The poly(A) polymerase PAPS1 mediates pollen maturation by regulating sperm cell differentiation in plants

Abstract In flowering plants, a haploid microspore undergoes an asymmetric division to produce the male germline that encounters a mitotic division to produce two germ cells. The resulting germ cells undergo a series of specialization events to produce the two sperm cells required for double fertilization. These events include to upregulate male germline‐specific while downregulating male germline‐nonspecific regulon, but how these specializations events are regulated, are still unresolved. To know how plant sperm cell is specialized, we mutagenized Arabidopsis double homozygous transgenic line (MGH3p‐MGH3::eGFP and ACTIN11p‐H2B::mRFP) by an ethyl methane sulfonate (EMS) treatment and isolated a mutant with sperms identity loss, resulting in a completely male defective plant. Second‐generation sequencing identified a point mutation G/A causing premature stop codon TGG/TGA in the poly(A) polymerase PAPS1 that is linked with phenotype. Further, we found that paps1 mutant fails to upregulate male germline‐specific regulon and to downregulate male germline‐nonspecific factors required for sperm cell differentiation and attaining pollen maturation. Previously, polyadenylation of pre‐mRNAs by PAPS1 has been found crucial for both RNA‐based silencing processes and the processing of pre‐mRNAs into mature mRNAs ready for translation. This study concludes that PAPS1 mediates sperm cell differentiation through upregulating specific while silencing the nonspecific factors of male germlines.


| INTRODUCTION
In flowering plants, the male gametophyte or pollen grain generates and delivers the male gametes to the embryo sac for double fertilization and thus plays a vital role in plant fertility and crop production.
Haploid unicellular microspores are produced during male gametogenesis through meiosis within specialized male reproductive organs, the stamens. Each microspore undergoes a highly asymmetric division to produce a small germ or generative cell encapsulated by large vegetative cell. The generative cell then undergoes a second round of mitotic division to create two sperm cells. Sperm cells are most divergent of all cell types as they accomplish their task outside of the male producing body in completely new environment of female body. One sperm cell fertilizes the central cell to produce the endosperm, and the other fertilizes the egg cell to form the embryo. Thus, the making of fully specialized sperm cells is critical for double fertilization and has significant consequences for seed production and crop fertility (Borg et al., 2009;Borg & Twell, 2010).
However, the successful fertilization needs the germ cells to undergo a series of differentiation events to attain fully mature pollen (Borg et al., 2011), but how these events are controlled remain unknown. In previous studies, five pollen-specific MIKC MADS box proteins expressed in the vegetative cell were found important in pollen maturation (Verelst et al., 2007a(Verelst et al., , 2007b. Armadillo BTB Arabidopsis protein 1 (ABAP1) regulates pollen differentiation by making a potential interaction with a transcription factor TCP16 (Cabral et al., 2021). Further, the distinct and diverse transcriptome of the plant male germ cells enlightening that germline development is transcriptionally tightly regulated (Borges et al., 2008;Engel et al., 2003).
However, in terms of male gamete functional specification, only the male germline-specific R2R3 MYB transcription factor DUO1 pollen1 (DUO1) has been found functioning in the regulation of sperm cell differentiation in Arabidopsis (Borg et al., 2011). Here, we found the new and significant role of poly(A) polymerase PAPS1 in mediating pollen maturation by activating a germline-specific differentiation program through downregulating male gamete nonspecific regulon and upregulating male gamete specific factors.
The poly(A) tail at the 3 0 end (polyadenylation) of pre-mRNAs by PAPS is a critical process in eukaryotic gene expression for both the RNA-based silencing processes, such as RNA-directed DNA methylation and the processing of pre-mRNAs into mature mRNAs ready for translation (Hunt, 2008;Millevoi & Vagner, 2010;Zhang et al., 2019).
The Arabidopsis thaliana genome encodes four PAPS proteins, termed PAPS1 to PAPS4, and all of them are essential for plant growth and development because homozygous transfer DNA (T-DNA) insertion mutants could not be obtained for any of the PAPS genes (Meeks et al., 2009). The paps1 mutant produces a male gametophytic defect phenotype and exhibits opposite effects on flower and leaf growth with increased flower growth and reduced leaf growth because of reduced activity of the small auxin-up RNA (SAUR) (Vi et al., 2013;Zhang et al., 2019). It was demonstrated that the opposite effects of PAPS1 on flower and leaf growth indicate the different identities of these organs and recognize a role for PAPS1 in the vital connection between growth pattern and organ identity. Furthermore, paps1-3 null-mutant results in depletion of 21-and particularly 24-nucleotidelong short interfering RNAs (siRNAs) and microRNAs (miRNAs), a strong overaccumulation of transposable element (TE) transcripts, and defects in the expression of pollen-differentiation genes. Moreover, PAPS1 was found to regulate pollen development by making a functional interaction with components of mRNA processing and RNAdirected DNA methylation (RdDM) pathways (Zhang et al., 2019).
However, the potential role of PAPS1 in the regulation of sperm cell differentiation and subsequent attaining of pollen maturation remained undetermined. Here, we found that PAPS1 mediates pollen maturation by regulating sperm cell differentiation in Arabidopsis.
To understand sperm differentiation mechanism in plants, we mutagenized by an ethyl methane sulfonate (EMS) treatment an Ara-

| Plant materials and growth conditions
For all experiments, A. thaliana ecotype Columbia-0 (Col-0) obtained from ABRC stock center (http://www.arabidopsis.org) was used as the wild type. The double transgenic line MGH3p-MGH3::eGFP and ACTIN11p-H2B::mRFP described previously (Borges et al., 2012) was used as control. In mature pollen, MGH3p-MGH3::eGFP is expressed specifically in sperm cells, whereas ACTIN11p-H2B::mRFP is expressed F I G U R E 1 paps1-4 is male gametophyte defective. Genetic crosses show that paps1-4 is completely male defective mutant (a). The comparison of numbers of seeds per silique produced in WT and paps1-4 shows that paps1-4 does not affect female gametophyte (b). WT = Col-0 in vegetative nucleus. For better vegetative growth of plant, shortday conditions in a growth chamber (light conditions: 8-h light/16-h dark; temperature: 22 AE 3 C and humidity: 55%) were used, while long-day growth conditions (light conditions: 16-h light/8-h dark; temperature: 22 AE 3 C and humidity: 55%) were used to achieve flowering.

| Mutagenesis and screen
Plants homozygous for the double transgene MGH3p-MGH3::eGFP and ACTIN11p-H2B::mRFP were selected for EMS mutagenesis. A total of 15,000 transgenic seeds were mutagenized by imbibition of .6% EMS for 12 h, .4% EMS for 8 h, and .3% EMS for 12 h (Kim et al., 2006). Well-dried seeds were soaked in a 50-ml plastic tube with 40 ml of 100 mM phosphate buffer (pH 7.5) at 4 C overnight and then treated with .3%, .4%, and .6% of EMS in a phosphate buffer

| Phenotypic analysis of mutants
Pollen grains of different developmental stages of Arabidopsis wild type as well as mutants were stained with a 4 0 ,6-diamidino-2-phenylindole (DAPI) solution (1 μg ml À1 DAPI and .1% Triton X-100) for 5 min before observation. The viability of pollen grains was assessed using Alexander staining. The paps1-4 mutant was transformed into quartet (qrt) mutant background to make tetrad pollen in which microspores fail to separate during pollen development (Preuss et al., 1994). For in vitro pollen germination, pollen was harvested from newly fully opened flowers and was placed onto pollen germination medium consisting of .01% (w/v) H 3 BO 3 , 1 mM MgSO 4 , 1 mM Ca (NO 3 ) 2 , 1 mM CaCl 2 and 18% (w/v) sucrose, pH 7.0, .8% (w/v) agar, and grown at 28 C in a growth chamber in the dark. The pollen grains were viewed with a laser scanning confocal microscope Zeiss LSM710 system, and images were collected by UltraView spinningdisc confocal scanner unit (PerkinElmer). The excitation wavelength of 359 nm and the emission wavelength of 461 nm were used for DAPI fluorescent signals analysis. However, the excitation and detection wavelengths for GFP and RFP were 488 and 558 nm for excitation and 505-530 and 583 nm for detection, respectively. As the main purpose of this project was to explain the mechanism of cell fate determination, therefore we focused mainly on the isolation of mutants in which the normal expressions of male germline-specific markers are defected. After EMS treatment, we also sequenced the whole regions of constructs to ensure that defects in the normal expression of markers do not result from any possible mutation in the original constructs.

| PAPS1 mediates sperm cell differentiation
The expression of male gamete-specific MGH3 promoter in VN and male gamete-nonspecific ACT11 promoter in SCs were strongly in wild-type and paps1-4 mutant. Like MGH3, the expression pattern of both TIP5;1 and DAW1 was also defective in paps1-4 as compared to wild type (Figure 4a,b). In paps1-4, the promoter activities of male gamete-specific genes MGH3, TIP5;1, and DAW1 were decreased in SCs but increased in VN (Figure 4c). However, the opposite case was found for male gamete-nonspecific gene ACT11, and the activity of its promoter was increased in SCs but decreased in VN in paps1-4 mutant. These data show that the activity of PAPS1 is essential for male gamete differentiation in A. thaliana.

| DISCUSSION
The abnormality in paps1-4 pollen developmental process appeared in tricellular stage, soon after pollen mitosis II (PMII). In control wildtype plant, when the pollen is at bicellular stage, the GC expresses both green and red markers, while the VN does not express any of them. Soon after PMII, the green marker is still expressed in the resulting sperm cells; however, the expression of red marker is . Promoter activity of selected male germline-specific genes MGH3, TIP5;1 and DAW1 and male germline-nonspecific gene ACT11. GFP signal was recorded in single insert hemizygous lines in the paps1/+ background. The promoters of male germline-specific genes show reduced activity in SCs while significantly increased activity in VN of paps1-4 compared with wild-type pollen grains. Each bar indicates the mean of three independent lines, and error bars display the SE (c). The DAPI fluorescence in qrt paps1-4 plant showing two wild-type tricellular pollen grains and two mutant-type smaller bicellular pollen grains with delayed cell division (d). Bar scale 10 μm regulon, an important activity to change the expression shift of proteins essential for gamete specificity and cell identity.
The importance of interaction among the cells of a pollen required for mediating SC differentiation is clarified by the presence of a cytoplasmatic bridge connecting the SCs as well as their positioning inside the VC of a pollen in tobacco and other species (Yu & Russell, 1993). Furthermore, small RNAs primarily originated from the VC were identified in SCs of Arabidopsis (Slotkin et al., 2009). Moreover, the promoter of ABA-hypersensitive germination3 (AHG3) is transcriptionally active in the VC, whereas a translational fusion protein, AHG3-GFP, driven by the same AHG3 promoter, was localized in SCs. These different localizations suggest that AHG3 transcripts or the AHG3 protein could move from the VC to SCs. These data show that both VC and SCs are linked and the transport of proteins between them is highly regulated.
The T-DNA insertion mutant paps1-3 was previously reported to exhibit similar phenotype of male gametophytic defect, as the mutant allele could not be transmitted through the pollen in reciprocal crosses (Vi et al., 2013). Furthermore, paps1-3 mutant was previously found to show considerably reduced relative expression levels of the key genes for pollen differentiation like the LEUCINE-RICH REPEAT/ EXTENSIN 10 receptor kinase or SKU5 SIMILAR 13 (SKS13) and SKS14 (Zhang et al., 2019); however, a proper causal link of male gametophyte defect with SC differentiation was not established.
Here, we found that paps1-4 mutant disturbs both male germlinespecific and germline-nonspecific peptides and hence disrupts the whole regulating mechanism needed for SC differentiation. The undifferentiated SCs leading male gametophyte defective phenotype and the resulting pollen grains fail to germinate.
Like previously described paps1-1 and paps1-3 mutant phenotypes (Vi et al., 2013;Zhang et al., 2019), paps1-4 mutant also exhibited opposite effects on flower and leaf growth, with larger flowers and smaller leaves. However, we also found another interesting phenotype of smaller pollen with inhibited pollen maturation.
These opposite effects of PAPS1 on pollen (reduced growth), flower (increased growth), and leaf (reduced growth) indicate the different identities of these organs that determine a role for PAPS1 in the vital connection between growth pattern and organ identity. Previous study shows that it did not result in a global loss of poly(A) tails when PAPS1 activity was severely compromised in paps1-1 seedlings grown at high temperatures (Vi et al., 2013), suggesting that PAPS1 influences the regulation of only specific peptides required for SC differentiation. Our results also confirm that loss-of-function mutant paps1-4 does not bring a global loss but merely influences the upregulation of male germline specific proteins while the downregulation of male germline-nonspecific peptides required for differentiation process.