Proline is required for male gametophyte development in Arabidopsis

Abstract

Background: In crosses between the proline-deficient mutant homozygous for p5cs1 and heterozygous for p5cs2 (p5cs1 p5cs2/P5CS2), used as male, and different Arabidopsis mutants, used as females, the p5cs2 mutant allele was rarely transmitted to the outcrossed progeny, suggesting that the fertility of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised.

Results: To confirm the fertility defects of pollen from p5cs1 p5cs2/P5CS2 mutants, transmission of mutant alleles through pollen was tested in two ways. First, the number of progeny inheriting a dominant sulfadiazine resistance marker linked to p5cs2 was determined. Second, the number of p5cs2/p5cs2 embryos was determined. A ratio of resistant to susceptible plantlets close to 50%, and the absence of aborted embryos were consistent with the hypothesis that the male gametophyte carrying both p5cs1 and p5cs2 alleles is rarely transmitted to the offspring. In addition, in reciprocal crosses with wild type, about 50% of the p5cs2 mutant alleles were transmitted to the sporophytic generation when p5cs1 p5cs2/P5CS2 was used as a female, while less than 1% of the p5cs2 alleles could be transmitted to the outcrossed progeny when p5cs1 p5cs2/P5CS2 was used as a male. Morphological and functional analysis of mutant pollen revealed a population of small, degenerated, and unviable pollen grains, indicating that the mutant homozygous for p5cs1 and heterozygous for p5cs2 is impaired in pollen development, and suggesting a role for proline in male gametophyte development. Consistent with these findings, we found that pollen from p5cs1 homozygous mutants, display defects similar to, but less pronounced than pollen from p5cs1 p5cs2/P5CS2 mutants. Finally, we show that pollen from p5cs1 p5cs2/P5CS2 plants contains less proline than wild type and that exogenous proline supplied from the beginning of another development can partially complement both morphological and functional pollen defects.

Conclusions: Our data show that the development of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised, and indicate that proline is required for pollen development and transmission.

Figure 1

Segregation of the p5cs2 mutant allele in a self-pollinated p5cs1 p5cs2/P5CS2 population. Percentage of resistant versus total plantlets (black left column) or susceptible versus total plantlets (black right column) grown under sulfadiazine selection are shown. The corresponding gray columns represent the percentages expected if the p5cs1 p5cs2/P5CS2 were not a gametophytic mutant. Values represent the means of six independent experiments ± SE.

Figure 2

Morphological analysis of seed defects in siliques of wild type, p5cs2/P5CS2 and p5cs1 p5cs2/P5CS2 plants. The percentage of aberrant versus normal seeds was scored in wild type, p5cs2/P5CS2 heterozygous and p5cs1 p5cs2/P5CS2 mutants. While siliques from heterozygous p5cs2 mutant (A and B, middle) present ~25% of aberrant seeds (white arrowheads) the percentage of seed abortion of p5cs1 p5cs2/P5CS2 siliques (A and B, right) is indistinguishable from wild type (A and B, left). Details, at higher magnification, of some of the aberrant seeds of figure B (middle) are shown in the insets. Values represent the means of four independent experiments ± SE.

Figure 3

Molecular analysis of reciprocal crosses between p5cs1 p5cs2/P5CS2 mutants and wild type. (A) Schematic drawing of the insertional mutant p5cs1(Salk_063517) and p5cs2 (Gabi-Kat _452G01). The position of the T-DNA insertion, the location of the PCR primers used for genotyping, and the expected length of the PCR products are shown for P5CS1 (At2G39800), and P5CS2 (At3G55610). (B) PCR amplification of T-DNA insertions associated to P5CS2 (PAC161 T-DNA, top panels) and P5CS1 (pROK T-DNA, bottom panels) in reciprocal crosses between p5cs1 p5cs2/P5CS2 and wild type. Samples from 24 randomly chosen outcrossed plantlets are shown. The Results shown on top panels indicate that the P5CS2 mutation is never transmitted to the progeny (0/24) when p5cs1 p5cs2/P5CS2 serves as a male (top panel left), but is normally segregated (12/24), when serves as a female. Negative and positive controls are shown in the leftmost panel, relative to the amplification of wt (− ,top and bottom leftmost panel), p5cs2/P5CS2 (+, top leftmost panel) and p5cs1 (+, bottom leftmost panel) parental genotypes. The numbers next to the PCR products represent the expected molecular weight expressed in Kb for the P5CS2-T-DNA (top panel), and P5CS1-T-DNA (bottom panel) junction fragments, respectively.

Figure 4

Morphological analysis of pollen from p5cs1 p5cs2/P5CS2 and wild type. Acetic orcein stain of pollen from wild type (A and C) and p5cs1 p5cs2/P5CS2 plants (B and D) at two different magnifications show the presence in the latter pollen of small and shriveled pollen grains (arrows) alongside normal-looking grains. Bars = 50 μm (A, B) and 25 μm (C,D).

Figure 5

Histological analysis of wild type and mutant pollen. Cross-sections of anthers of different developmental stages. Toluidine blue-stained cross-sections of anthers from wild type (A,C,E,G,I) and p5cs1 p5cs2/P5CS2 (B,D,F,H,J) from stage 9 to 13 are shown. The first clear differences between p5cs1 p5cs2/P5CS2 and wild type anthers appear from stage 11 (E,F), when two populations of pollen grains can be distinguished in the pollen sacs. Bar = 100 μm. (K-N) Details at higher magnification of anthers from wild type (K,L) and p5cs1 p5cs2/P5CS2 (M,N), relative to stage 10 (K,M) and 11 (L,N), respectively. Small misshaped pollen grains are clearly visible from stage 11 (E). No significant alterations, compared to wild type, are seen in anthers from p5cs1 p5cs2/P5CS2 before stage 11. Bar = 25 μm.

Figure 6

Alexander's stain of pollen from wild type and p5cs1 p5cs2/P5CS2 plants. Alexander's staining of pollen and anthers from wild type (A,C) compared to p5cs1 p5cs2/P5CS2 mutant (B,D) reveals that a fraction of the mutant pollen population, looking small and misshaped, is not viable, as does not assume Alexander's stain. In contrast the remaining fraction of the pollen population appears as red as, and indistinguishable from wild type pollen. A detail of pollen grains from p5cs1 p5cs2/P5CS2 mutant is shown in the inset at higher magnification. Bars = 50 μm.

Figure 7

DAPI analysis of pollen from wild type and p5cs1 p5cs2/P5CS2. (A) DAPI staining of mature pollen from p5cs1 p5cs2/P5CS2. The small misshaped pollen of the mutant pollen population appears highly degenerated and depleted of nucleus. In contrast the larger and wild-type looking pollen grains display up to three nuclei, as in normal wild type pollen. (B) Bright-field image of the same picture showing large and small mutant pollen grains. Bar = 25 μm.

Figure 8

PCR analysis of large and small pollen grains from p5cs1 p5cs2/P5CS2 plants. DNA extracted from pools of large and small pollen grains from p5cs1 p5cs2/P5CS2 plants was genotyped by PCR for the presence or absence of insertional mutations in P5CS1 and P5CS2. No DNA could be amplified from the small pollen population (bottom panel), while both p5cs1 and p5cs2 mutant alleles, as well as the wild type P5CS2 allele, were amplified from large pollens (top panel).

Figure 9

Morphological analysis of pollen from p5cs1 homozygous mutants. In support of a proline requirement for pollen development, morphologic analysis of pollen from p5cs1 homozygous mutants revealed the presence of degenerated pollen grains (A-D), unstained with Alexander's stain (D) and lacking visible nuclei with DAPI staining (C, and bright-field control in B). Up to three nuclei are visible in pollen from wild type control (F, and bright-field control in E). Bars= 50 μm (A), 25 μm (B-D).

Figure 10

In vitro germination assays of pollen from p5cs1 p5cs2/P5CS2 plants. To assess the viability of pollen from p5cs1 p5cs2/P5CS2 plants, pollen from wild type (A and, at higher magnification, C) and p5cs1 p5cs2/P5CS2 (B and, at higher magnification, D) was incubated in vitro on germination medium and scored for successful germination (pollen tube fully or partially elongated). In panel (E) the percentage of germinated versus total pollen is shown for wild type (left column) and mutant pollen (right column). In (F) the percentage of germination over total pollen is given for either large (middle column) or small (rightmost column) mutant pollen, compared to wild type (leftmost column). Bars = 100 μm (A, B) and 25 μm (C,D). Values in (E) and (F) represent the means of four independent experiments ± SE.

Figure 11

Exogenous proline treatment of anthers and pollen from p5cs1 p5cs2/P5CS2 plants. To confirm a proline requirement for pollen development and functionality 10 μM proline was supplied either in vitro to germinating pollens (C,D,G,H) or in planta to developing anthers (A,B,E,F). While in vitro proline treatment of pollen from p5cs1 p5cs2/P5CS2 plants produces no differences in germination percentage (E, F), in planta supplementation gives rise to a significant improvement of germination efficiency (G, H). Bars = 50 μm. Values in (E to F) represent the means of four independent experiments ± SE.