Genome-wide identification, subcellular localization, and expression analysis of the phosphatidyl ethanolamine-binding protein family reveals the candidates involved in flowering and yield regulation of Tartary buckwheat (Fagopyrum tataricum).

Background: PEBP (phosphatidyl ethanolamine-binding protein) is widely found in eukaryotes including plants, animals and microorganisms. In plants, the PEBP family plays vital roles in regulating flowering time and morphogenesis and is highly associated to agronomic traits and yields of crops, which has been identified and characterized in many plant species but not well studied in Tartary buckwheat (Fagopyrum tataricum Gaertn.), an important coarse food grain with medicinal value. Methods: Genome-wide analysis of FtPEBP gene family members in Tartary buckwheat was performed using bioinformatic tools. Subcellular localization analysis was performed by confocal microscopy. The expression levels of these genes in leaf and inflorescence samples were analyzed using qRT-PCR. Results: Fourteen Fagopyrum tataricum PEBP (FtPEBP) genes were identified and divided into three sub-clades according to their phylogenetic relationships. Subcellular localization analysis of the FtPEBP proteins in tobacco leaves indicated that FT- and TFL-GFP fusion proteins were localized in both the nucleus and cytoplasm. Gene structure analysis showed that most FtPEBP genes contain four exons and three introns. FtPEBP genes are unevenly distributed in Tartary buckwheat chromosomes. Three tandem repeats were found among FtFT5/FtFT6, FtMFT1/FtMFT2 and FtTFL4/FtTFL5. Five orthologous gene pairs were detected between F. tataricum and F. esculentum. Seven light-responsive, nine hormone-related and four stress-responsive elements were detected in FtPEBPs promoters. We used real-time PCR to investigate the expression levels of FtPEBPs among two flowering-type cultivars at floral transition time. We found FtFT1/FtFT3 were highly expressed in leaf and young inflorescence of early-flowering type, whereas they were expressed at very low levels in late-flowering type cultivars. Thus, we deduced that FtFT1/FtFT3 may be positive regulators for flowering and yield of Tartary buckwheat. These results lay an important foundation for further studies on the functions of FtPEBP genes which may be utilized for yield improvement.

Exogenous melatonin improves germination rate in buckwheat under high temperature stress by regulating seed physiological and biochemical characteristics.

The germinations of three common buckwheat (Fagopyrum esculentum) varieties and two Tartary buckwheat (Fagopyrum tataricum) varieties seeds are known to be affected by high temperature. However, little is known about the physiological mechanism affecting germination and the effect of melatonin (MT) on buckwheat seed germination under high temperature. This work studied the effects of exogenous MT on buckwheat seed germination under high temperature. MT was sprayed. The parameters, including growth, and physiological factors, were examined. The results showed that exogenous MT significantly increased the germination rate (GR), germination potential (GP), radicle length (RL), and fresh weight (FW) of these buckwheat seeds under high-temperature stress and enhanced the content of osmotic adjustment substances and enzyme activity. Comprehensive analysis revealed that under high-temperature stress during germination, antioxidant enzymes play a predominant role, while osmotic adjustment substances work synergistically to reduce the extent of damage to the membrane structure, serving as the primary key indicators for studying high-temperature resistance. Consequently, our results showed that MT had a positive protective effect on buckwheat seeds exposed to high temperature stress, providing a theoretical basis for improving the ability to adapt to high temperature environments.

Strigolactones affect the yield of Tartary buckwheat by regulating endogenous hormone levels.

BACKGROUND: As a newly class of endogenous phytohormones, strigolactones (SLs) regulate crop growth and yield formation by interacting with other hormones. However, the physiological mechanism of SLs affect the yield by regulating the balance of endogenous hormones of Tartary buckwheat is still unclear. RESULTS: In this study, a 2-year field experiment was conducted on Tartary buckwheat (Jinqiao 2) to study the effects of different concentrations (0, 10, and 20 µmol/L) of artificial synthetic analogs of SLs (rac-GR24) and inhibitor of SL synthesis (Tis-108) on the growth, endogenous-hormone content, and yield of Tartary buckwheat. The main-stem branch number, grain number per plant, grain weight per plant, and yield of Tartary buckwheat continuously decreased with increased rac-GR24 concentration, whereas the main-stem diameter and plant height initially increased and then decreased. Rac-GR24 treatment significantly increased the content of SLs and abscisic acid (ABA) in grains, and it decreased the content of Zeatin (Z) + Zeatin nucleoside (ZR). Conversely, Tis-108 treatment decreased the content of SLs and ABA but increased the content of Z + ZR. Results of correlation analysis showed that the content of ABA and SLs, the ratio of SLs/(Z + ZR), SLs/ABA, and ABA/(Z + ZR) were significantly negatively correlated with the yield of Tartary buckwheat, and that Z + ZR content was significantly positively correlated with the yield. Regression analysis further showed that ABA/ (Z + ZR) can explain 58.4% of the variation in yield. CONCLUSIONS: In summary, by adjusting the level of endogenous SLs in Tartary buckwheat, the balance of endogenous hormones in grains can be changed, thereby exerting the effect on yield. The results can provide a new agronomic method for the high-yield cultivation of Tartary buckwheat.

Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Embryo Rescue Technique in Buckwheat Fagopyrum esculentum Moench.

Culture of immature buckwheat embryos is an excellent explant for tissue culture and regeneration in vitro, as well as a method of embryo rescue in case of fruit abortion especially in interspecific crosses. Here, we describe the method of immature embryo rescue using a differentiated approach to different age groups. The method involves the development of embryos along the path of direct embryogenesis bypassing the callus stage.

Visualization of Fagopyrum esculentum and Fagopyrum tataricum Chromosomes and Micronuclei.

This chapter presents the squash chromosome preparation technique for Fagopyrum esculentum and F. tataricum, using the root tips as the source of the material. Using an optimized version of this method, the chromosomes are free of cytoplasmic debris and are spread evenly on the glass slide. What comes of it is the possibility to make observations of the chromosome number and structure at the metaphase stage. This technique's modified version allows micronuclei analysis in interphase cells of buckwheats.

Tartary buckwheat rutin: Accumulation, metabolic pathways, regulation mechanisms, and biofortification strategies.

Rutin is a significant flavonoid with strong antioxidant property and various therapeutic effects. It plays a crucial role in disease prevention and human health maintenance, especially in anti-inflammatory, antidiabetic, hepatoprotective and cardiovascular effects. While many plants can synthesize and accumulate rutin, tartary buckwheat is the only food crop possessing high levels of rutin. At present, the rutin content (RC) is regarded as the key index for evaluating the nutritional quality of tartary buckwheat. Consequently, rutin has become the focus for tartary buckwheat breeders and has made considerable progress. Here, we summarize research on the rutin in tartary buckwheat in the past two decades, including its accumulation, biosynthesis and breakdown pathways, and regulatory mechanisms. Furthermore, we propose several strategies to increase the RC in tartary buckwheat seeds based on current knowledge. This review aims to provide valuable references for elevating the quality of tartary buckwheat in the future.

Potential relation between starch granule-associated proteins and retrogradation properties of buckwheat starch.

To elucidate the effect of starch granule-associated proteins (SGAPs) on retrogradation properties of buckwheat starch, the retrogradation properties of Tartary buckwheat starch (TBS) and common buckwheat starch (CBS) before and after removal of SGAPs were systematically investigated, with wheat starch (WS) as reference. A significant decrease in gel strength of starches and density of starch aggregates were observed after removing SGAPs. The results were in line with the changes in retrogradation enthalpy of starches and short-range ordered structure of starch aggregates. After removing SGAPs, the retrogradation enthalpy of TBS decreased from 4.16 J/g to 3.74 J/g, CBS decreased from 4.05 J/g to 3.35 J/g and WS decreased from 3.27 J/g to 2.81 J/g, respectively. Taken together the results of LF-NMR, FTIR and rheological analysis, it can be concluded that SGAPs could promote the hydrogen bond interactions between starch molecules by competitively binding with water molecules, enhancing the rearrangement of starch molecules and forming a more ordered structure. Overall, the study suggested that the presence of SGAPs could enhanced the interaction between starch molecules chains, thus accelerated the retrogradation process. The research results provide more information about SGAPs in buckwheat starch and support further study for manipulation of starch properties.

Genomic insight into the origin, domestication, dispersal, diversification and human selection of Tartary buckwheat.

BACKGROUND: Tartary buckwheat, Fagopyrum tataricum, is a pseudocereal crop with worldwide distribution and high nutritional value. However, the origin and domestication history of this crop remain to be elucidated. RESULTS: Here, by analyzing the population genomics of 567 accessions collected worldwide and reviewing historical documents, we find that Tartary buckwheat originated in the Himalayan region and then spread southwest possibly along with the migration of the Yi people, a minority in Southwestern China that has a long history of planting Tartary buckwheat. Along with the expansion of the Mongol Empire, Tartary buckwheat dispersed to Europe and ultimately to the rest of the world. The different natural growth environments resulted in adaptation, especially significant differences in salt tolerance between northern and southern Chinese Tartary buckwheat populations. By scanning for selective sweeps and using a genome-wide association study, we identify genes responsible for Tartary buckwheat domestication and differentiation, which we then experimentally validate. Comparative genomics and QTL analysis further shed light on the genetic foundation of the easily dehulled trait in a particular variety that was artificially selected by the Wa people, a minority group in Southwestern China known for cultivating Tartary buckwheat specifically for steaming as a staple food to prevent lysine deficiency. CONCLUSIONS: This study provides both comprehensive insights into the origin and domestication of, and a foundation for molecular breeding for, Tartary buckwheat.

Accumulation of the bitter substance quercetin mediated by the overexpression of a novel seed-specific gene FtRDE2 in Tartary buckwheat.

Tartary buckwheat (Fagopyrum tataricum) is frequently employed as a resource to develop health foods, owing to its abundant flavonoids such as rutin. However, the consumption of Tartary buckwheat (TB) is limited in food products due to the strong bitterness induced by the hydrolysis of rutin into quercetin. This transformation is facilitated by the degrading enzyme (RDE). While multiple RDE isoenzymes exist in TB, the superior coding gene of FtRDEs has not been fully explored, which hinders the breeding of TB varieties with minimal bitterness. Here, we found that FtRDE2 is the most abundant enzyme in RDE crude extracts, and its corresponding gene is specifically expressed in TB seeds. Results showed that FtRDE2 has strong rutin hydrolysis activity. Overexpression of FtRDE2 not only significantly promoted rutin hydrolysis and quercetin accumulation but also dramatically upregulated genes involved in the early phase of flavonoid synthesis (FtPAL1、FtC4H1、Ft4CL1, FtCHI1) and anthocyanin metabolism (FtDFR1). These findings elucidate the role of FtRDE2, emphasizing it as an endogenous factor contributing to the bitterness in TB and its involvement in the metabolic regulatory network. Moreover, correlation analysis revealed a positive relationship between the catalytic activity of RDE extracts and the expression level of FtRDE2 during seed germination. In summary, our results suggest that FtRDE2 can serve as a promising candidate for the molecular breeding of a TB variety with minimal bitterness.

Growth performance, lipid metabolism, and systemic immunity of weaned piglets were altered by buckwheat protein through the modulation of gut microbiota.

Tartary buckwheat protein (BWP) is well known for the wide-spectrum antibacterial activity and the lipid metabolism- regulating property; therefore, BWP can be applied as feed additives to improve the animal's nutritional supply. With the aim to investigate the bioactive actions of the BWP, growth performance, lipid metabolism and systemic immunity of the weaned piglets were measured, and the alterations of pig gut microbiota were also analyzed. According to the results, the growth performances of the weaned piglets which were calculated as the average daily gain (ADG) and the average daily feed intake (ADFI) were significantly increased when compared to the control group. Simultaneously, the serum levels of the total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were decreased, while the levels of high-density lipoprotein cholesterol (HDL-C) were increased in the BWP group. Moreover, the relative abundances of Lactobacillus, Prevotella_9, Subdoligranulum, Blautia, and other potential probiotics in the gut microbiota of weaned piglets were obviously increased in the BWP group. However, the relative abundances of Escherichia-Shigella, Campylobacter, Rikenellaceae_RC9_gut_group and other opportunistic pathogens were obviously decreased in the BWP group. In all, BWP was proved to be able to significantly improve the growth performance, lipid metabolism, and systemic immunity of the weaned piglets, and the specific mechanism might relate to the alterations of the gut microbiota. Therefore, BWP could be explored as a prospective antibiotic alternative for pig feed additives.

Fabrication and characterization of alginate-zein core-shell microcapsules for controlled release of buckwheat honey.

In this study, extrusion method was employed to fabricate alginate-zein core-shell microcapsules loaded with buckwheat honey by dropping alginate and buckwheat honey mixture solution into a 70.0 % zein ethanol solution(v/v) containing 5.0 % CaCl2 solution (wt%). The microcapsules were constructed by two parts: 1) the formation of hydrophilic beads through the crosslinking of alginate chains with Ca2+; 2) the introduction of alginate beads into the aqueous zein ethanol solution which decreased the ethanol concentration, prompting the precipitation of zein and the deposition of zein nanoparticles onto the surfaces of alginate beads. Comparing with the alginate beads, the prepared microcapsules not only possessed better water-holding capacity, but also achieved controlled release of buckwheat honey. Importantly, the microcapsules significantly retained the antioxidant activity of the buckwheat honey. Therefore, this innovative method for fabricating alginate-zein core-shell microcapsules can suggest a promising approach to broaden the application of buckwheat honey in the food field.

Effect of high moisture extrusion on the structure and physicochemical properties of Tartary buckwheat protein and its in vitro digestion.

Tartary buckwheat is rich in nutrients and its protein supports numerous biological functions. However, the digestibility of Tartary buckwheat protein (TBP) poses a significant limitation owing to its inherent structure. This study aimed to assess the impact of high moisture extrusion (HME, 60 % moisture content) on the structural and physicochemical attributes, as well as the in vitro digestibility of TBP. Our results indicated that TBP exhibited unfolded and amorphous microstructures after HME. The protein molecular weight of TBP decreased after HME, and a greater degradation was observed at 70 °C than 100 °C. In particular, HME at 70 °C caused an almost complete disappearance of bands near 35 kDa compared with HME at 100 °C. In addition, compared with native TBP (NTBP, 44.53 µmol/g protein), TBP subjected to HME at 70 °C showed a lower disulfide bond (SS) content (42.67 µmol/g protein), whereas TBP subjected to HME at 100 °C demonstrated a higher SS content (45.70 µmol/g protein). These changes endowed TBP with good solubility (from 55.96 % to 83.31 % at pH 7), foaming ability (20.00 %-28.57 %), and surface hydrophobicity (8.34-23.07). Furthermore, the emulsifying activity (EA) and in vitro digestibility are closely related to SS content. Notably, extruded TBP (ETBP) obtained at 70 °C exhibited higher EA and digestibility than NTBP, whereas ETBP obtained at 100 °C showed the opposite trend. Consequently, HME (especially at 70 °C) demonstrated significant potential as a processing technique for improving the functional and digestive properties of TBP.

Pseudocereal Oils, Authenticated by Fourier Transform Infrared Spectroscopy, and their Chemopreventive Properties.

Amaranth, quinoa, and buckwheat are the representatives of pseudocereals, different parts and by-products of which are used in daily nutrition and food processing industry. However, only scarce information exists on the bioactivity of their oils. Thus, oils obtained from amaranth, buckwheat, and red, yellow, and white quinoa seeds were evaluated in terms of their nutritional (fatty acid profile, squalene), cytotoxic (against normal and neoplastic gastrointestinal, prostate, and skin cells), anti-inflammatory and antiradical (interleukin 6, TNF-alpha, nitric oxide, DPPH, Total phenolics, and superoxide dismutase) potential in the in vitro model. Linoleic (42.9-52.5%) and oleic (22.5-31.1%) acids were the two main unsaturated, while palmitic acid (4.9-18.6%) was the major saturated fatty acid in all evaluated oils. Squalene was identified in all evaluated oils with the highest content in amaranth oil (7.6 g/100 g), and the lowest in buckwheat oil (2.1 g/100 g). The evaluated oils exerted a high direct cytotoxic impact on cancer cells of different origins, but also revealed anti-inflammatory and antiradical potentials. Yellow quinoa oil was the most active, especially toward skin (A375; IC50 6.3 µg/mL), gastrointestinal (HT29 IC50 4.9 µg/mL), and prostate cancer cells (LNCaP IC50 7.6 µg/mL). The observed differences in the activity between the oils from the tested quinoa varieties deserve further studies. High selectivity of the oils was noted, which indicates their safety to normal cells. The obtained results indicate that the oils are good candidates for functional foods with perspective chemopreventive potential.

The extraction of buckwheat protein and its interaction with kappa-carrageenan: Textural, rheological, microstructural, and chemical properties.

Current plant-based foods use plant proteins as a key structuring and texturing ingredient. The use of water for extraction can replace conventional protein extraction methods. Water extraction of protein is environmentally friendly and could prevent the loss of protein functionality due to extreme pH changes. This study demonstrates an aqueous extraction method, coupled with ultrasound as pre-treatment, to obtain buckwheat protein (BWPE) and assess its gelling property and composited gel with kappa-carrageenan (k-carr). Textural and rheological analyses showed that the hardness and storage modulus of the composited gel containing 1 % w/w BWPE and 1 % w/w k-carr was 4.2-fold and 100-fold, respectively, higher than k-carr gel at 1 % w/w. Light microscopy showed a mixed bi-continuous gel system, with k-carr reinforcing the protein gel network. Besides volume exclusion effects, chemical bond and FTIR analyses revealed that adding k-carr to BWPE altered the protein's secondary structure and mediated protein denaturation during heating. This results in greater ß-sheet content, which creates a more organised gel structure. These results demonstrated that ultrasound-assisted water-extracted BWPE, together with varying concentrations of k-carr, can be used to develop composited gels of tailorable textural and rheological properties to suit different food applications.

Tartary buckwheat protein-phenol conjugate prepared by alkaline-based environment: Identification of covalent binding sites of phenols and alterations in protein structural and functional characteristics.

Tartary buckwheat protein-rutin/quercetin covalent complex was synthesized in alkaline oxygen-containing environment, and its binding sites, conformational changes and functional properties were evaluated by multispectral technique and proteomics. The determination of total sulfhydryl and free amino groups showed that rutin/quercetin can form a covalent complex with BPI and could significantly reduce the group content. Ultraviolet-visible spectrum analysis showed that protein could form new characteristic peaks after binding with rutin/quercetin. Circular dichroism spectrum analysis showed that rutin and quercetin caused similar changes in the secondary structure of proteins, both promoting ß-sheet to α-helix, ß-ture and random coil transformation. The fluorescence spectrometry results showed that the combination of phenols can cause the fluorescence quenching, and the combination of rutin was stronger than the quercetin. Proteomics showed that there were multiple covalent binding sites between phenols and protein. Rutin had a high affinity for arginine, and quercetin and cysteine had high affinity. Meanwhile, the combination of rutin/quercetin and protein had reduced the surface hydrophobic ability of the protein, and improved the foaming, stability and antioxidant properties of the protein. This study expounded the mechanism of the combination of BPI and rutin/quercetin, and analysed the differences of the combination of protein and phenols in different structures. The findings can provide a theoretical basis for the development of complexes in the area of food.

Assessment of tropane alkaloid levels in Brazilian buckwheat flour products: A novel LC-UHPLC-MS/MS approach using solid-liquid extraction at low temperature.

In recent years, the monitoring of tropane alkaloids, specifically hyoscyamine and scopolamine, in food has become a pressing concern. This is due to increasing reports of food contamination with these compounds worldwide, raising awareness about the potential risks associated with their consumption. A novel method is proposed here for the determination of the sum of (+)-hyoscyamine, (-)-hyoscyamine, and (-)-scopolamine in buckwheat-based matrices, using solid-liquid extraction at low temperature and quantification by bidimensional chromatography coupled to tandem mass spectrometry. The validated method presented a linear response in the concentration range of 2.5-15 µg kg-1 (r > 0.99). The precision and accuracy were in the ranges from 0.8 to 11.0 % and from 96 to 103 %, respectively. The limit of quantification (LOQ) was 2.5 µg kg-1. No contamination was found at levels above the LOQ in any of the 18 samples analyzed (buckwheat flour, grains, and gluten-free mix).

Platforms of graphene/MXene heterostructure for electrochemical monitoring of rutin in drug and Tartary buckwheat tea.

The use of two-dimensional heterostructure composite as electrode modification material has become a new strategy to improve the electrocatalytic activity and electroactive sites of electrochemical sensor. Herein, a soluble heterostructure, namely rGO-PSS@MXene, was designed and synthesized by integrating poly (sodium p-styrenesulfonate)-functionalized reduced graphene oxide into MXene nanosheets via ultrasonic method. The interactive heterostructure can effectively alleviate the self-stacking of MXene and rGO, endowing them with superior electron transfer capacity and large specific surface area, thereby producing prominent synergistic electrocatalytic effect towards rutin. In addition, the excellent enrichment effect of rGO-PSS@MXene for rutin also plays an important role through the electrostatic and π-π stacking interactions. The electrochemical characteristics of rutin on the sensor were examined in detail and a sensitive sensing method was proposed. Under optimized conditions, the method showed satisfactory linear relationship for rutin in the concentration range of 0.005-10.0 µM, with limit of detection of 1.8 nM (S/N = 3). The quantitative validation results in herbal medicine and commercial Tartary buckwheat tea were highly consistent with the labeled quantity and the results of HPLC determination, respectively, suggesting the sensor possessed excellent selectivity and accuracy. This proposed strategy for rutin determination is expected to expand the application of MXene heterostructure in electrochemical sensors, and is envisioned as a promising candidate for quality monitoring of drugs and foods.

Comparative study of dietary phenols with Tartary buckwheat protein (2S/13S): impact on structure, binding sites and functionality of protein.

BACKGROUND: This research was to investigate the interaction mechanism between 2S albumin and 13S globulin (2S and 13S, the most important storage proteins in Tartary buckwheat seeds) and three phenols (rutin, quercetin and myricetin) regarding the structural and antioxidant properties of their complexes. RESULTS: There are differences in the binding affinity of phenols for 2S and 13S. Rutin had a higher binding affinity for 2S, myricetin had a higher binding affinity for 13S, and 13S exhibited a higher affinity toward phenols than did 2S. Binding with phenols significantly changed the secondary and tertiary structures of 2S and 13S, decreased the surface hydrophobic value and enhanced the antioxidant capacity. Molecular docking and isothermal titration calorimetry showed that the binding processes were spontaneous and that there were hydrogen bonds, hydrophobic bonds and van der Waals force interactions between phenols and proteins. CONCLUSION: These findings could provide meaningful guidance for the further application of buckwheat protein complex. © 2023 Society of Chemical Industry.

Variations in chemical composition of common buckwheat (Fagopyrum esculentum Moench) as a result of different environmental conditions.

BACKGROUND: Common buckwheat (Fagopyrum esculentum Moench) is a pseudo cereal that is gaining interest in the world. The chemical profile of common buckwheat determines its high nutritional and health-promoting value. The accumulation of these valuable ingredients depends on many factors, such as: variety, location of cultivation and related weather and agrotechnical conditions. Due to the growing interest in common buckwheat as a natural plant material for food production, it is important to know the factors affecting the quantitative and qualitative composition of its grains. The aim of the research was to determine the effect of the genotype (G), environment (E) and G × E interaction on the content of nutrients (protein, starch, ash, lipids) and bioactive components [dietary fiber (DF), total phenolic content (TPC)] in the common buckwheat grains. The study covered four cultivars grown in three locations for three consecutive vegetation seasons (2016/2017, 2017/2018, 2018/2019). RESULTS: Based on the obtained results, a significant influence of the environment and G × E interaction on the content of the studied parameters was found. The greatest impact on the diversity of the content of nutrients had environmental conditions, which in the case of protein and ash determined these features in more than 80%, and in the case of starch, 70%. With regard to bioactive compounds, the greatest influence of the environment was observed for the amount of TPC (78%), lignin (51%) and the DF complex (56%). CONCLUSION: The obtained results are useful for breeders working on expanding the pool of common buckwheat genotypes, stable in changing environmental conditions. © 2023 Society of Chemical Industry.