In this article we will discuss about:- 1. Meaning of Self-Incompatibility 2. Molecular Basis 3. Determinants 4. Significance.
Meaning of Self-Incompatibility:
Self-incompatibility or intraspecific incompatibility is a well-designed genetic mechanism by which certain plants recognize and reject their own pollen thus forcing outbreeding. It is defined as “inability of the plant producing functional gametes to set seed upon self-pollination”,. Lundqvist (1964) defined it as “the inability of fertile hermaphrodite seed plant to produce zygotes after self-pollination”.
Its genetic system is based on a single locus, the sterility (S) locus, with multiple alleles. Pollen germination or pollen tube growth is blocked when the pollen grain and the stigma upon which it lands have the same allele at the same locus. Besides the genetic factors, intraspecific incompatibility is also associated with different lengths of stamens and style in flowers on same plant.
This self- incompatibility is acquired nearly one or two days before anthesis as well as in open flowers. Nearly two-thirds of the families of angiosperms exhibit self- incompatibility. The significance of SI in the evolutionary context cannot be overstated, since its possession leads to obligate outbreeding and the maintenance of heterozygosity within a species.
In the crop and ornamental plants, most of the perennial grasses, forage, legumes, and members of Brassicaceae, Asteraceae, Rosaceae, and Solanaceae have SI mechanism of varying kinds and degrees of effectiveness. Fig. 6.8 shows a diagrammatic representation of the behaviour of compatible and incompatible pollination.
Molecular Basis of Self-Incompatibility:
The overwhelming support for the presence of specific proteins that arbitrate in self-incompatibility, the major arena of research then centered on the isolation of incompatibility genes, identification of the protein products (S- proteins), and characterization of S-gene expression in wild type and transgenic plants.
i. Molecular Basis of Gametophytic Self-Incompatibility:
The molecular biology of self-incompatibility has been worked most extensively in Nicotiana alata. Sequential expression of S-genes and their protein products in the female floral parts perfectly matches with the site and the time of incompatibility.
Northern blot analysis of RNA from flower buds of different ages probed with a cDNA clone encoding S-linked proteins showed that, gene expression is maximum in mature styles, comparatively weak in the ovary and totally absent in the immature styles.
Similar analysis at the tissue and cell level showed that the gene transcripts are localized in the stigmatic papillae and the stylar transmitting tissue of mature flowers and in the epidermis of the placenta.
In fact the gene transcripts are localized in those tissues and cells, through which the pollen tube makes its journey to reach the embryo sac. S- gene expression and self-incompatibility in Nicotiana alata by immunocytochemical localization of allele-specific antibodies has removed all doubt about this relation. It showed a remarkable coincidence between accumulation of gene products and gene transcripts in the same tissue and cells of the carpel.
The S-gene products were found to be homologous to the fungal RNases, thus the stylar product of the S-gene was designated as S-RNases. Several physiological factors and molecular approaches have succeeded in holding S-RNase responsible for self- incompatibility.
The high expression of S- RNases in a transgenic hybrid developed between two self-compatible line of Nicotiana, viz., Nicotiana alata and N. langsdorfii leaves no doubt that pollen rejection is due to RNase action. On the other hand, antisense suppression of S-RNase activity in a transgenic hybrid between Nicotiana plumbaginifolia and N. alata allowed the acceptance of self pollen.
Besides antisense suppression of S-RNase and prevention of self pollen rejection, there is another way to do so by introducing a S-gene construct, into Petunia inflata where one of the histidine residues, which is the heart of RNase molecule essential for its catalytic activity, is replaced by asparagine by site-directed mutagenesis.
The transgenic plant produced the modified protein lacking the S-RNase activity but failed to reject the self pollen bearing the same allele. Mc-Clure (1990) explained the mechanism of RNase function in a self-incompatible system.
They found that RNA extracted from styles of incompatibly pollinated Nicotiana alata flowers suffered a massive degradation and appeared as a streak of low molecular mass in an agarose gel run under denaturating condition.
Since S- RNase is easily taken up by the pollen tubes grown in vitro, cytotoxic effects of the enzymes on the pollen tube RNA may be responsible for RNA degradation. Thus ensuring the death of the pollen tube in an incompatible pollination.
Changes in the level of cytosolic Ca2+ have been found to be responsible for self- incompatibility reaction in Petunia rhoeas. It has been seen that by raising the endogenous level of Ca2+ by ultra violet activation, the growth of pollen tube was inhibited even in the absence of incompatible stigmatic extract. This elevated Ca2+ level induced by the S-locus proteins might moderate the transcriptional activation of genes necessary for the initiation of self- incompatibility reactions in the species.
ii. Molecular Basis of Sporophytic Self-Incompatibility:
Sporophytic self-incompatibility has been best characterized in Brassica oleracea and Brassica campestris and the stigmatic protein responsible for the incompatibility is a S-locus- specific protein called glycoproteins (SLGs). Separation of cDNA clones encoding SLGs from different homozygous lines of Brassica oleracea initiated the molecular analysis of sporophytic self-incompatibility.
The SLG genes transcribe closely related glycoproteins that are secreted into the walls of the stigmatic papillae. Experimental regulation of an SLG gene in a transgenic plant gives support to the existence of a correlation between cellular and developmental expression of the gene with the site and the timing of inhibition of pollen germination.
An SLG gene from Brassica oleracea incorporated into Nicotiana tabacum is expressed at the protein level in the stylar transmitting tissue. Southern blot analysis of genomic DNA of Brassica oleracea has exposed the existence of several sequences with different degrees of homology to the SLG gene.
A gene designated as S-locus related (SLR) is not linked to S-locus gene series, but like the SLG gene its expression is restricted to the papillar cells of the stigma concurrent with the achievement of self- incompatibility.
In spite of considerable similarity with the SLGs in supposed signal sequence, N-glycosylation sites and a cystein- rich domain has a subsidiary role in incompatibility. It appears that another gene designated as S-locus receptor kinase (SRK) maybe involved in the mechanism that activates self-pollen rejection in Brassica oleracea.
This gene is genetically linked to the SLG gene at the S-locus and has a complex structure consisting of an SLG-like domain, presumed serine/threonine protein kinase domain, and a stretch of transmembrane domain linking the SLG-like region with the kinase moiety.
Hiscock (2002) proposes a model for the mechanism of SSI in Brassica oleracea. Inhibition of incompatible pollen occurs at the stigma surface, usually before germination of the pollen, and appears to involve a deregulation of water flow to the pollen from the stigma during hydration of the pollen grain (Dickinson, 1995). Here, pollen from an S1S2 individual is inhibited on an S1S3 stigma as a consequence of a haplotype-specific interaction between male (pollen) and female (stigma) products of the S1, haplotype (Fig. 6.15).
This S proteins are colour coded: S1, green; S2, blue; and S3, pink. SCR1, a pollen S protein is recognized by, and binds to the extracellular receptor domain of SRK1, a stigma S protein, thereby inducing dimerization of SRK1, and autophosphorylation denoted by P, on serine and threonine residues in the kinase domain (shown as a zig-zag tail). ‘Activation’ of the SRK protein then initiates an intracellular signaling cascade within the stigma that leads to localized rejection of the pollen.
SLG acts as an accessory protein in the formation of the receptor complex PCP-A1, a small cysteine-rich pollen-coat protein similar to SCR, but not genetically linked to S, binds nonspecifically to SLGs from all haplotypes and may function as an additional accessory protein alongside SLG during formation of the receptor complex.
Signaling downstream of SRK has not yet been characterized, but essential for the SI response is ARC1, an arm-repeat protein, that binds to the kinase domain of SRK in a phosphorylation- dependent manner, but whose function is as yet unknown (Fig. 6.16).
Nasrallah (1994) provided evidence that the binding of a pollen-borne cell surface ligand to the SLG-SLK receptor leads to a series of resultant reactions mediated by a protein kinase, which regulate the sporophytic incompatibility system. The strongest support is that the amino acid sequence of the SRK gene product has characteristics of a plasma membrane based protein.
The specific targeting of the SRK protein to the plasma membrane of the stigmatic cells of the transgenic tobacco further support the view the molecular role of protein kinase as pivotal in the incompatibility response.
In an another experiment it was found that the spontaneous mod (“modifier”) mutation in Brassica campestris produce self-compatible plants characterized by the absence of an aquaporin-like gene encoding a water protein channel.
Ikeda (1997) from this observation concluded that aquaporin regulates the self- incompatibility reaction in wild -type plants as a component of the SRK signaling system, by obstructing the flow of water from the stigmatic papillae into pollen grains.
Determinants of Self-Incompatibility:
The self-incompatibility phenomenon is controlled by a complex and polymorphic locus.
Among the genes at this S-locus is a pair of sequence related genes, the cell wall localized S- locus glycoprotein (SLG) gene and the plasma membrane-spanning receptor protein kinase (SRK gene), both of which are expressed specifically in the stigma epidermal cells. Different variants of the S-locus, designated as haplotypes, are characterized by highly polymorphic alleles for SLG and SRK.
Although one function of SLG is to stabilize SRK, it itself is viewed as a ligand- activated receptor kinase and the primary female determinant of self-incompatibility. Selective binding of pollen-borne ligand is thought to initiate a signal transduction cascade resulting in the SI pollen rejection reaction.
Recombination analysis of the Brassica campestris S8 haplotype has shown that the male and the female SI determinant are contained in a 65-kb chromosomal segment encompassing SLG8 and SRK8.
During sequence analysis of the 13-kb region between SLG8 and SRK8 it was found that the 400-base pair Hind III-Xba I restriction fragment indicated in Figure 1A (arrange sequence) contained a small segment with an unusual high frequency of cystein residue in one of the deducible reading frames. RNA gel blot analysis with the 400 bp Hind III- Xba I fragment as probe revealed that this segment is part of a transcribe gene that is expressed specifically in anthers.
Screening of an Ss anther cDNA library allowed the isolation of a 450-bp full length cDNA clone. Sequence comparison of this clone and the SLG8– SRK8 intergenic segment determined that the gene consists of two exons (110 and 300 bp) separated by an unusually large intron of 4.1 kb (figure below A).
In DNA gel blot analysis, the 400-bp Hind III-Xba I probe detected restriction fragment with sizes predicted by the restriction map (figure B, below) Thus a single copy, 5 locus-encoded, anther expressed gene, which was named 5 locus cystein-rich protein (SCR) gene.
To test that whether SCR is indeed that male determinant of SI, a detail analysis of both its expression and function was done by Schopfer,1999. They analyzed the expression of wild type self-incompatibility plants by RNA gel blot analysis.
SCR shows an anther specific, developmentally regulated expression. SCR transcripts can be detected in anthers only after the generation of haploid microspores, with transcripts accumulating in the microspores. Therefore, the SCR gene is active postmeiotically and gametophytically.
SCR transcripts in anthers isolated from a self-incompatible mutant Brassica oleracea strain, designated ml600, generated by gamma- irradiation of an S13S13homozygote. Reciprocal pollination between ml600 resulted in the loss of S13 specificity in pollen but not in stigma.
Gel blot analysis of anther RNA demonstrated that in contrast to plants carrying the wild type S13 haplotype, ml600 anthers lacked detectable SCR transcripts (Figure above 3A, lanes 8 to 10). The correlation between loss of pollen S13 specificity and absence of detectable SCR,, transcripts in the ml 600 mutants strain provides strong evidence that SCR is necessary for pollen SI specificity.
In order to establish the function of SCR as the pollen determinants of SI Schopfer (1999) transformed a B. oleracea S2S2 homozygous strain with a chimeric gene consisting of the SCR6 promoter fused to the SCR6 cDNA. Among the 14 hygromycin resistant plants, 12 plants expressed the SCR6 transgene and were designated S2S2/SCR+6 where two plants failed to produce detectable levels of SCR6 transcripts and were designated S2S2/SCR+6 (Figure 3A, lanes 11 to 14).
Pollen from the two S2S2/SCR+6 plants germinated and produced pollen tubes on S6S6 stigmas. In contrast pollen from each of the 12 S2S2/SCR+6 plants was inhibited by S6S6 stigmas, even though pollen was viable as demonstrated by its ability to produce pollen tubes on stigmas homozygous for an unrelated S-haplotype such as S22. Thus pollen of S2S2 /SCR+6 transformants had acquired S6 specificity not only proves the involvement of SCR in SI, but also demonstrates that this gene is sufficient for determining male SI specificity.
Significance of Self-Incompatibility:
Self-incompatibility is by far the most efficient outbreeding mechanism. It has been envisaged as one of the main causes for the rapid evolution of angiosperms. In many crop plants the major limitation to exploit hybrid vigour is the lack of male sterile line, and the absence of suitable chemicals to induce effective male sterility without affecting the female fertility.
Thus, here self-incompatibility can be used effectively in producing hybrid seeds, without undergoing emasculation, nuclear or cytoplasmic sterility or restoring to gametocides. Thus it is very essential to select strictly self- incompatible lines, which will not demonstrate pseudo-compatibility under reasonably varied environmental conditions.
Besides its importance in breeding programmes, it sometimes becomes essential to overcome self-incompatibility. These include, production of pure lines of the selected parents in the plant breeding programmes; maintaining hybrid characters in species which are difficult to propagate vegetatively or susceptible to viral infection; in fruit orchards self-incompatible is a rule, however, it becomes necessary to include varieties that with cross-compatible, because lack of proper weather conditions for the activity of pollinating insects may drastically reduce yield.
After pollination the pistil faces a formidable task of selecting compatible pollen and rejecting the incompatible ones. Studies in the last two decades have proved convincingly that the molecules present on the pollen grains and the stigmas are intimately involved in the recognition reactions that determine the fate of the compatible pollens.
The major breakthrough in the study of pollen-pistil interactions has come from the cloning of self-incompatibility genes and the recognition of stylar components that mediates in pollen rejection.