A novel endo-beta-mannanase gene in tomato LeMAN5 is associated with anther and pollen development

Endo-beta-mannanase (EC 3.2.1.78) is involved in cell wall disassembly and the weakening of plant tissues by degrading mannan polymers in the cell walls. Endo-beta-mannanase genes are expressed in tomato (Lycopersicon esculentum) seeds (LeMAN1 and LeMAN2) and fruits (LeMAN3 and LeMAN4). A novel endo-beta-mannanase gene (termed LeMAN5) was found in the tomato genome by genome-walking PCR and bacterial artificial chromosome library screening. The 5'-upstream region of this endo-beta-mannanase gene contained four copies of the pollen-specific cis-acting elements POLLEN1LELAT52 (AGAAA). A GUS-reporter gene driven with the putative LeMAN5 promoter (-543 to +38) was activated in anthers and pollen of transgenic Arabidopsis, with the highest beta-glucuronidase activity detected in pollen. beta-Glucuronidase expression was detected in mature pollen retained in sporangia, discharged pollen, and elongating pollen tubes in transgenic Arabidopsis. Consistently, expression of LeMAN5 mRNA and endo-beta-mannnanase activity was detected in tomato anthers and pollen. In anthers, the highest mRNA expression and endo-beta-mannanase activity were detected during late stages of anther development, when pollen maturation occurred. Endo-beta-mannanase activity was present in discharged pollen, which was easily eluted in a buffer, indicating that the enzyme proteins are probably secreted from, and deposited on, the surface of pollen. These data suggest that the LeMAN5 endo-beta-mannanase is associated with anther and pollen development.     

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.     

Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems

Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accessions: OSIPA cDNA, AF220610; OSIPK cDNA, AF312920; OSIPA partial gene and upstream promoter region, AY166659; OSIPK gene-specific and upstream sequence, AY168440.     

F-actin forms mobile and unwinding ring-shaped structures in germinating Arabidopsis pollen expressing Lifeact

The flowering plant pollen tube is the fastest elongating plant cell and transports the sperm cells for double fertilization. The highly dynamic formation and reorganization of the actin cytoskeleton is essential for pollen germination and pollen tube growth. To drive pollen-specific expression of fluorescent marker proteins, commonly the strong Lat52 promoter is used. Here we show by quantitative fluorescent analysis that the gametophyte-specific ARO1 promoter from Arabidopsis drives an about 3.5 times weaker transgene expression than the Lat52 promoter. In one third of the pollen of F-actin-labeled ARO1p:tagRFP-T-Lifeact transgenic lines we observed mobile ring-shaped actin structures in pollen grains and pollen tubes. Pollen tube growth, transgene transmission and seed production were not affected by tagRFP-T-Lifeact expression. F-actin rings were able to integrate into emerging actin filaments and they may reflect a particular physiological state of the pollen or a readily available storage form provided for rapid actin network remodeling.     

In the right place at the right time: Parnassia resolves the herkogamy dilemma by accurate repositioning of stamens and stigmas

Background and Aims

Spatial (herkogamy) and temporal (dichogamy) separation of pollen presentation and stigma receptivity have been interpreted as reducing interference between male and female functions in hermaphroditic flowers. However, spatial separation leads to a potential conflict: reduced pollination accuracy, where pollen may be placed in a location on the pollinator different from the point of stigma contact.

Methods

To understand better how herkogamous flowers resolve this conflict, a study was made of a subalpine herb, Parnassia epunctulata, the nectariferous flowers of which exhibit sequential anther dehiscence (staggered pollen presentation) and stamen movements; usually one newly dehisced anther is positioned each day over the central gynoecium, while the older stamens bend away from the central position.

Key Results

The open flowers were visited by a variety of pollinators, most of which were flies. Seed set was pollinator-dependent (bagged flowers set almost no seeds) and pollen-limited (manual pollination increased seed set over open pollination). Analyses of adaptive accuracy showed that coordinated stamen movements and style elongation (movement herkogamy) dramatically increased pollination accuracy. Specifically, dehiscing anthers and receptive stigmas were positioned accurately in the vertical and horizontal planes in relation to the opposite sexual structure and pollinator position. By contrast, the spatial correspondence between anthers and stigma was dramatically lower before the anthers dehisced and after stamens bent outwards, as well as before and after the period of stigmatic receptivity.

Conclusions

It is shown for the first time that a combination of movement herkogamy and dichogamy can maintain high pollination accuracy in flowers with generalized pollination. Staggered pollen and stigma presentation with spatial correspondence can both reduce sexual interference and improve pollination accuracy.