Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter.

SBgLR (Solanum tuberosum genomic lysine-rich) is a pollen-specific gene cloned from potato (Solanum tuberosum L.). The region from -269 to -9 (The A of translation start site "ATG" as +1) of the SBgLR promoter was identified as critical for gene specific expression in pollen grains. Sequence analysis indicates a palindromic sequence "TTTCTATTATAATAGAAA" in the -227 to -209 region, in which two pollen-specific motifs TTTCT and AGAAA surround a unique putative TATA box. Moreover, nine putative pollen-specific motifs are located in the region between the TATA box and ATG. We placed the -227 to -9 region (reserving the palindrome) and the -222 to -9 region (breaking the palindrome) downstream of the CaMV35S enhancer, respectively, to construct two fusion promoters. Histochemical assays in transgenic plants demonstrated that the region from -222 to -9 is necessary and sufficient for pollen-specific expression of the uidA gene. However, the region of -227 to -9 is incapable of driving GUS expression in pollen grains and parts of vegetative tissues. A series of 5' deletions from -269 to -9 of SBgLR promoter were constructed. A transient expression assay indicated that the region from the -227 to -9 suppressed gfp gene expression in pollen, and a positive regulatory element was present in the region of -253 to -227. The function of the palindromic sequence as a repressor inhibiting gene expression in pollen was further confirmed by the mutated promoter, breaking the palindrome by substituting its 3'-flanking five base pairs, which resumes the reporter gene expression in mature pollen.

RNA silencing mediated by direct repeats in maize: a potential tool for functional genomics.

It has been shown in tobacco and Arabidopsis that transgenes with multiple direct repeats induce RNA silencing at high frequency. In this study, we tried to establish a direct repeat-induced RNA silencing system in maize and evaluate whether it can be developed as a high throughput tool for functional genomics. Our results showed that the construct phC4, which carries four direct repeats of a chloramphenicol acetyl-transferase (CAT) gene, was able to induce silencing of itself with high efficiency in maize. Using a transient expression system, we further demonstrated that construct phC3G with a beta-glucuronidase (GUS) gene located downstream of three direct repeats of CAT gene silenced not only itself in maize calli but also an "endogenous" GUS gene, which was stably expressed in maize calli. Most importantly, when constructs with the maize iojap (ij) gene inserted in either sense or antisense orientation into the downstream of four direct repeats of CAT gene were transformed into maize plants, co-suppression of endogenous and transgenic ij genes was detected in majority of transgenic maize plants. Our co-suppression results suggest that with improvements, this new approach has the potential to become an efficient research tool for high throughput functional genomics.

Zm401, a short-open reading-frame mRNA or noncoding RNA, is essential for tapetum and microspore development and can regulate the floret formation in maize.

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. Previously, we cloned a pollen specific gene, zm401, from a cDNA library generated from the mature pollen of Zea mays. Expression of partial cDNA of zm401 in maize and ectopic expression of zm401 in tobacco suggested it may play a role in anther development. Here we present the expression and functional characterization of this pollen specific gene in maize. Zm401 is expressed primarily in the anthers (tapetal cells as well as microspores) in a developmentally regulated manner. That is, it is expressed from floret forming stage, increasing in concentration up to mature pollen. Knockdown of zm401 significantly affected the expression of ZmMADS2, MZm3-3, and ZmC5, critical genes for pollen development; led to aberrant development of the microspore and tapetum, and finally male-sterility. Zm401 possesses highly conserved sequences and evolutionary conserved stable RNA secondary structure in monocotyledon. These data show that zm401 could be one of the key growth regulators in anther development, and functions as a short-open reading-frame mRNA (sORF mRNA) and/or noncoding RNA (ncRNA).

Seed-specific expression of the lysine-rich protein gene sb401 significantly increases both lysine and total protein content in maize seeds.

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants, and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli by microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis, and its expression was confirmed by Western blot analysis. Quantification of the lysine and protein contents in R1 maize seeds showed that, compared with the nontransgenic maize control, the lysine content increased by 16.1% to 54.8% and the total protein content increased by 11.6% to 39.0%. There were no visible morphological changes in the vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programs aimed at improving the nutritional value of maize.

Ectopic expression of foxtail millet zip-like gene, SiPf40, in transgenic rice plants causes a pleiotropic phenotype affecting tillering, vascular distribution and root development.

Plant architecture determines grain production in rice (Oryza sativa) and is affected by important agronomic traits such as tillering, plant height, and panicle morphology. Many key genes involved in controlling the initiation and outgrowth of axillary buds, the elongation of stems, and the architecture of inflorescences have been isolated and analyzed. Previous studies have shown that SiPf40, which was identified from a foxtail millet (Setaria italica) immature seed cDNA library, causes extra branches and tillers in SiPf40-transgenic tobacco and foxtail millet, respectively. To reconfirm its function, we generated transgenic rice plants overexpressing SiPf40 under the control of the ubiquitin promoter. SiPf40-overexpressing transgenic plants have a greater tillering number and a wider tiller angle than wild-type plants. Their root architecture is modified by the promotion of lateral root development, and the distribution of xylem and phloem in the vascular bundle is affected. Analysis of hormone levels showed that the ratios of indole-3-acetic acid/zeatin (IAA/ZR) and IAA/gibberellic acid (IAA/GA) decreased in SiPf40-transgenic plants compared with wild-type plants. These findings strongly suggest that SiPf40 plays an important role in plant architecture.

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.

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.

Functional characterization of the pollen-specific SBgLR promoter from potato (Solanum tuberosum L.).

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5'deletions of SBgLR promoter were fused to the beta-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region -345 to -269 relative to the translation start site. This 76 bp (-345 to -269) fragment enhanced GUS expression in leaves, stems and roots when fused to -90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region -345 to -311. Further study indicated that the -269 to -9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer.

SB401, a pollen-specific protein from Solanum berthaultii, binds to and bundles microtubules and F-actin.

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.

Characterization and functional analysis of a pollen-specific gene st901 in Solanum tuberosum.

A pollen-specific gene, sb401, which was isolated from a cDNA library of in vitro geminated pollen of the diploid potato species Solanum berthaultii, belongs to the class of genes expressed late during pollen development. Using sb401 as a probe, a pollen-specific gene st901 was isolated from the genomic library of a potato species Solanum tuberosum cv. Desiree. Sequencing and RT-PCR analysis showed that the st901 genomic gene is 2,889 bp long, contains three exons and two introns, and encodes a putative polypeptide of 217 residues. The predicted protein sequence contains four imperfect repeated motifs of V-V-E-K-K-N/E-E; the core sequence of the repeats (K-K-N/E-E) resembles a microtubule-binding domain of the microtubule-associated protein MAP1B from mouse. The examination of a promoter-reporter construct in transgenic potato plants revealed that the st901 is expressed exclusively in mature pollen grains, which is consistent with the results of Northern blot and RT-PCR. For analysis of the function of st901, transgenic plants harboring antisense copies of st901 cDNA driven by a native st901 promoter were generated. Suppression of st901 gene in potato resulted in aberrant pollen at maturation and pollen viability of transgenic plants ranged from 4.4 to 14.8%, while that of control plants were more than 90%. These results strongly suggest that st901 has an essential role in pollen development.