concentration was much lower in the node-associated leaf, although the Zn signal was slightly elevated in the phloem of major leaf veins (Fig. 2C). The highest Zn concentration in the vascular bundles of stem nodes reached 0.4 mg cm 22 , which was much higher than that in the node-associated leaves (0.27 mg cm 22 ), suggesting that Zn was preferentially allocated to these nodes.
Analysis of Zn concentration in the internodes, nodes, and associated leaves confirmed these results (Fig. 3). Zn concentration was highest in the nodal parts of the lower section of stem nodes. Conversely, Zn concentration in the internodes was very low regardless of position. These results thus indicate that Zn preferentially distributes to the nodal regions of appletree stems.

Distribution and Speciation of Zn in Buds
Having identified the preferential allocation of Zn to stem nodes and axillary buds, we then examined the distribution and speciation of stored Zn in buds during winter (Figs. 4 and 5). We selected dormant terminal buds, rather than axillary buds, as experimental material for better structural observation and higher resolution of XRF scanning; further, by working with terminal buds, we avoided the risk of unsuccessful budbreak of axillary buds owing to apical dominance. Dormant vegetative buds consist of three major tissues: bud scales, leaf primordium, and pith with procambium tissue that will differentiate into xylem and Figure 2. m-XRF images of the cross sections of stem nodes with axillary buds and node-associated leaf from apple trees. A, Schematic showing the collection sites of samples. B and C, Zn distribution in the cross sections of a stem node (B) and the corresponding attached leaf (C) collected from an apple tree. The color-merge images show the relative locations of Zn (red), potassium (K; blue), and calcium (Ca; green). Fluorescence intensity values of Zn were normalized, such that the brightest spots correspond to the highest concentrations. Images were digitally extracted and made into a composite for comparison. Xy, Xylem; Ph, phloem.
phloem vessels to form vascular bundles. Zn preferentially allocated to the leaf primordium (Fig. 4). Then, we prepared transverse sections of terminal buds under high-pressure freezing (HPF) and analyzed these sections by nano-XRF to further reveal the details of Zn distribution at higher resolution. Figure 5A illustrates the incipient procambium region at the shoot apex of a dormant apple vegetative bud. Interestingly, there was a very strong association between the intensity of Zn and phosphorus (P) signals around the incipient procambium within cells. We acquired a second nano-XRF map from the transverse sections cut from near the base of the dormant bud (Fig. 5B). In XRF imaging, procambial tissue was particularly noticeable with a narrow band of closely arranged cells containing localized concentrations of Zn. Bands of xylem cells close to the procambium can be observed on the right-hand side of the image. As was observed in the terminal bud imagery (Fig. 5B), a close co-occurrence of Zn and P localization occurred in the intercellular spaces of procambium cells, whereas K predominantly localized in the cell walls of all types of cells within the mapped region ( Fig. 5B). Higher-resolution elemental maps obtained from the incipient procambium region further revealed the presence of multiple, variously sized spots containing significant colocalization of Zn and P within incipient procambium cells. Statistical analysis revealed a significant linear correlation (P , 0.05, r 2 5 0.516) between the intensity of Zn and P signals in this region (Fig. 5C). Based on the strong colocalization of Zn and P (Fig. 5, A-C), we inferred that Zn is likely chelated by P in the procambial cells. Further, the presence of Zn and P as "hotspots" suggested that P may be present in organic forms during seasonal storage.
We performed XANES analysis on powdered frozenhydrated samples to acquire overall information about speciation of Zn in both dormant and vegetative buds during budbreak. Figure 5D shows the Zn K-edge, K3weighted XANES spectra for bud tissues at the dormant and bud flush stages, and the spectra of eight model compounds. Linear-combination fitting revealed that Zn was present as Zn-phytate, Zn-nicotinamide, Zn-His, and Zn-polygalacturonic acid (Fig. 5D). During dormancy, Zn was present almost exclusively Figure 3. Zn concentration in leaves, internodes, and nodes in an apple tree. Samples were collected from the upper, middle, and lower sections of apple tree stems. Data are means 6 SE of five biological replicates. Lowercase letters indicate significant differences in Zn concentration among the leaf, internode, and node at P , 0.05. DW, Dry weight. Fluorescence intensity values of Zn were normalized such that the brightest spots correspond to the highest concentrations. Images were digitally extracted and made into a composite for comparison. BS, Bud scales; LP, leaf primordium; Pi, pith.
We further analyzed the concentration of different P fractions in buds/leaves during budbreak (Fig. 6). We selected four different budbreak stages (dormant bud, Figure 5. Distribution and speciation of Zn in a bud on an apple tree. A, Nano-XRF images of the incipient procambial region within a terminal dormant apple bud showing Zn accumulation within the cells around incipient procambium, with P localized in the same cells. Transverse sections were cut from the shoot apex (inset). B, Nano-XRF images of the vascular tissues within a terminal dormant apple bud showing strong localization of Zn in the procambial region and colocalization of P with Zn; transverse sections were cut at the bottom of a dormant bud (inset). C, The corresponding localization of Zn and P in a dormant bud and the correlation between XRF intensity of Zn versus P. Pixel brightness is displayed in RGB, such that the brightest spots correspond to the highest contents for the element depicted. D, Zn K-edge XANES recorded for Zn model compounds and apple buds at dormant and bud flush stages (solid lines), and the corresponding linear combination fits (dotted lines). E, Proportion (percent mole fraction) of Zn species in the samples. Xy, Xylem; Ph, phloem; Pr, procambium. swollen bud, bud flush, and new emerged leaves) for the measurement of total P (P tot ), inorganic phosphate (P i ), and organic phosphate (P org ). P tot was significantly enhanced after bud flush (Fig. 6A). P i was very low in dormant buds and increased significantly toward bud burst, with its maximum concentration during bud flush (Fig. 6B). In contrast, the concentration of P org in buds peaked in the dormant season and gradually declined after bud dormancy break (Fig. 6C). We also determined the variation in Zn concentration during budbreak. Zn level was relatively low during dormancy, increased significantly after bud flush in the early spring, and reached its highest concentration in the newly developing apple leaves (Fig. 6D).

Movement of Zn during Bud Development
To further explore the relationship between Zn and budbreak, we determined dynamic changes in stored Zn in terminal buds at different budbreak stages during winter and early spring (Fig. 7). We selected five budbreak stages (from dormant to total bud opening) for investigation (Fig. 7, A and B). XRF analysis showed a rapid budbreak-associated movement of Zn during bud development (Fig. 7C). At dormant stage, Zn preferentially distributed in the leaf primordium and, to a lesser extent, in two very narrow bands of the section that corresponds to the procambial strands (Fig. 7, dormant bud). During the initial stage of budbreak, the Zn signal decreased slightly around the leaf primordium, likely due to dilution by the growth and expansion of leaf primordia (Fig. 7, initiated development). During bud swelling, Zn localized in both leaf primordia and at a high concentration within procambial strands. These changes in localization may depend on the formation of functional vascular connections between bud and stem and the delivery of Zn via phloem tissue (Fig. 7, swollen bud and green tip). Following leaf emergence from the bud, Zn was most abundant in the vascular tissues subtending the emerged leaf (Fig. 7, bud flush). Preferential distribution of Zn in the vascular tissue was increasingly obvious after bud swell and especially pronounced at the bud flush stage (i.e. extended leaves visible), at which time the Zn signal in the vascular connection was ;2to  5-fold higher than in the same zone in buds at other developmental stages (Fig. 7D, L1 and L2). Conversely, the Zn signal in the leaf primordium peaked during the dormant stage and then remained steady after budbreak (Fig. 7D, L1 and L2).

Buds Are a Valuable Seasonal Zn-Storage Site
Here, we found that terminal buds and axillary buds are vital Zn storage sites in apple trees. To determine whether this is a common phenomenon among deciduous fruit species, we determined the Zn distribution patterns in dormant terminal buds collected from four other different deciduous fruit trees that represent most of the high-value fruit types (pome, drupe, berry, and nut; Fig. 8). The results confirmed that buds are highdensity sites for mineral accumulation. Consistent with our observations in apple, leaf primordium and procambium within buds were preferential storage sites for Zn during dormancy in all four fruit species.

Zn Preferentially Distributes in Stem Nodes
The leaf-stem node is a crucial junction for the transfer of mineral nutrients to developing and dormant buds. A significant increase in Zn concentration was observed in nodal parts, specifically in the vascular tissues of stem nodes, indicating that nodes may act as a hub to reduce Zn flow for its preferential distribution to developing tissues and reproductive organs (Figs. 1-3). Additionally, we observed a node-based switch for the redistribution of Zn between xylem and phloem tissues (Fig. 1B). The transfer of Zn from the phloem in the lower stem to the xylem in the upper stem suggests that Zn is readily remobilized in apple trees and that some of the Zn in the older leaves can be remobilized to the new growing tissues. This finding is consistent with previous observations that suggest a high efficiency of the Zn phloem-transport system in some woody species (Tian et al., 2014;Saa et al., 2018;Xie et al., 2019). The potential intervascular transfer of Zn may also prolong the retention time of Zn, resulting in the enhanced Zn concentration observed in the node vascular tissues. This preferential distribution of Zn in the nodal parts (Figs. 2 and 3) is likely essential for the effective delivery of Zn to sink organs with high meristematic activity at times of limited vascular activity. The critical role of stem and reproductive nodes in the allocation of nutrients to reproductive structures has been previously described in rice (Oryza sativa), for which the nodes accumulate Zn to concentrations 10 times higher, or more than those in other tissues during both vegetative and reproductive stages. Various transporters localized at different cells in the vascular bundles also play important roles in the preferential distribution of nutrients in rice nodes (Yamaji et al., 2013;Ma, 2014, 2017).

Zn Is Likely Sequestered as Phytate in Buds
We applied HPF followed by freeze substitution for sample preparation, as previous studies have shown that this sample preparation protocol can preserve the in vivo localization of mobile elements (Smart et al., 2010;Moore et al., 2011). Here, we observed that Zn strongly colocalized with P inside procambium cells (Fig. 5, A-C). Most stored P in plants exists as P org (e.g. RNA, DNA, ATP, and membrane phospholipids), and ribosomal RNAs account for the largest P org pool (Ticconi and Abel, 2004;Péret et   For each type, a microscopic image is shown (left) with its corresponding color-merge image indicating the relative location of Zn and other elements. Pixel brightness is displayed in red, green, and blue such that the brightest spots correspond to the highest content of the element depicted. LP, Leaf primordium; IP, inflorescence primordium; Pr, procambium; Pi, pith. acid (InsP6) accounts for a very small fraction of total P org , it reportedly acts as the major phosphate storage compound in plant seeds and twigs (Brinch-Pedersen et al., 2002;Xue et al., 2007;Hu and Chu, 2017) and shows very high affinity for heavy metals in plants (Sarret et al., 2003;Bohn et al., 2008;Regvar et al., 2011;Kyriacou et al., 2014). Therefore, we hypothesized that Zn may exist as a phytate salt in primary procambium cells in the buds. Our study of the ligand environment of Zn at different budbreak stages confirmed this hypothesis (Fig. 5, D and E). Furthermore, the highest concentration of P org observed in buds during dormancy suggested a seasonal storage of P in an organic compound in bud tissues. The increased concentration of P i , combined with the gradual decline of P org after budbreak, further indicated a transfer from P org to P i during budbreak (Fig. 6). This finding provides further evidence that Zn may be sequestered as Zn-phytate during bud dormancy and that this Zn-phytate is decationized and hydrolyzed under growth-promoting conditions. While many studies have revealed the chelation of InsP6 with Zn in seeds (Lönnerdal, 2000;Urbano et al., 2000;Jiang et al., 2001;Ficco et al., 2009), the presence of Zn-phytate has not been previously reported in buds of deciduous trees. The occurrence of phytic acid in leaves is considered negligible, which makes it difficult to analyze its role as a Zn chelator in vegetative tissues. Although most plant cells, including those in vegetative tissues, can synthesize InsP6 (Raboy, 2003(Raboy, , 2009Hadi Alkarawi and Zotz, 2014;Kurita et al., 2017), information about the presence and storage of InsP6 within plant leaves and vegetative buds is still limited. The formation of Zn phytate in leaves was previously reported only in some hyperaccumulator species or in circumstances in which plants were exposed to conditions of high exogenous Zn. The binding of Zn to phytic acid reportedly helps plants in limiting Zn mobility and reducing toxicity (Neumann and zur Nieden, 2001;Sarret et al., 2003;Dinh et al., 2015;Gupta et al., 2016;Doolette et al., 2018). In poplar (Populus alba), InsP6 is abundant in overwintering twigs, where it is essential for long-term P storage in bark storage proteins together with various cations (calcium, magnesium, zinc, sodium, potassium, and lead); InsP6 rapidly decreased in early spring (Kurita et al., 2017). The decrease in speciation of Zn-phytate observed here (Fig. 5,D and E) suggests that this mechanism may be common for Zn reserves in apple buds during dormancy and that the accumulation of InsP6 at the very early stages of terminal bud formation may play an important role in seasonal Zn storage. At budbreak, InsP6 is likely decationized and hydrolyzed by phytases, followed by the release of P i , inositol, and Zn.
After bud flush, a substantial proportion of Zn (;48.7%) was contained in polygalacturonic acid, indicating that most of the Zn in newly emerged leaves is retained in the cell walls. Polygalacturonic acid is a major component of pectin (pectic polysaccharides), which is most abundant in plant primary cell walls and the middle lamella (Caffall and Mohnen, 2009). Various studies have shown that this major component of pectin in cell walls has a high binding capacity for Zn 21 (Khotimchenko et al., 2008) and thus limits the translocation of positively charged Zn 21 (Fernández and Brown, 2013;Doolette et al., 2018). Our own results indicated an increase of Zn species as Zn-nicotianamine after bud flush, suggesting a possible positive role for nicotianamine-mediated Zn remobilization via the phloem tissues in apple trees. This observation is consistent with results of previous studies of a functional role for nicotianamine in long-distance transport of Zn in plants (Curie et al., 2009;Sinclair and Krämer, 2012;Xie et al., 2019).

Zn Is Efficiently Released during Budbreak
Our results demonstrate that leaf primordium and procambium within buds are sites for high-density Zn storage. Both leaf primordium and procambium show very high levels of meristematic activity. The high level of Zn allocation in these tissues is consistent with the high Zn requirement of highly metabolically active differentiating cells, whereby it is more efficient for plants to store mineral elements needed directly at the growing point than to use energy for moving these elements from other storage locations when needed. The preferential distribution of Zn in the meristematic and vascular tissues has been observed in other plant tissues, such as in the plumule and radicle of germinating rice seeds (Wang et al., 2011;Lu et al., 2013a).
The dynamic spatial and temporal distribution of Zn described herein reflect the patterns of metabolism and development that would occur during bud germination Figure 9. Conceptual model of Zn storage and utilization in deciduous fruit tree species. A, During active growth, Zn inflow from the roots is allocated to the stem nodes via phloem vessels, where it is available for developing buds. B, During bud development and dormancy, Zn is sequestered as Zn-phytate, thus remaining as an overwintering Zn storage pool. C, When growth-promoting conditions are restored in early spring, Zn-phytate is decationized and hydrolyzed concomitantly with the initiation of budbreak. The growth-related function of Zn further mediates the development of new vascular system connections, thereby ultimately promoting efficient nutrient flow into the bud to support the continuation of growth. Xy, Xylem; Ph, phloem; LP, leaf primordium; Pr, procambium. and subsequent organ development. During bud development, Zn may be stored as Zn-phytate, which is then metabolized to provide Zn to the developing organs. Following the breaking of dormancy, new vascular connections form, and enhanced Zn delivery occurs (Fig. 7C). Some Zn derives from the relocation of Zn from the stem nodes, as seasonal storage of nutrients occurs in various perennial plant species. Woody plants must maintain an adequate storage pool for initial growth in the following spring, and nutrients are usually delivered from annual to perennial organs, such as stems or higher-order fine roots, to storage organs before dormancy (Estiarte and Peñuelas, 2015;Zadworny et al., 2015;Netzer et al., 2017). Secondly, Zn may come via direct root absorption.
Overall, our data confirmed that a large amount of Zn is required at budbreak for specific growth-related functions, such as biosynthesis of protein and chlorophyll, enzyme activation (Coleman, 1998) and maintenance of membrane structure and functionality (Broadley et al., 2007;Palmgren et al., 2008). Further, Zn is required for the synthesis of auxin through its role in the biosynthesis of Trp (the amino acid precursor of auxin synthesis; Li et al., 2013). Recently, cross-talk between nutrients and auxin is receiving increasing attention. Some evidence seems to suggest that Zndeficient plants show decreased auxin production, while other studies have shown that auxin signaling is closely associated with plant P status (Kobayashi et al., 2006;Rai et al., 2015;Begum et al., 2016;Sofo et al., 2017). Ferguson and Beveridge (2009) proposed that auxin is not in itself the internal cause of budbreak, but that auxin triggers budbreak by inducing the differentiation of the vascular system, thereby directing an adequate nutrient supply to the bud. The same authors found that the extent of sustained growth post budbreak is limited by the ability of buds to continually attract nutrients (Ferguson and Beveridge, 2009). Other reports suggest that nutrients stimulate bud outgrowth by inducing the biosynthesis of hormones involved in the process (Umehara et al., 2010;Zhu and Kranz, 2012). In our study, spatial and temporal imaging of Zn demonstrated that budbreak precedes the development of new vascular connections (as indicated by new Zn flow into the buds) and that a highly efficient remobilization of Zn occurred during budbreak, which was accompanied by the differentiation of procambium into new functional vascular tissues. This is in agreement with the viewpoint that bud outgrowth, but not budbreak per se, is associated with the development of vascular connections to deliver nutrients where sink demand is high (Husain and Linck, 1966;Ferguson and Beveridge, 2009). The findings described above warrant re-evaluation of the stimulating function of hormones in budbreak and plant development. Here, we propose that the presence of Zn in stem nodes is critical to provide Zn to developing buds and that this Zn supply is essential for budbreak and to support initial growth and development, which subsequently enhances vascular connections and nutrient flow to continue the nourishment of new developing organs.
This study reveals a likely universal adaptation strategy in deciduous perennials to store and regulate Zn flow during the seasonal cycles of growth and dormancy. As illustrated in Figure 9, during the active growth period, Zn flow is preferentially allocated to the stem nodes, which act as a hub to effectively redirect Zn to the developing buds to supply Zn for subsequent budbreak and development of new plant organs early in the following spring (Fig. 9A). Our data also indicate that Zn in the buds may be sequestered in the form of Zn phytate, a highly stable Zn storage pool. Subsequently, Zn phytate is used for budbreak and initial growth in the subsequent spring (Fig. 9B). During budbreak, Zn phytate is decationized and hydrolyzed, thus releasing Zn to trigger the initiation of budbreak and subsequent meristematic growth (Fig. 9C).

Plant Material
Experiments were conducted on 5-year-old healthy apple trees (Malus domestica 'Golden Delicious') planted in a greenhouse at Zhejiang University (latitude 36°289 N; longitude 120°159 E). Each grafted tree was potted in a 20-L container filled with a mixture of soil:perlite (1:1) and watered with Hoagland nutrient solution as needed. Trees were grown under natural light supplemented with lamps (16 h/8 h photoperiod at 350 to 500 mmol m 22 s 21 ), under a 26°C/20°C day/night temperature regime and relative humidity 50% to 70%.
During the active growth stages, stem-petiole connections (nodes) were harvested to analyze Zn distribution patterns in stems at different developmental stages. At the end of winter, completely ecodormant apical buds were collected to investigate Zn seasonal storage status. To further study the dynamic nutrient status during budbreak, vegetative buds at different budbreak stages were selected for sampling, based on the previous determination of five stages of budbreak by macroscopic observation of bud morphology: dormant bud, initiated development, swollen bud (inner leaf scales visible), green tip, and bud flush (extended leaves visible). Four additional types of deciduous fruit trees, peach (Amygdalus persica), grape (Vitis vinifera), pistachio (Pistacia vera), and blueberry (Vaccinium spp.), were also sampled to investigate Zn distribution patterns in dormant terminal buds to determine whether the patterns observed in apple buds are common across deciduous fruit-tree species.

Mineral Element Mapping by m-XRF and Nano-XRF
Samples for m-XRF analysis were cut at a thickness of 180 mm using a cryotome (CM1950, Leica Biosystems) at 220°C (Tian et al., 2010); m-XRF imaging was performed at the Stanford Synchrotron Radiation Laboratory using Beam Lines 2 and 3. Experiments were recorded at 13,500 eV, using a 20 3 20-mm beam spot size, 20 3 20-mm pixel size, and 100 ms dwell time per pixel. Fluorescence signal intensities for Zn, K, and Ca were calculated using SMAK software (http://www.ssrl.slac.stanford.edu/;swebb/smak.htm). As it is rather difficult to obtain reliable spatial images of P in plant samples under the experimental conditions used here, P mapping was further addressed by using the Nano-XRF technique as described below. The Micro-XRF P-imaging results are shown in Supplemental Figure S1. The integrated intensities of Zn and other elements were calculated from XRF spectra and normalized by the intensity of the Compton scattering peak. Element mapping for the area measured was obtained from the normalized intensity for each element. Quantification of fluorescence yield counts was normalized by I0 and dwell time. Then, the normalized XRF intensities were scaled to different color brightness for individual elements, such that the brightest spots corresponded to the highest element fluorescence. The scale used for fluorescence counts for individual elements was the same for each map. Samples for Nano-XRF mapping were cut at different parts of the terminal bud; a preparation procedure involving HPF, followed by freeze substitution (FS) with acetone was used to keep the ultrastructure of samples and the in vivo spatial distribution of elements as intact as possible during sample processing and analysis. Nano-XRF was performed at a helium atmosphere on the Advanced Photon Source 2-ID-D and 2-ID-E hard x-ray microprobe beamline (Cai Z et al., 2003). Incident x-rays of 10 keV were chosen to excite elements from K to Zn. A Fresnel zone plate focused the x-ray beam to a spot size of 0.2 3 0.2 mm and 0.1 3 0.1 mm on the sample, which was raster-scanned at 1-mm step increments in the sample image, and 20 ms per pixel dwell time. Sample x-ray fluorescence was captured with an energy dispersive silicon drift detector. The resulting element maps were visualized and analyzed using MAPS (Vogt, 2003).

Speciation of Zn measured by K-edge XANES
Fresh, dormant and bursting apple buds were randomly sampled and ground into a powder for XANES analysis. Samples were prepared according to the method of Tian et al. (2011a). The methods used to prepare standard Zn compounds, including ZnSO 4 , Zn-citrate, Zn-His, Zn-nicotianamine, Zn-cell wall, Zn-polygalacturonic acid, and Zn-phytate, were based on previous studies (Kopittke et al., 2011;Lu et al., 2013b;Doolette et al., 2018). XANES data were collected at the SSRL with the storage ring SPEAR-3 operating at 3 GeV and with ring currents of 80-100 mA. x-ray absorption spectroscopy of bulk tissues was carried out on SSRL beam lines 7-3 using the same equipment conditions described previously (Tian et al., 2010). Multiple scans (8-16, depending on Zn concentration) were collected and averaged for each sample to improve the signal-to-noise ratio. Principal component analysis and target transform were carried out using the SIXpack program. XANES data analysis was carried out using the Athena program suite according to standard methods (https://bruceravel.github.io/demeter/documents/Athena/index.html). Spectra analysis was performed using linear combination fit analysis, which was carried out by calculating all possible component fits based on all reference spectra selected from principal component analysis and target transform. The best component fit was judged by the R-factor [sum(data-fit)2/sum(data2)]. Best fits were derived by stepwise increase of the number of fit components and further optimized by minimizing fit residuals, which were defined as the normalized root-square difference between the data and the fit (Tian et al., 2011b).

Measurement of Total Zn
Leaves, internodes, and nodes were collected from the upper, middle, and lower sections of the experimental apple trees for Zn measurement. Samples were dried in an oven for 72 h until constant weight was reached, and dry weight was measured. The dried plant materials were ground, and 0.1-g samples were put into the glass digestion tubes. Then, 5.0 mL HNO 3 was added, and samples were left overnight. The solution was then heated at 180°C using the graphite digester, and 1-mL H 2 O 2 additions were made until the digestion was complete. Then the digested solution was transferred to a volumetric flask, brought up to 50 mL with deionized water, and filtered. The concentration of Zn in the filtrates was determined using inductively coupled plasma mass-spectroscopy (Agilent 7500a). The apple composition analysis certified reference materials (Aoke Biotechnology) was used as standard reference material.

Measurement of Different P Fractions
Samples were dried in an oven for 72 h until constant weight was reached, and dry weight was measured. The dry, ground plant samples (0.15 g) were put into glass digestion tubes and 5.0 mL of 98% (v/v) H 2 SO 4 was added. The solution was preheated at 180°C using the graphite digester and then digested at 280°C. Then 1 mL H 2 O 2 was added several times until digestion was complete. Concentration of total P was analyzed by the molybdenum blue method. The measurement of P i followed previously described methods (Zhou et al., 2008). The apple composition analysis software CRM (Aoke Biotechnology) was used as standard reference material. P org was then calculated by subtracting the P i values from the P tot values.

Statistical Analysis
All data were statistically analyzed using the SPSS package (version 11.0). ANOVA was performed on the data sets. The mean and SE of each treatment were calculated, and Fisher's LSD test was used to determine significant differences (P , 0.05 for significant and P , 0.01 for highly significant) for each set of data.

Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Distribution patterns of P in apple buds during different bud break stages.