SWEET11 and 15 as key players in seed filling in rice

Jungil Yang*, Dangping Luo*, Bing Yang, Wolf B. Frommer and Joon-Seob Eom Institute for Molecular Physiology, Heinrich-Heine University Duesseldorf, 40225 Duesseldorf, Germany; Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany; Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA; Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011,


Introduction
Population growth is expected to lead to an increasing need for rice production, especially in Africa (Sharma, 2014). A key question is thus how to optimize yield potential. Rice grains are composed mainly of starch (over 90% in many cases; www.knowledgebank.irri.org/ricebreedingcourse/Grain_ quality.htm), which derives from imported soluble carbohydrates. These carbohydrates are produced and delivered from leaves (source tissues) to caryopses (sink tissues) via the phloem (Patrick, 1997;. With a concentration of c. 600 mM, sucrose is the major sugar translocated in rice phloem sap (Fukumorita & Chino, 1982). To fill the seeds, massive amounts of sucrose have to be imported into developing caryopses. Sucrose is unloaded from the phloem strands that enter the seed coat, from where sugars are transferred into the developing caryopsis to supply cells with nutrients, in particular sugars as sources of energy and as carbon skeletons for cell wall and starch biosynthesis (Oparka & Gates, 1984;Zhang et al., 2007).
In plants, cell-to-cell transport of sugars is thought to be mediated by apoplasmic (export from one cell by a plasma membrane transporter and subsequent import into the adjacent cell by another transport protein) or by symplasmic transfer via plasmodesmata. Other routes are conceivable, but evidence for vesicular transport processes is sparse (van den Broek et al., 1997). Two of the key processes for long-distance translocation are phloem loading and seed filling. In maize, SUT1 and SWEET13a,b,c are essential for efficient phloem loading (Slewinski et al., 2009;Bezrutczyk et al., 2018). The transport processes that ultimately lead to cell wall synthesis and storage product accumulation in seeds, in particular in cereal caryopses are not fully understood. To delineate sym-and apoplasmic pathways in rice caryopses, Oparka carried out a combination of ultrastructural and dye tracer studies (Oparka & Gates, 1981a,b, 1982, 1984. The rice caryopsis is supplied by three vascular bundles that pass through the pericarp. The dorsal vascular bundle is the major route for sugar delivery to the developing caryopsis (Oparka & Gates, 1981a;Krishnan & Dayanandan, 2003). Symplasmic connections were found from the parenchyma of the dorsal vascular bundle throughout the nucellar projection and the nucellar epidermis. By contrast, the apoplasmic connection between the nucellar epidermis and pericarp (inner integument) is blocked by a lipid barrier. Furthermore, nucellar epidermis and aleurone are symplasmically isolated, thus requiring transporters at the maternal/filial boundary for release from maternal tissues and subsequent uptake into filial tissues. Comprehensive analyses that included radiotracer analyses led Oparka to propose that sucrose, after unloading from the phloem, diffuses along the nucellar epidermis from where a yet-unknown set of sugar transporters transfers the sugars into the developing endosperm (Oparka & Gates, 1981b). Candidates for such transporters include two classes of plasma membrane sucrose transporters (SUTs and clade 3 SWEETs) (Aoki et al., 2003;Chen et al., 2012) and three classes of plasma membrane hexose transporters (MSTs (STPs), ERDs, and clade 1 and 2 SWEETs) (Toyofuku et al., 2000;Johnson & Thomas, 2007;Chen et al., 2015a).
In legume seeds, during early developmental stages, cwINVs produce hexoses from the incoming sucrose. These hexoses are thought to stimulate mitotic activity to increase cell number, whereas subsequently at later stages the cwINVs are switched off and sucrose transporters are induced. Sucrose is thought to then act as a differentiation signal that triggers storage product accumulation (Weber et al., 2005). Specialized sucrose facilitators (SUFs, members of the SUT family), PsSUF1, PsSUF4 and PvSUF1 contribute to sucrose efflux from seed coats of pea and common bean . In monocots, specifically in maize and rice, cwINVs also play important roles in seed filling (Cheng & Chourey, 1999;Wang et al., 2008a). Because sucrose, upon arrival in the caryopsis through the phloem of the dorsal vascular bundle, is partially hydrolyzed by cell wall invertases (cwINVs; in particular OsGIF1/OsCIN2) into glucose and fructose, one would predict the adjacent expression of hexose transporters (Wang et al., 2008a). Several hexose transporters possibly involved in import of cwINV-derived hexoses into the caryopsis or endosperm have been identified. Rice MST4 and MST6 are expressed in maternal tissues including the dorsal vascular bundle, nucellus including nucellar projection and nucellar epidermis, and the aleurone layer of the filial endosperm (Wang et al., 2007(Wang et al., , 2008b. However, sucrose transporters of the SUT family also have been identified as important for seed filling in rice (Hirose et al., 1997(Hirose et al., , 2010Furbank et al., 2001;Aoki et al., 2003;Scofield et al., 2007a,b;Eom et al., 2012;Reinders et al., 2012). OsSUT1 was found to be expressed in the aleurone of developing caryopses (Furbank et al., 2001;Hirose et al., 2002). OsNF-YB1, an aleurone-specific transcription factor, directly regulates OsSUT1, OsSUT3 and OsSUT4 (Bai et al., 2016). Notably, antisense inhibition of OsSUT1 caused seedfilling defects (Scofield et al., 2002). SWEETs are a class of seven transmembrane hexose and sucrose uniporters that function as oligomers (Chen et al., 2010;Xuan et al., 2013). It has been proposed that AtSWEET13 functions as a 'revolving door' mechanism to accelerate the transport efficacy (Feng & Frommer, 2015;Han et al., 2017;Latorraca et al., 2017). Roles of SWEETs include phloem loading and nectar secretion, and they have been shown to act as susceptibility factors for pathogen infections (Chen et al., 2010(Chen et al., , 2012Lin et al., 2014). ZmSWEET4c in maize is expressed in the basal endosperm transfer layer (BETL) and necessary for seed filling and BETL differentiation. The apparent orthologue OsSWEET4 also appears to have a role in grain filling, although its detailed cell specificity has yet to be determined (Sosso et al., 2015). Because we also found that several sucrose transporting SWEETs contribute to seed filling in Arabidopsis (Chen et al., 2015b), we speculated that one or several rice orthologues may play analogous roles in supplying sucrose to either the rice SUTs or cwINVs.
Here, we show that similar to the situation in legumes, the hexose transporter OsSWEET4 is predominantly expressed during early stages of caryopsis development, whereas OsSWEET11 and 15 mRNA levels gradually increased from early stages and accumulated at higher levels during later developmental stages. Expression was found in the ovular vascular trace, nuclear epidermis and endosperm. We show that ossweet11 single and ossweet11;15 double mutants show defects in endosperm development and filling. Our findings are supported by work performed in parallel, which also identified OsSWEET11 as critical for seed filling (Ma et al., 2017). We found that the phenotype of ossweet11;15 double mutants was more severe in having an empty seed phenotype, which was accompanied by accumulation of more starch in the pericarp. Together, these results indicate that OsSWEET11 and OsSWEET15 are necessary for sugar efflux from the maternal nuclear epidermis as well as efflux from the ovular vascular trace to the apoplasm and also may contribute to sucrose influx into the aleurone.

RNA isolation and transcript analyses
Total RNA was isolated using Spectrum TM Plant total RNA kits (Sigma) or the Trizol method (Invitrogen), and first strand cDNA was synthesized using the Quantitect reverse transcription kit (Qiagen). qRT-PCR was performed using a LightCycler 480 (Roche), with the 2 ÀDCt method for relative quantification (Livak & Schmittgen, 2001). Primers for OsSWEET11, OsSWEET15 and OsUBI1 (Takahashi et al., 2005) are listed in Table S1.
Generation of OsSWEET11 and OsSWEET15 reporter constructs The 2334-bp GUSplus (GUS, b-glucuronidase) coding sequence fused to the nopaline synthase terminator was amplified by PCR from pC1305.1 (Cambia, Canberra and Brisbane, Australia). The amplified fragment was subcloned into the pJET2.1/blunt vector (Thermo Fisher, Waltham, MA, USA) and sequencing was performed for validation. After SacI-EcoRI double restriction the resulting fragment was transferred into the plant transformation vector pC1300intC to generate a promoterless GUSplus vector. For tissue specificity analysis, a 4354-bp genomic fragment containing 2106 bp of the 5 0 upstream region and 2248 bp comprising the entire coding region of OsSWEET11, and a 4193-bp genomic fragment containing 2069 bp of the 5 0 upstream region and 2124 bp comprising the entire coding region of OsSWEET15 (without stop codon) was amplified by PCR using Kitaake genomic DNA as a template, respectively (primers: Table S1). The amplified product was subcloned into a pJET2.1/blunt vector and confirmed by sequencing. The cloned fragment digested with HindIII and BamHI for OsSWEET11, or XbaI and KpnI for OsSWEET15, was inserted in front of the GUSplus coding sequence (previously restricted with HindIII/BamHI and XbaI/ KpnI, respectively). The resulting pOsSWEET11:gOsSWEET11-GUSplus and pOsSWEET15:gOsSWEET15-GUSplus constructs were used to transform O. sativa japonica cv Kitaake. Eighteen and 13 independent lines were obtained for pOsSWEET11: gOsSWEET11-GUSplus and pOsSWEET15:gOsSWEET15-GUSplus, respectively, with similar expression patterns.

FRET sucrose sensor analysis in HEK293T cells
For functional analyses in HEK293T cells, OsSWEET11 and OsSWEET15 coding sequences were cloned into the Gateway Fig. 1 Relative expression of OsSWEET4, OsSWEET11 and OsSWEET15 during rice seed development after pollination. Expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in wild-type glasshouse-grown seeds. Data shown as mean AE SEM, n = 3; expression levels were normalized to rice Ubiquitin1 levels.

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New Phytologist entry vector pDONR221f1, then transferred into pcDNA3.2V5 by LR recombination reactions. HEK293T cells were cotransfected with a plasmid carrying the OsSWEET11 or OsSWEET15 and the sucrose sensor FLIPsuc90l-sCsA, using Lipofectamine 2000 (Invitrogen). For FRET imaging, HBSS medium with and without sucrose was used to perfuse FLIPsuc90l-sCsA-expressing HEK293T cells. Image acquisition and analysis were performed as described previously (Chen et al., 2012).

Identification of OsSWEET11 and OsSWEET15 in rice caryopses
The elongation of caryopses occurs in the first 6 DAP, whereas width and thickness expand mainly between 4 and 12 DAP (Xu et al., 2008;Wu et al., 2016b). To identify SWEETs expressed specifically in rice caryopses, we analysed public microarray data from RiceXPro (ricexpro.dna.affrc.go.jp). Among the five clade 3 SWEETs analysed, OsSWEET11 and 15 had the highest mRNA levels in the endosperm between 7 and 14 DAP ( Fig S2). To validate the microarray data, we harvested immature seeds at different developmental stages from glasshouse-grown plants and reanalysed mRNA levels of OsSWEET11 and 15 by quantitative reverse transcription polymerase chain reaction (qRT-PCR). For comparison, we used OsSWEET4, which had been shown to play a role as a hexose transporter in seed development (Sosso et al., 2015; Fig. 1). At 1 DAP, OsSWEET4 mRNA levels were at a maximum, and levels declined c. three-fold at 3 DAP. In comparison, OsSWEET11 was low at 1 DAP, but equal to OsSWEET4 at 3 DAP. OsSWEET11 gradually increased throughout development. Although the microarray showed that OsSWEET15 was only 2-39 lower compared to OsSWEET11, our analysis indicated a much lower relative level. Nevertheless, the developmental pattern of OsSWEET15 mRNA levels was similar to that of OsSWEET11. Although the c. two-fold increase in OsSWEET11 mRNA levels between 9 and 11 DAP coincides with the onset of rapid biomass accumulation, substantial mRNA levels were also detected at both earlier and later stages, possibly implicating OsSWEET11 and 15 in both rapid size expansion of the caryopses and in processes before and after.

OsSWEET11 plays a key role in seed filling
OsSWEET11 had previously been shown to function as a plasma membrane sucrose transporter (Chen et al., 2012). Because OsSWEET11 was by far the most highly expressed clade 3 SWEET gene in the caryopsis, we hypothesized that knockout mutants might be affected in seed filling. Two independent ossweet11 mutants, one carrying a single base pair deletion that led to a frameshift was created by CRISPR-Cas9, together with a second TALEN-derived mutant carrying a 489-bp deletion were characterized phenotypically (Fig. S1). Under glasshouse conditions, both mutants had incompletely filled seeds at the time the WT had reached maturity (Fig. 2). At 40 DAP, WT panicles were fully mature with brown spikelets, whereas ossweet11 mutants had immature panicles that still contained chlorophyll (Fig. 2c,d). Full maturity of the ossweet11 mutants, when there was complete loss of chlorophyll, was reached only much later (> 60 DAP). Even after the extended maturation period, mutants showed significantly reduced yield (both percentage of mature seeds after harvest and 1000-grain weight; Fig. S3a,b). Depending on the growth conditions, the phenotype was more or less severe (Figs 2, S3); defects became more severe in paddy field conditions (single field experiment in 2016; similar also as in the parallel study with multiple field trials; Ma et al., 2017). Of note, however, plant height, spikelet number and panicle length appeared unaffected in both glasshouse and paddy field conditions (Figs 2c, S3c,d).
OsSWEET11 in the nucellar projection, nucellar epidermis and aleurone Oparka had predicted symplasmic diffusion of sugar in the pericarp and apoplasmic transport at the nucellar epidermis-aleurone interface all around the endosperm. Notably, this pattern is different from that found in developing barley seeds, where the main import route was through the nucellar projection (Oparka & Gates, 1984;Melkus et al., 2011). To determine whether OsSWEET11 exports sucrose at the nucellar projection or the nucellar epidermis/aleurone interface, we analysed transgenic rice plants expressing translational GUS fusions containing a 2-kb promoter fragment and the whole coding region including all introns. Crude histochemical GUS analysis of caryopses showed comparable GUS staining in seeds in eight of 18 independent transformants. Two independent lines were used for a more detailed analysis. In early stages (up to 3 DAP), we observde GUS activity in maternal tissues including the ovular vascular trace and the nucellus, possibly indicating a role in remobilization of carbohydrates during nucellar degradation (Fig. S4). At 5 DAP, GUS activity was detected in the ovular vascular trace, the nucellar projection, the nucellar epidermis surrounding the developing endosperm, the remaining nucellar layers and also in the aleurone layer of the endosperm (Fig. 3a-c). To our surprise, we found OsSWEET11 expression in nucellar projection as well as the nucellar epidermis, and in addition also in the outermost endosperm cell layer, the aleurone, providing a potential path for sucrose export out of the nucellar projection into the endosperm, and a parallel pathway for export from the circumferential nucellar epidermis and then subsequently a potential import via OsSWEET11 into the aleurone. A parallel study obtained similar expression patterns using a transcriptional GUS fusion (Ma et al., 2017).

Potential compensation for ossweet11 deficiency by other SWEETs
The relatively weak phenotype of the ossweet11 mutants may either indicate the existence of alternative genes and pathways, or indicate compensation. Analysis of the expression levels of clade 3 SWEET genes in the ossweet11 mutant showed that OsSWEET13 mRNA levels were slightly increased, but that the absolute levels were extremely low. The mRNA levels of OsSWEET15 were c. two-fold higher in ossweet11 seeds compared to the WT (Fig. 4). Because, depending on the experiment, OsSWEET15 was expressed at only slightly lower levels compared to OsSWEET11 and was furthermore candidate that may contribute to compensation in the mutant, we first tested whether it functions as a sucrose transporter, determined its expression pattern in developing caryopses and then analysed knockout mutants. As one may have predicted, OsSWEET15 also functioned as a sucrose transporter when co-expressed with a sucrose sensor in HEK293T cells (Fig. S5). Translational GUS fusions of the OsSWEET15 gene driven by their native promoter showed similar tissue specificity as compared to OsSWEET11 (13 independent lines): GUS activity was detected at early stages in all regions of the nucellus, and later in the ovular vascular trace, the nucellar projection and the aleurone (Fig. 3d,e). At 9 DAP, OsSWEET15 GUS activity was also detected in the nucellar epidermis (Fig. 3f). The two SWEET transporters exhibit similar expression patterns in developing seeds, especially the ovular vascular and the interface between the nucellar epidermis and the aleurone, intimating redundant roles during seed development. However, on their own, two independent ossweet15 knockout mutants generated via CRISPR-Cas9 (frameshift mutations that prevent production of a functional OsSWEET15 protein; Fig. S1) did not show any detectable phenotypic differences compared to WT in four independent experiments (Fig. 2b).

OsSWEET11 and 15 are essential for seed filling
Because the seed filling of ossweet11 mutants was only partially affected relative to ossweet4 mutants (Sosso et al., 2015), and OsSWEET15 appeared to be expressed in the same cell types to substantial levels, and even possibly compensates in part for OsSWEET11 deficiency in the mutant, we generated ossweet11;15 double mutants for both alleles of the two loci. In glasshouse conditions both at ISU and Stanford, the double mutant phenotype was very severe, much more than in the single ossweet11 mutants (Fig. 5). The differences were even more severe in the ISU glasshouses, where ossweet11 showed only a minor phenotype, whereas the caryopses of the double mutant were severely affected (Fig. S6). A detailed time series showed that phenotypic differences became apparent at c. 5 DAP (Fig. 5a). Differences became bigger at 7 DAP, a time point at which ossweet11 mutants had already started to develop a wrinkled grain morphology, whereas ossweet11;15 was characterized by grains that were flattened with a smaller diameter (Fig. 5a,b). Sections through the grain showed that the mutants were endosperm-deficient and either had only remnants of the endosperm or had lost the endosperm completely (Fig. 5c). Histological analyses of resinembedded sections showed that the endosperm developed at c. 3 DAP in both WT and ossweet11, whereas a functional endosperm did not form in the ossweet11;15 mutant (Fig. S7). The cellularization of the endosperm was completed and nucellar

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New Phytologist cells (adjacent to the nucellar epidermis) had degenerated in the WT c. 5-7 DAP (Fig. S7a) (Wu et al., 2016a). In the ossweet11 mutant, the endosperm was defective and degeneration of the nucellar cells was delayed by c. 2-3 d (Fig. S7b). OsSWEET11 and 15 are both essential for seed filling as evidenced by a poorly developed endosperm and a defect in nucellar degeneration (Fig. S7c).
Starch accumulation in the pericarp of ossweet11;15 double mutants Based on the localization of OsSWEET11 and 15, we predicted that inhibition of the transporters would lead to starch accumulation in cells that export sucrose and cells peripheral to the endosperm. In the WT, starch is stored transiently in the pericarp until 7-9 DAP. Starch degradation in the pericarp correlated with starch accumulation in the endosperm starting c. 7 DAP (Wu et al., 2016b). As expected, we also found starch in the endosperm, as well as residual amounts in the pericarp of WT caryopses (Fig. 6a,d). By contrast, starch accumulated to high levels in the pericarp of the ossweet11 mutant (Fig. 6b,e). In ossweet11;15, starch accumulated to even higher concentrations in the pericarp, whereas the endosperm did not show substantial starch grains (Fig. 6c,f). The accumulation of starch in the pericarp of ossweet11 and ossweet11;15 double mutants supports the critical roles of OsSWEET11 and OsSWEET15 in sucrose translocation and mobilization towards the developing endosperm.

Discussion
We draw five key conclusions from the results of a combination of analyses comprising gene expression, translational reporters to map tissue-specific protein accumulation, and knockout mutants (2) if we assume that they mark apoplasmic import routes, sucrose can enter both directly below the vein via the nucellar projection as well as via the circumferential nucellar epidermis; (3) SWEET11 and 15 may not only play roles in cellular efflux at these two sites, but may also be responsible for importing sucrose into the aleurone; (4) OsSWEET11 and 15 both contribute to seed filling with seemingly redundant roles. Because the single ossweet15 mutants had no apparent phenotypic differences to wild-type under the tested conditions, OsSWEET11 appears to function as the dominant transporter, consistent with having higher relative levels of mRNA. (5) Because OsSWEET4 is predominantly expressed at early stages of development, it may cooperate with cell wall invertase cwINV2 (OsCIN2, GIF1) in hexose import into cells surrounding the dorsal vein, whereas OsSWEET11/15 may play more important roles at later stages. The findings made here for OsSWEET11 in Oryza sativa cv Kitaake are similar to those from a parallel study that used O. sativa cv Nipponbare (Ma et al., 2017). Notably, the phenotype of the ossweet11 mutant was more severe in field vs glasshouse conditions.

Pathways for seed filling
The tissue-specific expression of OsSWEET11 and 15 in parenchymatic cells of the vascular bundle, the nucellar projection, the nucellar epidermis and the aleurone indicates specific roles of OsSWEET11 and 15 in sucrose translocation into developing caryopses. Here we propose a possible model for sucrose translocation from the vascular bundle to the endosperm (Fig. 7). We propose four sites where sucrose transport occurs: (1) vascular parenchyma cells in the vascular bundle: SWEET effluxers may be required to supply OsCIN2 with sucrose (Wang et al., 2008a). Of note, the oscin2/gif1 cwINV mutant has a clearly distinct phenotype with markedly greater grain chalkiness and is thus not similar to ossweet11 (Wang et al., 2008a).
(2) Nucellar epidermis: SWEET expression in the nucellar epidermis is compatible with Oparka's anlyses which indicated that there is no symplasmic pathway to the filial tissues, thus requiring sugar transporters at the nucellar epidermis (Oparka & Gates, 1981a,b, 1982, 1984. (3) Aleurone: an unexpected location for SWEET-mediated efflux because the aleurone likely requires sucrose influx. Because SWEETs appear to function as uniporters, a sucrose gradient across nucellar epidermis and aleurone, driven by a high rate of delivery from the maternal side and rapid conversion in the endosperm, would allow the use of the same uniporters on both cell types. This situation is remotely similar to the human intestine, where transcellular transport across the intestinal epithelia is mediated by GLUT2 on both the apical and basal membrane under conditions where the glucose concentrations in the lumen exceed those of the blood stream (Kellett et al., 2008). In addition to the two SWEETs, SUT sucrose transporters, which are expressed in the aleurone, may contribute to secondary active sucrose import into the aleurone (Scofield et al., 2002;Bai et al., 2016). (4) Nucellar projection: the presence of OsSWEET11 and 15 in the nucellar projection may appear as the most surprising site, because plasma membrane sucrose transport is not in line with radiotracer import studies, which had indicated that in rice, the import of sugars occurs exclusively via The model is made based on observations from this and previous studies (Oparka & Gates, 1981a,b;Wu et al., 2016a). Sucrose may move from the phloem to parenchyma cells in the ovular vascular bundle and then to the nucellar projection and nucellar epidermis through symplasmic pathways via plasmodesmata (a). We surmise that OsSWEET11 and OsSWEET15 mediate sucrose export from vascular parenchyma into the apoplasmic space (b). In addition, OsSWEET11 and OsSWEEET15 may be involved in sucrose export from cells belonging to the nucellar projection and the nucellar epidermis to the apoplasm, followed by import into the aleurone (endosperm) (c). SWEET-mediated transfer of sucrose across the nucellar epidermis/aleurone interface would require a sucrose gradient across both cell types. al, aleurone; en, endosperm; ne, nucellar epidermis; np, nucellar projection; pa, parenchyma.

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New Phytologist the nucellar epidermis-aleurone pathway (Oparka & Gates, 1981b). However, others had suggested that the nucellar projection may contribute to sugar import into the developing endosperm (Krishnan & Dayanandan, 2003). Notably, the nucellar projection pathway appears to be the main pathway for sugar import in barley caryopses as shown by magnetic resonance imaging(MRI) (Melkus et al., 2011). We are aware that in contrast to Oparka's radiotracer studies, we did not measure the actual translocation of assimilates, but rather the presence of a transport protein, and we do not know whether the two SWEETs are active at the plasma membrane of these cells. Nevertheless, we suggest that it may be useful to reassess sugar entry pathways, for example by MRI at different stages and in different varieties. One possible cause for the discrepancy could be that Oparka used an indica rice variety (IR 2153-338-3), whereas the japonica variety Kitaake was used for all of the experiments described here; Nipponbare was used by the parallel study that localized a transcriptional GUS fusion of the OsSWEET11 promoter to the same cells as the translational fusions in our work (Ma et al., 2017). Alternatively, the two pathways may be used at different stages of development. Notably, the three Arabidopsis transporters SWEET11, 12 and 15 which play critical roles for sucrose efflux from the seed coat showed very complex changes in cellular expression during seed development (Chen et al., 2015b).

Starch in the pericarp as a buffer
During the development of the rice caryopsis, starch grains accumulated in pericarp at 6 days after pollination (DAP), which disappeared at c. 7-9 DAP (Wu et al., 2016b). Transient starch accumulation also had been observed in the pericarp of barley and wheat (Radchuk et al., 2009;Xiong et al., 2013). Starch accumulation in the pericarp was also observed in ossweet11 single and ossweet11;15 double mutants at 9 DAP (Fig. 6), but it remains to be determined whether this accumulation was caused by defects in sucrose import at the nucellar projection or at the nucellar epidermis-aleurone interface. Ma et al. (2017) also localized transcriptional reporter fusions of OsSWEET11 to the pigment strand close to the main vascular trace and found a severe seed-filling defect in field-grown plants. In our glasshouse experiments, the phenotypic effect of ossweet11 mutations was a lot less severe, in some cases even marginal, intimating a strong effect of the growth conditionspossibly light and nutritionon the phenotype. Our work indicates, based on the similarity in steady-state RNA levels, timing of mRNA accumulation, and tissue specificity and the combined effect observed in double knockout mutants, that OsSWEET15 partially compensates for OsSWEET11 deficiency.

Relevance for pathogen susceptibility
The finding that OsSWEET11 and 15 play important roles in seed filling also is relevant with respect to the role of OsSWEET11 in rice blight susceptibility (Yang et al., 2006;Antony et al., 2010;Chen et al., 2010;Yuan et al., 2010). Ectopic expression of OsSWEET11 is activated by pathovar-specific effectors of the blight pathogen Xanthomonas oryzae pv oryzae (Xoo). Mutations in the effector binding site of the OsSWEET11 promoter lead to resistance to Xoo (Yang et al., 2006;Antony et al., 2010;Yuan et al., 2010). It will therefore be important to ensure that genome editing of the promoter of OsSWEET11with the purpose of engineering resistancedoes not impact proper OsSWEET11 expression in seeds, to ensure that resistant lines do not carry a yield penalty. This goal appears feasible because apparently mutants (xa13) that are used by breeders do not show yield deficiencies (Laha et al., 2016).

Conclusions
The analysis of SWEET gene expression in rice caryopses together with the characterization of knockout mutants demonstrates that OsSWEET11 and 15 play central roles in seed filling. The cellular expression patterns of OsSWEET11 and 15 may indicate the presence of two parallel apoplasmic pathways for sugar entry into the endosperm. A careful analysis of the timing and localization of other sugar transporters of the SWEET, SUT and MST families. as well as the cell wall invertases, and an analysis of sucrose import by MRI in a variety of rice cultivars may help to further delineate the precise sugar import pathways and contribute to the knowledgebase for engineering improved yield potential in rice.        New Phytologist is an electronic (online-only) journal owned by the New Phytologist Trust, a not-for-profit organization dedicated to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews and Tansley insights.
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