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

Abstract

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.

Figure 1.

A, Sequence of the 5′ region of tomato seed germinationassociated endo-β-mannanase LeMAN2 cDNA. The first ATG in the coding region is highlighted by reverse shading. The 5′-untranslated region is indicated by double underlining. The sequences used to design GSP1 and GSP2 for genome-walking PCR are highlighted in bold, and their positions are indicated by arrows. The sequence that was also conserved in the untranslated region of genome-walking PCR product (see below) is highlighted by gray shading. B, DNA sequence of a portion of the 0.7-kb product obtained by genome-walking PCR. The predicted TATA-box and transcription initiation site are highlighted by a square and an asterisk, respectively. The DNA sequence in the untranscribed region is shown in small letters and the untranslated region in the putative cDNA is shown by double underlining. The sequence highlighted by gray shading (AAATAATA) was also found in the 3′ end of untranslated region LeMAN2 cDNA (Fig. 1A). The 5′-upstream sequence of the promoter region is not shown. C, The structure of LeMAN2 and LeMAN5 genes. The positions of GSP1 and GSP2 primers in LeMAN2 gene are shown with arrows.

Figure 2.

Activation of LeMAN5 promoter in Arabidopsis flowers. Expression of the GUS-reporter gene driven with the 5′-upstream region (-543 to +38) of LeMAN5 was characterized. A, GUS staining of inflorescence of transgenic Arabidopsis. B, GUS staining in a cross section of anther of transgenic Arabidopsis. C, GUS staining in a longitudinal section of anther and pistil of transgenic Arabidopsis. D, GUS staining of the pistils of transgenic (T) and wild-type (W) Arabidopsis cross-pollinated with wild-type (Wp) and transgenic pollen (Tp), respectively. E and F, GUS staining of germinated pollen of transgenic Arabidopsis. Note that callose plugs are not stained (arrows). G, Germinated pollen of wild-type Arabidopsis in the GUS staining solution.

Figure 3.

Expression of LeMAN5 mRNA and endo-β-mannanase activity in developing tomato anther. A, Northern blot of total RNA extracted from anther at stages I through IV (top two panels show developmental stages of tomato flowers and anthers). The same amounts (5 μg) of total RNA were applied and the blot was probed with LeMAN2 riboprobe. Ethidium bromide-stained ribosomal RNA bands are shown under the RNA gel blots to indicate RNA loading in each lane. B, Comparison of the DNA sequence of 5′-RACE product obtained from stage III anther with LeMAN5 genomic DNA and LeMAN2 cDNA sequences. Only 5′ regions of the DNA sequences are shown. The first ATG in the coding region is highlighted by reverse shading. The sequence conserved in the three sequences is highlighted by gray shading. An asterisk in LeMAN5 gene indicates the predicted transcription initiation site. The DNA sequence in the untranscribed region is shown in small letters. C, Specific activities of endo-β-mannanase in developing anther during stages I through IV. The diameters of clearing zones in gel diffusion assay were measured and quantified using a commercial fungal (Aspergillus niger) mannanase (Megazyme, Wicklow, Ireland) as a standard. The data represent the averages of the two experiments. Vertical bars represent variations between two data points.

Figure 4.

Endo-β-mannanase activity in pollen. A, Gel diffusion assay. Pollen grains were extracted from stage IV tomato anthers by forceps and were then suspended in 50 mm K-phosphate buffer, pH 6.8. The sample was centrifuged at 10,000g for 2 min. The supernatant (S) and the resuspended pollen (P) were frozen, thawed, and applied to a gel diffusion assay plate. B, Endo-β-mannanase activities in the supernatant and pollen fraction were calculated as described above.