JAGGER, an AGP essential for persistent synergid degeneration and polytubey block in Arabidopsis

ABSTRACT

A precise control of sperm cells delivery and fusion to the egg cell and the central cell is fundamental for the accomplishment of successful double fertilization in flowering plants. This is mostly regulated by female gametophyte egg and central cells, which control the timing of synergids cell degeneration. We recently identified an arabinogalactan protein, AGP4, named JAGGER, that impairs the persistent synergid degeneration, and consequently leads to the attraction of more than one pollen tube into one embryo sac, a situation termed polytubey. jagger mutants revealed an increased rate of polytubey and persistent synergids that do not degenerate. This persistent synergid, is, as we suggested, the cell responsible for attracting an extra pollen tube into the embryo sacs.

The production of viable offsprings is the main goal of all flowering plants. Therefore all the efforts a plant lays in growing and producing flowers will be reflected in its seed set. To achieve successful fertilization a pollen grain must alight on a receptive stigma of a flower. After this, it hydrates and germinates producing a pollen tube. The pollen tube will carry the sperm cells and will grow into the pistil transmitting tract until it reaches the ovule and the female gametophyte deeply embedded in the sporophytic female tissues. Once there, the pollen tube penetrates one of the 2 synergids. This occurs in an area formed by synergids plasma membrane invaginations and thickened cell wall – the filiform apparatus. After synergid penetration, the pollen tube bursts releasing the sperm cells. Subsequently, double fertilization takes place: one sperm cell fuses with the egg cell, originating the embryo, while the other fuses with the central cell, initiating endosperm development .^1-3

The synergid cells surrounding the egg cell at the embryo sac entrance, with its filiform apparatus, are the cells responsible for pollen tube attractants production, such as AtLURE1.⁴ Once the pollen tube penetrates one of the 2 synergids, this cell degenerates and ceases producing the attraction molecules. The second synergid, called the persistent synergid, will degenerate only after successful fertilization is guaranteed.⁵

The success of double fertilization is not only dependent on the control of sperm cells fusion with the female gametes, but also it is indirectly dependent on controlling the fusion of the persistent synergid with the developing endosperm to cease pollen tube attractants production and avoid the attraction of extra pollen tubes – polytubey.⁶

All the steps of the reproductive cycle depend on the precise crosstalk and coordinated growth of the developing pollen tube with the female sporophytic and gametophytic tissues ¹. The final step of this whole process, sperm cells delivery and fusion with the egg and the central cell is determinant to guarantee the seed formation. And it is during this final step that the final control is orchestrated to prevent the entrance of more than one pair of sperm cells into the embryo sac, avoiding polyspermy.^5,6

Arabinogalactan proteins, known as AGPs, comprise a large family of hydroxyproline rich proteins. Their protein backbones are massively decorated with carbohydrates, largely defining their interacting molecular surface.⁷ Most of the AGPs are predicted to be tethered to the plasma membrane by a GPI-anchor,⁸ placing them in a perfect position to eventually interact with other molecules in several signaling pathways. AGPs are abundant in cell walls, plasma membranes and extracellular secretions, being present in many stigma exudates, style transmitting tissues and pollen itself.^9,10 These glycoproteins have long been related to plant reproduction, being implicated in different steps of the reproductive process.^11-13 We have shown the differential expression of a subset of AGPs in specific Arabidopsis female tissues, suggesting their possible roles during reproduction.¹⁴

In a recent study we have shown the importance of another AGP, JAGGER (AGP4), for persistent synergid degeneration and polytubey block.¹⁵ This study revealed that a specific sporophytic AGP is somehow responsible for regulating the elimination of the persistent synergid, the one that is not invaded by the pollen tube upon fertilization. In jagger ovules this synergid keeps producing molecular attractants allowing more than one pollen tube to grow into one embryo sac. That is why we named this AGP4 as JAGGER, the one who cannot get no satisfaction. Although JAGGER is expressed inside the embryo sac, a knock-down mutant driven by the SEEDSTICK (STK) promoter, allowed us to verify the sporophytic origin of the defect we observed. STK is expressed in the ovule integuments, in the septum tissues but not in the transmitting tract or inside the embryo sac,¹⁶ proving that it is the JAGGER present in the ovular integuments that is responsible for the phenotype observed. In pSTK:RNAi:JAGGER lines, more than one pollen tube could be observed growing toward the embryo sac (Fig. 1A) opposed to the wild-type ones (Fig. 1B), which correlated with a decrease in JAGGER expression in these lines.

Figure 1.

Aniline blue staining of reciprocal crosses between pSTK:JAGGER_RNAi and wild-type flowers. (A) Cross between a pSTK:JAGGER_RNAi mutant female flower and wild-type pollen where a polytubey occurrence can be observed as indicated by the yellow asterisks. (B) Cross between a female wild-type flower and pSTK:JAGGER_RNAi mutant pollen where normal single pollen tubes enter each ovule as pointed out by the pink asterisks.

AGPs biological mode of action is starting to be unravelled now. Very recently, loss of function mutants of AGPs specific hydroxyproline-O-β-galactosyltransferases, GALT2, GALT3, GALT4, and GALT5^17-19 and HPGT1, HPGT2 and HPGT3²⁰ indicated the important contributions of the carbohydrate moieties of AGPs toward its function, as speculated before, making them strong candidates for mediation of cell-cell communication.

Earlier studies from Lamport and Várnai²¹ had already “identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with 2 short branched sidechains each with a single glucuronic acid residue that binds Ca²⁺ when paired with its adjacent sidechain." This trisaccharide is able to bind external Ca²⁺ acting as a capacitator that releases the Ca²⁺ in an auxin dependent manner. This binding ability, allows AGPs to co-ordinate developmental processes in plants, involving exocytosis of AGPs and recycled Ca²⁺. This is somehow in accordance with the work from Mizukami and colleagues²² who discovered one more important player in the pollen tube attraction system of Torenia fournieri. In a very elegant investigation, Mizukami and colleagues have demonstrated that a disaccharide, methyl-glucuronosyl galactose from AGPs, is essential to induce competence in Torenia pollen tubes, making them capable of recognizing the embryo sac molecular attractants, LUREs. This disaccharide is produced by the sporophytic tissues surrounding the embryo sac and allows pollen tubes to become competent to grow inside it and accomplish double fertilization. This work proved what was proposed for a long time, that the embryo sac, although essential for pollen tube attraction, it is not sufficient to accomplish this task.²³

Admitting Lamport and Vanái’s²¹ theory all AGPs would function in the same way, with no differences between their carbohydrates moieties. So why would plants have such a big family of glycoproteins differentially expressed in time and space and specific for different tissues? It is important to clarify if “the glycosyltransferases act on all or only a subset of AGPs? What is the peptide or glycan substrate specificity for each AGP glycosyltransferase?” as Showalter and Basu²⁴ already questioned. Older studies hypothesized that different types of cells and tissues would have different glycosylation machineries or enzymes, determining the AGP glycosylation pattern in these cells or tissues and also cell fate.²⁵ Demesa-Arévalo and Vielle-Calzada²⁶ showed that this was the case for AGP18, as discussed in more detail in Pereira et al., (2015).

It is possible that some AGPs may be acting as calcium chelators as proposed by Lamport and Várnai,²¹ it is known that calcium oscillations are extremely important for pollen tube reception, sperm cell release, and fusion with the female gametes. Being AGPs so strongly expressed in this tissues,²⁷ it is plausible to ponder about such a mechanism of action.

So far, the specific functions of only a few AGPs in Arabidopsis reproduction has been well described, such as the involvement of AGP6 and AGP11 in pollen tube germination and growth,²⁸ the importance of AGP18 for the female megaspore development²⁶ and now JAGGER, an AGP required for persistent synergid degeneration and polytubey block.¹⁵

In accordance with all these recent results, we hypothesize that JAGGER carbohydrates mediate this blockage. One hypothesis is that JAGGER is recognized by specific pollen tube receptors, most probably receptor like kinases with lectin domains, as proposed by Mizukami et al.²² for the AMOR disaccharide, acting as a signal or as a ligand/co-receptor. In this way, pollen tubes primed by JAGGER would act as carriers of a sugar signal for persistent synergid degeneration. This JAGGER in turn, once inside the embryo sac could be used as a molecular marker for persistent synergid cell elimination. According to Maruyama et al.,⁶ after successful fertilization, this persistent synergid degenerates by cell fusion with the endosperm and elimination of its nuclear contents. It is known that these 2 steps involve Polycomb Repressive Complex 2 (PRC2), chromatin-remodelling factors responsible for gene silencing, and an ethylene signaling cascade, leading to the death of the persistent synergid and blocking the production of pollen tube attractants.^29,6 What remains to be known is how the persistent synergid is recognized as a target for elimination.³⁰ JAGGER might be one of the cell death markers for the persistent synergid, destining it for elimination. AGPs have already been described as molecular markers during development.^27,31

It remains to be shown, what is the function of the AGP4 present along the transmitting tract of the pistil. JAGGER might be important for persistent synergid elimination, but it may also be involved, along with other AGPs, or even other non-AGP molecules, in preparing pollen tubes for entering the embryo sac. In this situation, being such a big family of proteins, the chance of having genetic redundancy is very high³² as already been shown for 2 male expressed AGPs, AGP6 and AGP11.⁸

Further questions need also to be answered such as:

jagger mutants are not fully penetrant, suggesting the involvement of other players in the molecular network controlling persistent synergid cell death. Further studies need to be carried out in order to identify and characterize these players.

Is the pollen tube the JAGGER “carrier” into the embryo sac or somehow the sperm cells are involved in this process after being released from the pollen tube?

How is JAGGER marking the persistent synergid for death? Is it before or after cell fusion with the endosperm? Is it marking the cell itself or its nucleus?

May different AGPs be acting in different pathways, some acting as repulsive and others as attraction agents during reproduction?

These results will expand our knowledge regarding Arabinogalactan Proteins biological function, whose molecular mechanism of action is still elusive. The importance of these glycoproteins in Angiosperms reproduction and its possible involvement in attracting/repulsing PTs into the embryo sac is of great significance,³³ since fertilization drives formation and development of seeds, the most important source of food in our planet.

Disclosure of potential confllicts of interest.

No potential conflicts of interest were disclosed.

Funding

This work was financed by FEDER through the COMPETE program, and by Portuguese National funds through FCT, Fundação para a Ciência e Tecnologia (Project PTDC/AGR-GPL/115358/2009), and from an FCT PhD grant SFRH/BD/60995/2009 awarded to A.M.P. This project also benefited from financial support from COST Action FA0903: "Harnessing Plant Reproduction for Crop Improvement."