Biofortification of iron content by regulating a NAC transcription factor in maize.

Iron (Fe) deficiency remains widespread among people in developing countries. To help solve this problem, breeders have been attempting to develop maize cultivars with high yields and high Fe concentrations in the kernels. We conducted a genome-wide association study and identified a gene, ZmNAC78 (NAM/ATAF/CUC DOMAIN TRANSCRIPTION FACTOR 78), that regulates Fe concentrations in maize kernels. We cultivated maize varieties with both high yield and high Fe concentrations in their kernels by using a molecular marker developed from a 42-base pair insertion or deletion (indel) in the promoter of ZmNAC78. ZmNAC78 expression is enriched in the basal endosperm transfer layer of kernels, and the ZmNAC78 protein directly regulates messenger RNA abundance of Fe transporters. Our results thus provide an approach to develop maize varieties with Fe-enriched kernels.

Maize Dek407 Encodes the Nitrate Transporter 1.5 and Is Required for Kernel Development.

The kernel serves as the storage organ and harvestable component of maize, and it plays a crucial role in determining crop yield and quality. Understanding the molecular and genetic mechanisms of kernel development is of considerable importance for maize production. In this study, we obtained a mutant, which we designated defective kernel 407 (dek407), through ethyl methanesulfonate mutagenesis. The dek407 mutant exhibited reduced kernel size and kernel weight, as well as delayed grain filling compared with those of the wild type. Positional cloning and an allelism test revealed that Dek407 encodes a nitrate transporter 1/peptide transporter family (NPF) protein and is the allele of miniature 2 (mn2) that was responsible for a poorly filled defective kernel phenotype. A transcriptome analysis of the developing kernels showed that the mutation of Dek407 altered the expression of phytohormone-related genes, especially those genes associated with indole-3-acetic acid synthesis and signaling. Phytohormone measurements and analysis indicated that the endogenous indole-3-acetic acid content was significantly reduced by 66% in the dek407 kernels, which may be the primary cause of the defective phenotype. We further demonstrated that natural variation in Dek407 is associated with kernel weight and kernel size. Therefore, Dek407 is a potential target gene for improvement of maize yield.

The long non-coding RNA PILNCR2 increases low phosphate tolerance in maize by interfering with miRNA399-guided cleavage of ZmPHT1s.

The open reading regions of ZmPHT1s (inorganic phosphate [Pi] transporters) in maize possess target sites of microRNA399 (miR399). However, the relationship between miR399 and ZmPHT1s and its functional importance in response to Pi deficiency remain to be explored. We show here that ZmPHT1;1, ZmPHT1;3, and ZmPHT1;13 are the targets of ZmmiRNA399. We found that a long non-coding RNA, PILNCR2 (Pi-deficiency-induced lncRNA 2), is transcribed from the opposing DNA strand of ZmPHT1;1 and predominantly localized in the cytoplasm. A ribonuclease protection assay and an RNA-RNA binding assay showed that PILNCR2 and ZmPHT1s could form the RNA/RNA duplexes in vivo and in vitro. A co-expression assay in N. benthamiana revealed that the PILNCR2/ZmPHT1 RNA/RNA duplexes interfere with miR399-guided cleavage of ZmPHT1 mRNAs. Overexpression of PILNCR2 increased low-Pi tolerance in maize, whereas its knockout and knockdown decreased low-Pi tolerance in maize. Consistently, ZmPHT1;3 and ZmPHT1;13 mRNA abundance was increased in transgenic plants overexpressing PILNCR2 but reduced in its knock-out mutants, suggesting that PILNCR2 positively regulates the mRNA abundance of ZmPHT1;3 and ZmPHT1;13 in maize. Collectively, these results indicate that PILNCR2 plays an important role in maize Pi homeostasis by interfering with miRNA399-guided cleavage of ZmPHT1 mRNAs.

MicroRNA408 negatively regulates salt tolerance by affecting secondary cell wall development in maize.

Although microRNA408 (miR408) is a highly conserved miRNA, the miR408 response to salt stress differs among plant species. Here, we show that miR408 transcripts are strongly repressed by salt stress and methyl viologen treatment in maize (Zea mays). Application of N, N1-dimethylthiourea partly relieved the NaCl-induced down-regulation of miR408. Transgenic maize overexpressing MIR408b is hypersensitive to salt stress. Overexpression of MIR408b enhanced the rate of net Na+ efflux, caused Na+ to locate in the inter-cellular space, reduced lignin accumulation, and reduced the number of cells in vascular bundles under salt stress. We further demonstrated that miR408 targets ZmLACCASE9 (ZmLAC9). Knockout of MIR408a or MIR408b or overexpression of ZmLAC9 increased the accumulation of lignin, thickened the walls of pavement cells, and improved salt tolerance of maize. Transcriptome profiles of the wild-type and MIR408b-overexpressing transgenic maize with or without salt stress indicated that miR408 negatively regulates the expression of cell wall biogenesis genes under salt conditions. These results indicate that miR408 negatively regulates salt tolerance by regulating secondary cell wall development in maize.

The auxin signaling pathway contributes to phosphorus-mediated zinc homeostasis in maize.

Although the interaction between P and Zn has long been recognized in plants, the physiological and molecular mechanisms underlying P and Zn interactions are poorly understood. We show here that P supply decreases the Zn concentration in maize shoots and roots. Compared to +P + Zn (addition of both P and Zn), +P-Zn reduced and -P-Zn increased the total length of 1° lateral roots (LRs). Under +P + Zn, both P and Zn concentrations were lower in the sl1 mutant roots than in wild-type (WT) maize roots, and P accumulation did not reduce the Zn concentration in ll1 mutant roots. Transcriptome profiling showed that the auxin signaling pathway contributed to P-mediated Zn homeostasis in maize. Auxin production and distribution were altered by changes in P and Zn supply. Cytosolic Zn co-localized with auxin accumulation under +P + Zn. Exogenous application of 1-NAA and L-Kyn altered the P-mediated root system architecture (RSA) under Zn deficiency. -P-Zn repressed the expression of miR167. Overexpression of ZmMIR167b increased the lengths of 1° LRs and the concentrations of P and Zn in maize. These results indicate that auxin-dependent RSA is important for P-mediated Zn homeostasis in maize.HighlightAuxin-dependent RSA is important for P-mediated Zn homeostasis in maize.

Nitrogen supply affects ion homeostasis by modifying root Casparian strip formation through the miR528-LAC3 module in maize.

Although nitrogen (N) is known to affect mineral element homeostasis in plants, the molecular mechanisms of interactions between N and other nutrients remain largely unclear. We report here that N supply affects ion homeostasis in maize. Berberine hemisulfate staining and a propidium iodide penetration assay showed that N luxury significantly delayed Casparian strip (CS) formation in maize roots. We further demonstrated that N-mediated CS formation in maize was independent of RBOHF-activated H2O2 production. N luxury induced the expression of ZmmiR528 in whole roots and root tips. Knockdown and loss-of-function of ZmmiR528 promoted CS formation under both N-luxury and N-deficient conditions. Both ZmMIR528a and ZmMIR528b contribute to early CS formation under different N conditions. RNA-seq and real-time RT-PCR analysis demonstrated that ZmLAC3, but not ZmLAC5, responded to N treatments. Consistent with results obtained with ZmmiR528 TM transgenic maize and mir528a/b loss-of-function mutants, transgenic maize overexpressing ZmLAC3 displayed early CS formation under different N conditions. Under field conditions, K, Ca, Mn, Cu, Mg, and Zn concentrations were greater in the ear leaf of ZmLAC3-overexpressing transgenic maize than in the wild type. These results indicate that ZmmiR528 affects CS formation in maize by regulating the expression of ZmLAC3, and modification of CS formation has the potential to improve maize quality.

MIR164b represses iron uptake by regulating the NAC domain transcription factor5-Nuclear Factor Y, Subunit A8 module in Arabidopsis.

Recent findings have revealed the important roles of microRNAs (miRNAs) in the secondary responses to oxidative damage caused by iron (Fe) excess. However, the functional importance of miRNAs in plant responses to Fe deficiency remains to be explored. Here, we show that the expression level of miR164 in Arabidopsis (Arabidopsis thaliana) roots was repressed by Fe deficiency. Primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2) were higher in the mir164b mutant than in the wild-type (WT) under Fe-deficient conditions. Analysis of the Fe concentrations and ferric reductase activities in the roots of miR164 knockdown transgenic plants showed that members of the miR164 family had different functions in Fe-deficiency responses. Promoter::GUS analysis showed that NAM/ATAF/CUC (NAC) domain transcription factor5 (NAC5) is regulated at both transcriptional and posttranscriptional levels under Fe-deficient conditions. Transgenic Arabidopsis plants overexpressing NAC5 were more tolerant of Fe deficiency than the WT. NAC5 has transactivation activity and directly transactivates the expression of Nuclear Factor Y, Subunit A8 (NFYA8), as demonstrated by chromatin immunoprecipitation followed by quantitative polymerase chain reaction, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay. Like overexpression of NAC5, overexpression of NFYA8 increases primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRT1 and FRO2 under Fe-deficient conditions. Thus, MIR164b is important for Fe-deficiency responses by its regulation of the NAC5-NFYA8 module.

Biomechanical study of extramedullary and intramedullary fixation in the treatment of unstable intertrochanteric reversed-tilt fractures of the femur.

Background: To investigate the efficacy of the 135° hip screw, 95° intramedullary hip screw (IMHS) and 95° hip screw in the treatment of intertrochanteric reverse dip fracture of the femur. Methods: We retrospectively analyzed 125 matched pairs of human femurs (median age 64 years) which were osteotomized at a 33° angle in the left femur and extended downward from the minor trochanter to simulate a reverse oblique intertrochanteric fracture. The right femur served as a control. The left femur (n=4) was implanted with a 135° hip screw, 95° hip screw, or IMHS. A strain detector was placed distal to the fracture site to monitor fragment strain. The lateral displacement of the proximal femur was measured by a linear variable differential transformer. An Instron tester measured stiffness, strain, and lateral displacement at 25° adduction, and 90° adduction with vertical loads on the femoral head. A 2 cm gap was then formed at the fracture site to simulate comminution and the mechanical test was repeated. Results: Before the formation of the gap, there was no significant difference in stiffness among different bone structures (P>0.05), but after the formation of the gap, the stiffness of all the adduction structures decreased (P=0.03), and the difference in adduction was statistically significant (135° hip screw: 46.6%±3%; 95° hip screw: 22.9%±2%; IMHS: 53.7%±7.8%; P<0.05). Similar results were found for the abduction and buckling positions. There was no significant difference in the lateral displacement of the gap before (P=0.92) and after (P=0.26), but a significant difference in the failure load was found (135° hip screw: 1,222±560 N; 95° hip screw: 2,566±283 N; IMHS: 4,644±518 N; P=0.02). Conclusions: There was no statistically significant difference in stiffness among different structures (P>0.05). However, in the presence of gaps, IMHS bone implant structures are much stiffer than 135° and 95° structures and have a greater destructive load.

A restored humoral environment ameliorates acute brain injury after Angiostrongylus cantonensis infection.

Circulating factors in the circulatory system support important functions of living tissues and the body. Parabiosis is a condition in which two living animals are connected using surgical methods and share a single circulatory system. Angiostrongylus cantonensis is the major cause of infectious eosinophilic meningitis, which causes severe damage to the central nervous system (CNS) and immune system. However, the mechanism of immunopathology remains largely unknown. We hypothesize that a restored humoral environment can help relieve damage to the CNS and immune system. In the present study, we found that administration of normal serum significantly reduced mortality, alleviated thymic atrophy and reduced inflammation in the brains of mice infected with A. cantonensis. We further generated parabiotic pairs between two healthy mice, one of which was then orally infected with A. cantonensis. The results showed that compared with singleton mice, mice connected with a healthy parabiotic partner were protected against CNS and immune system damage, as revealed by significantly reduced inflammation in the brain, alleviated thymic atrophy, and decreased expression of proinflammatory cytokines. These findings revealed that a healthy systemic environment can relieve damage to the CNS and immune system in infected mice, suggesting novel therapeutic approaches for diseases involving severe brain and immune system damage.

Identification of major QTL for waterlogging tolerance in maize using genome-wide association study and bulked sample analysis.

Waterlogging has increasingly become one of the major constraints to maize (Zea mays L.) production in some maize growing areas as it seriously decreases the yield. Waterlogging tolerance in maize germplasm provides a basis for maize waterlogging improvement. In this study, nine seedling traits, plant height (PH), root length (RL), shoot dry weight (SDW), root dry weight (RDW), adventitious root number (ARN), node number of brace root (BRNN), brace root number (BRN), brace root dry weigh (BRDW), survival rate (SR), and the secondary traits that were defined as relative phenotypic value of seedling traits under waterlogging and control treatments were used in a natural population that contain 365 inbred lines to evaluate the waterlogging tolerance of tropical maize. The result showed that maize waterlogging tolerance was genetically controlled and seedling traits were significantly different between the control and waterlogging treatments. PH, RL, SDW, and RDW are important seedling traits for waterlogging tolerance identification. Some tropical maize inbred lines were identified with extreme waterlogging tolerance that can provide an important germplasm resource for breeding. Population structure analysis showed that two major phylogenetic subgroups in tropical maize could be identified. Genome-wide association study (GWAS) using 39,266 single nucleotide polymorphisms (SNPs) across the whole genome identified 49 trait-SNPs distributed on over all 10 chromosomes excluding chromosome 10. Seventy-one significant SNPs, distributed on all 10 chromosomes excluding chromosome 5, were identified by extend bulked sample analysis (Ext-BSA) based on the inbred lines with extreme phenotypes. GWAS and Ext-BSA identified the same loci on bin1.07, bin6.01, bin2.09, bin6.04, bin7.02, and bin7.03. Nine genes were proposed as potential candidate genes. Cloning and functional validation of these genes would be helpful for understanding the molecular mechanism of waterlogging tolerance in maize.

Complex Genetic System Involved in Fusarium Ear Rot Resistance in Maize as Revealed by GWAS, Bulked Sample Analysis, and Genomic Prediction.

Fusarium ear rot (FER) caused by Fusarium verticillioides is one of the most prevalent maize diseases in China and worldwide. Resistance to FER is a complex trait controlled by multiple genes highly affected by environment. In this paper, genome-wide association study (GWAS), bulked sample analysis (BSA), and genomic prediction were performed for understanding FER resistance using 509 diverse inbred lines, which were genotyped by 37,801 high-quality single-nucleotide polymorphisms (SNPs). Ear rot evaluation was performed using artificial inoculation in four environments in China: Xinxiang, Henan, and Shunyi, Beijing, during 2017 and 2018. Significant phenotypic and genetic variation for FER severity was observed, and FER resistance was significantly correlated among the four environments with a generalized heritability of 0.78. GWAS identified 23 SNPs that were associated with FER resistance, 2 of which (1_226233417 on chromosome 1 and 10_14501044 on chromosome 10) were associated at threshold of 2.65 × 10-7 [-log(0.01/37,801)]. Using BSA, resistance quantitative trait loci were identified on chromosomes 3, 4, 7, 9, and 10 at the 90% confidence level and on chromosomes 3 and 10 at the 95% confidence level. A key region, bin 10.03, was detected by both GWAS and BSA. Genomic prediction for FER resistance showed that the prediction accuracy by trait-related markers was higher than that by randomly selected markers under different levels of marker density. Marker-assisted selection using genomic prediction could be an efficient strategy for genetic improvement for complex traits like FER resistance.

ZmEHD1 Is Required for Kernel Development and Vegetative Growth through Regulating Auxin Homeostasis.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize.

Major depressive disorder in Chinese persons with speech disability: High rates of prevalence and perceived need for mental health care but extremely low rate of use of mental health services.

BACKGROUND: In recent years, there has been increasing awareness on the importance of mental health services for persons with disability in China, but data on mental health of persons with speech disability (PwSD) are scarce. This study examined prevalence and correlates of major depressive disorder (MDD) among Chinese PwSD, as well as their perceived need for and utilization of mental health services. METHODS: A total of 227 community-residing adult PwSD were successfully recruited by using multi-stage sampling approach, and interviewed with the Mini-international Neuropsychiatric Interview 5.0. Depressed PwSD's perceived need for and utilization of mental health services were also measured. RESULTS: 29.5% of the PwSD suffered from MDD during the month before the interview and, of the depressed PwSD, 44.8% perceived a need for mental health care but only 1.5% had sought help from mental health specialists. Factors significantly associated with MDD included female gender (OR=2.42), marital status of "non-married" (OR=2.27), having the disability during childhood (OR=4.60) and adulthood (OR=10.99) (vs. at birth), co-occurring other types of disabilities (OR=2.29), major medical conditions (OR=2.62), and impaired ability of activities of daily living (OR=3.23). LIMITATIONS: Findings can only be generalized to PwSD who register with the Disabled People's Federation (DPF) in China, because the sampling frame was based on the registration system of DPF. CONCLUSIONS: There is a large unmet need for mental health services among Chinese PwSD. It is urgently needed to integrate the management of MDD and other common mental disorders into the services system for PwSD in China.

Circadian misalignment alters insulin sensitivity during the light phase and shifts glucose tolerance rhythms in female mice.

Shift work and jet lag, characterized by circadian misalignment, can disrupt several physiological activities, but whether they affect the rhythm of glucose uptake and insulin sensitivity remain unclear. In the present study, female C57BL/6J mice were maintained for four weeks under the condition of 8-hour phase advance and delay every 3-4 days to mimic shift work. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed repeatedly at Zeitgeber time (ZT) 0, ZT6, ZT12, and ZT18. Glucose-stimulated insulin secretion (GSIS) test was performed at ZT6. We found that the average level of daily glucose tolerance did not decrease but the phase of glucose tolerance advanced by 2.27 hours and the amplitude attenuated by 20.4% in shift work mice. At ZT6, IPITT showed blood glucose at 30 min after insulin injection decreased faster in shift work mice (-3.50±0.74mmol/L, -61.58±7.89%) than that in control mice (-2.11±1.10mmol/L, -33.72±17.24%), but IPGTT and GSIS test showed no significant difference between the two groups. Food intake monitor showed that the feeding time of shift work mice continued to advance. Restricting feed to a fixed 12-hour period alleviated the increase of insulin sensitivity induced by shift-work. We also observed that an increase of blood glucose and liver glycogen at ZT0, as well as a phase advance of liver clock genes and some glucose metabolism-related genes such as forkhead box O1 (Foxo1) and peroxisome proliferator activated receptor alpha (Pparα) in shift work mice. Our results showed that light change-simulated shift work altered insulin sensitivity during the light phase and shifted glucose tolerance rhythms in female mice, suggesting a causal association between long-term shift work and type 2 diabetes.

Changes of serum inflammatory factors and miR-145 expression in patients with osteoarthritis before and after treatment and their clinical value.

BACKGROUND: Osteoarthritis is a chronic degenerative disease with an incidence of 50% in people over 65 years old and 80% in people over 80 years old worldwide. It is the second leading reason of loss of working capacity after cardiovascular diseases and severely affects the society and families. Therefore, finding biological markers related to the diagnosis and treatment of osteoarthritis is of great significance in clinical practice. AIM: To observe the changes and clinical value of serum inflammatory factors and miR-145 expression in patients with osteoarthritis before and after treatment. METHODS: Eighty-three patients with knee osteoarthritis (observation group) who were admitted to our hospital from April 2013 to June 2015, and 60 healthy people (control group) during the same period were selected. After 4 wk of treatment, the levels of miR-145, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were compared between the control group and the observation group before treatment. The correlation of miR-145, TNF-α, IL-6, and IL-10 levels with visual analogue scale (VAS), Lysholm, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores was assessed by Pearson correlation analysis. The correlation of the expression of miR-145, TNF-α, IL-6, and IL-10 with Kellgren-Lawrence (K-L) grades was assessed by Spearman correlation analysis. The critical levels of miR-145, TNF-α, IL-6, and IL-10 in distinguishing different K-L grades were determined by receiver operating characteristic (ROC) curve analysis. RESULTS: The expression level of miR-145 in the observation group was significantly higher than that in the control group before treatment (P < 0.05). After treatment, the expression level of miR-145 in the observation group was significantly lower than that before treatment (P < 0.05). The levels of TNF-α and IL-6 in the observation group were significantly higher than those in the control group (P < 0.05), and the level of IL-10 was significantly lower than that in the control group (P < 0.05). After treatment, the levels of TNF-α and IL-6 in the observation group were significantly lower than those before treatment (P < 0.05), and IL-I0 level was significantly higher than that before treatment (P < 0.05). VAS and WOMAC scores were both positively correlated with miR-145, TNF-α, and IL-6 (P < 0.05), and negatively correlated with IL-10 (P < 0.05), while Lysholm scores were negatively correlated with miR-145, TNF-α, and IL-6 (P < 0.05), and positively correlated with IL-10 (P < 0.05). K-L grades were positively correlated with miR-145, TNF-α, and IL-6 (P < 0.05), and negatively correlated with IL-10 (P < 0.05). The area under the ROC curve (AUC) and specificity of TNF-α in differentiating K-L grades I-II were the highest, which were 0.785 and 97.45%, respectively, and miR145 had the highest sensitivity of 94.59%; the AUC and sensitivity of IL-6 in differentiating K-L grades II-III were the highest, which were 0.766 and 97.30%, respectively, and TNF-α had the highest specificity of 86.68%. CONCLUSION: MiR-145 and inflammatory factors have certain diagnostic value in osteoarthritis, and they are expected to become potential indicators for the diagnosis and evaluation of osteoarthritis in the future.

The PILNCR1-miR399 Regulatory Module Is Important for Low Phosphate Tolerance in Maize.

The regulation of adaptive responses to phosphorus (P) deficiency by the microRNA399 (miR399)/PHOSPHATE2 (PHO2) pathway has been well studied in Arabidopsis (Arabidopsis thaliana) but not in maize (Zea mays). Here, we show that miR399 transcripts are strongly induced in maize by phosphate (Pi) deficiency. Transgenic maize plants that overexpressed MIR399b accumulated excessive amounts of P in their shoots and displayed typical Pi-toxicity phenotypes. We reannotated ZmPHO2 with an additional 1,165 bp of the 5' untranslated region. miR399-guided posttranscriptional repression of ZmPHO2 was mainly observed in the P-efficient lines. We identified Pi-deficiency-induced long-noncoding RNA1 (PILNCR1) from our strand-specific RNA libraries. Transient expression assays in Nicotiana benthamiana and maize leaf protoplasts demonstrated that PILNCR1 inhibits ZmmiR399-guided cleavage of ZmPHO2 The abundance of PILNCR1 was significantly higher in P-inefficient lines than in P-efficient lines, which is consistent with the abundance of ZmmiR399 transcripts. These results indicate that the interaction between PILNCR1 and miR399 is important for tolerance to low Pi in maize.

Genome-wide association study dissects yield components associated with low-phosphorus stress tolerance in maize.

KEY MESSAGE: Phosphorus deficiency in soil is a worldwide constraint threatening maize production. Through a genome-wide association study, we identified molecular markers and associated candidate genes and molecular pathways for low-phosphorus stress tolerance. Phosphorus deficiency in soils will severely affect maize (Zea mays L.) growth and development, thus decreasing the final yield. Deciphering the genetic basis of yield-related traits can benefit our understanding of maize tolerance to low-phosphorus stress. However, considering that yield-related traits should be evaluated under field condition with large populations rather than under hydroponic condition at a single-plant level, searching for appropriate field experimental sites and target traits for low-phosphorus stress tolerance is still very challenging. In this study, a genome-wide association analysis using two natural populations was performed to detect candidate genes in response to low-phosphorus stress at two experimental sites representative of different climate and soil types. In total, 259 candidate genes were identified and these candidate genes are mainly involved in four major pathways: transcriptional regulation, reactive oxygen scavenging, hormone regulation, and remodeling of cell wall. Among these candidate genes, 98 showed differential expression by transcriptome data. Based on a haplotype analysis of grain number under phosphorus deficiency condition, the positive haplotypes with favorable alleles across five loci increased grain number by 42% than those without favorable alleles. For further verifying the feasibility of genomic selection for improving maize low-phosphorus tolerance, we also validated the predictive ability of five genomic selection methods and suggested that moderate-density SNPs were sufficient to make accurate predictions for low-phosphorus tolerance traits. All these results will facilitate elucidating genetic basis of maize tolerance to low-phosphorus stress and improving marker-assisted selection efficiency in breeding process.

MicroRNA528 Affects Lodging Resistance of Maize by Regulating Lignin Biosynthesis under Nitrogen-Luxury Conditions.

Lodging under nitrogen (N)-luxury conditions substantially reduces crop yield and seed quality. However, the molecular mechanisms of plant lodging resistance remain largely unclear, especially in maize. We report here that the expression of ZmmiR528, a monocot-specific microRNA, is induced by N luxury but reduced by N deficiency. We show by the thioacidolysis and acetyl bromide analysis that N luxury significantly reduces the generation of H, G, and S monomers of the lignin as well as its total content in maize shoots. We further demonstrate that ZmLACCASE3 (ZmLAC3) and ZmLACCASE5 (ZmLAC5), which encode the copper-containing laccases, are the targets of ZmmiR528. In situ hybridization showed that ZmmiR528 is mainly expressed in maize vascular tissues. Knockdown of ZmmiR528 or overexpression of ZmLAC3 significantly increased the lignin content and rind penetrometer resistance of maize stems. In contrast, transgenic maize plants overexpressing ZmmiR528 had reduced lignin content and rind penetrometer resistance and were prone to lodging under N-luxury conditions. RNA-sequencing analysis revealed that ZmPAL7 and ZmPAL8 are upregulated in transgenic maize lines downregulating ZmmiR528. Under N-luxury conditions, the expression levels of ZmPALs were much higher in ZmmiR528-knockdown lines than in the wild type and transgenic maize lines overexpressing ZmmiR528. Taken together, these results indicate that, by regulating the expression of ZmLAC3 and ZmLAC5, ZmmiR528 affects maize lodging resistance under N-luxury conditions.

Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing.

Ribosome biogenesis is a fundamental process in eukaryotic cells. Although Urb2 protein has been implicated in ribosome biogenesis in yeast, the Urb2 domain is loosely conserved between plants and yeast, and the function of Urb2 protein in plants remains unknown. Here, we isolated a maize mutant, designated as urb2, with defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that urb2 encodes an Urb2 domain-containing protein. Compared with the wild-type (WT), the urb2 mutant showed decreased ratios of 60S/40S and 80S/40S and increased ratios of polyribosomes. The pre-rRNA intermediates of 35/33S rRNA, P-A3 and 18S-A3 were significantly accumulated in the urb2 mutant. Transcriptome profiling of the urb2 mutant indicated that ZmUrb2 affects the expression of a number of ribosome-related genes. We further demonstrated that natural variations in ZmUrb2 are significantly associated with maize kernel length. The overall results indicate that, by affecting pre-rRNA processing, the Urb2 protein is required for ribosome biogenesis in maize.