Time-series transcriptomics reveals a drought-responsive temporal network and crosstalk between drought stress and the circadian clock in foxtail millet.

Drought stress is a serious factor affecting crop growth and production worldwide. The circadian clock has been identified as key to improving regional adaptability of plants. However, our understanding of the contribution of the circadian clock to drought response and the impacts of drought stress on the circadian clock in plants is still limited. To explore the interactions between the circadian clock and drought stress, foxtail millet seedlings were treated with simulated drought (20% polyethylene glycol-6000) treatment starting at the day (DD) onset zeitgeber time 0 (ZT0, lights on) and at the night (DN) onset zeitgeber time 16 (ZT16, lights off). A high temporal-resolution transcriptomic investigation was performed using DD and DN samples collected at intervals of 2 or 4 h within a 24-h drought-treatment period. Overall, we identified 13 294 drought-responsive genes (DRGs). Among these DRGs, 7931 were common between DD and DN samples, 2638 were specific to DD, and 2725 were specific to DN. Additionally, we identified 1257 circadian genes, of which 67% were DRGs. Interestingly, with drought treatment starting at the day for 8, 12 or 16 h, the circadian phase shifted to 12 h. We also found that the circadian clock led to different day and night drought-responsive pathways. The identification of DRG_Clock (DRG and circadian clock) and DRG_NonClock (DRG and not circadian clock) genes provides a reference for selecting candidate drought resistance genes. Our work reveals the temporal drought-response process and crosstalk between drought stress and the circadian clock in foxtail millet.

Maize WRKY28 interacts with the DELLA protein D8 to affect skotomorphogenesis and participates in the regulation of shade avoidance and plant architecture.

Competition for light from neighboring vegetation can trigger the shade-avoidance response (SAR) in plants, which is detrimental to their yield. The molecular mechanisms regulating SAR are well established in Arabidopsis, and some regulators of skotomorphogenesis have been found to be involved in the regulation of the SAR and plant architecture. However, the role of WRKY transcription factors in this process has rarely been reported, especially in maize (Zea mays). Here, we report that maize Zmwrky28 mutants exhibit shorter mesocotyls in etiolated seedlings. Molecular and biochemical analyses demonstrate that ZmWRKY28 directly binds to the promoter regions of the Small Auxin Up RNA (SAUR) gene ZmSAUR54 and the Phytochrome-Interacting Factor (PIF) gene ZmPIF4.1 to activate their expression. In addition, the maize DELLA protein Dwarf Plant8 (D8) interacts with ZmWRKY28 in the nucleus to inhibit its transcriptional activation activity. We also show that ZmWRKY28 participates in the regulation of the SAR, plant height, and leaf rolling and erectness in maize. Taken together, our results reveal that ZmWRKY28 is involved in GA-mediated skotomorphogenic development and can be used as a potential target to regulate SAR for breeding of high-density-tolerant cultivars.

The m6 A reader SiYTH1 enhances drought tolerance by affecting the messenger RNA stability of genes related to stomatal closure and reactive oxygen species scavenging in Setaria italica.

Foxtail millet (Setaria italica), a vital drought-resistant crop, plays a significant role in ensuring food and nutritional security. However, its drought resistance mechanism is not fully understood. N6 -methyladenosine (m6 A) modification of RNA, a prevalent epi-transcriptomic modification in eukaryotes, provides a binding site for m6 A readers and affects plant growth and stress responses by regulating RNA metabolism. In this study, we unveiled that the YT521-B homology (YTH) family gene SiYTH1 positively regulated the drought tolerance of foxtail millet. Notably, the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H2 O2 under drought stress. Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m6 A modification related to stomatal closure and reactive oxygen species (ROS) scavenging under drought stress. SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1-GFP transgenic foxtail millet. It formed dynamic liquid-like SiYTH1 cytosol condensates in response to drought stress. Moreover, the cytoplasmic protein SiYTH1 was identified as a distinct m6 A reader, facilitating the stabilization of its directly bound SiARDP and ROS scavenging-related transcripts under drought stress. Furthermore, natural variation analysis revealed SiYTH1AGTG as the dominant allele responsible for drought tolerance in foxtail millet. Collectively, this study provides novel insights into the intricate mechanism of m6 A reader-mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

ZmDof3, a maize endosperm-specific Dof protein gene, regulates starch accumulation and aleurone development in maize endosperm.

KEY MESSAGE: To explore the function of Dof transcription factors during kernel development in maize, we first identified Dof genes in the maize genome. We found that ZmDof3 was exclusively expressed in the endosperm of maize kernel and had the features of a Dof transcription factor. Suppression of ZmDof3 resulted in a defective kernel phenotype with reduced starch content and a partially patchy aleurone layer. The expression levels of starch synthesis-related genes and aleurone differentiation-associated genes were down-regulated in ZmDof3 knockdown kernels, indicating that ZmDof3 plays an important role in maize endosperm development. The maize endosperm, occupying a large proportion of the kernel, plays an important role in seed development and germination. Current knowledge regarding the regulation of endosperm development is limited. Dof proteins, a family of plant-specific transcription factors, play critical roles in diverse biological processes. In this study, an endosperm-specific Dof protein gene, ZmDof3, was identified in maize through genome-wide screening. Suppression of ZmDof3 resulted in a defective kernel phenotype. The endosperm of ZmDof3 knockdown kernels was loosely packed with irregular starch granules observed by electronic microscope. Through genome-wide expression profiling, we found that down-regulated genes were enriched in GO terms related to carbohydrate metabolism. Moreover, ZmDof3 could bind to the Dof core element in the promoter of starch biosynthesis genes Du1 and Su2 in vitro and in vivo. In addition, the aleurone at local position in mature ZmDof3 knockdown kernels varied from one to three layers, which consisted of smaller and irregular cells. Further analyses showed that knockdown of ZmDof3 reduced the expression of Nkd1, which is involved in aleurone cell differentiation, and that ZmDof3 could bind to the Dof core element in the Nkd1 promoter. Our study reveals that ZmDof3 functions in maize endosperm development as a positive regulator in the signaling system controlling starch accumulation and aleurone development.

The 5' untranslated region of potato SBgLR gene contributes to pollen-specific expression.

MAIN CONCLUSION: The 5'UTR of SBgLR enhances gene expression by regulating both its transcription and translation. SBgLR (Solanum tuberosum genomic lysine rich) is a pollen-specific gene in Solanum tuberosum that encodes a microtubule-associated protein. The region from -85 to +180 (transcription start site at +1) was determined to be critical for specific expression in pollen grains. Transient and stable expression assays showed that the 5'UTR (from +1 to +184) enhanced gene expression in all detected tissues of transgenic tobacco. Deletion analysis demonstrated that the secondary structure of the 5'UTR had no effect on pollen-specific SBgLR expression, while the region from +31 to +60 was crucial. Further investigation indicated that mRNA expression was slightly decreased when the +31 to +60 region was deleted, but the mRNA decay rate remained unchanged. Mutation analysis also confirmed that the pollen-specific element TTTCT, located at +37, played an important role in pollen-specific expression. Using yeast one-hybrid screening, we isolated a DNA-binding with one finger (Dof) protein gene (StDof23) and an AT-hook motif nuclear-localized (AHL) protein gene (StAHL) from potato pollen. Further investigation indicated that StDof23 interacted with and positively regulated the +31 to +60 region; moreover, StAHL interacted with and negatively regulated the -49 to +60 region. These results demonstrate that the 5'UTR not only enhanced gene expression but also altered the tissue-specific expression pattern by regulating both transcription and translation.

The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles.

Over the past two decades, Zea mays (maize) has been established as a model system for the study of indirect plant defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles, mainly composed of sesquiterpenes, to attract the natural enemies of the herbivores. This is associated with a swift transcriptional induction of terpene synthases such as TPS10; however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure. Here, by exploiting the finding that the maize TPS10 promoter retained its full responsiveness to herbivory in Arabidopsis, we identified the region from -300 to -200 of the TPS10 promoter as both necessary and sufficient for its herbivore inducibility through 5' deletion mapping. A high-throughput screening of an Arabidopsis transcription factor library using this promoter region as the bait identified seven AP2/ERF family transcription factors. Among their close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Meanwhile, EREB58 was also responsive to Jasmonate. In vivo and in vitro assays indicated that EREB58 promotes TPS10 expression by directly binding to the GCC-box within the region from -300 to -200 of the TPS10 promoter. Transgenic maize plants overexpressing EREB58 constitutively over-accumulate TPS10 transcript, and also (E)-ß-farnesene and (E)-α-bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles was abolished in EREB58-RNAi transgenic lines. In sum, these results demonstrate that EREB58 is a positive regulator of sesquiterpene production by directly promoting TPS10 expression.

Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm.

The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

Global analysis of uncapped mRNA changes under drought stress and microRNA-dependent endonucleolytic cleavages in foxtail millet.

BACKGROUND: mRNA degradation plays an important role in the determination of mRNA abundance and can quickly regulate gene expression. The production of uncapped mRNAs, an important mechanism of mRNA degradation, can be initiated by decapping enzymes, endonucleases or small RNAs such as microRNAs (miRNAs). Little is known, however, about the role of uncapped mRNAs in plants under environmental stress. RESULTS: Using a novel approach called parallel analysis of RNA ends (PARE), we performed a global study of uncapped mRNAs under drought stress in foxtail millet (Setaria italica [L.] P. Beauv.). When both gene degradation (PARE) and gene transcription (RNA-sequencing) data were considered, four types of mRNA decay patterns were identified under drought stress. In addition, 385 miRNA-target interactions were identified in the PARE data using PAREsnip. The PARE analysis also suggested that two miRNA hairpin processing mechanisms--loop-last and loop-first processing--operate in foxtail millet, with both miR319 and miR156 gene families undergoing precise processing via the unusual loop-first mechanism. Finally, we found 11 C4 photosynthesis-related enzymes encoded by drought-responsive genes. CONCLUSIONS: We performed a global analysis of mRNA degradation under drought stress and uncovered diverse drought-response mechanisms in foxtail millet. This information will deepen our understanding of mRNA expression under stressful environmental conditions in gramineous plants. In addition, PARE analysis identified many miRNA targets and revealed miRNA-precursor processing modes in foxtail millet.

Spatial and temporal activity of the foxtail millet (Setaria italica) seed-specific promoter pF128.

MAIN CONCLUSION: pF128 drives GUS specifically expressed in transgenic seeds of foxtail millet and Zea mays with higher activity than the constitutive CaMV35S promoter and the maize seed-specific 19Z promoter. Foxtail millet (Setaria italica), a member of the Poaceae family, is an important food and fodder crop in arid regions. Foxtail millet is an excellent C4 crop model owing to its small genome (~490 Mb), self-pollination and availability of a complete genome sequence. F128 was isolated from a cDNA library of foxtail millet immature seeds. Real-time PCR analysis revealed that F128 mRNA was specifically expressed in immature and mature seeds. The highest F128 mRNA level was observed 5 days after pollination and gradually decreased as the seed matured. Sequence analysis suggested that the protein encoded by F128 is likely a protease inhibitor/seed storage protein/lipid-transfer protein. The 1,053 bp 5' flanking sequence of F128 (pF128) was isolated and fused to the GUS reporter gene. The corresponding vector was then transformed into Arabidopsis thaliana, foxtail millet and Zea mays. GUS analysis revealed that pF128 drove GUS expression efficiently and specifically in the seeds of transgenic Arabidopsis, foxtail millet and Zea mays. GUS activity was also detected in Arabidopsis cotyledons. Activity of pF128 was higher than that observed for the constitutive CaMV35S promoter and the maize seed-specific 19 Zein (19Z) promoter. These results indicate that pF128 is a seed-specific promoter. Its application is expected to be of considerable value in plant genetic engineering.

Microtubule-Associated Protein SBgLR Facilitates Storage Protein Deposition and Its Expression Leads to Lysine Content Increase in Transgenic Maize Endosperm.

Maize (Zea mays) seed is deficient in protein and lysine content. Many studies have been made to improve the nutritional quality of maize seeds. Previously, we reported the role of a natural lysine-rich protein gene SBgLR in increasing protein and lysine content. However, how the SBgLR improves lysine and protein content remains unclear. Here, the reasons and possible mechanism for SBgLR in protein and lysine improvement have been analyzed and discussed. Through seed-specific expression of SBgLR, we obtained transgenic maize with the simultaneously increased lysine and protein contents. High-protein and high-lysine characters were stably inherited across generations. The expression of SBgLR in maize kernels increased the accumulation of both zeins and non-zein proteins. Transmission electron microscopy showed that the number of protein bodies (PBs) was increased obviously in SBgLR transgenic immature endosperms with the morphology and structure of PBs unchanged. The proteinaceous matrix was more abundant in transgenic mature endosperms under scanning electron microscopy. The stabilities of zein and lysine-rich non-zein genes were also increased in transgenic endosperms. Finally, the potential application of SBgLR in maize nutrient improvement was evaluated. This study shows that a cytoskeleton-associated protein has potential applicable value in crop nutrient improving, and provided a feasible strategy for improvement of maize grain quality.

SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet.

BACKGROUND: Late embryogenesis abundant (LEA) proteins are involved in protecting higher plants from damage caused by environmental stresses. Foxtail millet (Setaria italica) is an important cereal crop for food and feed in semi-arid areas. However, the molecular mechanisms underlying tolerance to these conditions are not well defined. RESULTS: Here, we characterized a novel atypical LEA gene named SiLEA14 from foxtail millet. It contains two exons separated by one intron. SiLEA14 was expressed in roots, stems, leaves, inflorescences and seeds at different levels under normal growth conditions. In addition, SiLEA14 was dramatically induced by osmotic stress, NaCl and exogenous abscisic acid. The SiLEA14 protein was localized in the nucleus and the cytoplasm. Overexpression of SiLEA14 improved Escherichia coli growth performance compared with the control under salt stress. To further assess the function of SiLEA14 in plants, transgenic Arabidopsis and foxtail millet plants that overexpressed SiLEA14 were obtained. The transgenic Arabidopsis seedlings showed higher tolerance to salt and osmotic stress than the wild type (WT). Similarly, the transgenic foxtail millet showed improved growth under salt and drought stresses compared with the WT. Taken together, our results indicated that SiLEA14 is a novel atypical LEA protein and plays important roles in resistance to abiotic stresses in plants. CONCLUSION: We characterized a novel atypical LEA gene SiLEA14 from foxtail millet, which plays important roles in plant abiotic stress resistance. Modification of SiLEA14 expression may improve abiotic stress resistance in agricultural crops.

An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress.

The DREB (dehydration-responsive element binding)-type transcription factors regulate the expression of stress-inducible genes by binding the DRE/CRT cis-elements in promoter regions. The upstream transcription factors that regulate the transcription of DREB transcription factors have not been clearly defined, although the function of DREB transcription factors in abiotic stress is known. In this study, an abscisic acid (ABA)-responsive DREB-binding protein gene (SiARDP) was cloned from foxtail millet (Setaria italica). The transcript level of SiARDP increased not only after drought, high salt, and low temperature stresses, but also after an ABA treatment in foxtail millet seedlings. Two ABA-responsive elements (ABRE1: ACGTGTC; ABRE2: ACGTGGC) exist in the promoter of SiARDP. Further analyses showed that two ABA-responsive element binding (AREB)-type transcription factors, SiAREB1 and SiAREB2, could physically bind to the ABRE core element in vitro and in vivo. The constitutive expression of SiARDP in Arabidopsis thaliana enhanced drought and salt tolerance during seed germination and seedling development, and overexpression of SiARDP in foxtail millet improved drought tolerance. The expression levels of target genes of SiARDP were upregulated in transgenic Arabidopsis and foxtail millet. These results reveal that SiARDP, one of the target genes of SiAREB, is involved in ABA-dependent signal pathways and plays a critical role in the abiotic stress response in plants.

Maize ZmRACK1 is involved in the plant response to fungal phytopathogens.

The receptor for activated C kinase 1 (RACK1) belongs to a protein subfamily containing a tryptophan-aspartic acid-domain (WD) repeat structure. Compelling evidence indicates that RACK1 can interact with many signal molecules and affect different signal transduction pathways. In this study, we cloned a maize RACK1 gene (ZmRACK1) by RT-PCR. The amino acid sequence of ZmRACK1 had seven WD repeats in which there were typical GH (glycine-histidine) and WD dipeptides. Comparison with OsRACK1 from rice revealed 89% identity at the amino acid level. Expression pattern analysis by RT-PCR showed that ZmRACK1 was expressed in all analyzed tissues of maize and that its transcription in leaves was induced by abscisic acid and jasmonate at a high concentration. Overexpression of ZmRACK1 in maize led to a reduction in symptoms caused by Exserohilum turcicum (Pass.) on maize leaves. The expression levels of the pathogenesis-related protein genes, PR-1 and PR-5, increased 2.5-3 times in transgenic maize, and reactive oxygen species production was more active than in the wild-type. Yeast two-hybrid assays showed that ZmRACK1 could interact with RAC1, RAR1 and SGT1. This study and previous work leads us to believe that ZmRACK1 may form a complex with regulators of plant disease resistance to coordinate maize reactions to pathogens.

Seed-specific expression of a lysine-rich protein gene, GhLRP, from cotton significantly increases the lysine content in maize seeds.

Maize seed storage proteins are a major source of human and livestock consumption. However, these proteins have poor nutritional value, because they are deficient in lysine and tryptophan. Much research has been done to elevate the lysine content by reducing zein content or regulating the activities of key enzymes in lysine metabolism. Using the naturally lysine-rich protein genes, sb401 and SBgLR, from potato, we previously increased the lysine and protein contents of maize seeds. Here, we examined another natural lysine-rich protein gene, GhLRP, from cotton, which increased the lysine content of transgenic maize seeds at levels varying from 16.2% to 65.0% relative to the wild-type. The total protein content was not distinctly different, except in the six transgenic lines. The lipid and starch levels did not differ substantially in Gossypium hirsutum L. lysine-rich protein (GhLRP) transgenic kernels when compared to wild-type. The agronomic characteristics of all the transgenic maize were also normal. GhLRP is a high-lysine protein candidate gene for increasing the lysine content of maize. This study provided a valuable model system for improving maize lysine content.

Genome-wide annotation of genes and noncoding RNAs of foxtail millet in response to simulated drought stress by deep sequencing.

Drought is a major abiotic stress that affects plant growth, production, and survival. Plants have evolved sophisticated and highly complex reactions to drought stress, including large-scale transcriptome reconfiguration. Foxtail millet (Setaria italica) is a member of the Poaceae family. Because of its outstanding tolerance to drought stress foxtail millet has the potential to become a new model organism. To enrich our knowledge of the processes that contribute to drought resistance, we have used a deep sequencing approach to generate a genome-wide transcriptome of foxtail millet after exposure to simulated drought stress. A large number of differentially expressed genes were characterized; in particular, we examined the roles of small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNAs) in response to a water-deficit condition. These RNAs have remained largely unexplored in previous studies of stress-induced transcriptomes. We found that the reduced levels of 24-nt siRNA flanking genes were associated, for the most part, with proximal up-regulated genes, indicating a potential effect of 24-nt siRNAs on drought-regulated gene expression. Several lncRNAs that responded to the simulated drought stress were also identified, and we found that one of them shared sequence conservation and colinearity with its counterpart in sorghum (Sorghum bicolor). Our findings provide new insights into drought-induced changes in the foxtail millet transcriptome.

Genome-wide characterization of microRNA in foxtail millet (Setaria italica).

BACKGROUND: MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet (Setaria italica) have, until now, been poorly understood. RESULTS: In this study, two replicate small RNA libraries from foxtail millet shoots were sequenced, and 40 million reads representing over 10 million unique sequences were generated. We identified 43 known miRNAs, 172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet. Some miRNA*s of the known and novel miRNAs were detected as well. Further, eight novel miRNAs were validated by stem-loop RT-PCR. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. Of the predicted target genes, 79% (351) had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing. CONCLUSIONS: We predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally.

Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize.

Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription-PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein-protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.

Improved nutritive quality and salt resistance in transgenic maize by simultaneously overexpression of a natural lysine-rich protein gene, SBgLR, and an ERF transcription factor gene, TSRF1.

Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9-10 and 19-11, ZmMYC1 in line 19-11 and ZmSDR in line 19-11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt treatment, the expressions of the ten stress-related genes were significantly induced in both wild-type (WT) and transgenic lines. However, compared to WT, the increases of ZmP5CS1 in all these three transgenic lines and ZmP5CS2 in line 9-10 were less than WT plants. This study provides an effective approach of maize genetic engineering for improved nutritive quality and salt tolerance.

ZmAdSS1 encodes adenylosuccinate synthetase and plays a critical role in maize seed development and the accumulation of nutrients.

Adenylosuccinate synthetase (AdSS, EC.6.3.4.4) is a key enzyme in the de novo synthesis of purine nucleotides in organisms. Its downstream product AMP plays a critical role in the process of energy metabolism, which can affect the content of ADP and ATP. However, impacts of its loss-of-function on plant metabolism and development has been relatively poorly reported. Here, we report the identification and analysis of a maize yu18 mutant obtained by mutagenesis with ethylmethane sulfonate (EMS). The yu18 is a lethal-seed mutant. Map-based cloning and allelic testing confirmed that yu18 encodes adenylosuccinate synthetase and was named ZmAdSS1. ZmAdSS1 is constitutively expressed. In the yu18 mutant, the activity of the ZmAdSS1 enzyme was decreased, which caused AMP content reduced 33.62%. The yu18 mutation significantly suppressed endoreduplication and disrupted nutrient accumulation, resulting in lower starch and protein contents that are responsible for seed filling. Further transcriptome and metabolome analysis revealed dramatic alterations in the carbohydrate metabolic pathway and amino acid metabolic pathway in yu18 kernels. Our findings demonstrate that ZmAdSS1 participates in the synthesis of AMP and affects endosperm development and nutrient accumulation in maize seeds.