EXO70A2 is critical for the exocyst complex function in Arabidopsis pollen

Abstract

Pollen development, pollen grain germination and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to fertilize ovules. Pollen grains undergo two major developmental switches: dehydration characterized by metabolic quiescent state, and rehydration upon pollination that leads to extraordinary metabolic and membrane trafficking activity, resulting in germination and rapid tip growth of pollen tubes. To sustain these processes, many plant housekeeping genes evolved their pollen-specific paralogs. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we describe that EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in Arabidopsis pollen, which governs the conventional secretory function of the exocyst, analogically to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination and pollen tube growth. GFP:EXO70A2 was localized similarly to other exocyst subunits to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for the EXO70A1 function in the sporophyte, indicating functional redundancy of these two closely related isoforms. Phylogenetic analysis revealed that the ancient duplication of EXO70A to two (or more) paralogs, one of which is highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in the Arabidopsis pollen required for efficient plant sexual reproduction. KEY WORDSexocyst, EXO70A2, EXO70A1, pollen development, pollen tube, tip growth, polarized secretion ABBREVIATIONS CDS -coding sequence GFP -green fluorescent protein NBT -nitroblue tetrazolium PM -plasma membrane PME -pectin methylesterases ROS -reactive oxygen species WT -wild type

Redundant and Diversified Roles Among Selected Arabidopsis thaliana EXO70 Paralogs During Biotic Stress Responses

“…While EXO70B1 and H7 remained the most expressed paralogs in both WT and exo70A1 seedlings, loss of EXO70A1 leads to further increase in relative transcript abundance of H-group paralogs, especially H1and H2, and to a lesser extent also of F1, as well as to a slight drop in relative abundance of E2 ( Figure 3B). Even though the pollen specific isoform EXO70A2 mRNA levels were in our experiments under the detection limit, recent work by Markovic et al, 2020 indicates the importance of a sporophytic EXO70A2 function of in the exo70A1 plants. Interestingly, the increase of H1 and H2 in both biotic stresses and in the absence of A1 implies that H1 and H2 might compensate functionally for loss of basal A1 level during pathogen response.…”

Genome-wide analysis and transcript profiling of PSKR gene family members in Oryza sativa

“…Likewise, OsPSKR12 and OsPSKR15 were also upregulated in response to abiotic stress as depicted in expression analysis done by RNA-seq and real time data. The strong correlation between OsPSKR12 and Exo70 (Fig 7) further supports the role of PSK-PSKR signalling in abiotic stress response as Exo70 is involved in stress tolerance besides its involvement in pollen maturation, germination and pollen tube growth and [54][55][56]. Moreover, the co-expression analysis of PSKR gene, as in Fig 7, showed that their expression is highly correlated to genes encoding for WRKY, MADS-box, NB-ARC, etc.…”

“…Other co-expressing genes include Exo70, WRKY, MADS-box and ABC transporters. Exo70, a component of the exocyst complex, is involved in vesicle trafficking like exocytosis and play crucial role in plant immunity and pollen growth and maturation [53][54][55][56]. While WRKY and MADS-box are the most important class of transcription factors involved in abiotic stress response [57][58][59].…”

Fluorescence visualization of cellulose and pectin in the primary plant cell wall

“…5A). This distal–apical signal gradient is consistent with the results reported for Arabidopsis pollen tubes stained with CFW (Marković et al ., 2019). Signal at the tip was absent in some cells and present in others.…”