Convergent evolution of immune receptors underpins distinct elicitin recognition in closely related Solanaceous plants.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

Insights into ZmWAKL in maize kernel development: genome-wide investigation and GA-mediated transcription.

BACKGROUND: The functional roles of the Wall Associated Kinase (WAK) and Wall Associated Kinase Like (WAKL) families in cellular expansion and developmental processes have been well-established. However, the molecular regulation of these kinases in maize development is limited due to the absence of comprehensive genome-wide studies. RESULTS: Through an in-depth analysis, we identified 58 maize WAKL genes, and classified them into three distinct phylogenetic clusters. Moreover, structural prediction analysis showed functional conservation among WAKLs across maize. Promoter analysis uncovered the existence of cis-acting elements associated with the transcriptional regulation of ZmWAKL genes by Gibberellic acid (GA). To further elucidate the role of WAKL genes in maize kernels, we focused on three highly expressed genes, viz ZmWAKL38, ZmWAKL42 and ZmWAKL52. Co-expression analyses revealed that their expression patterns exhibited a remarkable correlation with GA-responsive transcription factors (TF) TF5, TF6, and TF8, which displayed preferential expression in kernels. RT-qPCR analysis validated the upregulation of ZmWAKL38, ZmWAKL42, ZmWAKL52, TF5, TF6, and TF8 following GA treatment. Additionally, ZmWAKL52 showed significant increase of transcription in the present of TF8, with ZmWAKL52 localizing in both the plasma membrane and cell wall. TF5 positively regulated ZmWAKL38, while TF6 positively regulated ZmWAKL42. CONCLUSIONS: Collectively, these findings provide novel insights into the characterization and regulatory mechanisms of specific ZmWAKL genes involved in maize kernel development, offering prospects for their utilization in maize breeding programs.

Epi-Brassinolide Regulates ZmC4 NADP-ME Expression through the Transcription Factors ZmbHLH157 and ZmNF-YC2.

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO2 from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the -1616 bp and -1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Outcomes of hospital-acquired pressure injuries and present-on-admission pressure injuries: A propensity score matching analysis.

BACKGROUND: Pressure injuries (PIs) continue to present significant challenges. In recent years, the number of patients with present-on-admission pressure injury (POA-PI) has increased, but researchers have devoted little attention to it, and little is known about its clinical outcome. AIMS: To compare the clinical outcomes of POA-PI and hospital-acquired pressure injury (HAPI) patients. METHODS: In this study, hospitalized patients with pressure injuries were divided into two groups based on whether they acquired the injury in the hospital or already present at the time of their admission. The disease prognosis, duration of stay, and healthcare costs of patients with HAPI and POA-PI were evaluated using propensity score matching analysis (PSM), t-tests, and Mann-Whitney U tests. RESULTS: The information on 1871 patients was retrieved from the electronic case system retroactively. A total of 305 pairs of patients were effectively matched between the two groups using propensity score matching (HAPI group = 305, POA-PI group = 305). There was no statistically significant difference at characteristics between the two groups (P > 0.05). The percentage of POA-PI group patients who were discharged from the hospital was greater than that of the HAPI group (P < 0.05). Conversely, the percentage of POA-PI group patients who died, ceased receiving treatment, or transferred to the hospital was lower than that of the HAPI group. Patients in the POA-PI group had shorter hospital stays than those in the HAPI group (P < 0.05). Patients in the POA-PI group had lower healthcare costs than those in the HAPI group (P < 0.05). CONCLUSIONS: Patients with POA-PI have superior clinical outcomes than patients with HAPI, but make up the overwhelming majority of hospitalized patients. It is imperative that future research focuses on the reduction of POA-PI and HAPI incidence and the identification of therapies that will enhance patient prevention for these conditions.

Coordinated regulation of starch synthesis in maize endosperm by microRNAs and DNA methylation.

Starch synthesis is an essential feature of crop filling, but knowledge of the molecular mechanisms regulating the expression of starch synthesis genes (SSGs) is currently limited to transcription factors (TFs). Here, we obtained transcriptome, small RNAome, and DNA methylome data from maize (Zea mays) endosperms during multiple developmental stages and established a regulatory network atlas of starch synthesis. Transcriptome analysis showed a sharp transition at 9-10 days after pollination, when genes involved in starch and sucrose metabolism are upregulated and starch accumulates rapidly. Expression pattern analysis established a comprehensive network between SSGs and TFs. During maize endosperm development, the miRNAs with preferential repression of the expression of TFs, particularly the TFs regulating SSG expression, were extensively downregulated. Specifically, ZmMYB138 and ZmMYB115 affected the transcriptional activities of Du1/Wx and Ae1/Bt2 genes at their respective promoter regions. Remarkably, the two TFs were negatively regulated by the copious expression of Zma-miR159k-3p at the post-transcriptional level. This suggests that miRNAs are important regulators of starch synthesis. Moreover, with the exclusion of the TFs, the expression of both SSGs and miRNAs was globally regulated by DNA methylation. Altogether, the present results (i) establish the regulatory functions of miRNAs and DNA methylation in starch synthesis and (ii) indicate that DNA methylation functions as a master switch.

Comparative Study of Starch Phosphorylase Genes and Encoded Proteins in Various Monocots and Dicots with Emphasis on Maize.

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. "True" orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.

Microbe-derived non-necrotic glycoside hydrolase family 12 proteins act as immunogenic signatures triggering plant defenses.

Plant pattern recognition receptors (PRRs) are sentinels at the cell surface sensing microbial invasion and activating innate immune responses. During infection, certain microbial apoplastic effectors can be recognized by plant PRRs, culminating in immune responses accompanied by cell death. However, the intricated relationships between the activation of immune responses and cell death are unclear. Here, we studied the glycoside hydrolase family 12 (GH12) protein, Ps109281, secreted by Phytophthora sojae into the plant apoplast during infection. Ps109281 exhibits xyloglucanase activity, and promotes P. sojae infection in a manner dependent on the enzyme activity. Ps109281 is recognized by the membrane-localized receptor-like protein RXEG1 and triggers immune responses in various plant species. Unlike other characterized GH12 members, Ps109281 fails to trigger cell death in plants. The loss of cell death induction activity is closely linked to a sequence polymorphism at the N-terminus. This sequence polymorphism does not affect the in planta interaction of Ps109281 with the recognition receptor RXEG1, indicating that cell death and immune response activation are determined using different regions of the GH12 proteins. Such GH12 protein also exists in other Phytophthora and fungal pathogens. Taken together, these results unravel the evolution of effector sequences underpinning different immune outputs.

Genome-wide identification of ZmSnRK2 genes and functional analysis of ZmSnRK2.10 in ABA signaling pathway in maize (Zea mays L).

BACKGROUND: Phytohormone abscisic acid (ABA) is involved in the regulation of a wide range of biological processes. In Arabidopsis, it has been well-known that SnRK2s are the central components of the ABA signaling pathway that control the balance between plant growth and stress response, but the functions of ZmSnRK2 in maize are rarely reported. Therefore, the study of ZmSnRK2 is of great importance to understand the ABA signaling pathways in maize. RESULTS: In this study, 14 ZmSnRK2 genes were identified in the latest version of maize genome database. Phylogenetic analysis revealed that ZmSnRK2s are divided into three subclasses based on their diversity of C-terminal domains. The exon-intron structures, phylogenetic, synteny and collinearity analysis indicated that SnRK2s, especially the subclass III of SnRK2, are evolutionally conserved in maize, rice and Arabidopsis. Subcellular localization showed that ZmSnRK2 proteins are localized in the nucleus and cytoplasm. The RNA-Seq datasets and qRT-PCR analysis showed that ZmSnRK2 genes exhibit spatial and temporal expression patterns during the growth and development of different maize tissues, and the transcript levels of some ZmSnRK2 genes in kernel are significantly induced by ABA and sucrose treatment. In addition, we found that ZmSnRK2.10, which belongs to subclass III, is highly expressed in kernel and activated by ABA. Overexpression of ZmSnRK2.10 partially rescued the ABA-insensitive phenotype of snrk2.2/2.3 double and snrk2.2/2.3/2.6 triple mutants and led to delaying plant flowering in Arabidopsis. CONCLUSION: The SnRK2 gene family exhibits a high evolutionary conservation and has expanded with whole-genome duplication events in plants. The ZmSnRK2s expanded in maize with whole-genome and segmental duplication, not tandem duplication. The expression pattern analysis of ZmSnRK2s in maize offers important information to study their functions. Study of the functions of ZmSnRK.10 in Arabidopsis suggests that the ABA-dependent members of SnRK2s are evolutionarily conserved in plants. Our study elucidated the structure and evolution of SnRK2 genes in plants and provided a basis for the functional study of ZmSnRK2s protein in maize.

Identification of quantitative trait loci for kernel-related traits and the heterosis for these traits in maize (Zea mays L.).

Heterosis has been extensively applied for many traits during maize breeding, but there has been relatively little attention paid to the heterosis for kernel size. In this study, we evaluated a population of 301 recombinant inbred lines derived from a cross between 08-641 and YE478, as well as 298 hybrids from an immortalized F2 (IF2) population to detect quantitative trait loci (QTLs) for six kernel-related traits and the mid-parent heterosis (MPH) for these traits. A total of 100 QTLs, six pairs of loci with epistatic interactions, and five significant QTL × environment interactions were identified in both mapping populations. Seven QTLs accounted for over 10% of the phenotypic variation. Only four QTLs affected both the trait means and the MPH, suggesting the genetic mechanisms for kernel-related traits and the heterosis for kernel size are not completely independent. Moreover, more than half of the QTLs for each trait in the IF2 population exhibited dominance, implying that dominance is more important than other genetic effects for the heterosis for kernel-related traits. Additionally, 20 QTL clusters comprising 46 QTLs were detected across ten chromosomes. Specific chromosomal regions (bins 2.03, 6.04-6.05, and 9.01-9.02) exhibited pleiotropy and congruency across diverse heterotic patterns in previous studies. These results may provide additional insights into the genetic basis for the MPH for kernel-related traits.

Genetic dissection of yield-related traits and mid-parent heterosis for those traits in maize (Zea mays L.).

BACKGROUND: Utilization of heterosis in maize could be critical in maize breeding for boosting grain yield. However, the genetic architecture of heterosis is not fully understood. To dissect the genetic basis of yield-related traits and heterosis in maize, 301 recombinant inbred lines derived from 08 to 641 × YE478 and 298 hybrids from the immortalized F2 (IF2) population were used to map quantitative trait loci (QTLs) for nine yield-related traits and mid-parent heterosis. RESULTS: We observed 156 QTLs, 28 pairs of loci with epistatic interaction, and 10 significant QTL × environment interactions in the inbred and hybrid mapping populations. The high heterosis in F1 and IF2 populations for kernel weight per ear (KWPE), ear weight per ear (EWPE), and kernel number per row (KNPR) matched the high percentages of QTLs (over 50%) for those traits exhibiting overdominance, whereas a notable predominance of loci with dominance effects (more than 70%) was observed for traits that show low heterosis such as cob weight per ear (CWPE), rate of kernel production (RKP), ear length (EL), ear diameter (ED), cob diameter, and row number (RN). The environmentally stable QTL qRKP3-2 was identified across two mapping populations, while qKWPE9, affecting the trait mean and the mid-parent heterosis (MPH) level, explained over 18% of phenotypic variations. Nine QTLs, qEWPE9-1, qEWPE10-1, qCWPE6, qEL8, qED2-2, qRN10-1, qKWPE9, qKWPE10-1, and qRKP4-3, accounted for over 10% of phenotypic variation. In addition, QTL mapping identified 95 QTLs that were gathered together and integrated into 33 QTL clusters on 10 chromosomes. CONCLUSIONS: The results revealed that (1) the inheritance of yield-related traits and MPH in the heterotic pattern improved Reid (PA) × Tem-tropic I (PB) is trait-dependent; (2) a large proportion of loci showed dominance effects, whereas overdominance also contributed to MPH for KNPR, EWPE, and KWPE; (3) marker-assisted selection for markers at genomic regions 1.09-1.11, 2.04, 3.08-3.09, and 10.04-10.05 contributed to hybrid performance per se and heterosis and were repeatedly reported in previous studies using different heterotic patterns is recommended.

Maize brachytic2 (br2) suppresses the elongation of lower internodes for excessive auxin accumulation in the intercalary meristem region.

BACKGROUND: Short internodes contribute to plant dwarfism, which is exceedingly beneficial for crop production. However, the underlying mechanisms of internode elongation are complicated and have been not fully understood. RESULTS: Here, we report a maize dwarf mutant, dwarf2014 (d2014), which displays shortened lower internodes. Map-based cloning revealed that the d2014 gene is a novel br2 allele with a splicing variation, resulting in a higher expression of BR2-T02 instead of normal BR2-T01. Then, we found that the internode elongation in d2014/br2 exhibited a pattern of inhibition-normality-inhibition (transient for the ear-internode), correspondingly, at the 6-leaf, 12-leaf and 14-leaf stages. Indeed, BR2 encodes a P-glycoprotein1 (PGP1) protein that functions in auxin efflux, and our in situ hybridization assay showed that BR2 was mainly expressed in vascular bundles of the node and internode. Furthermore, significantly higher auxin concentration was detected in the stem apex of d2014 at the 6-leaf stage and strictly in the node region for the ear-internode at the 14-leaf stage. In such context, we propose that BR2/PGP1 transports auxin from node to internode through the vascular bundles, and excessive auxin accumulation in the node (immediately next to the intercalary meristem) region suppresses internode elongation of d2014. CONCLUSIONS: These findings suggest that low auxin levels mediated by BR2/PGP1 in the intercalary meristem region are crucial for internode elongation.

The miR164-dependent regulatory pathway in developing maize seed.

MicroRNA164 (miR164) plays a key role in leaf and flower development, lateral root initiation, and stress responses. However, little is known about the regulatory roles of miR164 during seed development, particularly in maize. The aim of this study was to discover the developmental function of miR164 in maize seed. Small RNA sequencing (sRNA-seq) was performed at two key stages. The results indicated that miR164 was down-regulated during maize seed development. In addition, degradome library sequencing and transient expression assays identified the target genes for miR164. Two microRNA (miRNA) pairs, miR164-NAM, ATAF, and CUC32 (NAC32) and miR164-NAC40, were isolated. The developmental function of miR164 was determined by analyzing the differentially expressed genes (DEGs) between the wild-type and miR164 transgenic lines using RNA sequencing (RNA-seq) and by screening the DEGs related to NAC32 and NAC40 via co-expression and transient expression analysis. These results identified two beta-expansin genes, EXPB14 and EXPB15, which were located downstream of the NAC32 and NAC40 genes. This study revealed, for the first time, a miR164-dependent regulatory pathway, miR164-NAC32/NAC40-EXPB14/EXPB15, which participates in maize seed expansion. These findings highlight the significance of miR164 in maize seed development, and can be used to explore the role of miRNA in seed development.

Loss of p53 Sensitizes Cells to Palmitic Acid-Induced Apoptosis by Reactive Oxygen Species Accumulation.

Palmitic acid, the most common saturated free fatty acid, can lead to lipotoxicity and apoptosis when overloaded in non-fat cells. Palmitic acid accumulation can induce pancreatic ß-cell dysfunction and cardiac myocyte apoptosis. Under various cellular stresses, the activation of p53 signaling can lead to cell cycle arrest, DNA repair, senescence, or apoptosis, depending on the severity/type of stress. Nonetheless, the precise role of p53 in lipotoxicity induced by palmitic acid is not clear. Here, our results show that palmitic acid induces p53 activation in a dose- and time-dependent manner. Furthermore, loss of p53 makes cells sensitive to palmitic acid-induced apoptosis. These results were demonstrated in human colon carcinoma cells (HCT116) and primary mouse embryo fibroblasts (MEF) through analysis of DNA fragmentation, flow cytometry, colony formation, and Western blots. In the HCT116 p53-/- cell line, palmitic acid induced greater reactive oxygen species formation compared to the p53+/+ cell line. The reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and reduced glutathione (GSH) partially attenuated apoptosis in the HCT116 p53-/- cell line but had no obvious effect on the p53+/+ cell line. Furthermore, p53 induced the expression of its downstream target genes, p21 and Sesn2, in response to ROS induced by palmitic acid. Loss of p21 also leads to more palmitic acid-induced cell apoptosis in the HCT116 cell line compared with HCT116 p53+/+ and HCT116 p53-/-. In a mouse model of obesity, glucose tolerance test assays showed higher glucose levels in p53-/- mice that received a high fat diet compared to wild type mice that received the same diet. There were no obvious differences between p53-/- and p53+/+ mice that received a regular diet. We conclude that p53 may provide some protection against palmitic acid- induced apoptosis in cells by targeting its downstream genes in response to this stress.

The Proteomic Analysis of Maize Endosperm Protein Enriched by Phos-tagtm Reveals the Phosphorylation of Brittle-2 Subunit of ADP-Glc Pyrophosphorylase in Starch Biosynthesis Process.

AGPase catalyzes a key rate-limiting step that converts ATP and Glc-1-p into ADP-glucose and diphosphate in maize starch biosynthesis. Previous studies suggest that AGPase is modulated by redox, thermal and allosteric regulation. However, the phosphorylation of AGPase is unclear in the kernel starch biosynthesis process. Phos-tagTM technology is a novel method using phos-tagTM agarose beads for separation, purification, and detection of phosphorylated proteins. Here we identified phos-tagTM agarose binding proteins from maize endosperm. Results showed a total of 1733 proteins identified from 10,678 distinct peptides. Interestingly, a total of 21 unique peptides for AGPase sub-unit Brittle-2 (Bt2) were identified. Bt2 was demonstrated by immunoblot when enriched maize endosperm protein with phos-tagTM agarose was in different pollination stages. In contrast, Bt2 would lose binding to phos-tagTM when samples were treated with alkaline phosphatase (ALP). Furthermore, Bt2 could be detected by Pro-Q diamond staining specifically for phosphorylated protein. We further identified the phosphorylation sites of Bt2 at Ser10, Thr451, and Thr462 by iTRAQ. In addition, dephosphorylation of Bt2 decreased the activity of AGPase in the native gel assay through ALP treatment. Taking together, these results strongly suggest that the phosphorylation of AGPase may be a new model to regulate AGPase activity in the starch biosynthesis process.

[A case of severe obstructive sleep apnea hypopnea syndrome with urinary and anal incontinence].

A case of a young male patient, who came to the Second Xiangya Hospital, Central South University because of snoring for 10 years and nocturnal gatism for half month, was analyzed retrospectively. He was diagnosed as obstructive sleep apnea hypopnea syndrome (OSAHS) finally. The patient had been diagnosed and treated as stroke in the local hospital, while urinary and anal incontinence were not relieved. It was a dilemma for him to be properly diagnosed and treated. Polysomnography in our hospital revealed apnea hypopnea index (AHI) at 44.7 events/h, oxygen desaturation index (ODI) at 70.8 events/h and the longest apnea time at 185 seconds while the lowest blood oxygen saturation reduced to 31%. In addition, 413 events of apnea accounted for 61.2% of the sleep time and the minimal heart rate was 23 times/min. The patient was diagnosed as severe OSAHS with hypoxia metabolic brain disease, moderate pulmonary arterial hypertension, secondary polycythemia and obesity hypoventilation syndrome finally. He received the treatment of positive airway pressure non-invasive ventilator with an average pressure at 11.7 cmH2O with reduced AHI and increased blood oxygen saturation. The urinary and anal incontinence disappeared during the first night of treatment and it has been totally resolved so far. We considered that gatism was secondary to OSAHS with severe hypoxia resulted from attenuated regulation of primary defecation in the night. Physicians should pay attention to OSAHS when accepting obese patients with nocturnal incontinence, obvious daytime sleepiness and night snoring. Urinary and anal incontinence could be completely disappeared under therapy of positive airway pressure.

Transcriptome profiling of spleen provides insights into the antiviral mechanism in Schizothorax prenanti after poly (I: C) challenge.

Schizothorax prenanti (S. prenanti) is an important economical cold-water fish species in southwestern China, but it is susceptible to various pathogens infection. In order to clearly elucidate the antiviral mechanism, in this study, we have analyzed the transcriptome of S. prenanti spleen after challenge with the virus mimic, poly (I:C) (pIC), using next generation sequencing technology (RNA-seq). A total of 313 differential expressed genes (DEGs) in spleen at 12 h were obtained after pIC treatment, including 268 significantly up-regulated unigenes (fold change > 2) and 45 significantly down-regulated unigenes (fold change > 2). Through the immune-related DEGs (IRDs) screening, 47 IRDs were used to establish heat map, which intuitively showed a significantly difference after pIC treatment. To validate the RNA-seq data and observe gene expression, the expression levels of 14 IRDs were detected by qPCR after pIC treatment at 0, 4, 8, 12, and 24 h. The results indicated that the qPCR data presented a positive line correlation with RNA-seq data, and the 14 IRDs were responsive to pIC stimulation except IL-1ß. Thus, based on the RNA-seq and qPCR data, we inferred that MDA5- and Jak-mediated signaling pathways may involve in the antiviral signaling transduction, and induce type I IFNs and ISGs to block virus invasion, respectively. Unfortunately, TLR3 and TLR22, as receptors of virus dsRNA, were no significantly expressed in this study. Nonetheless, our study still provides useful mRNA sequences of antiviral immunity for further immunological research, and facilitates improving disease restriction in S. prenanti.

Identification and characterization of microRNAs in maize endosperm response to exogenous sucrose using small RNA sequencing.

Sucrose acts as a signaling molecule for genes critical to starch biosynthesis in maize endosperm. Previously, we showed that sucrose could regulate starch biosynthesis in maize via transcription factors. To better understand the complex regulation of starch biosynthesis, the 10days after pollination endosperm from Zea mays L. B73 inbred line was collected and treated with sucrose for small RNA sequencing. The sequencing results revealed that 24 known miRNAs and 190 novel miRNAs were significantly differentially expressed in response to sucrose. In addition, most of target mRNAs were characterized as transcription factors, mainly including, MYB, ARF, NAC, AP2/ERF, WRKY, and GRAS, which play important roles in starch biosynthesis and seed development in maize endosperm. The expression profiles of miR398a/b and miR159b/j/k followed opposite expression trends to their target genes when analyzed by qPCR. In conclusion, these results show that sucrose regulates the expression of starch synthetic genes through miRNAs.

Genome-wide characterization of non-reference transposons in crops suggests non-random insertion.

BACKGROUND: Transposons (transposable elements or TEs) are DNA sequences that can change their position within the genome. A large number of TEs have been identified in reference genome of each crop(named accumulated TEs), which are the important part of genome. However, whether there existed TEs with different insert positions in resequenced crop accession genomes from those of reference genome (named non-reference transposable elements, non-ref TEs), and what the characteristics (such as the number, type and distribution) are. To identify and characterize crop non-ref TEs, we analyzed non-ref TEs in more than 125 accessions from rice (Oryza sativa), maize (Zea mays) and sorghum (Sorghum bicolor) using resequenced data with paired-end mapping methods. RESULTS: We identified 13,066, 23,866 and 35,679 non-ref TEs in rice, maize and sorghum, respectively. Genome-wide characterization analysis shows that most of non-ref TEs were unique and non-ref TE classes shows different among rice, maize and sorghum. We found that non-ref TEs have a strong positive correlation with gene number and have a bias toward insertion near genes, but with a preference for avoiding coding regions in maize and sorghum. The genes affected by non-ref TE insertion were functionally enriched for stress response mechanisms in all three crops. CONCLUSIONS: These observations suggest that transposon insertion is not a random event and it makes genomic diversity, which may affect the intraspecific adaption and evolution of crops.

ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters.

Starch synthesis is a key process that influences crop yield and quality, though little is known about the regulation of this complex metabolic pathway. Here, we present the identification of ZmbZIP91 as a candidate regulator of starch synthesis via co-expression analysis in maize (Zea mays L.). ZmbZIP91 was strongly associated with the expression of starch synthesis genes. Reverse tanscription-PCR (RT-PCR) and RNA in situ hybridization indicated that ZmbZIP91 is highly expressed in maize endosperm, with less expression in leaves. Particle bombardment-mediated transient expression in maize endosperm and leaf protoplasts demonstrated that ZmbZIP91 could positively regulate the expression of starch synthesis genes in both leaves and endosperm. Additionally, the Arabidopsis mutant vip1 carried a mutation in a gene (VIP1) that is homologous to ZmbZIP91, displayed altered growth with less starch in leaves, and ZmbZIP91 was able to complement this phenotype, resulting in normal starch synthesis. A yeast one-hybrid experiment and EMSAs showed that ZmbZIP91 could directly bind to ACTCAT elements in the promoters of starch synthesis genes (pAGPS1, pSSI, pSSIIIa, and pISA1). These results demonstrate that ZmbZIP91 acts as a core regulatory factor in starch synthesis by binding to ACTCAT elements in the promoters of starch synthesis genes.

A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes.

KEY MESSAGE: Conditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein-protein interaction, and the investigation of PCD-related processes. Plant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein-protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, ß-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes.