Genomic structure of the ZmMADS2 gene. a, The ZmMADS2 gene consists of eight exons (E1–E8) and seven introns (I1–I7) with an unusually large second intron (I2). The transcription start point was determined using a primer extension assay. The ATG START codon is located 312 bp downstream of the transcription start point. The context sequence around the translational initiation codon ATG is typical for monocots. Intron sizes are given in base pairs above the sequence. b, ZmMADS2 belongs to the MIKC type of MADS box proteins. ZmMADS2 contains of a highly conserved MADS box at the N terminus, a linker sequence, a keratine box, and a highly variable C terminus. A putative bipartite nuclear localization signal is found inside the MADS box. Distribution of exons is shown above protein domains. c, Clustering of putative cis-acting elements and repetitive sequences upstream of the ATG START codon. Putative cis elements responsible for sugar starvation induction are displayed in green (ACGTA or AATAGAAAA), those responding to water stress and abscisic acid are displayed in blue (TAACTG and ACACNNG, respectively), putative root-specific elements are shown in red (ATATT), and sequences homologous to motifs responsible for late pollen-specific expression are given in orange (AGAAA or GTGA). Sequence repeats are shown below the horizontal bar. Yellow, Direct repeats (GTATGTAACATA... –... GTATGTAACATA in box V, AGTTATATATTTAT-AGTTATATATTTAT in box IV, and CTCAAATACGTAA... –... CTCAAATACGTAA in box III), brown, inverted repeats (TAATAATTATTA); dark orange, dyad repeats (ATTGAAAAGTTA). Arrow, Transcription start site; arrowhead, position of the start codon.

ZmMADS2 is expressed in anther tissues shortly before anther dehiscence and in mature pollen. a, Northern blot showing expression of ZmMADS2 in vegetative tissues and during anther development. Transcripts are first detected in the middle of stage VIII, when anthers reach their final size but are still green (see also Fig. 1c). Highest transcript amounts are visible in anthers at late stage VIII (red color; shortly before opening of the anterior pore) and in mature pollen. Very weak signals were observed in roots with and without tips. An expression in other vegetative tissues could not be detected. b, RT-PCR analysis of microdissected anther tissues. Connective tissue (CT) and endothecium with epidermis (EE) were isolated from anther stages indicated. Forty PCR cycles were conducted to amplify ZmMADS2 transcripts and 35 PCR cycles for GAPDH as a control. Expression of ZmMADS2 is visible in maternal anther tissues at the last stages of anther development (stages VIII and IX).

ZmMADS2-GFP expression in cross and longitudinal sections of anthers from transgenic maize plants at 2 d before anthesis. a, Cross section of a late stage VIII anther of a transgenic maize line expressing the ZmMADS2-GFP-fusion protein under control of the ZmMADS2 promoter (Fig. 3) in connective tissue (arrow) and endothecial cells (arrowheads). b, Close-up of an anther locule; arrow marks nucleus in connective tissue and arrowheads in endothecial cells. c, WT anther of the same stage lacking green fluorescence. d, DAPI staining of c. e and g, Longitudinal section to show expression of the ZmMADS2-GFP-fusion protein in nuclei of endothecial cells. e, Expression of the fusion protein is not restricted to the region of unopened anterior pore. Note that nuclei showing bright GFP-fluorescence display only faint DAPI signals. g, Close-up of a longitudinal section showing expression of the ZmMADS2-GFP fusion protein in connective tissue. f and h, DAPI staining of images shown in e and g. Note that the stronger the GFP fluorescence, the weaker the DAPI signal. Arrowheads point to strong GFP fluorescence and weak DAPI staining; arrow points toward stronger GFP fluorescence and weaker GFP signals. Bars = 100 μm.

Phenotypes of transgenic maize plants expressing ZmMADS2 cDNA in antisense orientation under control of the constitutive ubiquitin promoter of maize. Development of tassels, anthers, and pollen is arrested at 1 d before anthesis. a, Tassel of a transgenic plant at the latest stage of development. b, Tassel of a WT plant at anthesis. c, Anthers of a WT plant (left) and that of a transgenic antisense (AS) plant (right) at 2 d before anthesis (middle stage VIII), 1 d before anthesis (late stage VIII), and at anthesis (stage IX). Light microscopic (d) and UV microscopic (e) images of pollen of transgenic plants at the latest stage of development. Pollen were stained with 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) and acetic orcein. Note that a vacuole is still visible (asterisk), and sperm cells (arrowheads) are round instead of sickle shaped. Light microscopic (f) and UV microscopic (g) image of a mature WT pollen stained with DAPI and acetic orcein. Vacuoles are no longer visible, and sperm cells (arrowheads) are sickle shaped. Arrows point toward the vegetative nucleus. Bars = 20 μm.

ZmMADS2 promoter deletion analyses with transiently transformed pollen and leaves of maize and tobacco. a, Relative expression levels of luciferase directed by ZmMADS2 promoter deletion constructs in pollen and leaves of maize and tobacco, respectively. Ten experiments were conducted with pDNS-5, -6, -6b, -7, and -8, whereas three experiments were conducted with pDNS-9 and -10. The promoterless control was used in more than 30 experiments. Vertical lines within bars indicate relative minimum and maximum rates, and bars indicate average expression levels. For a better comparison, expression levels of pDNS-6b were set to 100% for each individual experiment. b, Deletion constructs consist of boxes and putative cis elements as shown in Figure 2c. Relative luciferase expression in pollen of maize and tobacco as an average of 10 experiments is shown. n.d., Not determined.

ZmMADS2 forms homodimers in the yeast two-hybrid system. Three independent clones were each pipetted on agar plates containing full medium without selection (a); selection medium without His, Trp, Lys, and Ura with 300 μg mL^-1 Zeocin (b), and the β-galactosidase assay (c). Top row, ZmMADS2 fused to the LexA BD (DNA-binding domain), bait. Second row, ZmMADS2 fused to the B42 activation domain (AD), prey. Third row, ZmMADS2 fused to LexA BD (bait) and ZmMADS2 fused to B42 AD (prey). Fourth row, Fos fused to LexA BD (bait) and Jun fused to B42 AD (prey) as positive control. Fifth row, Fos fused to LexA BD (prey) and Lamin fused to B42 AD (prey) as a negative control.

ZmMADS2 marks degrading nuclei of endothecial cells during anther maturation. a, ZmMADS2-GFP fluorescence of an intact nucleus within an endothecial cell. b, DAPI staining of the image shown in a. Note that the nucleus shows compartmentation. c, The ZmMADS2-GFP-fusion protein is visible, whereas most DNA is degraded. The structure of the nucleus is lost. d, DAPI staining of c. Few intact DNA are left. e, ZmMADS2-GFP is detectable in apoptotic bodies of an endothecial nucleus. f, DAPI staining of e. DNA is almost completely degraded. Bars = 10 μm.