PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP’s activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

The delivery of nonmotile sperm to the embryo sac via a pollen tube is a key innovation that allowed flowering plants to carry out sexual reproduction without the need for water (Friedman, 1993; Lord and Russell, 2002). Both the speed and signal responsiveness of pollen tube growth are critical for successful fertilization (Johnson et al., 2019). The typical shape of a growing pollen tube cell protruding from a pollen grain is a cylinder with a dome-shaped tip (Geitmann, 2010). Maintaining such a typical tube shape during pollen tube growth is fundamental to support its ability for fast growth (Michard et al., 2017), and a plasticity range of tubular growth rates allows a pollen tube to optimize directional growth along its journey from the stigma to the ovule (Luo et al., 2017). The pollen tube cell extends mainly by tip growth, requiring huge amounts of secretion/exocytosis at the tip (McKenna et al., 2009; Grebnev et al., 2017). The newly secreted cell wall at the tip is mainly composed of esterified pectin, which is expandable, whereas cell wall remodeling at the lateral region (including pectin deesterification and callose deposition) limits expansion (Grebnev et al., 2017). The tip width of a growing pollen tube actually reflects the size of the secretion zone capped by an expandable membrane and cell wall, as a collective result of multiple pollen tube growth machineries (Luo et al., 2017).The tip-localized exocytosis of a growing pollen tube is supported by a spatiotemporal tightly controlled actin cytoskeleton network (Hepler, 2016). The actin cytoskeleton configuration in a pollen tube includes highly dynamic fine actin filaments in the apical and subapical regions and parallel longitudinal actin bundles in the shank region (Qu et al., 2017). Various actin-binding proteins, such as actin nucleation factors, actin-severing proteins, and actin-bundling factors, are responsible for organizing the dynamic actin cytoskeleton network (Ren and Xiang, 2007). For example, the actin-bundling proteins fimbrin and LIM (Lin-1, isl1, Mec3) domain-containing proteins function in shank-localized actin bundles in pollen tubes (Zhang et al., 2019). For another example, the actin nucleator formin (formin3 in Arabidopsis [Arabidopsis thaliana] and formin1 in lily [Lilium longiflorum]) functions in actin polymerization in the pollen tube tip (Li et al., 2017; Lan et al., 2018). The branched actin nucleator Actin-Related Protein2/3 (ARP2/3) complex is an evolutionarily conserved, seven-subunit complex consisting of the actin-related proteins ARP2 and ARP3 (Machesky et al., 1994). The ARP2/3 complex initiates the formation of branches on the side of preexisting actin filaments, locally creating a force-generating branched actin network that underlies cellular protrusion and movement (Blanchoin et al., 2000; Amann and Pollard, 2001; Molinie and Gautreau, 2018). The phenotypes of mutants in ARP2/3 in the moss Physcomitrella patens (Harries et al., 2005; Perroud and Quatrano, 2006), in Arabidopsis (Le et al., 2003; Li et al., 2003; Mathur et al., 2003; Brembu et al., 2004; Deeks et al., 2004), in maize (Zea mays; Frank and Smith, 2002), and in tomato (Solanum lycopersicum; Chang et al., 2019) demonstrated the broad importance of the ARP2/3 complex and its activation during cellular morphogenesis, including tip-growing cells. Perhaps surprisingly, in Arabidopsis, null ARP2/3 alleles are transmitted normally through pollen and there is no obvious root hair phenotype (Le et al., 2003; Djakovic et al., 2006).These cell growth machineries are tightly coordinated by multiple signaling pathways, including membrane-localized receptor kinases and Rho-of-Plants (ROP) GTPases (Li et al., 2018). The tomato pollen-specific and membrane-localized receptor kinases LePRK1 and LePRK2 mediate signaling during pollen tube growth (Muschietti et al., 1998). LePRK2 perceives several extracellular growth-stimulating factors, including a Cys-rich extracellular protein (Late-Anther-Specific52 [LAT52]), a Leu-rich repeat protein from pollen, and two pistil/stigma molecules, Style Interactor for LePRKs and Stigma-Specific Protein1 (Tang et al., 2002, 2004; Wengier et al., 2003, 2010), which increase the speed of pollen tube growth (Zhang et al., 2008b; Huang et al., 2014). LePRK2 antisense and RNA interference (RNAi) pollen tubes grow slower (Zhang et al., 2008b), consistent with a positive role for LePRK2 in regulating the speed of pollen tube growth. LePRK1 binds LePRK2 (Wengier et al., 2003), but LePRK1 plays a negative role in pollen tube growth by controlling a switch from a fast tubular mode to a slow blebbing mode (Gui et al., 2014). LePRK1 RNAi pollen tubes burst more often than wild-type pollen tubes, implicating a role for LePRK1 in maintaining plasma membrane integrity (Gui et al., 2014). An Arabidopsis paralog of these LePRKs, PRK6, also localized on the tip membrane, perceives Arabidopsis attraction cues from the female, AtLURE1s, to guide pollen tube growth (Takeuchi and Higashiyama, 2016; Zhang et al., 2017).Rho family small guanine nucleotide-binding proteins called ROPs or RACs, which can switch between a GDP-bound inactive form and a GTP-bound active form, are regulators of polar growth in pollen tubes (Cheung and Wu, 2008; Yang, 2008). In Arabidopsis, ROP1-dependent signaling controls tip growth. Active ROP1 defines a cap region in the apical plasma membrane as an exocytosis zone (Luo et al., 2017). Overexpression of ROP1 or of a constitutively active version resulted in pollen tube tip swelling (i.e. increased tip width) and slower growth (i.e. reduced tube length), while overexpressing a dominant negative version of ROP1 inhibited pollen tube growth (i.e. shorter but normal width tubes). The size of the pollen tube tip reflects the aggregate activity of membrane-associated ROP at the tip (McKenna et al., 2009; Luo et al., 2017). Tomato ROPs have been reported to be associated with the LePRK1-LePRK2 complex (Wengier et al., 2003) and therefore presumably play similar roles as the Arabidopsis homologs in pollen tube growth, yet their biological roles have not been directly investigated.Guanine nucleotide exchange factors (GEFs) activate ROPs by promoting the conversion of ROP/RAC GTPases from a GDP-bound inactive form to a GTP-bound active form. Plants possess a plant-specific ROPGEF family whose members contain a highly conserved GEF catalytic domain, the PRONE (plant-specific ROP nucleotide exchanger) domain (Berken et al., 2005; Gu et al., 2006). The intracellular portions of LePRK1 and LePRK2 interact with Kinase Partner Protein (KPP; Kaothien et al., 2005), whose Arabidopsis homologs were later shown to belong to the PRONE-type ROPGEF family (Berken et al., 2005; Gu et al., 2006). Pollen tubes overexpressing nearly full-length KPP (missing eight amino acids at the N terminus) developed swollen tips with abnormal cytoplasmic streaming and F-actin arrangements (Kaothien et al., 2005). An Arabidopsis homolog of receptor kinase, AtPRK2a (also named AtPRK2), interacts with AtROPGEF12 (Zhang and McCormick, 2007) and with AtROPGEF1 (Chang et al., 2013) to affect ROP activity. Based on the in vitro catalytic activity of full-length and truncated AtROPGEF1, an autoinhibition conferred by the C-terminal variable region was proposed (Gu et al., 2006). AtROPGEF12 was also shown to interact with the guidance receptor kinase PRK6 (Takeuchi and Higashiyama, 2016).Increased expression of full-length KPP increased the speed of pollen tube growth without significantly affecting pollen tube shape. We show biochemically that the PRONE domain of KPP does have ROPGEF activity on several class I ROPs, with highest activity on ROP4. The N-terminal domain of KPP inhibits its own GEF activity, while its C-terminal domain enhances its own GEF activity. The C-terminal domain of KPP is also required for its interactions with LePRK1, LePRK2, and an actin-bundling protein, Pollen-expressed LIM2a (PLIM2a), while the C-terminal domain alone is sufficient to bind LePRK1 but insufficient to bind LePRK2. Furthermore, self-associated KPP colocalized with the actin nucleation proteins ARP2/3 complex during pollen tube growth and enriched the membrane localization of ARP2/3 in the pollen tube. Interfering with ARP2/3 activation by coexpressing a dominant negative version of ARP2 reduced the speed of pollen tube growth and alleviated the defects caused by the overexpression of truncated KPP. CK-666, a specific small molecule inhibitor of ARP2/3 activation, canceled the promotive effect of full-length KPP on the speed of pollen tube growth. These results indicate that during pollen germination and tube growth, KPP not only links pollen receptor kinase and ROP signaling but also links the actin network to the pollen tube plasma membrane, thereby directly affecting the cellular morphology and efficiency of pollen tube growth.