Qian Zhao

Characterization of microRNAs expression during maize seed development

BackgroundMicroRNAs (miRNAs) are approximately 20-22 nt non-coding RNAs that play key roles in many biological processes in both animals and plants. Although a number of miRNAs were identified in maize, the function of miRNA in seed development was merely discussed.ResultsIn this study, two small RNA libraries were sequenced, and a total reads of 9,705,761 and 9,005,563 were generated from developing seeds and growing leaves, respectively. Further analysis identified 125 known miRNAs in seeds and 127 known miRNAs in leaves. 54 novel miRNAs were identified and they were not reported in other plants. Additionally, some miRNA*s of these novel miRNAs were detected. Potential targets of all novel miRNAs were predicted based on our strict criteria. In addition to deep-sequencing, miRNA microarray study confirmed the higher expression of several miRNAs in seeds. In summary, our results indicated the distinct expression of miRNAs during seed development.ConclusionsWe had identified 125 and 127 known miRNAs from seeds and leaves in maize, and a total of 54 novel miRNAs were discovered. The different miRNA expression profile in developing seeds were revealed by both sequencing and microarray studies.

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

BackgroundLate 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.ResultsHere, 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.ConclusionWe 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.

Culturing of immature inflorescences and Agrobacterium -mediated transformation of foxtail millet ( Setaria italica )

In previous reports, we developed an Agrobacterium -mediated transformation system for foxtail millet. Here, we report optimization of the system through improvement of the regeneration system efficiency and optimization of conditions for gene delivery. Immature inflorescences explants of foxtail millet cv. Jigu 11 varying in length (0.5 to 1.0, 1.1 to 1.5, 1.6 to 2.0 and >2.0 cm) were cultured on modified MS medium for callus induction and regeneration. The highest embryogenic callus-formation efficiency (90.72%) was achieved with 0.5 to 1.0 cm long inflorescences and 25 days old calli induced from 0.5 to 1.0 cm long immature inflorescences gave rise to the highest differentiation frequency (90.93%). In addition, factors affecting T-DNA delivery were examined by transient β-glucuronidase (GUS) expression. Calli induced from younger explants (0.5 to 1.0 cm immature inflorescences) were optimal. Agrobacterium tumefaciens strain LBA4404 performed significantly better than EHA105. Co-cultivation at 22°C with 0.15 g/l dithiothreitol (DTT) in the infection solution and co-cultivation medium led to higher GUS transient expression efficiency than with other treatments. Using this optimizedxa0 procedure, the lysine-rich protein encoding gene SBgLR from potato was transformed into foxtail millet cv. Jigu 11 with 5.5% transformation efficiency. The procedure described here will be useful for genetic improvement of foxtail millet. Key words : Foxtail millet, regeneration, Agrobacterium-mediated transformation, temperature, dithiothreitol (DTT).

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.

Maize transcription factor Zmdof1 involves in the regulation of Zm401 gene

Zmdof1 is a member of the maize Dof transcription factor family genes and participates in the regulation and control of the PEPC gene. The Zm401 gene, which contains short open reading frames (ORFs), has been cloned from maize, and its promoter contains several Zmdof1 recognition sites (DOFCORE, AAAG). Zm401 has an important role in anther development, and the protein encoded by the longest ORF, Zm401p10, localizes in the nucleus and is essential for maize anther development. In this study, we cloned Zmdof1, and expression pattern assay suggested that Zmdof1 has a role not only in nutrition organ development but also in maize pollen maturation. Transient expression of a Zmdof1::GFP fusion protein in onion epidermal cells showed a nuclear localization. 5′ deletion analysis of the Zm401 promoter showed that the region of −670 to −510 is important for promoter activity. Trans-activation assays in the yeast one-hybrid system confirmed that Zmdof1 had a strong interaction with AAAG elements in the Zm401 promoter. Co-transformation of a PZm401::Gus construct with UBI::Zmdof1 resulted in an approximately 40% decrease in GUS expression. Decrease of pollen viability resulting from ectopic expression of Zm401 controlled by its native promoter was recovered when Zmdof1 was transformed. Quantitative RT-PCR analysis showed that in PZm401::Zm401/UBI::Zmdof1 transgenic tobacco, the expression of Zm401 decreased significantly, coupled with an increase of Zmdof1 expression. The results indicated that Zmdof1 interacts with the Zm401 promoter in vitro and downregulates Zm401 in transgenic tobacco pollen. A probable regulatory mechanism of Zmdof1 to Zm401 in pollen was proposed.

Overexpression of millet ZIP-like gene (SiPf40) affects lateral bud outgrowth in tobacco and millet.

A SiPf40 gene was identified from an immature seed cDNA library of foxtail millet (Setaria italica). This gene encodes for a 29.4 KDa protein containing eight potential transmembrane domains and a highly conserved ZIP signature motif typical of ZIPs (zinc or iron transporter proteins) family. Other SiPf40 potential homologous genes have also been identified in rice, maize, wheat and Arabidopsis by Southern analysis. Expression data showed that this gene is preferentially expressed in millet hypocotyl and bud; however, a minimal level of constitutive expression could be detected in other foxtail millet tissues. Overexpression of SiPf40 gene causes extra branches in tobacco and extra tillering in millet associated with vessel enlarging and xylary fibers increasing, whereas the tiller number decreases in SiPf40 gene silenced plants. Moreover, IAA content decreased significantly in shoot apex of the transgenic tobacco overexpressing SiPf40 gene. All together, these morphological alterations indicate that SiPf40 gene is essential for lateral shoots growth.

Zm401p10, encoded by an anther-specific gene with short open reading frames, is essential for tapetum degeneration and anther development in maize

In flowering plants, the tapetum is proposed to play a vital role in the early stages of pollen development. Disruptions to tapetum development and degeneration typically result in male sterility. The present study characterised a maize (Zea mays L.) anther-specific gene, Zm401, which only contains short open reading frames (sORFs). The longest ORF of the Zm401 gene encodes a small protein designated Zm401p10 that accumulates in the nucleus. Overexpression of Zm401p10 in maize retarded tapetal degeneration and caused microspore abnormalities. A microarray analysis identified 278 downregulated and 150 upregulated genes in anthers overexpressing Zm401p10. These results indicate that the Zm401 gene is one of the major components of the molecular network regulating maize anther development and male fertility, and that Zm401p10 is expressed from the longest ORF of the gene.

Characterization and Functional Analysis of the Potato Pollen-Specific Microtubule-Associated Protein SBgLR in Tobacco

Microtubule-associated proteins play a crucial role in the regulation of microtubule dynamics, and are very important for plant cell and organ development. SBgLR is a potato pollen-specific protein, with five imperfect V-V-E-K-K-N/E-E repetitive motifs that are responsible for microtubule binding activity. In present study, SBgLR showed typical microtubule-associated protein characteristics; it bound tubulin and microtubules, and colocalized with microtubules in vitro. We also found that SBgLR could form oligomers, and that both the SBgLR monomers and oligomers bundle microtubules in vitro. Constitutive expression of SBgLR in tobacco caused curving and right-handed twisting root growth, abnormal directional cell expansion and cell layer arrangement, and pollen abortion. Immunofluorescence staining assays revealed that microtubule organization is altered in root epidermal cells in SBgLR-overexpressing lines. These suggest that SBgLR functions as a microtubule-associated protein in pollen development. Our results indicate that normal organization of MTs may be crucial for pollen development.

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.

A DREB-Like Transcription Factor From Maize (Zea mays), ZmDREB4.1, Plays a Negative Role in Plant Growth and Development

The DREB (dehydration-responsive element binding)-type transcription factors are classified into six subgroups, named A-1 to A-6. The members of DREB A-1 and A-2 subgroups have been reported to be involved in response to various abiotic stresses. However, there were only a few genes belonging to A-3 to A-6 subgroups to be reported. In this study, we cloned a DREB A-4 subgroup gene from maize (Zea mays), ZmDREB4.1, and analyzed its characteristics and functions. ZmDREB4.1 was expressed in roots, stems, and leaves at very low levels. It was not induced by any biotic or abiotic treatment. ZmDREB4.1 was located in the nucleus, could directly bind to the DRE element and functioned as a transcriptional activator. The constitutive expression of ZmDREB4.1 in tobacco (Nicotiana tabacum L.) repressed leaf extension and hypocotyl, petiole and stem elongation. In maize, overexpression of ZmDREB4.1 repressed calli growth and regeneration. Further analysis showed that the smaller leaves of transgenic tobacco resulted from inhibition of cell division. The contents of cytokinin and auxin in transgenic leaves were severely decreased. The shorter hypocotyls, stems and petioles of transgenic tobacco were caused by inhibition of cell elongation. The transgenic hypocotyls, stems and petioles contained reduced gibberellin levels. Application of exogenous GA3 rescued the shorter hypocotyls, stems and petioles, but not the smaller leaves. These results demonstrated that ZmDREB4.1 plays an important role in the negative regulation of plant growth and development.