Plants were harvested before full maturity for analysis after growth periods of 2, 4, 6, and 8 weeks

For culture on agar plates, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water three times, and sown on the Murashige and Skoog medium containing 2% sucrose and 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically in the growth chamber at 22 ◦C under 12 h light/12 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agar-solidified MS media supplemented with Na+ at the indicated concentrations and were grown at 22 ◦C under 12 h light/12 h dark photoperiod. For hydroponic culture, after germination and being grown on the MS plate for ten days, the seedlings were transferred to 1/6-strength MS liquid solutions and were grown under the 12 h light/12 h dark photoperiod in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with a range of NaCl concentrations and were grown under 12 h light/12 h dark photoperiod. We show a representative A. mangium root under hydroponic condition in Figure 1a. A cap-like structure, which was apparently consisted of the plant tissues, covered the entire root apex up to the 5 mm region behind the tip. After the structure was readily sloughed off , the root was covered again with a new one of similar size in a week after the detachment . To evaluate roles of the structure on root growth,stacking pots supplier the whole structure and resulting tissues on root surface were carefully removed with forceps, and then the roots were exposed to 0.5 mM calcium solution containing 0, 100, or 500 μM Al for 48 h.

Al induced a root bending in both roots immediately after the exposure, and the proportion in the occurrence of root bending was higher in Al-treated roots without the cap-like structure . The addition of Al slightly reduced the elongation of roots both with and without a cap-like structure, and the degree in the reduction was larger in structure-less roots . In the absence of Al, the removal of a cap-like structure hardly decreased the root elongation, although it slightly increased the occurrence of root bending . In roots without cap-like structures, this facilitation of root bending may result in a sight decrease in the Al-resistant root elongation. The present study revealed that the presence of a cap-like structure around root apices in A. mangium ameliorates an Al-facilitated bending of root growth direction. Root caps play major roles in root gravitropism through the localization of amyloplast granules and/or the basipetal flow of auxin . Our further analysis of the cap-like structure identified that a major attached position of a structure to the root was confined to columella root cap region, and the rigidity between this connection was enhanced during prolonged Al exposure periods . The undetached state of a cap-like structure may have a role in protecting inner root cap cell function from instant Al-caused damages. An apparent root bending induced by Al seems to require high concentrations, since no similar root bending in Al-sensitive plants has been reported. It is likely that a much lower concentration of Al may be sufficient for the complete arrest of root elongation until root cap cell dysfunction become recognizable as a root bending.

The present study did not find any detrimental role of a floating, cap-like structure around the root in Al-resistant root elongation, similar to previous findings on root caps . In the present study, however, we did not evaluate potential roles of undetached tissues on Al tolerance mechanisms. A cycle of detachment and formation of caplike structures imply that non-detached original tissues in the root surface area of root apex may play roles in the Al-resistant root elongation in A. mangium. Another study of A. mangium revealed that more than half of fibrillary tissue initials separated from the root surface were present beyond the entire root elongation region . In summary, we have found a novel cap-like structure of tissues for the protection of root cap cells at the root apex of A. mangium. Further characterization of the detachment pattern of the tissues from the root surface should provide better understanding of their roles in high Al tolerance mechanisms. Potassium and calcium are key elements for plant physiology, as evidenced by being the highest concentration cations in plants and, together with N and P, among the most crucial elements for plant growth and productivity. K is centrally involved in a wide range of crucial biophysical and biochemical processes in plants, including cell osmotic pressure regulation, growth, regulation of photosynthesis, ion homeostasis, control of cell membrane polarization, enzyme conformation/activation, water conservation and salt resilience. Ca has its own crucial roles including as a significant component in cellular walls, the maintenance of membrane integrity against the passive transfer of H+ , K+ , Na+ , and other monovalent cations, and biochemical signaling. Due to these various essential roles, much research has focused on the mechanisms of K and Ca uptake by and transport within plants. For Ca and Mg, another key nutrient element, isotopic fractionation by plants has been investigated both experimentally in the lab to elucidate processes of acquisition and transport, but also in the field ) as monitors of the local and global biogeochemical cycling of these elements.

The isotopic fractionation by plants of micro-nutrients such as Fe, Cu, Zn, and Mo have also been measured. In the case of K, it has been suggested that terrestrial plants represent a significant reservoir for K, with as much as 40– 70% or more of the dissolved K in the world’s rivers coming from the decay of plant matter, resulting from its crucial role as a plant nutrient. Therefore, potassium isotopic systematics potentially provides a new tool for tracking and quantifying nutrient cycling in ecological systems ; a proxy for global geochemical cycling; and a research avenue for understanding the optimal fertilization for efficient agricultural food production. To fulfill that potential, the degree of K isotopic fractionation by plants needs to be established. Two published studies do suggest that plants may fractionate K isotopes. Deviations in the 41K/39K ratio of ∼1‰ from an in-house reference material favoring the light isotope have been measured by Li et al. in several plant materials , but with no measurement of the K sources. Likewise, in their broad survey of natural geologic and organic materials Morgan et al. found that a banana and a potato purchased in a Scottish grocery had 41K/39K ∼ 0.4‰ lighter and ∼0.1 ‰ heavier respectively than seawater K, but again with no measurements of the K sources. Here we use hydroponically grown plants, with isotopically characterized K and Ca nutrient sources, to quantify and compare the isotopic fractionation of K and Ca by three species of vascular plants: soybean , rice , and wheat . These three plants were chosen to cover a selection of important food crops; and each are C3 plants. We choose to compare K and Ca isotopic fractionation for several reasons. First, the atomic weights of K and Ca are relatively close so that mass fractionations can be easily compared. Second, K and Ca have contrasting roles in plant physiology as described above, and have contrasting valencies. Third, there is a body of experimental data on Ca isotopic fractionation by plants that we can compare our Ca isotopic results to . Methods Soybean, rice, and wheat were grown from seeds and cultivated in a large hydroponic system at the U.C. Berkeley Oxford Facility greenhouse. The hydroponic solution, a modified Hoagland’s solution replete with Ca nitrate and K nitrate was replenished periodically to provide an isotopically constant source of Ca and K during plant growth.Harvested plants were divided broadly for soybeans into root, stem,grow lights supplier and leaf samples, and for rice and wheat plants into samples of roots and leaves. The plant samples were rinsed, dried, and weighed, and then ashed before complete dissolution in high-purity nitric acid. Aliquots were taken of the sample solutions for K and Ca isotopic analyses. K was separated from the sample aliquots using AG50×8 cation resin and 1 M HNO3. The K isotopic analyses were conducted on an IsoProbe MC-ICPMS at Lawrence Berkeley National Lab using a sample-standard bracketing technique where Ar-based mass interferences were removed by the introduction of Ne + H2 gas to the hexapole collision cell. Results are reported as per mil deviations of the 41K/39K ratio relative to an inhouse K standard using a spectroscopic concentration standard . We estimate that our in-house standard on the Bulk Silicate Earth scale of Wang and Jacobsen has an approximate δ41KBSE of +0.5‰. For Ca isotopic analysis, the sample aliquots were spiked with a 42Ca–48Ca double spike before chemical separation using Eichrom DGA resin eluted with 3 N HNO3 and separated Ca collected with DI H2O. The spiked Ca separates were then analyzed for isotopic composition using thermal ionization mass spectrometry with a Triton multicollector instrument using a multi-dynamic Faraday cup routine. The Ca isotopic results are reported as per mil deviations of the 44Ca/40Ca ratio from the Bulk Silicate Earth 44Ca/40Ca of 0.0212035. On this scale our long-term average δ44Ca of SRM915A is −1.0 ± 0.1 2s. Further details of the K and Ca separation and isotopic analyses are presented in the Supporting Information.

Another possible effect to consider with transport across cellular membranes is that ions would need to be desolvated before their passage. Hoffman et al. suggest through molecular dynamics simulations, that water exchange rates for solvated ions has a rate dependence on the mass of the solvated ion such that the lighter isotope is favored in desolvation, resulting in a predicted ∼2‰ fractionation in 41K/39K between a precipitating solid and the solution. However, a further feature of K+ specific channels lies in the process of desolvation/solvation of K+ during transmembrane transport. Solvated K + enters the channel where the K+ is handed off to eight protein bonded oxygen atoms replacing the role of the solvating H2O molecules, minimizing the energy required for desolvation, with resolvation occurring with the exit of K+ from the channel.Therefore, we suggest that isotopic fractionation due to dehydration of K+ accompanying K+channel transport may be minimal. Instead we hypothesize that their size selectivity as argued above is the main source of fractionation of K isotopes. We propose that the HATS, operative under low external K+ , would fractionate K isotopes less that the LATS, which in large part depends on highly selective K+ ion channels. This suggestion is supported by experiments involving cells of a marine diatom where under low external Zn concentration , the observed isotopic fractionation of Zn is four times less than under high Zn external concentration . A difference between HA vs LA transport has also been called upon by Deng et al. to explain differences in Zn isotopic fractionation between Zn nonaccumulating and Zn hyperacumulating plants. For isotopic fractionation of K in plants, a test of this would be examining K isotopic fractionation as a function of external solution K+ concentration. Another possible test would be experiments involving genetically modified plants that are capable of only either HATS or LATS K+ transport to look for contrasts in K isotopic fractionation. Roots are not only vital for anchorage and for acquisition of water and nutrients from the soil, but are also engaged in complex physical and chemical interactions with the soil. Plant roots release approximately 11–40% of their photosynthetically fixed carbon, commonly known as root exudates, into the soil . Root exudates and mucilage act as nutrient sources and as signaling molecules for soil microorganisms, thus shaping the microbial community in the immediate vicinity of the root system . In turn, microbial processes promote plant growth by aiding in nutrient acquisition, plant growth hormone production and bio-control of plant pathogens . The physicochemical characteristics of the surrounding soil are also affected by interactions between roots and the microbial community. This interplay between the different rhizosphere components is affected by spatio-temporal processes, which culminates in dynamic feedback loops that maintain the complex rhizosphere environment with physical, chemical and biological gradients that are distinct from the bulk soil .