Overexpressed Tomosyn Binds Syntaxins and Blocks Secretion during Pollen Development

Abstract

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex formation is necessary for intracellular membrane fusion and thus has a key role in processes such as secretion. However, little is known about the regulatory factors that bind to Qa-SNAREs, which are also known as syntaxins (SYPs) in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) Tomosyn protein (AtTMS) and demonstrated that it is a conserved regulator of SYPs in plants. AtTMS binds strongly via its R-SNARE motif-containing C terminus to the Qa domain of PM-resident, pollen-expressed SYP1s (SYP111, SYP124, SYP125, SYP131, and SYP132), which were narrowed down from 12 SYPs. AtTMS is highly expressed in pollen from the bicellular stage onwards, and overexpression of AtTMS under the control of the UBIQUITIN10, MSP1, or LAT52 promoter all resulted in defective pollen after the microspore stage in which secretion was inhibited, leading to the failure of intine deposition and cell plate formation during pollen mitosis I. In tobacco (Nicotiana benthamiana) leaf epidermal cells, overexpression of AtTMS inhibited the secretion of secreted GFP. The defects were rescued by mCherry-tagged SYP124, SYP125, SYP131, or SYP132. In vivo, SYP132 partially rescued the pMSP1:AtTMS phenotype. In addition, AtTMS, lacking a transmembrane domain, was recruited to the plasma membrane by SYP124, SYP125, SYP131, and SYP132 and competed with Vesicle-Associated Membrane Protein721/722 for binding to, for example, SYP132. Together, our results demonstrated that AtTMS might serve as a negative regulator of secretion, whereby active secretion might be fine-tuned during pollen development.

Combining QTL Mapping and Gene Expression Analysis to Elucidate the Genetic Control of ‘Crumbly’ Fruit in Red Raspberry (Rubus idaeus L.)

“…SUMO proteins are involved in important processes related to plant reproduction such as flowering time regulation, GA signaling, and gametophyte development [21]. SYP 132 is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein that controls the ultimate fusion of a secretory vesicle with its target compartment [22]. The SYP132 protein is located inside the plasma membrane (PM) of many tissues including developing pollen.…”

“…The SYP132 protein is located inside the plasma membrane (PM) of many tissues including developing pollen. The gene is expressed in the early stages of pollen development and represents a putative candidate to mediate fusion processes during the mitotic divisions that are responsible for the formation of tricellular pollen [22]. Therefore, in raspberry, a significant reduction in the expression levels of these two genes could impede the normal development of pollen and then indirectly affect fertilization, potentially contributing to the formation of uneven fruit with reduced number of drupelets.…”

“…The second gene, AT5G08080.3, encodes the SYNTAXIN OF PLANTS 132 (SYP132), a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein that controls the ultimate fusion of a secretory vesicle with its target compartment [22]. The microarray probe CUST_2371_PI426541283, matching AT5G08080.3, was downregulated in the mostly crumbly samples with differences being statistically significant (p < 0.001) at both open flower and green berry stages (see Supplementary Table S1).…”

A GPAT1 Mutation in Arabidopsis Enhances Plant Height but Impairs Seed Oil Biosynthesis

“…To create a mutant of the GPAT1 gene with the CRISPR/Cas9 gene editing system, we designed an sgRNA target sequence using the online tool CRISPR-P2.0 ( ) [ 81 ]. The selected target sequence was cloned into the final pAtU6-26:sgRNA-23p35S:Cas9 pBlunt vector [ 82 ]. The construct was confirmed by sequencing and then introduced into WT Arabidopsis plants via the floral dip method [ 79 ].…”

Complex roles of Rabs and SNAREs in the secretory pathway and plant development: a never‐ending story

“…Interestingly, Li et al . (2019) have discovered a Tomosyn protein which can bind through its C‐terminal R‐SNARE‐like motif to several Qa‐SNAREs and act as a negative regulator of secretion to control pollen development (Li et al ., 2019).…”