miR156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis

Abstract

The SBP-box transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE8 (SPL8) is required for proper development of sporogenic tissues in Arabidopsis thaliana. Here, we show that the semisterile phenotype of SPL8 loss-of-function mutants is due to partial functional redundancy with several other members of the Arabidopsis SPL gene family. In contrast with SPL8, the transcripts of these latter SPL genes are all targeted by miR156/7. Whereas the introduction of single miR156/7-resistant SPL transgenes could only partially restore spl8 mutant fertility, constitutive overexpression of miR156 in an spl8 mutant background resulted in fully sterile plants. Histological analysis of the anthers of such sterile plants revealed an almost complete absence of sporogenous and anther wall tissue differentiation, a phenotype similar to that reported for sporocyteless/nozzle (spl/nzz) mutant anthers. Expression studies indicated a functional requirement for miR156/7-targeted SPL genes limited to early anther development. Accordingly, several miR156/7-encoding loci were found expressed in anther tissues at later stages of development. We conclude that fully fertile Arabidopsis flowers require the action of multiple miR156/7-targeted SPL genes in concert with SPL8. Either together with SPL/NZZ or independently, these SPL genes act to regulate genes mediating cell division, differentiation, and specification early in anther development. Furthermore, SPL8 in particular may be required to secure fertility of the very first flowers when floral transition-related miR156/7 levels might not have sufficiently declined.

Figure 1.

Phylogenetic Relationships among SPL Gene Family Members in Arabidopsis and Exposed Redundancy in Fertility. (A) Unrooted phylogram of all SPL genes obtained using the neighbor-joining algorithm and based on the conserved SBP domain. Nodes representing bootstrap values >50% from 10,000 replicates are encircled. Both subfamilies I and II are indicated by dashed lines. miR156/7-targeted SPL genes are depicted in green, whereas nontargeted large SPL genes and SPL8 are in blue and red, respectively. The alignment used to generate this tree is available as Supplemental Data Set 1 online. (B) and (C) Mean number of pollen sacs (B) and mean number of pollen (C) formed per anther of the wild type and mutants for SPL8, SPL2, SPL9, and SPL15 and combinations thereof, as labeled in the image. A total of 30 anthers from the 5th and 15th flowers of six primary inflorescences were analyzed. Small pollenless locules are referred to as underdeveloped. (D) Mean number of seeds set per silique of the wild type and multiple SPL gene mutants as labeled in the image. Siliques from the 5th and 15th flowers of a total of 20 primary inflorescences were examined. Error bars indicate sd.

Figure 2.

Reproductive Phenotypes of 35S:MIR156b spl8-1 Plants. (A) Morphology of 6-week-old 35S:MIR156b and 35S:MIR156b spl8-1 plants under long-day growth conditions. Homo- or hemizygosity for the 35S:MIR156b transgene is indicated by (+/+) or (+/−), respectively, and homo- or heterozygosity for the spl8-1 mutant allele respectively by (−/−) or (+/−). (B) 35S:MIR156b (+/+) flower at anthesis showing dehisced anthers releasing pollen to the stigma. Sepals carry trichomes on their abaxial side. Inset in the bottom left corner shows the pistil dissected from the same flower. (C) Flower at anthesis from a plant homozygous for both the 35S:MIR156b transgene and the spl8-1 mutant allele. The small anthers do not dehisce and are pollenless. Trichomes are largely absent from the abaxial side of the sepals. Inset in the bottom left corner shows the dissected pistil from the same flower with a swollen upper part and a short stalk (white arrow). (D) A sepal from the flower shown in (C), carrying adaxial trichomes.

Figure 3.

Effect of miR156 Target Mimicry on Seed Set in Wild-Type and spl8-1 Mutant Plants. (A) Primary inflorescences, cut off below the last cauline leaf, of wild-type and mutant plants as labeled in the image. Bar = 1 cm. (B) Siliques from the 1st to 6th flower (left to right) formed within the primary inflorescences (arranged top to bottom as shown from left to right in (A). Bar = 1 cm. (C) Mean number of seeds set per silique from the 1st, 5th, 10th, and 15th flower, respectively, formed within the primary inflorescences of wild-type and mutant plants as labeled in the image. Error bars indicate sd (n = 20).

Figure 4.

Comparative Histological Analysis of Anther Development. (A) to (T) Toluidine blue–stained cross sections through anthers at different developmental stages of wild-type (Col-0) ([A] to [E]), 35S:MIR156b ([F] to [J]), spl8-1 ([K] to [O]) and 35S:MIR156b spl8-1 ([P] to [T]) mutant plants. (A), (F), (K), and (P) Anthers at stage 2. Within their corners, subepidermal archesporial cells are still detectable or have just undergone a periclinal division to give rise to a PPC and a PSC (arrows). (B), (G), (L), and (Q) Anthers at stage 3. The PPC and PSC continued to divide to form additional cell layers (arrows) in the wild type (B), 35S:MIR156b transgenics (G), and spl8-1 mutants (L). In 35S:MIR156b spl8-1 double mutant plants (Q), this process seems to have come to a hold or to be delayed. (C), (H), (M), and (R) Anthers at stage 4. PSC-derived sporogenous cells are detectable in the center of developing locules surrounded by two cell layers derived from PPCs in the wild type (C), 35S:MIR156b transgenics (H), and the spl8-1 mutant (M). Sporogenous cells and structured locule layer formation seem to be absent in 35S:MIR156b spl8-1 plants (R). (D), (I), (N), and (S) Anthers at stage 5. PMCs are present and surrounded by distinct anther walls in the wild type (D), 35S:MIR156b transgenics (I), and spl8-1 mutants (N). Note that the lower left locule of this particular spl8-1 anther lacks typical PMCs (arrow). Anthers of 35S:MIR156b spl8-1 double mutants (S) lack both the PMCs and the distinct anther walls. Instead, much more vacuolated and connective tissue-like cells occupy their corresponding positions. (E), (J), (O), and (T) Anthers around stage 12 to 13, carrying pollen grains in each locule, albeit fewer in 35S:MIR156b transgenics (J) and spl8-1 mutants (O) compared with the wild type (E). The 35S:MIR156b spl8-1 anther (T) completely lacks pollen sacs, whereas the selected spl8-1 anther (O) lacks a pollen sac at its inner left side (asterisk). v, vascular bundle. Bar in the first panel of each column = 25 μm.

Figure 5.

Anther Cell Proliferation Visualized by H4 in Situ Hybridization. (A) to (H) Cross sections of flower buds at different developmental stages. (A) and (E) Stage 2 anthers showing little difference in H4 hybridization signal between wild-type (A) and 35S:MIR156b spl8-1 (E) plants. (B) and (F) Stage 3 anthers indicating a diminishing H4 hybridization signal in 35S:MIR156b spl8-1 (F) in comparison to the wild type (B). (C) and (G) Stage 5 anthers. H4 activity remains well detectable and spread in the wild type (C) but has become restricted mainly to the central vasculature in 35S:MIR156b spl8-1 ([G], arrow). (D) and (H) Stage 6 anthers. H4 expression continues in the wild type (D) with the developing tapetum showing the strongest signal. By contrast, 35S:MIR156b spl8-1 anthers at this stage are nearly devoid of detectable H4 activity. Note that H4 activity also strongly declined in the gynoecium, whereas it is still easy detectable in developing petals. an, anther; g, gynoecium; p, petal. Bars = 50 μm.

Figure 6.

Temporal and Spatial Expression of Selected SPL Genes in Anthers. (A) Graphic representation of plants grown in long days for 21 d (left) and 30 d (right) and used for collecting shoot tips with floral buds for cross-sectioning and in situ hybridization as shown in (B) to (K). (B) Cross section through one of the earliest flower buds formed in the primary inflorescence (see schematic on the left in [A]) after in situ hybridization with an SPL11 probe. No detectable expression of SPL11 was found in these stage 2 anthers. (C) to (K) Cross sections through buds and anthers of flowers formed relatively late in the primary inflorescence (see schematic on the right in [A]) and hybridized to SPL gene probes as indicated below. (C) Stage 2 anthers (comparable to [B]) showing clear expression of SPL11. (D) Stage 3 anther showing a broad expression of SPL11 in the anthers. (E) Stage 5 anther with strongest expression of SPL11 in pollen mother and tapetal cells. (F) Stage 2 anthers showing expression of SPL13. (G) Stage 3 anther showing weak SPL13 expression in sporogenous and parietal cells. (H) Stage 5 anther expressing SPL13 mainly in the tapetum. (I) Stage 4 anthers showing SPL15 expression in sporogenous cells and the surrounding cell layers. SPL15 is also clearly expressed in the central placental tissue of the early ovary. (J) Stage 3 anther showing SPL2 weakly expressed in sporogenous and parietal cells. (K) Stage 4 anther expressing SPL2 in sporogenous cells and surrounding cell layers. an, anther; g, gynoecium; t, tapetum. Bars = 25 μm.

Figure 7.

MIR156/7 Promoter-GUS Reporter Gene Expression at Early Anther Stages. (A) to (D) pMIR156h:GUS. (E) to (H) pMIR157c:GUS. (I) to (L) pMIR157d:GUS. (A), (E), and (I) Top views of inflorescences from 25-d-old plants with developing flower buds after GUS staining. The numbers indicate the anther stages in these buds. Note that a GUS signal appears in the pistils of pMIR156h:GUS flowers during late developmental stages ([A], arrows). (B) to (D), (F) to (H), and (J) to (L) Transverse sections of early, developing floral buds from 25-d-old plants after GUS staining. Crystalline GUS staining appears pink under dark-field illumination. (B) Stage 3 anthers with only weak GUS signal detectable. (C) Stage 5 anthers with GUS signal easily detectable. (D) Stage 6 anthers with strong GUS signal distributed over the entire anther. (F) Stage 2 anthers with no GUS signal detectable. (G) Stage 5 anthers. GUS signal appears in some anthers (arrow). (H) Stage 6 anthers. GUS signal accumulates widely in all anthers. (J) Stage 2 anthers. Weak GUS signal detectable in anthers and other floral organs. (K) Stage 5 anthers. Strong GUS signal distributed over the entire anther. (L) Stage 7 anthers. GUS signal located mainly in anther walls, but some can be detected in the tetrads as well. (M) Relative transcript levels of the GUS reporter genes on three time points for pMIR156h:GUS, pMIR157c:GUS, and pMIR157d:GUS transgenic plants. (N) Relative primary transcript levels of the endogenous MIR156h, MIR157c, and MIR157d loci determined at three different time points. an, anther; g, gynoecium. Bar in the first panel of each column = 1 mm for (A) and 0.1 mm for (B) to (D). Error bars in (M) and (N) indicate ± sd (n = 3).

Figure 8.

In Situ Hybridization of SPL8, SPL11, and SPL/NZZ mRNA in Early Anthers. (A) to (C) and (D) to (F) SPL8 expression in wild-type and spl-1 mutant anthers, respectively. (G) to (I) and (J) to (L) SPL11 expression in wild-type and spl-1 mutant anthers, respectively. (M) to (O) and (P) to (R) SPL/NZZ expression in wild-type and spl8-1 35S:MIR156b mutant anthers, respectively. (A) and (D) Stage 2 anthers. Whereas expression of SPL8 is limited mainly to L2-derived cells in the wild type ([A], arrows), spl-1 mutant anthers show a broadened SPL8 expression domain (D). (B) and (E) Stage 4 anthers. SPL8 expression is clearly observed in the sporogenous cells and the parietal cell layers of wild-type anthers ([B], arrows), but a less strong and more marginal expression is seen in spl-1 anthers ([E], arrows). (C) and (F) Stage 5 anthers. SPL8 expression is detected in PMCs and surrounding cell layers of wild-type anthers (C), and only a weak signal can be found in the corners of abnormal differentiating spl-1 anthers ([F], arrows). (G) and (J) Stage 2 anthers. Weak SPL11 expression is found primarily in L2-derived cells of wild-type anthers (G), and a stronger and more spotty hybridization signal seems to occur in spl-1 anthers ([J], arrows). (H) and (K) Stage 4 anthers. SPL11 is expressed in the sporogenous cells and surrounding cell layers but not in the epidermis of wild-type anthers (H), and a more spotty signal in spl-1 anthers seems to be excluded from the abaxial side (K). (I) and (L) Stage 5 anthers. SPL11 expression is detected in PMCs and surrounding cell layers of wild-type anthers (I). In spl-1 mutant anthers, the signals appears stronger and more spotty but outside the abaxial region (L). (M) and (P) Stage 2 anthers. SPL/NZZ expression is seen in L2-derived cells in the four corners of both wild-type ([M], arrows) and spl8-1 35S:MIR156b anthers ([P], arrows). (N) and (Q) Stage 4 anthers. SPL/NZZ is expressed in the sporogenous cells of the four lobes in wild-type anthers (N). However, markedly weaker signal is found in those of spl8-1 35S:MIR156b anthers ([Q], arrows). (O) and (R) Stage 5 anthers. SPL/NZZ expression is easily detectable in PMCs and surrounding cell layers of wild-type anthers (O), but only a very weak signal can be seen in the four lobes of spl8-1 35S:MIR156b anthers ([R], arrows). Bar in the first panel of each column = 25 μm.

Figure 9.

Comparative Expression Levels of miR156/7 and Several of Their Targets in the Wild Type and the spl-1 Mutant. Relative expression levels as determined by qRT-PCR on RNA from inflorescence apices of 40-d-old plants grown in long days, representing floral buds up until stage 12 (Smyth et al., 1990). (A) Relative expression levels of miR156/7-targeted SPL genes in the wild type (Ler) and spl-1. (B) Relative expression levels of mature miR156 and miR157 in the wild type (Ler) and spl-1. Error bars indicate ± sd (n = 3).

Figure 10.

A Model for SPL8 and miR156/7-Targeted SPL Gene Function in Early Anther Development in Arabidopsis. At the onset of the anther developmental program, the floral C function gene AG promotes the expression of SPL/NZZ. SPL/NZZ is required for proper expression of genes involved in cell division, differentiation, and specification and leads to PMC and tapetum formation. Equally critical at this stage appear to be the SPL8 and miR156/7-targeted SPL gene functions, either required to act together with SPL/NZZ or independently to regulate genes mediating cell division, differentiation, and specification. Lines ending with arrowheads indicate positive and those with rhombs negative genetic interactions. A question mark indicates a possible but yet unclear interaction.