In spite of extensive transport and spectroscopic studies on its exotic properties, the evidence of superconductivity in its surface states remains elusive. Here, we report the observation of proximity-induced surface superconductivity in Nb/Cd3As2 hybrid structures. Our four-terminal transport measurement identifies a pronounced proximity-induced pairing gap (gap size comparable to Nb) on the surfaces, which exhibits a flat conductance plateau in differential conductance spectra, consistent with our theoretical simulations. The surface supercurrent from Nb/Cd3As2/Nb junctions is also achieved with a Fraunhofer/SQUID-like pattern under out-of-plane/in-plane magnetic fields, respectively. The resultant mapping shows a predominant distribution on the top and bottom surfaces as the bulk carriers are depleted, which can be regarded as a higher dimensional analog of edge supercurrent in two-dimensional quantum spin Hall insulators. Our study provides the evidence of surface superconductivity in Dirac semimetals.Cd 3As2.Surface superconductivity in a Dirac semimetal remains rarely studied. Here, Huang and Zhou et al. report the evidence of proximity-induced surface superconductivity in a superconductor/Dirac semimetal hybrid system Nb/Cd Among the rich class of topological phases, topological Weyl and Dirac semimetals, which are characterized by discrete gapless nodes in the bulk spectra, have attracted wide attention5. Referred to as Weyl or Dirac points, these gapless nodes in the bulk can be connected by open strings formed by topologically protected surface states on the boundaries, called Fermi-arc states6. Due to their anomalous electromagnetic responses14, as well as the interesting interplay between bulk and surface Fermi arcs16, topological semimetals have been studied intensively in recent years5. In parallel to the rapid exploration of Dirac properties in topological semimetals, the study of their superconducting states has become an important topic aiming for possible unconventional superconductivity20. It was predicted that Fermi-arc states in a Dirac semimetal such as Cd3As2 could be used to realize Majorana fermions21. This is due to the fact that the Fermi-arc states in Cd3As2 stem from helical edge states of quantum spin Hall insulators embedded in the time-reversal invariant semimetal22. By proximitizing the Fermi-arc states with s-wave superconductors, Majorana flat band can be possibly created at the interface of a π-Josephson junction23. While the signature of superconductivity has been found in Cd3As226, the role of Fermi-arc states in these pairing phases remains unclear.The study of topological phases has been one of the central topics in condensed matter physics in the past decade21 that the Fermi arcs can acquire hard superconducting gaps from the parent superconductor. In topological insulator/superconductor (TI/SC) junctions, it is generally believed that the induced superconducting gap in the surface states is small due to high interfacial barriers and Fermi surface mismatch29. In a Dirac semimetal Cd3As2, the unavoidable coexistence of low-energy bulk excitations and Fermi-arc states5 causes further complications to distinguish the roles of bulk states and surface states in the superconducting state. Therefore, a prerequisite experimental evidence of strong superconducting pairing on the surface is clearly desirable. In particular, due to the highly non-uniform spatial distribution of surface states in Cd3As2, the superconducting proximity on the surface and bulk may exhibit distinctive anisotropic signatures.Despite the appealing proposals for creating Majorana fermions, the proposed scheme relies on the assumption3As2 based on two superconducting characteristics: the proximity-induced superconducting gap and the spatial distribution of supercurrents on surfaces. Using four-terminal transport measurements on Nb/Cd3As2 hybrid structures, we observe a pronounced proximity-induced gap (Δs) on the surfaces, with its size comparable to the parent superconducting gap (ΔNb) of Nb. The sizable surface proximity gap manifests itself as a flat conductance plateau in differential conductance (dI/dV) spectra. In contrast, the proximity gap in bulk states is a few times smaller (Δb ~ 0.14Δs) and featured by a zero-bias broad peak (ZBBP) in the dI/dV spectra. The relation between the observed conductance plateau/ZBBP and proximity-induced gaps in surface/bulk states are further confirmed by our theoretical calculations with a qualitatively good agreement. Upon changing surface-bulk contributions in Cd3As2 samples with different thicknesses, the conductance plateau/ZBBP exhibit evolutionary behaviors consistent with their surface/bulk origins. Furthermore, using superconducting quantum interference (SQI) measurements on Nb/Cd3As2/Nb Josephson junctions (JJs), we observe a SQUID pattern in the surface-state-dominated Cd3As2 JJs, which indicates that the supercurrent density is predominantly on top and bottom surfaces.Here, we report the observation of strong proximity-induced superconductivity on the surfaces of Cd3As2 hybrid structure is displayed in Fig. 3As2 nanoplate (blue color). A four-terminal measurement across the interface was performed to detect the interface resistance, denoted as RInterface (see Supplementary Note The proximity-induced superconductivity in Nb/CdR–T) curves at T ≥ 2.0 K across the junction are shown in Fig. Tc1 = 8.4 K, Tc2 ~ 8.2 K, and Tc3 ~ 4.0 K corresponding to Nb superconducting for Tc1 and proximity-induced superconductivity for Tc2 and Tc3, respectively, since Nb has been in zero-resistance state at 8.3 K  with a ZBBP around ±0.19 mV (Δb) and an above-gap dip at high bias (Δd). For temperatures lower than 4 K, a very sharp zero-bias conductance peak (ZBCP) emerges which is superimposed on the broad peak  and third transition (Tc3) in the R–T curve while the first transition (Tc1) mostly corresponds to the conductance plateau with a small resistance drop by the above-gap dip in Fig. 30 in Fig. 3As2. The BTK fittings yield important parameters of Δb = 0.19 meV and Z = 0 , in which an incoming electron from the Dirac semimetal is converted to a reflected hole at the Nb/Cd3As2 interface. Thus, the presence of both ZBBP and BICP for sub-gap voltage bias suggests two types of Andreev reflection channels in Cd3As2: one exists only within a narrow energy window given by the width of the broad peak, while the other has a much wider energy range measured by the width of the conductance plateau. Since the energy window for Andreev reflections is directly related to the interfacial coupling between the channels of Cd3As2 and Nb, the contrasting features of the broad peak and plateau also suggest very different proximity effects in two types of channels. By placing Cd3As2 next to the superconducting Nb, the superconducting proximity effect is expected to occur in both the bulk and the surface region, as shown in Fig. Next, we probe the relation between BICP, ZBBP and proximity-induced superconductivity. For voltage bias 3As2  domination. In contrast, thinner samples (# 03 and 04) exhibit low bulk domination. Based on the analysis of the SdH oscillations , and kF = 0.032 Å−1 in device #05 with negligible k0, thus the AR electron density in device #01 is much higher than that in device #05. Furthermore, scattering effects between the bulk and surface states can be significant due to disorders, and the surface states are no longer well-localized on the surfaces33 which reduce the effective coupling between the surface and Nb. Besides, for high Fermi levels, there could possibly exist a significant Fermi surface mismatch between the superconductor and the Cd3As2, which could further suppress the interfacial coupling.In the thick limit of Cd3As2. Two superconducting gaps against the relative amplitude of AS/AB are presented in Fig. 3As2, the transparent interface with enough electron states from surface channels ensures that the proximity effect arises. Hence, the bulk-state superconducting gap Δb drops fast while the surface superconducting gap Δs increases slightly with a larger surface domination. The penetration depth for each surface can be estimated with an assumption that the superconducting gap decreases following34We now analyze the proximity-induced superconducting gaps in CdξN is the superconducting coherence length in Cd3As2 . In contrast, Fermi-arc states with different energies are connected on the surface, thus the number of Fermi-arc channels on the junction interface is expected to depend weakly on the Fermi level. Therefore, tuning the chemical potential EF of Cd3As2 in our model allows us to theoretically investigate the roles of surface states and bulk states in the Andreev reflection processes.It is worth noting that due to the low-energy Dirac spectrum of CdEF = 0 meV), where the bulk density of states is very low and the Andreev reflections driven by bulk channels are negligible. This simulates the scenario in thin samples (devices #03 and #04) where the Fermi surface property is dominated by Fermi-arc states. As shown in Fig. EF = 70 meV from the Dirac points, the bulk density of states gets enhanced and its contribution to Andreev reflections cannot be ignored. This simulates the case of relative thick samples (devices #01 and #02) where the bulk states and surface Fermi arcs have comparable weights in transport. In this scenario, a ZBBP emerges on top of the plateau in the differential conductance spectrum , with twice of the gap size 2Δs corresponding to the width of the conductance plateau due to Fermi arcs, which is comparable to twice value of the parent superconducting gap. In contrast, the smaller induced gap is formed in states with lower density on the surface, and its size 2Δb matches the width of the zero-bias peak due to bulk states. This further confirms that the Fermi-arc states couple strongly to the superconductor, and thus acquire a sizable superconducting gap (~70% of parent gap Δ). In contrast, the states living in the bulk couple relatively weakly to the superconductor, which results in a smaller proximity gap. Therefore, the flat conductance plateau in the dI/dV curve indicates Fermi-arc superconductivity as a possible interpretation, while the zero-bias peak arises from relatively small proximity gap in the bulk states, which opens a narrower window near zero bias for Andreev reflections driven by bulk channels.To understand the physical mechanisms for the formation of zero-bias peak and flat plateau in the d3As2, we next build Nb/Cd3As2/Nb Josephson junctions to directly analyze the spatial distribution of the supercurrent. This is enabled by SQI measurements in different directions.Having established the proximity-induced surface superconductivity in Nb/Cd3As2 Josephson junctions with closely spaced superconducting Nb electrodes on the top surface. We choose 120-nm-thick Cd3As2 with 140-nm-thick Nb electrodes to study the surface dominated supercurrent. The inset of Fig. L = 500 nm and W = 7 μm, respectively. Figure R–T curve of the junction with two transitions Tc1 and Tc2 at zero magnetic field. Tc1 ~ 7 K originates from the Nb superconducting transition while Tc2(on) ~ 3 K comes from the superconducting proximity effect. The resistance continues to decrease as the junction cools down and reaches the zero-resistance state below Tc2(off) ~ 1 K. The tail of the resistance drop can be explained in terms of the BKT transition36 as shown in green line by Halperin–Nelson equation37Figure R0 and b are material parameters, which realizes a zero-ohmic-resistance state driven by the binding of vortex-antivortex pairs at the BKT transition temperature TBKT = 1.0 K. On the other hand, junction resistance decreases at the temperature above Tc2, leading to a broadened SC onset, which can be well reproduced by the Aslamazov–Larkin37 fit in red dashed line for the two-dimensional (2D) fluctuation conductivity. These behaviors are consistent with expectations for 2D superconductivity as an evidence for the 2D surface superconductivity in Cd3As2 JJ since the thickness of Cd3As2 is far away from the 2D condition. Figure I−V) characteristics and differential resistance (dV/dI) of the junction measured at 50 mK, respectively. From the slope of the I−V curve, the normal-state resistance Rn ~ 17 Ω is extracted. In the regime |I| < 1 μA, the voltage across the junction and the dV/dI are zero, indicating a robust Josephson effect. The IcRn product gives a characteristic voltage of ~17 μV which is lower than the transition temperature of 1 K (with superconducting gap Δ = 1.76kBTc ~ 150 μV). It indicates that the junction is in the long junction limit38, where the superconducting coherence length ξN is smaller than the effective width between two Nb electrodes, i.e., ξN < Leff  as shown, respectively, in Fig. B applied along the y direction, the supercurrent spreads across the junction , which accords well with the SQUID pattern in Fig. zs are extracted to be 13 and 14 nm, respectively, which is consistent with our experimental results on Andreev reflections across the Nb/Cd3As2 junctions. As control experiments, thick Cd3As2 is found to be hard to achieve Josephson effect while it performs multiple Andreev reflections (MARs) , for example, their anisotropic surface distribution and thickness-independent properties, show naturally consistent characteristic with the signatures of the observed surface superconductivity . Moreover, even eliminating bulk states in TI by chemical doping such as Bi1.5Sb0.5Te1.7Se1.3 system, the SQI result is different in Cd3As2 and TI because surface states cover all the surfaces of TI. Specifically, in TI, due to the uniform surface states in all surfaces, the supercurrent density is edge-dominated along all three directions which results in a mixed Fraunhofer and SQUID-like pattern rather than a pure SQUID pattern49. Nevertheless, the surface states are generally only on two side-surfaces along the y-axis of Cd3As2. This enables the observation of SQUID pattern upon magnetic fields applied along the principal z-axis, which is spatially resolved as edge supercurrent along the y-direction as shown in Fig. We further contrast our result with previous reports on superconductivity in surface states of a usual 3D topological insulator (TI). Topologically nontrivial surface states also exist in TI and similar BICP due to surface states has been reported in Bi3As2 originate from edge states of 2D TI embedded in the Dirac semimetal. In particular, Fermi arcs labeled by momentum kz ∈  stem from the slice of 2D TI indexed by kz. Thus, proximitizing the surface of Cd3As2 is equivalent to inducing pairing in different slices of 2D TI edges as displayed in Fig. kz. In other words, the surface supercurrent observed in our work can be regarded as a higher dimensional analog of edge supercurrent in 2D quantum spin Hall insulators (QSHIs). We note that with certain perturbations that break the C4-cymmetry of Cd3As2, Dirac points located along the principal axis can be gapped out. In this case, the system becomes a strong topological insulator50; in particular, the kz = 0 plane is still characterized by a nontrivial Z2 invariant of 2D QSHIs50. Thus, this subtlety does not affect our conclusion that the observed surface superconductivity provides signatures of higher dimensional edge supercurrents in 2D QSHIs.As we introduced earlier, the Fermi-arc states in Cd3As2, one pair of Majorana bound states can be created at the junction interface of each slice of 2D TI23. This can result in a large number of non-dispersive Majorana fermions, called Majorana flat bands, that connects the separated Dirac points located at 20. The Majorana flat bands created by superconducting Fermi arcs can be signified by a characteristic sudden jump at π phase in the Josephson current–phase relation. Similar to the single Majorana bound state at a π-junction interface on quantum spin Hall edges23, the Majorana flat bands on the surface of Cd3As2 are protected by time-reversal symmetry and thus remain robust against non-magnetic disorder. While evidence of single Majorana bound states and chiral Majorana edge states has been spotted in recent experiments52, experimental realization of Majorana flat bands still remains unexplored. We believe that our experimental finding of strong proximity effect on surface may establish Cd3As2 to be a new promising platform for realizing Majorana flat bands.The supercurrent distribution in our surface-dominated provides the first direct evidence of surface superconductivity in topological semimetals. While the observations in the current work cannot rule out alternative physical origins other than Fermi arcs, our results call for future efforts to elucidate the possible Fermi-arc origin of the surface superconductivity. Prospectively, when a short linear Josephson junction with π phase difference is formed on the surface of Cd3As2 nanoplates were grown using Cd3As2 powders as the precursor in a horizontal tube furnace, in which argon was a carrier gas. Before the growth, the furnace was pumped and flushed with argon several times to remove water and oxygen. The temperature was ramped to the growth temperature within 15 min, held constantly for 20 min, and then was cooled down naturally over ~2 h in a constant flow of argon before the substrates were removed at room temperature. The precursor boat was placed in the hot center of the furnace (held at 500 °C), while the smooth quartz substrates were placed in the down-stream within a very small temperature range from 200 to 350 °C. The argon flow rate is 50 SCCM (standard cubic centimeters per minute). The smooth quartz substrates then appeared shining to the naked eyes. The largest crystal plane of as-grown Cd3As2 nanoplates is [112].The Cd3As2 hybrid structures were fabricated by electron beam lithography (EBL) technique and wet-etched by standard buffered HF solution for 5 s in the electrode regime. For Nb/Cd3As2 device, we first fabricated Cr/Au (10/150 nm) bilayers electrodes on Cd3As2 side using magneton sputtering. Then, we use standard EBL method to deposit the Nb layer. For Nb/Cd3As2/Nb Josephson junction, we use the EBL method and magnetic sputtering to deposit 140 nm-thick Nb electrodes.The Nb/CdI/dV) spectra were captured by ac-modulation technique. For the PPMS measurements, lock-in amplifier provides ac input in series with Agilent exporting dc voltage. Through a large resistor (0.05–1 MΩ) the input voltage converts to a constant current. Agilent 2912 and lock-in amplifiers with a low frequency (<50 Hz) were used for dI/dV spectra measurements. The R–T curves were measured by dI/dV spectra while the Agilent dc voltage is set to zero.Four-terminal temperature-dependent transport measurements were carried out in a Physical Property Measurement System (PPMS) system (Quantum Design) (1.9 K) and dilute refrigerator (down to 35 mK) using lock-in amplifier (SR830) and Agilent 2912. The differential conductance (dS1/2, 1/2〉, |P3/2, 3/2〉, |S1/2, −1/2〉, |P3/2, −3/2〉}, the momentum-space tight-binding Hamiltonian of the Dirac semimetal Cd3As2 used in the theoretical calculations of Andreev reflections is given by22:In the Bloch basis formed by {|a = 3 Å, c = 5 Å refer to the lattice constants within the ab-plane and along the c-axis, respectively. For simplicity, we neglect off-block diagonal parts which only account for higher order or bulk inversion asymmetric terms22. The parameters C0, C1, C2, M0, M1, M2, and A0 are set in units of eV with their values given in Supplementary Table EF is the Fermi level measured from the Dirac points in the Cd3As2. More details of the numerical Green’s function method for transport calculations in Fig. Here, Supplementary Information


Drosophila melanogaster. Drosophila studies examining the influence of yeast quality in the diet have generally either provided the diet over the whole life span (larva to adult) or during the adult stage and have rarely focussed on the juvenile diet. Here, we tested the effect of yeast quality in the larval diet on pre-adult development and adult weight, survival, reproduction and food preference. The yeast Saccharomyces cerevisiae was added in three forms in three treatments—live, heated or dried—to food used as the juvenile diet or was not added (empty treatment). Adults resulting from the larvae raised on these four juvenile diets were all maintained on a similar standard laboratory food diet. Our data indicate that yeast quality in the juvenile diet affects larva-to-pupa—but not pupa-to-adult—development. Importantly, adult survival, food preference, mating behaviour and cuticular pheromones strongly varied with the juvenile diet. Therefore, the variation of yeast quality in the pre-adult Drosophila diet affects key adult life traits involved in food search, reproduction and survival.Diet quality is critical for animal development and survival. Fungi can provide nutrients that are essential to organisms that are unable to synthetize them, such as ergosterol in  Unlike vertebrates, insects are auxotrophic for sterols, which requires them to obtain them from their diet. In particular, insects need to ingest yeasts and yeast-like fungi at both the larval and adult stages to correctly develop3. The ingestion of the yeast Saccharomyces cerevisiae throughout life was shown to affect various life traits in Drosophila melanogaster: a decreased amount of yeast in the larval diet delayed pupal eclosion and produced smaller adults4. Similarly, yeast restriction in the adult diet has been shown to decrease resistance to external stresses 6. Moreover, a poor-yeast diet throughout life was found to decrease fertility8 and increase life span9; these two effects are linked10. Many D. melanogaster studies have shown that the quality of the fungal diet, provided either throughout life or only at the adult age, can affect adult life traits. For example, adults fed with frozen or heat-killed yeast showed altered development, lower fertility, shorter life span and increased sensitivity to stress than did adults fed on a fresh yeast diet11. Furthermore these effects persisted even when a 600-fold increased amount of heat-killed yeast was added to the diet12. A strong yeast restriction in the larval diet was also shown to increase adult life span13.Animal survival and fitness depends on the quantity and quality of the diet. Food must be consumed in sufficient amounts and contain essential nutrients to avoid cellular dysfunction and exposure to pathologiesDrosophila species  are attracted to food sources containing yeast, and each species shows a marked preference for different yeast species14. While larval food preference can vary according to their earlier experience and the presence of maternally transmitted microbes16, the relationship between yeast in the larval diet and adult life traits has rarely been investigated13. Here, we measured the influence of pre-adult fungal diet quality on various adult life traits. We discovered that D. melanogaster larvae fed on different diets containing or lacking S. cerevisiae yeast in different forms showed differences in pre-adult development and adult life traits including survival, weight, reproduction, cuticular pheromones and food preference.Larvae and adults of different D. melanogaster adult traits . Larvae were raised on three diets containing S. cerevisiae yeast subjected to different treatments  or on a yeast-free diet (øY). After adult emergence, all of the flies were maintained on “Ind” food. Fly groups were named according to their juvenile diet .We evaluated the effects of yeast quality in the juvenile diet both on pre-imaginal development and on several Each food vial was initially seeded with 50 fertilized eggs. For each treatment and vial, we recorded the number of eggs developing into pupae and the duration of this developmental process. Eggs raised on WT and Ind diets produced 88 and 85% pupae  and the slope . The survival curves of the WT, Ind and 90° flies were very similar  in Ind males, which copulated more often than did 90° males with 90° females, and (ii) in øY males, which copulated more often than did Ind males with øY females. In addition, copulation duration significantly differed between pairs: IndxInd and IndxøY pairs showed longer copulation durations and greater variation of this parameter than did øYxøY pairs , some of which have pheromonal roles, which might explain some of the mating differences described above Fig. . Both InD. melanogaster preimaginal development and several adult life traits.Drosophila studies examining the influence of diet have generally focussed on the diet over all life stages (larva to adult) or only during adult life, and rarely have they focussed on the diet during larval development alone. Our study reveals that both yeast quantity and quality (varying among the different inactivation processes) in the juvenile diet influence 20) but that a reduced amount of yeast in the larval diet can significantly increase adult life span13. Adult diet restriction can also increase life span22, which appears to be associated with the low amount of yeast7. Unlike the effect of larval diet restriction, the effect of adult diet restriction can be reversed when flies are returned to a yeast-rich medium23. Our results indicate that there is no direct relationship between preimaginal development and adult life span since 90° flies did not show an increased life span relative to Ind and WT flies. In the total absence of a sterol source, no Drosophila larvae can theoretically survive24. Therefore, the development of a few øY adults might have been due to the presence of sterols in the corn flour used in our basic food recipe. Moreover, the altered larva-to-pupa development induced by the 90° diet might be explained by the partial deterioration of sterols, possibly through oxidization caused by heating25. Indeed, the major sterol of yeast, ergosterol26, is sensitive to a 90° treatment27, whereas sterols remain unaltered in dehydrated inactive yeast such as that used in our “Ind” diet28. Alternatively, these differences could be caused by other non-sterol yeast components and/or to the transmission of yeast component(s) in the adult digestive tract varying between treatments. The persistence of live yeast from juvenile to adult stage could explain these differences  promotes a rapid and clear food preference for this diet in adult flies. This result has important implications for those laboratories that test olfactory responses in flies raised on Ind-type diet. Our data suggest that individuals developing in nature on live wild-type yeasts may show a different food preference response compared to flies raised in the laboratory.The nutritional requirements and preferences of Drosophila larvae and adults can differ, particularly in regard to sterols and fatty acids35. While we found that the quality of yeast in the larval diet can affect copulatory behaviour and CH composition, we did not find a linear relationship between the two traits: WT females showed a higher copulation frequency than did Ind females , while WT and 90° females paired with Ind males showed similar copulation rates. However, Ind flies of both sexes showed the highest CH amounts and the highest proportions of desaturated CHs , compounds known to have pheromonal effects. Note that no relationship was found between the weight and the total CH amount (∑CHs). Interestingly, a recent study revealed a relationship between CH quality and lifespan13: Drosophila raised on a low-yeast diet showed a decreased production of autotoxins, represented by alkenes (desaturated CHs), which was correlated with an increased life span. Here, we found that øY females and, to a lesser extent, øY males produced lower alkene/alkane (D:L) ratio; these flies showed increased lifespan relative to the flies reared on the other three larval diets.Some of the bacteria present in the adult diet can influence Drosophila mating preferences for many generations. This phenomenon might be related to variation in cuticular hydrocarbons (CHs), some of which are known to have pheromonal effects38, and the very early diet can change this microbiota over the long term39. Although most studies of microbiota have focused on bacteria, future studies should consider the interactions between bacteria and fungi, as recently shown34, to determine the roles of the dominant and persistent fungal species in these complex associations within the microbiota.Additional in-depth studies are required to better understand the influences of different yeast treatments on the composition of the Drosophila diet and gut microbiota. The bacterial microbiota is known to affect many life traits of its host600. The culture was performed at 25 °C with 250 rpm rotation to allow growth to the early stationary phase.We used the Saccharomyces cerevisiae strain BY4742 Wild Type (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) . Preculture was performed over 48 h on Yeast extract (10 g/l)-Peptone (20 g/l)-Dextrose (20 g/l) medium (YPD) at 25 °C with rotation at 250 rpm with each yeast colony in a Erlenmeyer flask containing 100 ml of sterile YPD. A volume of the subculture was then transferred to an Erlenmeyer flask containing 100 ml of fresh YPD medium to reach 0.05 OD8 yeast cells/ml of food corresponding to 65 g/L yeast dry weight). The first medium contained live BY4742 S. cerevisiae wild type yeasts (“WT” = live wild type). The second medium contained another wild type strain of S. cerevisiae yeast produced by vacuum concentration and inactivated by spray drying . The third medium contained BY4742 yeasts pasteurized at 90 °C for 15 min (“90°” = pasteurized treatment). The fourth medium received no yeast supplementation (“øY” = empty treatment). The different food types were consistently used within 48 h of production.All juvenile food media contained corn flour (65.4 g/l) and agar (9.2 g/l), with S. cerevisiae yeasts mixed into the food for some media . Unless indicated, flies were transferred every two days to avoid larval competition and to regularly provide abundant progeny for testing. We used Dijon2000 (Di2), a wild-type strain maintained in our lab for 15 years, which showed very stable behavioural performance. Young emerging flies were housed in groups of 50–100 flies to allow mating in a vial containing 4 g of plain food. After 2–3 days, adults were transferred to a new vial for 3–4 h to allow egg laying. All of the experiments were performed at 25 °C.Fertilized eggs were transferred with a fine brush in groups of 50 to a vial containing one of each type of diet. In each vial, we measured the duration of development and scored the number of newly formed (white) pupae and of newly emerging male and female adults. All emerging adults were transferred to the regular lab medium (“Ind” treatment) for further testing. In most experiments, the adults were maintained in same-sex groups of 8–12 flies, but the males tested in copulation and food preference tests were isolated.Adult survival was only determined for males since we did not control for the mating status of females and the presence of progeny affects female survival. Males held in small groups of 8–12 flies were transferred every 5–7 days to fresh food vials. The number of surviving males was scored at each transfer, and to maintain the group size as constant as possible, we pooled flies from vials with high mortality. Two measures of survivorship were recorded: LT50 (time at which 50% of the flies had died) and lethality slope (the steepness of this curve indicates the proportion of flies dying per hour).We weighed groups of 10 live anaesthetized females on a precision balance  to obtain their fresh weight. Each group of females was then placed for 24 h in an empty glass vial in a 37 °C dry incubator to allow complete desiccation. The dead, dry flies were then weighed to obtain the dry weight. The relative level of water in each group was estimated based on the fresh weight:dry weight ratio.3). After 10 min, a virgin female was introduced. Each test was performed, between 9 am and noon, for 60 min under white light, and the overall frequency of copulation events was measured for each pair. We calculated the cumulative proportion of flies mating during the observation period and the duration of copulation events (time between copulation onset and separation). Females mating within the one-hour observation period were transferred alone to a fresh food vial , while non-mating pairs were individually maintained in a food vial . In all cases, the presence (fertility), number (fecundity) and sex ratio of adult progeny were noted.We measured the copulatory ability of pairs of 4-day old flies. One male was aspirated (without anaesthesia) under a watch glass used as an observation chamber (1.6 cmvs. Ind or 90° vs. øY). The test was performed over a 240-min period under far-red light (LED bulbs). After the test start, every 30 min, the position of the fly in the device was noted relative to the arm (type of food) chosen. Flies were rarely seen retreating into the straight arm of the device. Flies from different yeast treatments were simultaneously tested. Between trials, the substance choices were alternated between the olfactometer arms and assays. All of the experiments were performed between 8 am and noon. Significant differences were determined with the Fisher exact test.For food preference assay, we used 5-day old males that had been individually maintained in the preceding 24 h in empty vial containing only a humid filter paper. After a brief anaesthesia on ice, individual males were individually introduced into the straight arm of a Y-shape olfactometer. Tests were always performed between 9 am and 1 pm. At the end of the straight arm, the device is divided in two additional arms, each one containing a filter paper previously incubated at 25 °C for 30 min with one of two types of food medium (WT n-hexacosane) and 3.33 ng/µl of C30 (n-triacontane) as internal standards. Cuticular hydrocarbons were quantified by gas chromatography using a Varian CP3380 gas chromatograph fitted with a flame ionization detector, a CP Sil 5CB column ; 0.1 µm film thickness; Agilent) and a split–splitless injector  with helium as the carrier gas (50 cm/sec at 120 °C). The temperature program began at 120 °C, ramping at 10 °C/min to 140 °C, then ramping at 2 °C/min to 290 °C and holding for 10 min. Individual CH profiles were determined by integration of 46 peak areas in males and females, representing all of the peaks that could consistently be identified in all individuals. The chemical identity of the peaks was determined using gas chromatography–mass spectrometry system equipped with a CP Sil 5CB column. The amount (ng/insect) of each component was calculated based on the readings obtained from the internal standards. Twenty flies were tested per condition.Five-day-old flies were frozen for 5 min at −20 °C and individually extracted for 5 min at room temperature using 30 µl of a mixture of hexane and methylene chloride  to extract a large variety of compounds. The solution also contained 3.33 ng/µl of C26  after excluding extreme outliers using Tukey’s method42.All statistical analyses were performed using XLSTAT 2012p = 0.05, with Bonferroni correction). Freque ncies were compared using either a Fisher’s exact test (pair comparison) or a Wilks G2 likelihood ratio test completed with a computation of significance by cell (Fisher’s exact test).The number of pupae and adults, the overall amount of CH (∑CH), the Desaturated:Linear ratio (D:L), the fresh and dry weights and their ratio, the total number of adult progeny and the sex ratio were also compared using the same statistical tests. Variances were compared using Levene’s test involving absolute deviations at the median followed by post-hoc multiple pairwise comparisons (Supplementary information


Vertical POC fluxes measured in deep waters between the shelf and the SCS basin were much higher than those estimated by Martin’s attenuation equation. These elevated POC fluxes in deep waters were attributed to lateral particle transport as opposed to enhanced settling out of the euphotic zone. SEM images of sinking particles at 150 m show abundant marine biogenic detritus, while those in deep waters contained a higher proportion of lithogenic material. A great deal of the spatial variability in POC fluxes across the twilight zone and deep waters of the SCS cannot be represented by current biogeochemical models.We measured particulate organic carbon (POC) fluxes from the euphotic zone into the twilight zone and deep waters (>1000 m) that occurred between the shelf and the basin in the South China Sea (SCS) and at the SouthEast Asia Time Series Station (SEATS) using floating sediment trap arrays. Additionally, selected sinking particles were imaged by scanning electron microscope (SEM) to reveal particle morphology and composition. Results showed large variations in POC fluxes with elevated values (32–104 mg-C m Such carbon transport is often simplified to one-dimensional vertical process due to the dominant of sinking POC flux5. It is believed that the carbon budget in the deep ocean is maintained at steady state by a balance between POC supply and metabolic activity. The Martin’s flux attenuation equation: POC flux = Flux100 (Z/100)−b, where b is the rate constant of POC flux attenuation, z is the trap depth, and Flux100 is the POC flux at a reference depth of 100 m or other depths (e.g. 120 or 150 m), is widely used to estimate downward POC flux at different depths6. However, some researchers have questioned the applicability of this flux equation because of discrepancies between observed and modeled carbon fluxes in deep waters. They have suggested that the POC flux attenuation may be controlled by the magnitude of the flux, the characteristics of sinking particles and the specifics of sampling locations8. Recently, Burd et al.9 reviewed numerous field studies and found that estimated metabolic activity in deep waters seemed to be higher than organic matter flux. The discrepancy between carbon fluxes and metabolic activity in the deep ocean can be as much as two orders of magnitude. Burd et al.9 concluded that the imbalance between sources and sinks was poorly understood, but that it pointed to lateral particle transport and slowly sinking particles as two important yet poorly quantified terms.The export of particulate organic carbon (POC) transfers anthropogenic carbon dioxide into the ocean interior10. Chen11 noted an eastward SCS intermediate water (SCSIW) outflow , at depths from 350 to 1350 m) into the western Philippine Sea suggesting that SCSIW was an important water mass carrying nutrients to the East China Sea (ECS) shelves and seas of south Japan.The South China Sea (SCS) is the largest marginal sea in the world and it has a complicated flow: with the surface waters of the Kuroshio flowing into the SCS and the surface water of the SCS flowing out to the Taiwan Strait and the Luzon Strait−2 d−1 in summer12, which is much less than the eutrophic water in the southern ECS (58–785 mg-C m−2 d−1)13. POC inputs into the oligotrophic waters of the SCS vary seasonally14. Besides, several transport mechanisms can carry organic carbon into deep waters in the SCS including internal waves-triggered organic carbon export15, eddies-driven organic matter flux16, etc. Moreover, a significant lateral transport of sedimentary carbon into the deep NSCS is thought to be occurring17 although this has yet to be confirmed. Frequent typhoons19, eddies20, earthquakes21 and a steep topography in the NSCS all facilitate the lateral transport of biogenic and terrestrial carbon across the shelf23. This transport may also occur sporadically between the continental shelf and deep ocean basin but data about this phenomenon is very scarce.The oligotrophic ocean of the northern South China Sea (NSCS), far from the influence of land-runoff, shows open ocean characteristics with a POC flux at the euphotic zone ranging from 32 to 73 mg-C mIn this study, we measured carbon fluxes from upper layers (150 m), twilight zones (150 to 1000 m) to deep waters (greater than 1000 m) at the border between the shelf and the basin, and in deep waters in the SCS using our floating sediment trap array. We estimated POC fluxes, measured trace metal concentrations and obtained scanning electron microscope (SEM) images of the sinking particles. We then attempted to discuss the origin and processes of the laterally transported POC observed in the boundary between the shelf area and the open ocean of the SCS.24. Distributions of Chl a concentrations at all stations showed a subsurface Chl a maximum at 75–100 m, below which Chl a decreased with depth to a background value (<0.1 mg m−3) deeper than 200 m  were observed in the twilight zone (~150 to 500 m) at Sts. T2 and T3, perhaps due to the more recalcitrant nature of the particles in the twilight zone. Elevated POC concentrations were also observed in deep waters at Sts. T1 and T2 (~30 mg m−3) when compared to background values (~20 mg m−3) at adjacent deep-water sites  against salinity (S) is shown in Fig. −2 d−1 (St. T2), which is slightly lower than the value (50 ± 7 mg-C m−2 d−1) reported for the NSCS25 as a whole. Interestingly, elevated POC fluxes (32 and 23 mg-C m−2 d−1 at 500 and 1000 m) were found at St. T1 which did not exponentially decrease with increasing depth based on Martin’s attenuation curve  was also observed at St. T2  were approximately 33 (St. T1) to 36 mg-C mrve Fig. . A simil T2 Fig. . An anal T2 Fig.  with 102−2 d−1 at 150, 1000, 2000 and 3200 m)  at St. T4 are reasonable since sinking particles collected by moored traps (collected time: ~six months) will be degraded, which can be inferred from Hung et al.26 assessment about the extent of degradation of sinking particles collected by sediment traps.However, POC fluxes at St. T4  decreased smoothly with increasing depth  Fig. . Althoug27.The distribution of POC concentrations in deep waters at Sts.T2 and T3  or its conversion to dissolved organic carbon (DOC)6. We used b values previously measured in the NSCS28 of 0.65  and 1.10  to estimate the POC flux attenuation. This calculation led to POC fluxes of 15–9 and 9–4 mg-C m−2 d−1 at water depth of 500 and 1000 m at St. T1, and 17–10, 11–5 and 7–2 mg-C m−2 d−1 at water depth of 500, 1000 and 2000 m at St. T2, respectively  under events of lateral transport.It is common practice to predict the attenuation of POC flux with increasing depth using Martin’s equationely Fig. . In addi29 and Hung et al.30, the e-ratio (POC flux/primary production (PP)) in open ocean water is around 0.1 (0.05~0.16). If we use e-ratio 16%  and previous annual PP values (280 mg-C m−2 d−1)14, the estimated POC fluxes (b = 0.65 and 1.10)28 at 500 and 1000 m would be 12–20 and 6–13 mg-C m−2 d−1, respectively. The estimated POC fluxes are much lower than observed values  suggesting POC fluxes in deep waters cannot be accounted for by biogenic particles settling from the upper ocean.According to Hung and Gong−2 d−1 at Sts. T1 and T2, respectively. Assuming the affected area was 106 m2  during each episodic event, it follows that the integrated vertical flux of POC in deep waters at Sts. T1 and T2 was 27 × 103 g-C d−1  and 35 × 103 g-C d−1 . The lateral POC flux can be estimated to be 28 × 108 g-C d−1 (=POC concentration (~17 mg-C m−3) at 1000 m × average flow rate at 1000 m) if we assume an eastward flow of 1.9 Sv (m3 s−1) at depth of 1000 m11. Hence, the lateral POC flux is several orders of magnitude higher than the integrated vertical flux estimated by sediment traps. It is worth noting, however, that this lateral transport likely occurs sporadically, when currents strong enough to resuspend settled material contact the continental slope seafloor. Such resuspension events may not last for the full 24 h period implied by our calculation. More studies are clearly needed to better understand real lateral carbon transport in deep waters in the SCS.The average POC fluxes in deep waters were 27 and 35 mg C m11. Other deep lateral POC flux can be stored in deep waters (>1300 m), potentially trapping the carbon for centuries, if not millennia, given the long residence time of the deep seawater31. In this way, the lateral carbon transport plays an important role for carbon sequestering in deep waters of the NSCS.We have good reasons to believe that some POC can be transported to the ECS and on to the western Pacific via the eastward flowing SCSIW, and that this transport occurs at depths of 350 to 1300 m32, mesoscale eddies33, earthquakes34 and episodic events35 such as “benthic storms” associated with current interaction with steep topography, may contribute to lateral transport in the SCS. For example, the funnel-like topography of a canyon off southwest Taiwan induces sediment transport that is mainly driven by flood events in the summer and tidal action in the winter season32. Part of our sampling was undertaken in September, i.e. during the typhoon season. Under those conditions, large quantities of suspended riverine material containing some lithogenic particles mixed with biogenic particles are delivered directly into the deep sea by high-energy rivers22. Our measurements showed the Al-normalized flux . Typhoons are not only physical agents for enhanced delivery of terrestrial material to the sea. Geological agents can also trigger gravity flows such as turbidity currents that eventually leave deposits on the seafloor and form large-scale deep-water depositional systems41. For instance, typhoon Tembin was a category-2 typhoon (sustained winds: 30 m s−1) which traveled at a speed of 3.0~5.5 m s−1 from 22–29 August, 2012. The typhoon affected the study area (Sts. T1 and T2) for several days and dumped high amounts of rainfall, peaking at ~580 mm in Taiwan and resulted in flooding in southern Taiwan . It is likely that typhoon-induced hyperpycnal turbidity currents drive lateral particle transport from the shelf to basin area in the SCS. The lower salinity water .Furthermore, frequent earthquakes may be another trigger factor facilitating lateral POC transport33. The mesoscale eddies can even carry materials to the open ocean, as evidenced by satellite image observations, but it is not evident that these materials can enter deep waters. We cannot definitely distinguish all factors based on current data set, since resuspended particles may occur in the coast or on the continental shelf, and then be re-located elsewhere due to episodic events. Therefore, the elevated POC fluxes in deep waters probably can be caused by typhoons, earthquakes, eddies, seasonal and interannual variability in currents and/or cooling effects as well as deep layers sediment resuspension46. One may ask whether the deep sea POC flux is unique to the northern South China Sea or whether it applies to the whole South China Sea. For example, Chen et al.11 used a box model to show that the eastward flow of the SCSIW (350~1300 m) can transport high-nutrient waters across the entire ECS and beyond. Lateral POC fluxes and POC concentrations could be measured directly but this would require intensive sampling and lots of ship time. Until now, very few cruises have captured lateral POC transport in the SCS, thus we are not sure if the lateral contribution to the POC export flux is present in all parts of the deep (>500 m) SCS. Such deep water lateral transport in the SCS definitely requires further study.Some researchers also reported that mesoscale eddies can carry particles from nearshore to offshoreOcean Researcher III (OR-III) and V (OR-V) were conducted in each of the following periods: Sep. 5–7 (T1), Sep. 25–28 (T2), 2012 and Jul. 14–17 (T3) and Sep. 12–18 , 2013. The first cruise was conducted approximately a week after a category 2 typhoon Tembin, and the third cruise was conducted ~2 days after typhoon Soulik (category 2). Sinking particles were collected at 150, 500 and 1000 m at St.T1  located at the boundary between the shelf and the basin using a drifting sediment trap array25. At the second cruise, the sediment trap was also deployed at the boundary between the shelf and the basin  with four traps at 150, 500, 1000 and 2000 m, respectively. During two additional cruises, sinking particles were collected from deep waters  by using the same approach with depths of 150, 500, 1000, 2000 and 3000 m on Jul. 14–17; with depths of 150, 1000, 2000 and 3200 on Sept. 12–18, 2013, respectively  materials were assessed based on Hung et al.25 using an elemental analyzer . Chl a was analyzed with a Turner Designs 10-AU-005 fluorometer after extraction with 90% acetone following by the non-acidification method47. Contents of metal elements in sinking particles were determined by quadrupole-based inductively coupled plasma mass spectrometer  after total digestion of the particles with a combination of HF, HNO3 and HClO4. Selected bulk sinking particles were filtered on polycarbonate filters and imaged by scanning electron microscopy (SEM) according to Hung et al.49.A SBE 9/11 plus CTD sensor was used to assess hydrographic conditions. Seawater samples were collected to measure POC and Chl


Statistical analysis of the chromosomal composition in a population of 210 primary plants regenerated from two intergeneric wheat–barley cross combinations revealed the random nature of uniparental elimination for barley chromosomes.Uniparental chromosome elimination is a common process in interspecific and intergeneric cereal hybrids. To characterize the frequency of paternal chromosomes, a population of 218 independent green plants was generated from two wheat (♀) × barley (♂) cross combinations via embryo rescue. The chromosomal composition of 210 primary plants was analyzed with chromosome-specific DNA markers representing all seven barley chromosomes. The analysis revealed an equal proportion of haploid and full hybrids , while the rest of the population contained hypoploids  with no preference for any possible numbers (one to six) of barley chromosome additions. Contrary to the previous reports, there was no statistical bias or preferential elimination for any individual barley chromosome (1H–7H) in this population. The reasons for the apparent contradiction and the implications of the above findings for cereal breeding are discussed.The online version of this article (10.1007/s00299-019-02405-1) contains supplementary material, which is available to authorized users. Complete or partial uniparental genome (or chromosome) elimination during initial mitotic divisions in embryos of ultrawide (interspecific and intergeneric) sexual hybrids is a general phenomenon in the plant kingdom  × Hordeum bulbosum hybrids is associated with the formation of micronuclei, abnormally condensed chromatin, and chromosome fragments , and the other four combinations being interspecific Hordeum hybrids again with H. vulgare as the male parent. All these 48 combinations (74%) required in vitro embryo rescue to generate viable plants. The remaining 17 hybrid types (26%) resulted in haploids , and full, but no partial, hybrids.Uniparental genome elimination in hybrids obtained between members of the botanical tribe of Triticeae is almost exclusively restricted to the male parent. A closer analysis of a recent literature survey on parental genome elimination  × barley , comprising maternal haploids (zero barley chromosomes) and partial (1–6 chr.) as well as full hybrids (7 chr.). Therefore, this combination was selected to study the frequency of chromosome elimination and stability of individual chromosomes in ultrawide hybrids.Complete genome elimination as well as its absence in ultrawide cereal hybrids are both of practical significance and desirable for genetic improvement. In the first case, the resulting maternal (doubled) haploid lines represent valuable breeding material, especially in hybrid maize programs Weber . PartialAs expected for paternal chromosome elimination, a high frequency of maternal haploids is often reported in wheat × barley hybrids. In four independent studies, the (relative) frequencies of wheat haploids were as follows: nine haploids in ten hybrid plants (90%) from two cross combinations  described by Polgári et al. (PhI (line C04-1), a ‘Chinese Spring’ (CS) derivative that contains the PhI gene from Aegilops speltoides Tausch, an epistatic suppressor of the Ph1 (pairing homoeologous) gene in wheat  cultivar ‘Morex’ , the spikes were treated by injection with 100 ppm 2,4-D  to promote pseudo-seed development. Germination and regeneration of embryos excised 14–16 DAP took place on modified N6 medium  from young plants before tillering  were homogenized with two 3-mm stainless steel beads in the TissueLyser II laboratory mixer-mill disruptor  for 2 × 1.5 min at 29 Hz. Total DNA was isolated by adding 100 µL of AquaGenomic™ solution  and subsequent precipitation with isopropanol according to the manufacturer’s instructions. Qualitative and quantitative evaluation of the purified DNA was done with ND-1000 spectrophotometer  to meet the following criteria: peak of absorbance (A) scan at 260 nm, A260/A280 ≥ 1.8, A260/A230 ≥ 2.0.® thermal cyclers  with the following conditions: denaturation at 98 °C for 5 min; 40 cycles at 98 °C for 5 s, X°C , SSR (simple sequence repeat), and gene-specific markers is listed and characterized in Table ® antifade medium . Hybridization signals were examined under an Axioskop-2 epifluorescence microscope  equipped with appropriate filter sets. Images were captured with a SPOT™ CCD camera  and processed with the Image-Pro® Plus software  for contrast and brightness. No further manipulations were performed with the images. At least five metaphases  were examined for each plant sample, which provided identical results with those of the marker analysis.Young roots from tillering wheat × barley hybrid plants in pots were collected before 10 am, and treated with nitrous oxide gas  at room temperature, as described by Kato . Treatedz test as well as Chi-square test were performed for the relative frequencies obtained.To determine whether the null hypothesis of no significant difference between the two wheat genotypes for embryo induction and plant yield after crossing with barley can be maintained, a two-tailed two-sample df = (number of rows–1) × (number of columns–1).The preferential elimination of any barley chromosome was evaluated by first determining the absence or presence of DNA markers for individual barley chromosomes in all 210 plants. The binary presentation of the data (0 for absence and 1 for presence of each barley chromosome) was then analyzed by cross tabulation (contingency table). The null hypothesis was that each barley chromosome has an equal chance  of elimination. For testing statistical bias between the Observed Count and Expected Count values, the nonparametric Chi-square goodness-of-fit test was applied. Critical sample size for Chi-square analysis was determined using MS Excel’s Goal Seek capability based on the following criteria: alpha (probability of Type I error) = 0.05, power  = 0.8, V test .Statistical associations between chromosomes (double combinations) for nonrandom elimination were investigated first with the Chi-square test of independence. The strength of association in significant cases was further analyzed with Cramér’s t Cramér . ComputiPhI—48 embryos (5.7%) and 19 plants (2.3%) obtained from 836 cross-pollinated florets. These figures correspond to and confirm the previous results of Polgári et al.  were collected, with an average of 70% frequency regenerating into 218 (8.5% of florets) viable plants. The corresponding figures for the two cross combinations separately were: ‘Sichuan’—1717 florets pollinated, 268 embryos (15.6%) excised, and 199 plants (11.6%) regenerated; CS i et al.  (Table 2z test was performed to determine whether there was a significant difference between the two wheat genotypes with respect to the relative frequencies of embryos and plants. The z statistic was highly significant (p < 0.001) at the 0.05 critical alpha level in all the cases  were characterized with established chromosome-specific STS, SSR and gene-specific markers  positive for all chromosome-specific markers is summarized side by side the parental genotypes as controls  = 7.429; p = 0.191] (Supplementary Table 2: column 7). In other words, no preference was apparent in this population for any particular hypoploid level. This is a first indication for the random nature of chromosome elimination in this intergeneric cross combination. The same conclusion can be drawn from the statistical analysis of 119 hypoploid plants derived from the ‘Sichuan’ × ‘Morex’ combination alone  = 9.218; p = 0.100], Supplementary Table 2: column 8), demonstrating that the two wheat genotypes (‘Sichuan’ and CS PhI) may not be different for this trait. Post hoc analysis revealed a slight underrepresentation of hypoploid class n + 1 , which may indicate that, once the elimination program ran down to + 1 paternal chromosome, then the remaining single chromosome will be lost more easily.As expected, the number of barley chromosomes in the tested 210 plants ranged between 0 and 7. A bimodal distribution of frequency was observed with statistically significant peaks for 0  and 7 (full hybrids) chromosomes  = 7.918; p = 0.244] (Supplementary Table 4: column 2), and thus, the original null hypothesis of no bias was maintained. A similar outcome was again obtained for the ‘Sichuan’ × ‘Morex’ population alone . In both cases, post hoc analysis indicated a weak overrepresentation of chromosome 3H  among all hypoploid classes. This slight effect turned out to be confined to the n + 1 and n + 2 hypoploid groups (Supplementary Table 4: columns 9–10), and, thus, did not prove to be a general, characteristic trend in the population. A comparative visual inspection of the data distributed according to various hypoploid groups  also confirms that this bias may have been caused by small sample sizes and restricted representations of chromosomes in groups of low hypoploid levels.After tabulation of the frequency of individual barley chromosomes separately in all hypoploid classes and in full hybrids (Supplementary Table 3), we addressed the question whether there was a preferential elimination for any individual barley chromosome in the hypoploid population  or identified indirectly by using genetic (DNA) markers. Cytological observation of chromosome elimination can be done in the developing proembryo and endosperm Finch , or in r1) hybrids , Secale (9), and Thinopyrum (15). All these translocations were either meiotically produced  × barley (♂) cross combinations, and analyzed in 210 plants by DNA markers as well as statistically the frequency and pattern of paternal chromosome elimination by mitosis during early embryogenesis and vegetative development. To our knowledge, this is the first systematic and large-scale analysis on uniparental chromosome elimination in intergeneric hybrids of cereals or any plant. The analysis of our data suggests no preference in the elimination of individual barley chromosomes in wheat background. Thus, the genome elimination process—in this intergeneric combination of species, at least—can initially be considered as random. For the characterization of subsequent meiotic elimination in the reproductive phase of ultrawide hybrids, detailed investigations are essential.DP performed crossings, embryo rescue, and the DNA marker analysis, EM carried out the cytogenetic investigation and statistical analysis, LS designed and coordinated the study, participated in crossings, embryo rescue, and statistical analysis, and wrote the paper. All authors read and approved the manuscript for publication.Below is the link to the electronic supplementary material.Supplementary Table 1: Detailed characterization for the absence or presence of each barley chromosome (1H-7H) in 210 plants from two wheat × barley cross combinations (DOCX 34 KB)Supplementary Table 2: Statistical analysis of different classes of hypoploids (from zero to seven barley chromosomes) in a population from two wheat × barley cross combinations (XLSX 20 KB)Supplementary Table 3: Distribution of individual barley chromosomes (1H-7H) in all hypoploid (and full hybrid) classes in a population from two wheat × barley cross combinations (DOCX 13 KB)Supplementary Table 4: Statistical analysis on the persistence of individual barley chromosomes (1H-7H) in a population from two wheat × barley cross combinations (XLSX 17 KB)Supplementary Figure 1: Representative screening for the absence (20) or presence (25) of the 3H barley chromosome Supplementary Figure 3: Distribution (A) and relative frequency (B) of individual barley chromosomes (1H-7H) in all hypoploid groups  in a population from two wheat × barley cross combinations (DOCX 29 KB)Supplementary Figure 4: Occurrence (A) and relative frequency (B) of individual barley chromosomes (1H-7H) among all hypoploid groups  in a population from two wheat × barley cross combinations (DOCX 3048 KB)


MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target β-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, suggesting that MeCP2 can be retained in the nucleus by its affinity for DNA. Consistent with these findings, we demonstrate that disease progression in a mouse model of RTT is unaffected by an inactivating mutation in the NLS of MeCP2. Taken together, our work reveals an unexpected redundancy between functional domains of MeCP2 in targeting this protein to the nucleus, potentially explaining why NLS-inactivating mutations are rarely associated with disease.  •Nuclear localization of MeCP2 does not require its NLS•DNA binding by MeCP2 contributes to its NLS-independent nuclear localization•MeCP2 NLS mutation does not affect pathology in a mouse model of Rett syndrome Lyst et al. report that nuclear localization of MeCP2 does not require its nuclear localization signal. Instead, this protein is efficiently partitioned in the nucleus via its affinity for DNA. Consistent with this finding, mutating the MeCP2 NLS does not affect pathology in a mouse model of Rett syndrome. Eukaryotic cells are defined by having their genetic material contained within a membrane-bound organelle known as the nucleus. The nuclear pore complex (NPC) is a large protein channel embedded in the nuclear membrane that mediates transport of macromolecules between the nucleus and the cytoplasm. Small proteins are able to diffuse passively through the NPC, whereas larger factors require a nuclear localization signal (NLS)—a short motif that binds to proteins mediating active transport through the NPC . One sucMeCP2 has been subjected to intense study since the discovery that mutations in this factor cause Rett syndrome (RTT)—a severe neurological disorder affecting approximately 1 in 10,000 girls . The oriIn this study, we reveal a redundancy in the mechanism by which MeCP2 is localized to the nucleus. We report that nuclear localization of MeCP2 is not exclusively dependent on its canonical NLS. Rather, we find that either an intact NLS or chromatin binding is sufficient for nuclear retention. Consistent with NLS-independent nuclear localization of MeCP2, we find that disease progression in a mouse model of Rett syndrome is not impacted by a mutation that abolishes binding of the MeCP2 NLS to the nuclear import factors Kpna3 and Kpna4. Taken together, our results reveal a functional redundancy between DNA binding and the NLS in targeting MeCP2 to the nucleus. This may rationalize the absence of pathogenic mutations in the NLS of MeCP2 and may also be considered as an explanation for the scarcity of disease-causing mutations in the NLS motifs of other factors .Mecp2-EGFP knockin mice and identified the nuclear import factors Kpna3 and Kpna4 as binding partners by mass spectrometry  or upregulated (Flrt3 and Arhgap10) across multiple brain regions and in several mouse models of RTT  was dounce homogenized in NE1 buffer . This homogenate was taken as whole-brain extract, and the supernatant following centrifugation for 5 min at 500 g was taken as cytoplasmic extract.All animal experiments were carried out by staff licensed by the UK Home Office in accordance with the Animal and Scientific Procedures Act 1986. Targeting vectors for generating mutant mice were based on one described elsewhere . For theBrains from wild-type and mutant male littermates aged 10–18 weeks were dissected, removing the olfactory bulb and trimming to remove any spinal cord. Brains were weighed and immersed in fixative . After 2 hr, brains were transferred to fresh fixative and stored overnight at 4°C. Brains were transferred to 30% sucrose solution in PBS and stored at 4°C for 48 hr, replacing the solution after 24 hr. Brains were then frozen in isopentane, chilled on dry ice, and stored at −80°C. A Leica CM1900 cryostat set to −26°C was used to cut 30-μm coronal sections, which were mounted on SuperFrost Plus slides and dried. Sections were photographed using an Olympus SZX10 microscope at 2.6× magnification. The location of each section was determined with reference to “The Mouse Brain in Stereotaxic Coordinates” . The priTotal RNA was purified from whole brains of male mice aged 7–14 weeks using TRI Reagent . The QuantiTect Reverse Transcription Kit (QIAGEN) was used for cDNA synthesis, and gene expression was quantified using the SensiMix SYBR & Fluorescein kit (Bioline) on a LightCycler 480.For quantification of the nuclear to cytoplasmic ratio of EGFP-MeCP2 mutants in NIH 3T3 cells, statistical significance was calculated using the Wilcoxon test. For qPCR data as well as analysis of brain weights and cortical thicknesses, p values were calculated using the Student’s t test. The Kolmogorov-Smirnov test was used to calculate p values for comparisons of body weights and phenotypic scores.Antibodies used were as follows: Histone H3 ; gamma-tubulin ; MeCP2 ; KPNA3 ; KPNA4 ; EGFP (CRUK and Chromotek gta-10); and FLAG .


Microplastics  are estimated to accumulate in agricultural soils in quantities that exceed the total MP burden in ocean waters. Despite a wealth of information relating to the accumulation of MPs in aquatic species, there is little information on the uptake of MPs by terrestrial plants. Information about location of MPs in plant tissues is critical to understand the modes of their interaction with plants. Polystyrene (PS) is one of the most commonly used plastic polymers worldwide and it is often found in MPs sampled in the environment. The performance of traditional detection methods  for nanoparticles is limited due to the extensive sample preparation and the limited field of view. Here we report an approach for the imaging of different sizes of PS plastic beads (ranging from submicrometer to micrometer-sized) within plant tissues by using confocal laser scanning microscope (CLSM). Fluorescent dye Nile blue or 4-chloro-7-nitro-1,2,3-benzoxadiazole were encapsulated into the PS microbeads through swelling method and they were used to detect the localization of PS beads in the root and the green tissue respectively.•This is a simple and rapid approach for imaging of MPs in plant.•The fluorescent dyes can produce bright and stable emission signals that are distinguishable from the autofluorescence background of plant tissues.•The dyes leakage in the aqueous phase can be assumed to be negligible. Specification Table−1 suspensions in ultrapure water . The PS beads had primary nominal sizes of ∼0.2 μm and ∼2.0 μm. The actual sizes of the 0.2 μm and 2.0 μm PS microbeads were determined to be 0.23 ± 0.04 μm and 1.98 ± 0.08 μm using SEM .Fluorescent PS particles  were customized synthesized from Da'e Scientific Co., Ltd. . The two sizes of PS microbeads were first synthesized by means of dispersion polymerization and a mini-emulsion polymerization process . Then, tThese two fluorescent dyes were used to detect the presence of PS beads because the red fluorescence from NB is indistinguishable from the red autofluorescence emitted by chloroplasts and the green fluorescence from NBD-Cl is indistinguishable from the green autofluorescence in root tissues . Thus, in this study PS-NB was used to detect PS beads in roots, and PS-NBD-Cl was used to detect PS beads in stems and leaves. To assess the stability of the fluorescence, we analyzed the loss of fluorescence intensity after exposure of the PS beads in Hoagland solutions within plants for 3, 6, 12, 24, 48, and 72 h using a Synergy H1 microplate reader  with excitation and emission wavelengths of 488 nm and 518 nm or 635 nm and 680 nm. The leakage of fluorescence was assessed by measuring the fluorescence before and after filtration through a 0.1-μm syringe filter at the end of the indicated incubation periods. For Nile Blue labeled 0.2 μm and 2.0 μm PS beads suspensions, the amount of dye after filtration was below to the microplate reader detection limit. The dissolved dye 4-chloro-7-nitro-1, 2, 3-benzoxadiazole release into the solution did not exceed 3.7 % for the PS beads . Consequently, the presence of fluorescent markers in the aqueous phase can be assumed to be quite negligible.The average hydrodynamic diameter and the zeta potential of the PS beads in Hoagland solution were determined by dynamic laser scattering (DLS) using a Malvern Zetasizer Nano-ZS90 . Both stock suspensions were sonicated for 2 min using a sonicator  and were then diluted to obtain the final concentrations of the beads used in the tests. The average PS beads hydrodynamic diameter was 0.27 ± 0.04 μm and 2.5 ± 0.3 μm for the 0.2-μm and 2.0-μm PS beads, respectively. The surface of the MPs was negatively charged, with the 0.2-μm beads having a more negative zeta potential (−42.7 ± 0.9 mV) than the 2.0-μm beads (−12.1 ± 0.6 mV).Triticum aestivum L., Zhongmai 9) were supplied by the Chinese Academy of Agricultural Sciences, Beijing, China. Wheat seeds of an almost uniform size were selected and surface sterilized by treatment with a 10 % NaClO solution for 5 min. Subsequently, the seeds were washed three times with deionized water to remove the residual NaClO solution and were then transferred to moistened filter paper and incubated in the dark at 25 °C to induce germination. After four days, four seedlings with uniform size were transferred to a 1-L beaker containing Hoagland solution. The beaker was covered with a cap and placed in a growth chamber . Each box was equipped with an aquarium aerator to provide oxygen to the roots and to maintain the PS beads in suspension. The wheat plants were exposed to PS microbeads for 10 days. The experiment was repeated three times.Wheat was chosen as a model plant in this study because of its wide production as one of the main world-wide foodstuffs in the world. Seeds of wheat  and stem (2 cm from the base of the stem) segments were collected and embedded in 4 % agarose. The root and stem samples and the leaf blades (with the primary vein) were sectioned into 40- and 100-μm thick sections, respectively, using a vibrating microtome . Semi-thin sections of the samples were placed on a glass slide covered with a coverslip, and a few drops of PBS were added to keep the sample hydrated. The sectioned tissue was inspected using a confocal laser scanning microscope  with excitation/emission wavelengths of 488/515 nm and 635/680 nm for NBD-Cl and NB, respectively. Transverse and longitudinal sections of at least three plants were examined from each treatment group before representative images were selected. Images were captured with a SPOT camera and imported into imaging software Fluoview FV 10 for the compound microscope. Pictures have brightness and contrast enhancement.Selected samples from the roots, stems and fully expanded leaves near the primary veins were excised, sectioned into small pieces and frozen in liquid nitrogen. The samples were then freeze-dried and coated with gold for 60 s (ca. 1 nm thickness of gold) by a Sputter Coater , and then detected by a scanning electron microscope . The cross sections were viewed at an accelerating potential of 20 kV under high vacuum mode with backscatter detection. Images were captured at different magnifications. For each species, at least three plants were examined for each treatment group. Digital photographs were taken using an EVO 40 scanning electron microscope (Zeiss).–1  were used for investigations in the hydroponic exposure medium. Significant fluorescence was observed in the roots, shoots and leaves of wheat exposed to 0.2 μm beads  to track PS beads in plant tissues and found fluorescence to be a sensitive and reliable detection method. Plants without exposure of fluorescent PS beads were used as a control to adjust the detector gain and establish the baseline. All images were acquired with the same detector gain to ensure comparable relative intensities. Under the imaging conditions used, sections from untreated control plants showed no detectable autofluorescence at specific excitation . When roots were treated with fluorescent PS microbeads, the beads could be identified by their light fluorescence. The roots of wheat grown in 0.2 μm microbead suspension showed clear concentration-dependent fluorescence in a wide range from 0.5–50 mg L–1 , thus prμm beads . Very feμm beads . The preμm beads . Once inμm beads . No fluoin vivo distribution into the stem and leaves. Our work reported a visualization method to detect the accumulation and distribution of MPs in plant tissues, which could be beneficial to profoundly understand the biological effects and the modes of interaction of MPs with plants.Briefly, our observations demonstrated the usefulness of using fluorescent labeled MPs for studying their localization within the whole plant tissues. The results presented here clearly demonstrate the uptake and transport of PS microbeads by plant roots and the Microplastics may present an attributable risk to ecosystem and human health, and their presence in the biosphere has become a global concern. Terrestrial edible plants are for instance directly exposed to MPs when organic manure, sewage sludge as fertilizer, or plastic mulching  are applThe authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


Over 60 years have passed since Taube deduced an orbital-mediated electron transfer mechanism between distinct metal complexes. This concept of an orbital pathway has been thoroughly explored for donor–acceptor pairs bridged by covalently bonded chemical residues, but an analogous pathway has not yet been conclusively demonstrated for formally outer-sphere systems that lack an intervening bridge. In our present study, we experimentally resolve at an atomic level the orbital interactions necessary for electron transfer through an explicit intermolecular bond. This finding was achieved using a homologous series of surface-immobilized ruthenium catalysts that bear different terminal substituents poised for reaction with redox active species in solution. This arrangement enabled the discovery that intermolecular chalcogen⋯iodide interactions can mediate electron transfer only when these interactions bring the donor and acceptor orbitals into direct contact. This result offers the most direct observation to date of an intermolecular orbital pathway for electron transfer. It is known that intermolecular interactions impact electron transfer rates, but the mechanisms involved are challenging to define experimentally. Here, the authors have developed a platform that enables atomic orbital resolution of electron transfer through an explicit intermolecular interaction. Typically, light-induced excitation of a photocatalyst creates an energetic electron or hole that enables the transformation of a substrate to a useful product. The discovery and optimization of such catalysts requires the optimization of factors that govern intermolecular electron transfer (IET). In particular, the roles that intermolecular interactions have in mediating electron transfer are very challenging to define. During the last two decades, a body of literature has shown that electrostatic interactions10, hydrogen bonding13, and other non-bonding interactions19 can enhance IET rates, but interactions that are too strong may suppress reactivity by lowering the driving force for reaction20. This scenario provides the imperative to understand how weak intermolecular interactions can increase IET rates without compromising chemical reactivity.Molecular photoredox catalysis is widely used for a range of applications, such as energy storageG°ET) and the reorganization energy (λ), whereas quantum mechanical effects are accounted for by an electronic coupling factor (HDA)23. While the chemical origins of ΔG°ET and λ are well understood in terms of intuitive chemical principles, HDA can only be rigorously defined in terms of the complete wave functions of the non-adiabatic donor–acceptor states before and after electron transfer  chloride and chromium(II) complexes was accompanied by quantitative chloride ligand transfer between the two metal centers , of the oxidized catalyst is delocalized onto the heteroaromatic ring, but not onto the chalcogen atom itself  were employed as indicated. All thin films were prepared on glass substrates coated with fluorine-doped tin oxide. The oxygen containing analogue of the X-Ar series, O-Ar, decomposed rapidly following saponification and was therefore not evaluated.The ruthenium compounds under investigation were prepared in low to moderate yields from (trimethoxycarbonylterpyridine) ruthenium trichloride either by direct cyclometalation of the N^C^N ligand  solution  transition of each catalyst was hypsochromically shifted ~20 nm relative to the methyl esters, with no other significant changes  and 45 nm (810 cm−1), respectively. In contrast the exchange of selenium for sulfur in X-Ar results in a much more modest 25 nm (240 cm−1) bathochromic shift.The optical and redox properties of the saponified catalysts anchored to metal oxide films were investigated through spectroelectrochemistry on nano-ITO . In the presence of NaI electrolyte, the photo-oxidized catalyst instead oxidizes iodide through an IET process. To accurately resolve IET kinetics, it is necessary that the rate constant for IET (kIET) be much greater than that for BET (kBET). On pure TiO2 thin films, the BET rates were found to be too similar to the IET rates to enable accurate determination of the true second-order kIET, and TiO2 was therefore not a good substrate to fully analyze the iodide oxidation kinetics  energy of the reduced RuII catalysts were appropriately positioned to inject electrons into TiO2 and to anchor the catalysts to the metal oxide substrates67. Previous studies have shown that incorporation of chalcogen atoms in positions with access to the metal oxide surface can encourage side reactions between the electrolyte and the semiconductor64, and therefore X-Me and X-Ar were substituted para- to the ruthenium center on the central ring to direct the chalcogen atoms away from the surface. The N^C^N cyclometalating motif was chosen such that, upon oxidation, the resulting positive hole will be delocalized to encompass the central ring and its substituents, ensuring that this hole will be exposed to the solution.The compounds under investigation were designed to ensure optimal electronic and geometric properties for the systematic study of both intermolecular interactions and electron transfer Fig. . The hig3 groups or heteroaromatic groups, the electronic properties of the RuII complexes were well conserved  band with significant contributions from the heteroatom for the X-Me series, or from the aryl ring (but not the heteroatom) for the X-Ar series  was determined using a Hirshfeld partitioning scheme  for our systems are low compared to the diffusion limit (~1010 M−1 s−1)73, and thus many transient catalyst···iodide interactions form and break within the lifetime of the oxidized complex. Stronger intermolecular interactions could therefore increase the lifetime of the initial [Ox···I–] adduct, increasing the likelihood that a second iodide will encounter that adduct to create the [Ox···I···I]2– encounter complex and complete electron transfer. While this mechanism is plausible, it would predict the same rate enhancement for both X-Ar and X-Me, which is clearly not the case. Nonetheless, to eliminate this possibility, these adducts were modeled via DFT methods. The optimized interaction geometries are presented in Supplementary Figure Eint) presented in Supplementary Table K1, between O-Me and the remaining compounds in the X-Me series, and actually predict a slight decrease in stability between S-Me and Se-Me. The initial [Ox···I–] adduct stability clearly cannot account for the observed trends in kIET. We have approximated the overall association constant KA using semi-empirical methods  by relating kIET directly to γ  in 0.1 M tetrabutylammonium tetrafluoroborate acetonitrile solutions.Acetonitrile was purchased from Sigma-Aldrich and purified on an MBraun solvent purification system prior to use. Tetrabutylammonium tetrafluoroborate was purchased from Alfa Aesar, recrystallized from boiling 50% water/ethanol, and dried under high vacuum prior to use. UV-visible spectra were recorded using ~30 μM acetonitrile solutions on a Varian Cary 5000 spectrophotometer at a resolution of 1 nm. Cyclic voltammograms were recorded using a CH Instruments 660D potentiostat at room temperature using a standard three-electrode configuration , referenced externally to ferrocene/ferrocenium (0.630 V vs NHE);4,>98%), and lithium perchlorate  were purchased from Sigma-Aldrich and used as received. UV-visible absorption spectra of the functionalized films were recorded on a Varian Cary 60 spectrophotometer with a resolution of 1 nm. Spectroelectrochemical measurements were performed using a WaveNow potentiostat  at room temperature (22 ± 1 °C) with an AvaSpec-2048 fiber-optic spectrometer (Avantes) and an AvaLight-DHc light source (Avantes). Measurements used a standard three-electrode configuration , referenced externally to ferrocene/ferrocenium (0.630 V vs NHE);75 counter electrode: platinum wire) in 0.5 M NaClO4. The reference electrode was mounted in a Vycor-tipped glass tube with electrolyte to avoid contamination.Acetonitrile was purchased from Honeywell  and used as received. Sodium iodide , sodium perchlorate  Brilliant B; 532 nm, 5–6 ns full width at half-maximum (fwhm), 1 Hz, ∼10 mm in diameter) or a laser of the same model, frequency tripled (355 nm) coupled to an optical parametric oscillator  tuned to 532 nm. The 532 nm beam was then directed through two Glan laser polarizers to attenuate the pulse fluence  and was directed 45° to the film surface. A 150 W xenon arc lamp (Applied Photophysics), pulsed with 70 V, served as the probe beam and was aligned orthogonally to the excitation laser. Before the sample, the light was focused through a monochrometer (GM 252) to minimize background excitation of the samples. Detection was achieved with a monochromator (Spex 1702/04) optically coupled to an R928 photomultiplier tube (Hamamatsu). Transient data were acquired on a computer-interfaced digital oscilloscope  with 2.5 ns resolution terminated at 50 Ω. For longer timescale data acquisition, the xenon arc lamp was run continuously (not pulsed or shuttered) and the oscilloscope was terminated at 50 kΩ. To completely model the full kinetic decays data taken at multiple timescales were stitched together.Transient absorption spectra and kinetics were acquired on an apparatus that has been described in literature4 and NaI electrolytes were prepared in acetonitrile and purged with argon for at least 15 min prior to use. The functionalized metal oxide thin films were placed in a 1 cm2 cuvette with a 24/40 ground glass joint and fully submerged in argon-purged electrolyte solutions. The cuvette was then purged with argon for an additional 5 min and argon was continuously purged through the headspace of the cuvette throughout the measurements.NaClO78. The decay fitting was performed in Origin 2016pro, and least-squares error minimization was accomplished using the Levenberg–Marquardt iteration method.All single wavelength kinetic decays were modeled through the Kolrausch-Williams-Watts stretched exponential function4/MeCN from −10 to 90 μs and collected every 10 nm between 400 and 800 nm. The laser power was ~2 mJ per pulse and 30 sequential laser pulses were averaged at each collected wavelength.Kinetic traces were monitored in 0.5 M LiClO2–TiO2 core-shell films submerged in fixed NaClO4/NaI acetonitrile solutions with a total salt concentration of 0.5 M. The ratio between the two was varied to monitor IET at varied iodide concentrations between 0 and 0.5 M. The laser power was attenuated to ensure the amount of oxidized dye produced upon excitation was low and similar between all compounds studied, between 0.5 and 1 mJ per pulse. Single wavelength kinetic decays were collected at 580 nm, close to an isosbestic point of the electric field effect for all compounds to ensure only IET was monitored. The final data were averaged between 90 and 150 laser pulses, to achieve an acceptable signal to noise ratio.IET and BET kinetics were investigated using the functionalized SnOThe kinetics of charge recombination between injected electrons and triiodide were monitored at 375 nm in 0.5 M NaI from −10 μs to 1 s after pulsed laser excitation. The laser power was between 1 and 3 mJ per pulse. The final data were averaged over 500 laser pulses to achieve an acceptable signal to noise ratio.X-Me and X-Ar compounds, the procedures to prepare functionalized metal oxide thin film samples, and the computational methods employed in this investigation can be found in Supplementary Methods section of Supplementary Information.Detailed descriptions of the synthetic procedures to prepare and characterize the Supplementary InformationDescription Of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2