Significance Pollen viability depends on a tough external barrier called the pollen wall. Pollen wall components are produced by tapetum cells, which surround developing pollen grains within the anther. Precise coordination of tapetum activity with pollen grain development is required to ensure effective pollen wall formation. Here, we reveal that this is achieved through a multidirectional dialogue involving three distinct cell types. We show that peptide precursors from the tapetum are activated by proteases produced stage specifically in developing pollen grains. Unexpectedly, we found that activated peptides are perceived not in the tapetum, but in the middle layer, which encloses the developing tapetum and pollen grains, revealing an unsuspected role for this enigmatic cell layer in the control of tapetum development.

Many peptide hormones and growth factors in plants, particularly the small post-translationally modified signaling peptides, are synthesized as larger precursor proteins. Proteolytic processing is thus required for peptide maturation, and additional post-translational modifications may contribute to bioactivity. To what extent these post-translational modifications impact on processing is largely unknown. Likewise, it is poorly understood how the cleavage sites within peptide precursors are selected by specific processing proteases, and whether or not post-translational modifications contribute to cleavage site recognition. Here we describe a mass spectrometry-based approach to address these questions. We developed a method using heavy isotope labeling to directly compare cleavage efficiency of different precursor-derived synthetic peptides by mass spectrometry. Thereby, we can analyze the effect of post-translational modifications on processing, and the specific sequence requirements of the processing proteases. As an example, we describe how this method has been used to assess the relevance of tyrosine sulfation for the processing of the Arabidopsis CIF4 precursor by the subtilase SBT5.4.

The surface of pollen grains is reinforced by pollen wall components produced non-cell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage-specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types therefore cooperate to coordinate pollen wall deposition through a multilateral molecular dialog.

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