Comparison of Different Chemical Extraction Methods for Predicting the Bioavailability and Phytotoxicity of Soil PAEs to Green Vegetables (Brassica Rapa Var. Chinensis).

The aim of the present study was to compare the predictive ability of four chemical extraction methods, i.e., Tenax, hydroxypropyl[ß]cyclodextrin (HPCD), n-butanol and low-molecular-weight-organic-acids (LMWOA), for predicting the bioavailability and phytotoxicity of soil phthalic acid esters to the green vegetable Shanghaiqing (SHQ). Results showed that the extraction ability of different extraction methods varies significantly. For dibutyl phthalate (DBP), the extraction ability followed the order of Tenax > LMWOA > HPCD > n-butanol. For di-(2-ethylhexyl) phthalate (DEHP), the order of the extraction ability was n-butanol > HPCD > Tenax > LMWOA. All the extraction methods underestimated the DBP concentration while overestimating the DEHP concentration accumulated by SHQ. The concentrations of DBP and DEHP extracted by Tenax were most related to the concentrations accumulated by SHQ and the phytotoxicity indicators of SHQ. Tenax can serve as a good chemical extractant to assess the bioavailability and phytotoxicity of soil DBP and DEHP to SHQ.

Effects of dibutyl phthalate contamination on physiology, phytohormone homeostasis, rhizospheric and endophytic bacterial communities of Brassica rapa var. chinensis.

Phthalates are plasticizers and are ubiquitously detected in the environment, frequently at mg/kg levels. The present study aimed to evaluate the effects of dibutyl phthalate (DBP) on germination, growth, enzyme activity, phytohormone homeostasis and bacterial communities of two cultivars of Brassica rapa var. chinensis. The germination rate was decreased up to 20% compared to the control, and the growth of the vegetables was severely inhibited at the early stage when exposed to DBP at 20 mg/kg. Antioxidant defense enzyme activities and malondialdehyde (MDA) content increased upon exposure to DBP. A dose-response of auxin (IAA) was observed after a 2 d exposure. Gibberellin (GA3) and abscisic acid (ABA) responded at day 10 under DBP stress. GA3 did not show a clear dose-response effect and ABA increased about 3 times as the DBP concentration increased from 2 to 20 mg/L. Microbial population shifts were observed, especially in rhizosphere soil and roots. No obvious change occurred for the α diversity of rhizospheric bacteria among different treatments. Chao1, Shannon and Simpson indices of the root endophytic bacteria showed a decreasing trend with increasing DBP supplementation, while all the indices increased in shoot endophytic bacteria in comparison to the control. The results indicated that exposure to DBP may compromise the fitness of the leafy vagetables and alter the endophytic and rhizospheric bacteria, which might further affect the nutrients of the vegetables and alter ecosystem functions.

Presence, distribution and risk assessment of phthalic acid esters (PAEs) in suburban plastic film pepper-growing greenhouses with different service life.

The widespread usage of plastic film increased the content of phthalic acid esters (PAEs) in the environment, causing PAE residue in vegetables and subsequently increasing health risks to humans when consuming them. In this work, the presence, distribution and risk assessment of 15 PAEs in soils and peppers from suburban plastic film pepper-growing greenhouses were investigated. The total PAE contents in soil and pepper samples ranged from 320.1 to 971.2 µg/kg (586.3 µg/kg on average) and from 196.6 to 304.2 µg/kg (245.4 µg/kg on average), respectively. Di (2-ethyl)hexyl, dibutyl and diisobutyl phthalates (DEHP, DnBP and DiBP, respectively) were the most abundant in both soil and pepper samples. Specifically, DEHP showed the highest content in soils, while the DnBP content was the highest in peppers. The total PAE content in soils from pepper-greenhouses was much lower than in the agricultural soils mulched with plastic films, but significantly higher than in the agricultural soils from open uncovered fields. The total PAE content in peppers decreased as the service life of plastic film greenhouses increased. Correlation analysis suggested that the difference in distribution and accumulation behaviors of individual PAEs in greenhouse systems was correlated with their physicochemical properties. The non-cancer and carcinogenic risks of priority PAEs show low risks of PAEs detected in pepper and soil samples from the suburban plastic film greenhouses to human health.

Use of Bacillus-siamensis-inoculated biochar to decrease uptake of dibutyl phthalate in leafy vegetables.

Dibutyl phthalate (DBP) is a frequently detected farmland contaminant that is harmful to the environment and human health. In this study, a DBP-degrading endophytic Bacillus siamensis strain T7 was immobilized in rice husk-derived biochar for bioremediation of DBP-polluted agricultural soils. The effects of this microbe-biochar composite on the soil prokaryotic community and the mechanism by which it regulates DBP degradation, were also investigated. A supplement of T7-biochar composite not only significantly boosted DBP biodegradation in soil by raising the DBP degradation rate constant and half-life from 0.1979 d-1 and 2.3131 d to 0.2434 d-1 and 2.1062 d, respectively, but also impeded DBP uptake by leafy vegetables. The general bioremediation effect of T7-biochar alliance excelled pure T7 suspensions and biochar, by trapping more DBP and boosting its complete degradation in soil. Besides, the combination of strain T7 and biochar can increase the proportion of some beneficial bacteria and boost the functional diversity of soil prokaryotic community, then to a certain extent may reverse the negative effect of DBP pollution on the agricultural soils. These results indicate that the rice-husk-derived biochar is a proper media when utilizing functional microbes into environmental treatment. Overall, T7-biochar composite is a promising soil modifier for soil bioremediation and the production of DBP-free crops.

Major factors dominating the fate of dibutyl phthalate in agricultural soils.

Dibutyl phthalate (DBP) is a ubiquitous soil contaminant. We have investigated the sorption, degradation and residue of DBP in 20 types of agricultural soils and aimed to identify the major soil properties that dominate the fate of DBP. Sorption isotherms of DBP in all soils were fitted well with the Freundlich model. The sorption coefficient (Kf) varied between 3.99 and 36.1 mg1-1/nL1/n/kg. Path analysis indicated that 59.9% of variation in Kf could be explained by the combination of pH, organic carbon (OC) and clay content. Degradation of DBP in the 20 soils was well described by the first-order kinetic model, with half-lives (t1/2) ranging from 0.430 to 4.99 d. The residual DBP concentration after 60 d of incubation (R60) ranged from 0.756 to 2.15 mg/kg and the residual rates ranged from 3.97% to 9.63%. The Kf value was significantly positively correlated with t1/2 and R60. Moreover, soil pH, microbial biomass carbon (Cmic) and OC were identified as dominating factors that explained 84.4% of variation in t1/2. The R60 data indicated 72.2% of its variability attributable to the combination of OC and Cmic. The orders of the relative importance of dominating factors on the Kf, t1/2 and R60 were OC > pH > clay, Cmic > pH > OC and OC > Cmic, respectively. This work contributes to better understand the fate of DBP in soils and make scientific decisions about accelerating its dissipation in different soils.

Di-n-butyl phthalate degrading endophytic bacterium Bacillus amyloliquefaciens subsp. strain JR20 isolated from garlic chive and its colonization in a leafy vegetable.

Di-n-butyl phthalate (DBP) is one of the primary PAEs (phthalate acid esters) pollutants. DBP can be absorbed by plants and threaten human health via the food chain. Some DBP-degrading bacteria have been successfully isolated from the environment (water, soil, etc.). However, only a few DBP-degrading plant endophytes have been isolated. In this study, an endophytic bacterium, Bacillus amyloliquefaciens subsp. strain JR20, which was found capable of degrading DBP, was isolated from garlic chive. We found that strain JR20 metabolized 89.74% of DBP at a 5 mg/L concentration within 4 d in liquid mineral salts medium (MSM). The optimized conditions for maximum removal of DBP were as follows: DBP concentration, 5 mg/L; pH, 7-8; temperature, 30-40 °C. The colonization of strain JR20 significantly improved the degradation rate of DBP in the roots, stems and leaves of leafy vegetables.

Genome-wide identification, phylogeny and expression analyses of SCARECROW-LIKE(SCL) genes in millet (Setaria italica).

As a member of the GRAS gene family, SCARECROW-LIKE (SCL) genes encode transcriptional regulators that are involved in plant information transmission and signal transduction. In this study, 44 SCL genes including two SCARECROW genes in millet were identified to be distributed on eight chromosomes, except chromosome 6. All the millet genes contain motifs 6-8, indicating that these motifs are conserved during the evolution. SCL genes of millet were divided into eight groups based on the phylogenetic relationship and classification of Arabidopsis SCL genes. Several putative millet orthologous genes in Arabidopsis, maize and rice were identified. High throughput RNA sequencing revealed that the expressions of millet SCL genes in root, stem, leaf, spica, and along leaf gradient varied greatly. Analyses combining the gene expression patterns, gene structures, motif compositions, promoter cis-elements identification, alternative splicing of transcripts and phylogenetic relationship of SCL genes indicate that the these genes may play diverse functions. Functionally characterized SCL genes in maize, rice and Arabidopsis would provide us some clues for future characterization of their homologues in millet. To the best of our knowledge, this is the first study of millet SCL genes at the genome wide level. Our work provides a useful platform for functional analysis of SCL genes in millet, a model crop for C4 photosynthesis and bioenergy studies.

Heavy metals in navel orange orchards of Xinfeng County and their transfer from soils to navel oranges.

This study investigated heavy metal concentrations in soils and navel oranges of Xinfeng County, a well-known navel orange producing area of China. The results showed that the average concentrations of lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As) and mercury (Hg) in orchard soils all increased compared to the regional background values, especially for Cd, which increased by 422%. When compared to the Chinese Environmental Quality Standard for soil (GB15618-1995), Pb, Cr and Hg concentrations in all orchard soil samples were below the limit standards, but Cd concentrations in 24 soil samples (21%) and As concentrations in 8 soil samples (7%) exceeded the limit standards. However, concentrations of all heavy metals in navel orange pulps were within the National Food Safety Standard of China (GB 2762-2012). Dietary risk assessment also showed that the exposure to these five heavy metals by consumption of navel oranges could hardly pose adverse health effects on adults and children. Since the range and degree of soil Cd pollution was widest and the most severe of all, Cd was taken as an example to reveal the transfer characteristics of heavy metals in soil-navel orange system. Cd concentrations in different organs of navel orange trees decreased in the following order: root>leaf>peel>pulp. That navel oranges planted in the Cd contaminated soils were within the national food safety standard was mainly due to the low transfer factor for Cd from soil to pulp (TFpulp). Further studies showed that TFpulp was significantly negatively correlated with soil pH, organic carbon (OC) and cation exchange capacity (CEC). Based on these soil properties, a prediction equation for TFpulp was established, which indicated that the risk for Cd concentration of navel orange pulp exceeding the national food limit is generally low, when soil Cd concentration is below 7.30 mg/kg. If appropriate actions are taken to increase soil pH, OC and CEC, Cd concentrations in navel orange pulps could be further reduced.

C4 rice engineering, beyond installing a C4 cycle.

C4 photosynthesis in higher plants is carried out by two distinct cell types: mesophyll cells and bundle sheath cells, as a result highly concentrated carbon dioxide is released surrounding RuBisCo in chloroplasts of bundle sheath cells and the photosynthetic efficiency is significantly higher than that of C3 plants. The evolution of the dual-cell C4 cycle involved complex modifications to leaf anatomy and cell ultra-structures. These include an increase in leaf venation, the formation of Kranz anatomy, changes in chloroplast morphology in bundle sheath cells, and increases in the density of plasmodesmata at interfaces between the bundle sheath and mesophyll cells. It is predicted that cereals will be in severe worldwide shortage at the mid-term of this century. Rice is a staple food that feeds more than half of the world's population. If rice can be engineered to perform C4 photosynthesis, it is estimated that rice yield will be increased by at least 50% due to enhanced photosynthesis. Thus, the Second Green Revolution has been launched on this principle by genetically installing C4 photosynthesis into C3 crops. The studies on molecular mechanisms underlying the changes in leaf morphoanatomy involved in C4 photosynthesis have made great progress in recent years. As there are plenty of reviews discussing the installment of the C4 cycle, we focus on the current progress and challenges posed to the research regarding leaf anatomy and cell ultra-structure modifications made towards the development of C4 rice.

Two aquaporins, PIP1;1 and PIP2;1, mediate the uptake of neonicotinoid pesticides in plants.

Neonicotinoids (NEOs), a large class of organic compounds, are a type of commonly used pesticide for crop protection. Their uptake and accumulation in plants are prerequisites for their intra- and intercellular movements, transformation, and function. Understanding the molecular mechanisms that underpin NEO uptake by plants is crucial for effective application, which remains elusive. Here, we demonstrate that NEOs enter plant cells primarily through the transmembrane symplastic pathway and accumulate mainly in the cytosol. Two plasma membrane intrinsic proteins discovered in Brassica rapa, BraPIP1;1 and BraPIP2;1, were found to encode aquaporins (AQPs) that are highly permeable to NEOs in different plant species and facilitate NEO subcellular diffusion and accumulation. Their conserved transport function was further demonstrated in Xenopus laevis oocyte and yeast assays. BraPIP1;1 and BraPIP2;1 gene knockouts and interaction assays suggested that their proteins can form functional heterotetramers. Assessment of the potential of mean force indicated a negative correlation between NEO uptake and the energy barrier of BraPIP1;1 channels. This study shows that AQPs transport organic compounds with greater osmolarity than previously thought, providing new insight into the molecular mechanisms of organic compound uptake and facilitating innovations in systemic pesticides.

A survey of immunohistochemical biomarkers for basal-like breast cancer against a gene expression profile gold standard.

Gene expression profiling of breast cancer delineates a particularly aggressive subtype referred to as 'basal-like', which comprises ∼15% of all breast cancers, afflicts younger women and is refractory to endocrine and anti-HER2 therapies. Immunohistochemical surrogate definitions for basal-like breast cancer, such as the clinical ER/PR/HER2 triple-negative phenotype and models incorporating positive expression for CK5 (CK5/6) and/or EGFR are heavily cited. However, many additional biomarkers for basal-like breast cancer have been described in the literature. A parallel comparison of 46 proposed immunohistochemical biomarkers of basal-like breast cancer was performed against a gene expression profile gold standard on a tissue microarray containing 42 basal-like and 80 non-basal-like breast cancer cases. Ki67 and PPH3 were the most sensitive biomarkers (both 92%) positively expressed in the basal-like subtype, whereas CK14, IMP3 and NGFR were the most specific (100%). Among biomarkers surveyed, loss of INPP4B (a negative regulator of phosphatidylinositol signaling) was 61% sensitive and 99% specific with the highest odds ratio (OR) at 108, indicating the strongest association with basal-like breast cancer. Expression of nestin, a common marker of neural progenitor cells that is also associated with the triple-negative/basal-like phenotype and poor breast cancer prognosis, possessed the second highest OR at 29 among the 46 biomarkers surveyed, as well as 54% sensitivity and 96% specificity. As a positively expressed biomarker, nestin possesses technical advantages over INPP4B that make it a more ideal biomarker for identification of basal-like breast cancer. The comprehensive immunohistochemical biomarker survey presented in this study is a necessary step for determining an optimized surrogate immunopanel that best defines basal-like breast cancer in a practical and clinically accessible way.

Uptake kinetics and subcellular distribution of three classes of typical pesticides in rice plants.

Food safety problems caused by pesticide residues have always been a concern for many people. In this study, we investigated the uptake, translocation and subcellular distribution of neonicotinoid insecticides, triazole fungicides, and sulfonylurea herbicides in rice plants (Oryza sativa L.). The time-dependent uptake kinetics of the three categories of pesticides with different molecular structures fit a first-order one-compartment kinetic model. The neonicotinoids (log Kow -0.66-0.8) were mainly concentrated in the leaves, and the triazoles (log Kow 3.72-4.4) were mainly concentrated in the roots. Neonicotinoid pesticides in the roots were preferentially transported across the membrane through the symplastic pathway; triazole pesticides except for triadimefon and myclobutanil preferentially passed through the symplastic pathway; and sulfonylurea pesticides (log Kow 0.034-2.89) were first transported upward through the apoplastic pathway. In the roots, neonicotinoids, triazoles, and sulfonylurea herbicides were mainly concentrated in the soluble fractions, cell wall and apoplast fractions, respectively. In addition, there was a high positive correlation between the subcellular distribution of pesticides in the roots, stems and leaves. Molecular weight and log Kow jointly affected the enrichment of triazole pesticides in the roots, stems and leaves and the transfer from stems to leaves, while water solubility and log Kow commonly affected neonicotinoids. There was a correlation between pesticide absorption and the molecular structures of pesticides. To develop pesticides with strong uptake and transport capabilities, it is necessary to consider that the electronegativity of some atoms is stronger, the sum of the topological indices of heteroatoms can be large, and the van der Waals volume increases accordingly.

Functional characterization of rice (Oryza sativa) thioredoxins for detoxification and degradation of atrazine.

Thioredoxins (TRXs) are a group of antioxidant enzymes that play a critical role in plant growth and resistance to stress. However, the functional role and mechanism of rice TRXs in response to pesticides (e.g. atrazine, ATZ) stress remain largely unexplored. Here, 24 differentially expressed TRX genes (14 up and 10 down) of ATZ-exposed rice were identified through high-throughput RNA-sequencing analysis. Twenty-four TRX genes were unevenly mapped to 11 chromosomes and some of the genes were validated by quantitative RT-PCR. Bioinformatics analysis revealed that ATZ-responsive TRX genes contain multiple functional cis-elements and conserved domains. To demonstrate the functional role of the genes in ATZ degradation, one representative TRX gene LOC_Os07g08840 was transformed into yeast cells and observed significantly lower ATZ content compared to the control. Using LC-Q-TOF-MS/MS, five metabolites were characterized. One hydroxylation (HA) and two N-dealkylation products (DIA and DEA) were significantly increased in the medium with positive transformants. Our work indicated that TRX-coding genes here were responsible for ATZ degradation, suggesting that thioredoxins could be one of the vital strategies for pesticide degradation and detoxification in crops.

Corrigendum: Genome-wide identification and characterization of the NPF genes provide new insight into low nitrogen tolerance in Setaria.

[This corrects the article DOI: 10.3389/fpls.2022.1043832.].

Effects of tebuconazole application at different growth stages on rice grain quality of rice-based untargeted metabolomics.

Tebuconazole (TEB) is a pesticide widely used in crops and has a strong control effect on fungal pathogens. TEB abuse has caused many food safety problems. In this study, the TEB residue in rice and the effect of TEB on white rice quality were investigated. The results showed that under two spraying concentrations, the TEB residue in rice was 11.21-19.05 µg/kg and 24.45-31.12 µg/kg, and there was no food safety risk of pesticide residue. When applying TEB according to the instructions, no significant effect was found. However, when 3 times the recommended TEB concentration was used at the filling stage, the protein content of white rice decreased significantly from 106.52 mg/g to 80.72 mg/g. At the jointing，heading and filling stage, the amylose content of white rice decreased to 53.95 mg/g, 48.77 mg/g and 49.04 mg/g from the blank control group. Plant metabolic analysis using LC-QTOF/MS revealed that the amino acid-related metabolic pathways in white rice were significantly affected by TEB. This is closely related to the decrease in protein accumulation in white rice and the stress response of rice plants. The increase in pantothenic acid content in white rice indicated that the glycolysis pathway of white rice plants was affected, and the consumption of starch and sucrose increased, leading to the inhibition of amylose accumulation in white rice. The increase in soluble sugar content and decrease in phosphocholine content in white rice suggested that rice plants were affected by TEB exposure, which produced similar effects under drought stress.

Isolation, characterization and application of the epoxiconazole-degrading strain Pseudomonas sp. F1 in a soil-vegetable system.

Epoxiconazole (EPX) has a long half-life in soil and causes various toxicological effects in both the ecosystem and mammals. In this study, eight strains of bacteria capable of degrading EPX were isolated from pesticide-contaminated soil, with strain F1 showing the best effect. This strain was identified as Pseudomonas sp. by 16S rRNA gene sequencing and physiological-biochemical analyses. Our results indicated that strain F1 has a high capacity to degrade EPX, removing 92.1% of EPX within 6 days. The temperature and pH were the two most important environmental factors affecting EPX degradation, followed by substrate concentration and inoculum dose. In addition, strain F1 has a high capacity to promote EPX degradation in soils, with a lower t1/2 value (2.64 d) in F1-inoculated soil compared to the control (t1/2 = 96.3 d) without strain F1. The strain could efficiently colonize rhizosphere soil and enhance degradation of EPX, leading to a significant decrease in the accumulation and translocation of EPX in vegetables, thereby alleviating the effects of EPX-induced stress on plants. Moreover, we observed that strain F1-gfp was able to colonize the roots, stems and leaves of Brassica rapa var. chinensis. Such colonization may play a role in the efficient degradation of EPX within plants. To our knowledge, this is the first study to demonstrate biodegradation of EPX in a soil-vegetable system using an EPX-degrading bacterium. This study indicates that strain F1 is a promising candidate for simultaneous bioremediation of soil contaminated with EPX and safe food production.

Sorption of N-acyl homoserine lactones on maize straw derived biochars: Characterization, kinetics and isotherm analysis.

Soil amendment with biochar may trigger a series of positive and negative biological effects, partly because it interferes quorum sensing (QS) signals synthesized by microorganisms for communication. However, the mechanisms through which biochar interacts with these QS signals remain elusive. This study explored the mechanisms of interactions between N-acyl homoserine lactones (AHLs) and two maize straw-derived biochars (MBs) with different pyrolysis temperature. Pseudo-second-order equation model best depicted AHLs sorption kinetics on MBs. The intra-particle diffusion model revealed that AHLs sorption onto MBs consists of several stages. The sorption isotherms data of AHLs on MBs were in well agreement with both Langmuir and Freundlich models, indicating the occurrence of energetic distribution of active sites on the heterogeneous biochar with multilayer sorption. However, the AHLs sorption capacity on MBs varied, with biochar pyrolyzed at 600 °C displaying a higher AHLs sorption capacity compared with biochar pyrolyzed at 300 °C. It may be attributed to a variety of physiochemical interactions such as pore filling, functional groups complexation, hydrogen bond, and hydrophobic action. The adsorption/partitioning model results and thermodynamic parameters of Gibbs free energy (ΔG) confirmed that physical and chemical sorption occurred concurrently throughout the whole AHLs sorption process, with physical partitioning playing a greater role than surface sorption. The findings suggest that soil amendment with biochar may have a variety of effects on intra/inter-cellular communication, further implying biochar can be specially prepared to mediate soil processes related to microbial communication, like pollutant biodegradation, and carbon/nitrogen cycling.

Biodegradation of dibutyl phthalate by a novel endophytic Bacillus subtilis strain HB-T2 under in-vitro and in-vivo conditions.

The environmental prevalence and potential toxicity of dibutyl phthalate (DBP) motivate the attempt to develop feasible strategies to deal with DBP contamination. In this study, a strain of endphytic bacteria HB-T2 was isolated from sorrel roots and identified as Bacillus sp. by analysing its morphology, physiology, biochemistry and 16S rDNA sequence. The degradation efficiency of DBP by HB-T2 was almost identical under the temperature of 30∼40°C, but was significantly enhanced as the culture pH and inoculum size increases from 6.0 to 8.0, and 1% to 5% respectively. The degradation kinetics of DBP could be well described by the first-order kinetic model, with the degradation half-life ranging from 1.59 to 7.61 h when the initial concentrations of DBP were in the range of 5-20 mg/L. LC-MS analysis of the culture samples taken at varying intervals revealed monobutyl phthalate, phthalic acid and protocatechuic acid as the major metabolic intermediates during the degradation process. HB-T2 exhibited an excellent capability to degrade a wide range of phthalate esters (PAEs), especially butyl benzyl phthalate (BBP), dipentyl phthalate (DPP), and diisobutyl phthalate (DIBP). Inoculation of HB-T2 into Chinese cabbage (Brassica chinensis L.) growing in DBP-contaminated soils could significantly reduce the DBP levels in plant tissues and relieve the phytotoxic effects of DBP. Results of this study highlighted the great potential of this novel endophytic Bacillus subtilis strain HB-T2 for bioremediation of PAEs contamination and improvement of agricultural product safety by reducing PAEs accumulation in edible crops.

Carotenoid Cleavage Dioxygenase 1 Catalyzes Lutein Degradation To Influence Carotenoid Accumulation and Color Development in Foxtail Millet Grains.

Foxtail millet is a minor but economically important crop in certain regions of the world. Millet color is often used to judge grain quality, yet the molecular determinants of millet coloration remain unclear. Here, we explored the relationship between SiCCD1 and millet coloration in yellow and white millet varieties. Carotenoid levels declined with grain maturation and were negatively correlated with SiCCD1 expression, which was significantly higher in white millet as compared to yellow millet during the color development stage. Cloning of the SiCCD1 promoter and CDS sequences from these different millet varieties revealed the presence of two additional cis-regulatory elements within the SiCCD1 promoter in white millet varieties, including an enhancer-like GC motif element associated with anoxic specific inducibility and a GCN4-motif element associated with endosperm expression. Dual-luciferase reporter assays confirmed that SiCCD1 promoter fragments containing these additional cis-acting elements derived from white millet varieties were significantly more active than those from yellow millet varieties, consistent with the observed SiCCD1 expression patterns. Further in vitro enzyme detection assays confirmed that SiCCD1 primarily targets and degrades lutein. Together, these data suggest that SiCCD1 promoter variation was a key factor associated with the observed differences in SiCCD1 expression, which in turn led to the difference in millet coloration.

Genome-wide identification and characterization of the NPF genes provide new insight into low nitrogen tolerance in Setaria.

Introduction: Nitrogen (N) is essential for plant growth and yield production and can be taken up from soil in the form of nitrate or peptides. The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) genes play important roles in the uptake and transportation of these two forms of N. Methods: Bioinformatic analysis was used to identify and characterize the NPF genes in Setaria. RNA-seq was employed to analyze time-series low nitrate stress response of the SiNPF genes. Yeast and Arabidopsis mutant complementation were used to test the nitrate transport ability of SiNRT1.1B1 and SiNRT1.1B2. Results: We identified 92 and 88 putative NPF genes from foxtail millet (Setaria italica L.) and its wild ancestor green foxtail (Setaria viridis L.), respectively. These NPF genes were divided into eight groups according to their sequence characteristics and phylogenetic relationship, with similar intron-exon structure and motifs in the same subfamily. Twenty-six tandem duplication and 13 segmental duplication events promoted the expansion of SiNPF gene family. Interestingly, we found that the tandem duplication of the SiNRT1.1B gene might contribute to low nitrogen tolerance of foxtail millet. The gene expression atlas showed that the SiNPFs were divided into two major clusters, which were mainly expressed in root and the above ground tissues, respectively. Time series transcriptomic analysis further revealed the response of these SiNPF genes to short- and long- time low nitrate stress. To provide natural variation of gene information, we carried out a haplotype analysis of these SiNPFs and identified 2,924 SNPs and 400 InDels based on the re-sequence data of 398 foxtail millet accessions. We also predicted the three-dimensional structure of the 92 SiNPFs and found that the conserved proline 492 residues were not in the substrate binding pocket. The interactions of SiNPF proteins with NO 3 - were analyzed using molecular docking and the pockets were then identified. We found that the SiNPFs- NO 3 - binding energy ranged from -3.8 to -2.7 kcal/mol. Discussion: Taken together, our study provides a comprehensive understanding of the NPF gene family in Setaria and will contribute to function dissection of these genes for crop breeding aimed at improving high nitrogen use efficiency.