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An Aquaporin-Like Gene Required for the Brassica Self-Incompatibility Response

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Science  06 Jun 1997:
Vol. 276, Issue 5318, pp. 1564-1566
DOI: 10.1126/science.276.5318.1564

Abstract

Self-incompatibility in Brassica refers to the rejection of self-related pollen and is mediated by a receptor protein kinase localized to the plasma membrane of the stigma epidermis in the flower. The recessive mutation mod eliminates self-incompatibility in the stigma. In mod mutants, self-compatibility was shown to be associated with the absence of transcripts encoded by an aquaporin-related gene. This observation suggests that a water channel is required for the self-incompatibility response ofBrassica, which is consistent with the concept that regulation of water transfer from the stigma to pollen is a checkpoint in the early events of pollination in the crucifer family.

In many species of flowering plants, self-fertilization is prevented or reduced by the mechanism of self-incompatibility (1). In the cruciferBrassica, this genetic system ensures that, in a field of wild mustard plants, every instance of self-pollination is inhibited whereas most cross-pollinations are not. This mating control is achieved within minutes after capture of pollen by the papillar cells of the stigma epidermis (Fig. 1) and is manifested by the disruption of pollen tube development (2). The recognition of pollen in self-incompatible crucifers is controlled by haplotypes of the S locus (designated S1, S2, … Sn), each of which consists of a cluster of genes that are inherited as a unit (3). Two genes within the cluster encode papillar cell–specific surface receptors that are required for the stigma to discriminate against self-related pollen (3-6). It is thought that, on self-pollination, the S locus–encoded and plasma membrane–localized receptor kinase SRK detects a pollen-borne ligand and initiates an intracellular phosphorylation cascade within the stigmatic epidermal cell that blocks the development of incompatible pollen (3). The specific events of the self-incompatibility signal transduction pathway are not known. Products encoded by two members of the thioredoxin gene family interact with the kinase domain of SRK in a yeast two-hybrid screen, but their function in self-incompatibility is unclear (7).

Figure 1

Hydration of pollen in a compatible pollination in Brassica. The scanning electron micrographs show the changes in shape and volume of pollen grains at 5 min (A and B) and 45 min (C) after self-pollination. In (C), pollen grains are fully hydrated and show a three- to fourfold increase in volume relative to the unhydrated pollen grains in (A) and (B). Po, pollen; SE, stigma epidermal cell. Magnification, ×135 (A) and ×325 (B and C).

Loci that are not linked to the S locus are also required for self-incompatibility in the stigma (8) and may encode proteins of the SRK signaling pathway. We analyzed one such genetic modifier or self-incompatibility, the spontaneousmod mutation of Brassica campestris(8). Reciprocal crosses (9) between the self-incompatible (SI) S8/S8homozygote and the self-compatible (SC) USDA C634 strain indicate that the mod mutation compromises stigma function but not pollen function. The F1 plants derived from these crosses are SI, which indicates that self-compatibility is the recessive trait. We forced self-fertilization of one F1 plant by manual pollination of immature buds to produce an F2 population of 260 plants. Pollination analysis of the F2 plants revealed that the ratio of SI:SC plants approximated a modified dihybrid ratio of 9:7 in the entire population and a ratio of 3:1 among S8/S8 homozygotes. These results are consistent with the segregation of a nonfunctional S haplotype derived from strain C634 (6) and of a recessive mutation (mod) that is epistatic and not linked to the S locus.

To identify RNA transcripts encoded by or controlled by the MOD locus, we used the technique of differential display combined with reverse transcription–polymerase chain reaction analysis (DDRT-PCR) (10) with RNA isolated from the stigmas of 10 S8/S8 SI plants (the SI pool, expected to be genotypically MOD/MOD orMOD/mod) and RNA isolated from the stigmas of 10 S8/S8 SC plants that exhibited >500 pollen tubes per stigma on self-pollination (the SC pool, expected to be genotypically mod/mod). This strategy minimizes interference from irrelevant polymorphisms of individual plants, because the pooled samples should be randomized for genomic sequences with the exception of those located near or at the target locus.

We analyzed the RNA pools by DDRT-PCR (11) and obtained 29 cDNA differences in PCR reactions with 240 primer combinations. The DDRT-PCR products were used to identify restriction fragment length polymorphisms (RFLPs) genetically linked to the MOD locus. We detected a 119–base pair (bp) fragment, DD33, that was amplified from the SI pool but not from the SC pool. In SC plants, DD33 hybridized to a 1.6-kb Bgl II fragment, whereas in SI plants it hybridized to an 8-kb Bgl II fragment or to both 8- and 1.6-kb fragments (Fig. 2A). A 1.1-kb cDNA was isolated by screening an S8/S8 stigma cDNA library with DD33 and was shown to hybridize to the 8- and 1.6-kb restriction fragments (designated DD33MOD and DD33mod, respectively) identified by the DD33 probe, as well as to a family of related genes (Fig. 2B). Sequence analysis revealed that the DD33 sequence was identical to the 3′ untranslated region of the 1.1-kb cDNA and to the corresponding genomic sequences contained on the 8-kb DD33MOD and 1.6-kb DD33mod restriction fragments.

Figure 2

DNA gel blot analysis of DD33 and related sequences in SI and SC plants. Gel blots of DNA digested with Bgl II were probed with either the 119-bp DD33 fragment (A) or the 1.1-kb DD33 cDNA (B through D). (A) DNA from four SI and four SC plants used for DDRT-PCR. The SC plants exhibit a 1.6-kb restriction fragment (and are DD33mod/DD33mod). One SI plant exhibits an 8-kb fragment (and is DD33MOD/DD33MOD), and three SI plants exhibit both 8- and 1.6-kb fragments (and are DD33MOD/DD33mod). (B) DNA from the 10 SI and 10 SC plants of the RNA pools. Arrowheads indicate the positions of the 8- and 1.6-kb fragments, and small squares mark the 8-kb fragment in each of the SI plants. The sixth lane from the left represents the plant whose progeny is shown in (C). (C) DNA from SI (I) and SC (C) F3 progenies. (D) DNA from an SI undeleted MOD/mod plant (lane 1) and an SC γ-irradiated mutant plant (lane 2) that is missing the 8-kb DD33MOD fragment (arrowhead).

To determine whether DD33 was derived from or linked to MOD, we extended RFLP analysis to other plants in the F2population (79 S8/S8 and 79 S8/Sf2 plants were tested). Although several restriction fragments identified by the 1.1-kb cDNA probe were polymorphic in this group of plants, only the DD33MOD and DD33modfragments cosegregated with pollination phenotype (Fig. 2B). All SI plants contained the DD33MOD fragment, and all plants were homozygous for DD33mod were SC. The cosegregation of the DD33 RFLP with pollination phenotype was also observed in the F3 generation (Fig. 2C) and in backcrosses to the C634 strain. The 10 plants of the SC pool produced only SC progenies (17 plants in each of 10 families). Two F2 plants of the SI pool, predicted to be MOD/mod on the basis of DD33 polymorphism, yielded expected ratios of 1:1 and 3:1 SI:SC plants for backcrossed and selfed progenies (12). Independent evidence for the linkage of the DD33 sequence to the MOD locus was also provided by analysis of SC mutants generated by pollinating a mod/modstrain with γ-irradiated MOD/MOD pollen (13); in this screen, eight independent SC mutants lacked the DD33MOD fragment (Fig. 2D).

The 119-bp DD33 probe detected a 1.1-kb transcript in polyadenylated [poly(A)+] RNA (14) from stigmas, leaves, and anthers of MOD/MOD plants, but only a weak signal was apparent after prolonged exposures of blots with RNA from mod/mod plants (Fig.3A), indicating that the DD33modallele is hypomorphic. The steady-state amounts of DD33 transcripts inMOD stigmas are at least 30 times those in modstigmas. No change in the abundance of the DD33 transcript was observed in stigma samples from MOD or mod plants in response to self-pollination (Fig. 3B) or cross-pollination.

Figure 3

Gel blot analysis of DD33 transcripts in SI (MOD/MOD) and SC (mod/mod) plants. Polyadenylated RNA was isolated from (A) SI stigmas (lane 1), SC stigmas (lane 2), SI leaves (lane 3), SC leaves (lane 4), SI anthers (lane 5), and SC anthers (lane 6), as well as from (B) unpollinated SI (lane 1) and SC (lane 2) stigmas and self-pollinated SI (lane 3) and SC (lane 4) stigmas. Blots were hybridized first with the 119-bp DD33 probe (upper panels) and subsequently with the 1.1-kb DD33 cDNA [lower panel in (A)] or with an actin probe [lower panel in (B)].

The 1.1-kb cDNA contained an open reading frame that encodes 286 amino acids (Fig. 4) similar in sequence to the MIP (major intrinsic protein) membrane proteins from plants, mammals, yeasts, and bacteria (15). MIP proteins form channels with six membrane-spanning domains and are thought to facilitate the transport of water and other small molecules across membranes. The DD33 coding region is most similar to those of the aquaporin genes, whose products transport water; in plants, these include genes that encode tonoplast intrinsic proteins (16) as well as plasma membrane intrinsic proteins such as Arabidopsis RD28-PIP, PIP1A, PIP1B, and PIP2 (17). The DD33 gene contains residues conserved in the MIP gene family (15). It shows the greatest sequence identity (98.6%) to PIP1B and it clearly belongs in the PIP1 subgroup of genes. Therefore, it is likely that the MOD protein forms a channel in the plasma membrane and transports water.

Figure 4

Sequence alignment of the predicted DD33MOD (MOD) protein (GenBank accession number AF004293) with three Arabidopsis plasma membrane aquaporins: PIP1B (GenBank Z17424), PIP1A (GenBank X75881), and PIP2A (GenBank X75883). Dashes represent gaps introduced to optimize alignment and dots indicate residues in the PIP sequences that are identical to the MOD sequence. The residues that are conserved in members of the MIP family (15) are underlined and the serine residues that are potential targets for phosphorylation are indicated by arrowheads. Residue numbers are indicated on the right.

Hydration is important for pollen germination and subsequent pollen tube development. Pollen grains, which are released from the anther in a relatively dessicated state, must draw water and perhaps other substances from the receptive stigmatic surface (Fig. 1). This stigma-to-pollen transfer of material is a regulated process in plants such as Brassica and other crucifers whose dry stigmas lack surface secretions (2, 18). Incompatible pollinations in this family are often manifested by the failure of pollen hydration (18), and certain male-sterile Arabidopsismutants produce pollen that fails to hydrate on the stigma (19). Therefore, water transfer from stigmatic papillar cells to pollen grains may be a checkpoint at which the outcome of pollen-stigma interactions in the crucifer family is determined.

We propose that the MOD protein functions in the regulation of water availability at the papillar cell surface. The water channel would be required for self-incompatibility as a component of the SRK signaling pathway. The activation of SRK on self-pollination would result in MOD activation and an increase in the flow of water into the papillar cell away from pollen, thus preventing adequate rates of pollen hydration. The SC phenotype of mod stigmas would result from a lack of channel molecules that can serve as targets of SRK activity. Although a mechanism of self-incompatibility based on the regulation of water availability is consistent with pollination biology, some MIPs are permeable to small molecules other than water (15). Therefore, the MOD channel might promote either the efflux and localized accumulation at the pollen–papillar cell interface of substances inhibitory to pollen germination and tube ingress, or the influx, and therefore localized depletion from the cell wall, of substances required for pollen germination and tube growth. The rapid modulation of membrane permeability in response to self-pollination could be brought about by reversible phosphorylation of MOD channel proteins, resulting in an increase in their transport activity (20) or in their rapid recruitment to the plasma membrane (21).

  • * To whom correspondence should be addressed. E-mail: men4{at}cornell.edu

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