Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

A polygalacturonase inhibitory protein gene (<Emphasis Type="Italic">BcMF19</Emphasis>) expressed during pollen development in Chinese cabbage-pak-choi

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.     

Characterization and Expression Pattern of a Putative Pectin Methylesterase Gene, <Emphasis Type="Italic">BcMF27</Emphasis>, in <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Pectin methylesterases (PMEs) play an important role in modifying cell wall. PMEs catalyze the de-esterification of pectin, an important compound of cell wall, to affect fertility in plant reproduction. However, little especially molecular mechanism about pectin methylesterase is studied in recent years despite its importance to reproductive development in flower plant. Here the bioinformatics analysis of BcMF27 (Brassica campestris Male Fertility 27) (BRAD: Bra000541 GenBank: KT600012) sequence isolated from Brassica campestris L. ssp. chinensis showed its highly and characteristically conserved structure as a pectin methylesterase. Transient expression analysis in the onion epidermal cells revealed the product of BcMF27 was a transmembrane protein. Real-time RT-PCR and in situ hybridization suggested that BcMF27 was expressed in pollen grain and pollen tube. This study demonstrates that BcMF27 encodes a transmembrane pollen- and pollen tube-specific PME gene, and is also considered to help further understand the biological function of pectin methylesterases and the molecular mechanism of pollen development, pollen tube growth as a genic tool.     

Cloning and expression analysis of a pollen preferential rapid alkalinization factor gene, <Emphasis Type="Italic">BoRALF1</Emphasis>, from broccoli flowers

Rapid alkalinization factors (RALFs) are recently reported active peptide hormones and are considered to play important roles in plant development. We previously identified a differentially expressed cDNA fragment between cabbage flower buds of sterility lines and its maintainer line, which showed significant homology to Arabidopsis RALFL9. The novel RALF cDNA (BoRALF1) was isolated from broccoli flower buds by EST assembly. The open reading frame (ORF) comprises 240 bp, encoding a small putative preprotein of 79 amino acids (molecular weight of 8.72 kDa and a pI of 7.8), which contains the mature polypeptide at its C terminus. BoRALF1 shares 70.3% identity with Arabidopsis RALFL9, but has only moderate similarity with functionally characterized RALFs (ranging from 16.2% to 38.0%). BoRALF1 shows typical features of RALFs, including the 28-aa signal peptide, typical arrangement of four position conserved cysteines, the YIXY motif and a similar secondary structure. RT-PCR studies of different tissues and promoter-GUS fusions confirmed that BoRALF1 is expressed strictly in mature pollen grains and in the anther cells around the loculi. Based on in vivo transient assays, we found that BoRALF1 appears to be largely localized in the plasma membrane. Although the function of BoRALF1 remains to be determined, our experiments confirm the presence of RALF peptide in broccoli, and suggest it could have a role in anther or pollen development.     

Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.