The evening complex promotes maize flowering and adaptation to temperate regions.

Maize (Zea mays) originated in southern Mexico and has spread over a wide latitudinal range. Maize expansion from tropical to temperate regions has necessitated a reduction of its photoperiod sensitivity. In this study, we cloned a quantitative trait locus (QTL) regulating flowering time in maize and show that the maize ortholog of Arabidopsis thaliana EARLY FLOWERING3, ZmELF3.1, is the causal locus. We demonstrate that ZmELF3.1 and ZmELF3.2 proteins can physically interact with ZmELF4.1/4.2 and ZmLUX1/2, to form evening complex(es; ECs) in the maize circadian clock. Loss-of-function mutants for ZmELF3.1/3.2 and ZmLUX1/2 exhibited delayed flowering under long-day and short-day conditions. We show that EC directly represses the expression of several flowering suppressor genes, such as the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) genes ZmCCT9 and ZmCCT10, ZmCONSTANS-LIKE 3, and the PSEUDORESPONSE REGULATOR (PRR) genes ZmPRR37a and ZmPRR73, thus alleviating their inhibition, allowing florigen gene expression and promoting flowering. Further, we identify two closely linked retrotransposons located in the ZmELF3.1 promoter that regulate the expression levels of ZmELF3.1 and may have been positively selected during postdomestication spread of maize from tropical to temperate regions during the pre-Columbian era. These findings provide insights into circadian clock-mediated regulation of photoperiodic flowering in maize and new targets of genetic improvement for breeding.

Local auxin biosynthesis regulates brace root angle and lodging resistance in maize.

Root lodging poses a major threat to maize production, resulting in reduced grain yield and quality, and increased harvest costs. Here, we combined expressional, genetic, and cytological studies to demonstrate a role of ZmYUC2 and ZmYUC4 in regulating gravitropic response of the brace root and lodging resistance in maize. We show that both ZmYUC2 and ZmYUC4 are preferentially expressed in root tips with partially overlapping expression patterns, and the protein products of ZmYUC2 and ZmYUC4 are localized in the cytoplasm and endoplasmic reticulum, respectively. The Zmyuc4 single mutant and Zmyuc2/4 double mutant exhibit enlarged brace root angle compared with the wild-type plants, with larger brace root angle being observed in the Zmyuc2/4 double mutant. Consistently, the brace root tips of the Zmyuc4 single mutant and Zmyuc2/4 double mutant accumulate less auxin and are defective in proper reallocation of auxin in response to gravi-stimuli. Furthermore, we show that the Zmyuc4 single mutant and the Zmyuc2/4 double mutant display obviously enhanced root lodging resistance. Our combined results demonstrate that ZmYUC2- and ZmYUC4-mediated local auxin biosynthesis is required for normal gravity response of the brace roots and provide effective targets for breeding root lodging resistant maize cultivars.

DWARF53 interacts with transcription factors UB2/UB3/TSH4 to regulate maize tillering and tassel branching.

Strigolactones (SLs) are a recently identified class of phytohormones that regulate diverse developmental processes in land plants. However, the signaling mechanism of SLs in maize (Zea mays) remains largely unexplored. Here, we identified the maize gene DWARF 53 (ZmD53) and demonstrated that ZmD53 interacts with the SL receptors DWARF 14A/B (ZmD14A/B) in a rac-GR24-dependent manner. Transgenic maize plants expressing a gain-of-function mutant version of Zmd53 exhibited insensitivity to exogenous rac-GR24 treatment and a highly pleiotropic phenotype, including excess tillering and reduced tassel branching, indicating that ZmD53 functions as an authentic SL signaling repressor in maize. In addition, we showed that ZmD53 interacts with two homologous maize SPL transcription factors, UB3 and TSH4, and suppresses their transcriptional activation activity on TB1 to promote tillering. We also showed that UB2, UB3, and TSH4 can physically interact with each other and themselves, and that they can directly regulate the expression of TSH4, thus forming a positive feedback loop. Furthermore, we demonstrated that ZmD53 can repress the transcriptional activation activity of UB3 and TSH4 on their own promoters, thus decreasing tassel branch number. Our results reveal new insights into the integration of SL signaling and the miR156/SPL molecular module to coordinately regulate maize development.

CRISPR/Cas9-mediated knockout and overexpression studies reveal a role of maize phytochrome C in regulating flowering time and plant height.

Maize is a major staple crop widely used for food, feedstocks and industrial products. Shade-avoidance syndrome (SAS), which is triggered when plants sense competition of light from neighbouring vegetation, is detrimental for maize yield production under high-density planting conditions. Previous studies have shown that the red and far-red photoreceptor phytochromes are responsible for perceiving the shading signals and triggering SAS in Arabidopsis; however, their roles in maize are less clear. In this study, we examined the expression patterns of ZmPHYC1 and ZmPHYC2 and found that ZmPHYC1, but not ZmPHYC2, is highly expressed in leaves and is regulated by the circadian clock. Both ZmPHYC1 and ZmPHYC2 proteins are localized to both the nucleus and cytoplasm under light conditions and both of them can interact with themselves or with ZmPHYBs. Heterologous expression of ZmPHYCs can complement the Arabidopsis phyC-2 mutant under constant red light conditions and confer an attenuated SAS in Arabidopsis in response to shading. Double knockout mutants of ZmPHYC1 and ZmPHYC2 created using the CRISPR/Cas9 technology display a moderate early-flowering phenotype under long-day conditions, whereas ZmPHYC2 overexpression plants exhibit a moderately reduced plant height and ear height. Together, these results provided new insight into the function of ZmPHYCs and guidance for breeding high-density tolerant maize cultivars.

Characterization of Maize Phytochrome-Interacting Factors in Light Signaling and Photomorphogenesis.

Increasing planting density has been an effective means of increasing maize (Zea mays ssp. mays) yield per unit of land area over the past few decades. However, high-density planting will cause a reduction in the ratio of red to far-red incident light, which could trigger the shade avoidance syndrome and reduce yield. The molecular mechanisms regulating the shade avoidance syndrome are well established in Arabidopsis (Arabidopsis thaliana) but poorly understood in maize. Here, we conducted an initial functional characterization of the maize Phytochrome-Interacting Factor (PIF) gene family in regulating light signaling and photomorphogenesis. The maize genome contains seven distinct PIF genes, which could be grouped into three subfamilies: ZmPIF3s, ZmPIF4s, and ZmPIF5s Similar to the Arabidopsis PIFs, all ZmPIF proteins are exclusively localized to the nucleus and most of them can form nuclear bodies upon light irradiation. We show that all of the ZmPIF proteins could interact with ZmphyB. Heterologous expression of each ZmPIF member could partially or fully rescue the phenotype of the Arabidopsis pifq mutant, and some of these proteins conferred enhanced shade avoidance syndrome in Arabidopsis. Interestingly, all ZmPIF proteins expressed in Arabidopsis are much more stable than their Arabidopsis counterparts upon exposure to red light. Moreover, the Zmpif3, Zmpif4, and Zmpif5 knockout mutants generated via CRISPR/Cas9 technology all showed severely suppressed mesocotyl elongation in dark-grown seedlings and were less responsive to simulated shade treatment. Taken together, our results reveal both conserved and distinct molecular properties of ZmPIFs in regulating light signaling and photomorphogenesis in maize.

Regulatory modules controlling early shade avoidance response in maize seedlings.

BACKGROUND: Optimization of shade avoidance response (SAR) is crucial for enhancing crop yield in high-density planting conditions in modern agriculture, but a comprehensive study of the regulatory network of SAR is still lacking in monocot crops. RESULTS: In this study, the genome-wide early responses in maize seedlings to the simulated shade (low red/far-red ratio) and also to far-red light treatment were transcriptionally profiled. The two processes were predominantly mediated by phytochrome B and phytochrome A, respectively. Clustering of differentially transcribed genes (DTGs) along with functional enrichment analysis identified important biological processes regulated in response to both treatments. Co-expression network analysis identified two transcription factor modules as potentially pivotal regulators of SAR and de-etiolation, respectively. A comprehensive cross-species comparison of orthologous DTG pairs between maize and Arabidopsis in SAR was also conducted, with emphasis on regulatory circuits controlling accelerated flowering and elongated growth, two physiological hallmarks of SAR. Moreover, it was found that the genome-wide distribution of DTGs in SAR and de-etiolation both biased toward the maize1 subgenome, and this was associated with differential retention of various cis-elements between the two subgenomes. CONCLUSIONS: The results provide the first transcriptional picture for the early dynamics of maize phytochrome signaling. Candidate genes with regulatory functions involved in maize shade avoidance response have been identified, offering a starting point for further functional genomics investigation of maize adaptation to heavily shaded field conditions.

Molecular cloning and characterization of a novel carotenoid cleavage dioxygenase 1 from Lycium chinense.

Carotenoids are key precursor for aroma compounds in plants. Although the fruit of Lycium chinense contains numerous carotenoids, the formation mechanism of aroma compounds in L. chinense is still poorly understood. In this study, a new carotenoid cleavage dioxygenase (termed LmCCD1) was identified from the leaves of L. chinense. Expression analysis by semiquantitative PCR reveals that LmCCD1 gene is expressed in different tissues of L. chinense, and dominant expression of LmCCD1 gene was found in leaves, flowers, and ripe fruits. In addition, the expression level of LmCCD1 in fruits is in accordance with the content of ß-ionone. Finally, recombinantly expressed LmCCD1 can cleave ß-carotene and lycopene to produce ß-ionone and pseudoionone in in vitro assays. These results indicate that LmCCD1 a novel carotenoids cleavage dioxygenase gene that may regulate the metabolic pathways responsible for aroma metabolite production (such as ß-ionone and pseudoionone) in L. chinense has been identified.

Cloning and characterization of a novel ß-carotene hydroxylase gene from Lycium barbarum and its expression in Escherichia coli.

Lycium barbarum contains high levels of zeaxanthin, which is produced by the conversion of ß-carotene into zeaxanthin. ß-Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding ß-carotene hydroxylase (Chyb) from the L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3'-rapid amplification of cDNA end assay encoding a deduced Chyb protein (34.8 kDa) with a theoretical isoelectric point of 8.36. A bioinformatics analysis showed that the L. barbarum Chyb was located in the chloroplast. Further, to investigate the catalytic activity of the L. barbarum Chyb, a complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that Chyb can catalyze ß-carotene to produce zeaxanthin. Thus, this study suggests that L. barbarum ß-carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in E. coli.

From Organelle Morphology to Whole-Plant Phenotyping: A Phenotypic Detection Method Based on Deep Learning.

The analysis of plant phenotype parameters is closely related to breeding, so plant phenotype research has strong practical significance. This paper used deep learning to classify Arabidopsis thaliana from the macro (plant) to the micro level (organelle). First, the multi-output model identifies Arabidopsis accession lines and regression to predict Arabidopsis's 22-day growth status. The experimental results showed that the model had excellent performance in identifying Arabidopsis lines, and the model's classification accuracy was 99.92%. The model also had good performance in predicting plant growth status, and the regression prediction of the model root mean square error (RMSE) was 1.536. Next, a new dataset was obtained by increasing the time interval of Arabidopsis images, and the model's performance was verified at different time intervals. Finally, the model was applied to classify Arabidopsis organelles to verify the model's generalizability. Research suggested that deep learning will broaden plant phenotype detection methods. Furthermore, this method will facilitate the design and development of a high-throughput information collection platform for plant phenotypes.

Identification of Soybean Mutant Lines Based on Dual-Branch CNN Model Fusion Framework Utilizing Images from Different Organs.

The accurate identification and classification of soybean mutant lines is essential for developing new plant varieties through mutation breeding. However, most existing studies have focused on the classification of soybean varieties. Distinguishing mutant lines solely by their seeds can be challenging due to their high genetic similarities. Therefore, in this paper, we designed a dual-branch convolutional neural network (CNN) composed of two identical single CNNs to fuse the image features of pods and seeds together to solve the soybean mutant line classification problem. Four single CNNs (AlexNet, GoogLeNet, ResNet18, and ResNet50) were used to extract features, and the output features were fused and input into the classifier for classification. The results demonstrate that dual-branch CNNs outperform single CNNs, with the dual-ResNet50 fusion framework achieving a 90.22 ± 0.19% classification rate. We also identified the most similar mutant lines and genetic relationships between certain soybean lines using a clustering tree and t-distributed stochastic neighbor embedding algorithm. Our study represents one of the primary efforts to combine various organs for the identification of soybean mutant lines. The findings of this investigation provide a new path to select potential lines for soybean mutation breeding and signify a meaningful advancement in the propagation of soybean mutant line recognition technology.

Genome-wide selection and genetic improvement during modern maize breeding.

Since the development of single-hybrid maize breeding programs in the first half of the twentieth century1, maize yields have increased over sevenfold, and much of that increase can be attributed to tolerance of increased planting density2-4. To explore the genomic basis underlying the dramatic yield increase in maize, we conducted a comprehensive analysis of the genomic and phenotypic changes associated with modern maize breeding through chronological sampling of 350 elite inbred lines representing multiple eras of germplasm from both China and the United States. We document several convergent phenotypic changes in both countries. Using genome-wide association and selection scan methods, we identify 160 loci underlying adaptive agronomic phenotypes and more than 1,800 genomic regions representing the targets of selection during modern breeding. This work demonstrates the use of the breeding-era approach for identifying breeding signatures and lays the foundation for future genomics-enabled maize breeding.

In vivo and in vitro anti-hepatitis B virus activity of total phenolics from Oenanthe javanica.

The traditional Chinese medicine Oenanthe javanica (OJ) has been used for many years, mainly for the treatment of inflammatory conditions including hepatitis. In this study, human hepatoma Hep G2.2.15 cells culture system and duck hepatitis B virus (DHBV) infection model were used as in vivo and in vitro models to evaluate the anti-HBV effects of total phenolics from Oenanthe javanica (OJTP). The HBeAg and HBsAg concentrations in cell culture medium were determined by using the enzyme immunoassay kit after Hep G2.2.15 cells were treated with OJTP for 9 d. DHBV-DNA in duck serum was analyzed by dot blot hybridization assay. In the cell model, OJTP could dose-dependently inhibit the production of the HBeAg and HBsAg, and the inhibition rates of OJTP on HBeAg and HBsAg in the Hep G2.2.15 cells were 70.12% and 72.61% on day 9, respectively. In the DHBV infection model, OJTP also reduced HBV DNA level in a dose-dependent manner. The DHBV-DNA levels decreased significantly after the treatment with 0.10 g kg(-1)d(-1) and 0.20 g kg(-1)d(-1) OJTP. The inhibition of the peak of viremia was at the maximum at the dose of 0.20 g kg(-1)d(-1) and reached 64.10% on day 5 and 66.48% on day 10, respectively. Histopathological evaluation of the liver revealed significant improvement by OJTP. In conclusion, our results demonstrate that OJTP can efficiently inhibit HBV replication in Hep G2.2.15 cells line in vitro and inhibit DHBV replication in ducks in vivo. OJTP therefore warrants further investigation as a potential therapeutic agent for HBV infections.

Synthesis and application of lignin-based copolymer LSAA on controlling non-point source pollution resulted from surface runoff.

In this article, alkali lignin separated from paper pulp waste was grafted into a novel copolymer LSAA (a copolymer of lignin, starch, acrylamide, and acrylic acid). Its practical application effect and environmental safety were studied. The results of field simulation experiment indicated that the application of LSAA significantly affected the output of the runoff and pollutants. The runoff quantity was decreased by 16.67%-47.00% and the loads of total suspended solids (TSS), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) were reduced by 17.78%-62.14%, 26.32%-59.91%, 15.25%-47.42%, and 22.18%-52.78%, respectively. The tests on its environmental safety showed that LSAA did no harm the soil. Compared with polyacrylamide (PAM), a dominant product in this field, LSAA exhibited similar effects and cheap cost. Thus, this study not only created a new product for controlling runoff water quality but also offered a beneficial application for industrial paper waste.

Inorganic nitrogen removal of toilet wastewater with an airlift external circulation membrane bioreactor.

Removal of inorganic nitrogen (inorganic-N) from toilet wastewater, using a pilot-scale airlift external circulation membrane bioreactor (AEC-MBR) was studied. The results showed that the use of AEC-MBR with limited addition of alkaline reagents and volumetric loading rates of inorganic-N of 0.19-0.40 kg inorganic-N/(m3 x d) helped achieve the desired nitrification and denitrification. Furthermore, the effects of pH and dissolved oxygen (DO) on inorganic-N removal were examined. Under the condition of MLSS at 1.56-2.35 g/L, BOD5/ammonia nitrogen (NH4+ -N) at 1.0, pH at 7.0-7.5, and DO at 1.0-2.0 mg/L, the removal efficiencies of NH4+ -N and inorganic-N were 91.5% and 70.0%, respectively, in the AEC-MBR. The cost of addition of alkaline reagent was approximately 0.5-1.5 RMB yuan/m3, and the energy consumption was approximately 0.72 kWh/m3 at the flux of 8 L/(m2 x h).

Performances of biological aerated filter employing hollow fiber membrane segments of surface-improved poly (sulfone) as biofilm carriers.

Using the surface of poly (sulfone) hollow fiber membrane segments as grafted layer, the hydrophilic acrylamide chain was grafted on by UV-photoinduced grafting polymerization. The gained improvement of surface wettability for the modified membrane was tested by measuring the contact-angle as well as FTIR spectra. Then correlation between the hydrophilic ability of support material and the biofilm adherence ability was demonstrated by comparing the pollutant removal rates from urban wastewater via two identical lab-scale up-flow biological aerated filters, one employed the surface wettability modified poly (sulfone) hollow fiber membrane segment as biofilm carrier and the other employed unmodified membrane segment as biofilm carrier. The experimental results showed that under the conditions of influent flux 5 L/h, hydraulic retention time 9 h and gas to liquid ratio (G/L) 10:1, the removal rates of chemical oxygen demand (COD) and ammonium nitrogen (NH4(+)-N) for the modified packing filter and the unmodified packing filter was averaged at 83.64% and 96.25%, respectively, with the former filter being 5%-20% more than the latter. The effluent concentration of COD, NH4(+)-N and turbidity for the modified packing filter was 25.25 mg/L, 2 mg/L and 8 NTU, respectively. Moreover, the ammonium nitrogen removal performance of the filter packing the modified PSF was compared with the other bioreactor packing of an efficient floating medium. The biomass test indicated that the modified membrane matrixes provided better specific adhesion (3310-5653 mg TSS/L support), which gave a mean of 1000 mg TSS/L more than the unmodified membrane did. In addition, the phenomenon of simultaneous denitrification on the inner surface of the support and nitrification on the outer surface was found in this work.

Ultravilolet-radiation-induced graft polymerization of acrylamide onto the melt-blown polypropylene filter element by dynamic method.

By dynamic method under UV irradiation, commercial melt-blown polypropylene (PPMB) filter element was modified with acrylamide (AAm) using benzophenone (BP) as initiator. Attenuated total reflection-Fourier transform infrared spectroscopy and scanning electron microscope verified that polyacrylamide chain was grafted on the fiber surface of PPMB filter element. Elemental content analysis with energy dispersive X-ray of fibers revealed that the polymerization content in the inner part of filter element was relatively higher than that in the outer. Degree of grafting changed with initiator concentration, monomer concentration, reaction temperature and reached 2.6% at the reaction condition: CBP=0.06 mol/L, CAAm=2.0 mol/L, irradiation time: 80 min, temperature: 600 degrees C. Relative water flux altered with the hydrophilicity and pore size of filter element. In the antifouling test, the modified filter gave greater flux recovery (approximately 70%) after filtration of the water extract of Liuweidihuang, suggesting that the fouling layer was more easily reversible due to the hydrophilic nature of the modified filter.

Phytochrome-interacting factors directly suppress MIR156 expression to enhance shade-avoidance syndrome in Arabidopsis.

Plants have evolved a repertoire of strategies collectively termed the shade-avoidance syndrome to avoid shade from canopy and compete for light with their neighbors. However, the signaling mechanism governing the adaptive changes of adult plant architecture to shade is not well understood. Here, we show that in Arabidopsis, compared with the wild type, several PHYTOCHROME-INTERACTING FACTORS (PIFS) overexpressors all display constitutive shade-avoidance syndrome under normal high red to far-red light ratio conditions but are less sensitive to the simulated shade, whereas the MIR156 overexpressors exhibit an opposite phenotype. The simulated shade induces rapid accumulation of PIF proteins, reduced expression of multiple MIR156 genes, and concomitant elevated expression of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family genes. Moreover, in vivo and in vitro assays indicate that PIFs bind to the promoters of several MIR156 genes directly and repress their expression. Our results establish a direct functional link between the phytochrome-PIFs and miR156-SPL regulatory modules in mediating shade-avoidance syndrome.Plants employ developmental strategies to avoid shade and compete with neighbors for light. Here, Xie et al. show that phytochrome-interacting factors, which are regulated in a light-dependent manner, directly repress MIR156 genes and promote the expression of SPL genes to enhance shade-avoidance responses.

Gas-initiation under UV and liquid-grafting polymerization on the surface of polysulfone hollow fiber ultrafiltration membrane by dynamic method.

Using the inner-surface of polysulfone hollow fiber ultrafiltration membranes as grafted layer, the method of gas-initiation and liquid-polymerization has been studied, which aimed to adjust the diameter of the pores in the membranes. The degree of polymerization varied with the changes of the parameters, such as irradiation time, monomer concentration, temperature and time of polymerization and soon. The results indicated that using benzophenone (BP) which is in a gaseous condition as photo-initiator, acrylamide as graft monomer, the polyacrylamide chain was grafted on the surface of membranes. After the surface membrane being modified, the water flux and retention altered, and thus it can be seen that the diameter of the pores in the membrane was altered. These experiments contribute to finding a new way to produce the hollow fiber membrane with the small pore size and are extraordinarily worth developing and studying.

A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.

The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast.

Photochemical surface modification of poly(arylsulfone) ultrafiltration membrane and covalent immobilization of enzyme.

The sensitivity of poly(arylsulfone) (PSf) for UV irradiation in different solvents (water and ethanol) was investigated. It is confirmed that acrylic acid (AA) and acrylamide (AAm) are grafted only onto the surface of the membrane instead of the interior by FTIR and scanning electron microscope (SEM). The membrane performance (deltaJ/J0 and contact angle theta) after photografting was studied. In the range of conditions used, the grafting yield increases with irradiation time and monomer concentration growing. After photografting and N-3-dimethylaminopropyl-N'-ethycarbodiimide hydrochloride (EDC) activation, PSf membrane was immobilized with hydrogen peroxide oxidoreductase, and showed a higher activity than the control membrane.