Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development

Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development Chenyu Rong, Renren Zhang, Yuexin Liu, Zhongyuan Chang, Ziyu Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3801577/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Apr, 2024 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract The distribution of cytokinins in plant tissues determines plant growth and development and is regulated by several cytokinin transporters. Purine permease (PUP) is a cytokinin transporter found in plants. Although 13 PUP genes have been identified in the rice genome, however, most of their functions remain unknown. We found that pup11 mutants showed extremely low seed setting rates and a unique filled seed distribution. Our research revealed that pup11 mutants showed seed formation arrest because the accumulated starch disappeared 10 days after flowering. PUP11 has two major transcripts with different expression patterns and subcellular locations, and further studies revealed that they have redundant positive roles in regulating the seed setting rate. We also found that type-A RR genes were upregulated in the developing grains of the pup11 mutant compared with the wild type. The results also showed that PUP11 altered the expression of several sucrose transporters and significantly upregulated certain starch biosynthesis genes. In summary, our results indicate that PUP11 influences the rice seed setting rate by regulating sucrose transport and starch accumulation during grain filling. This research provides new insights into the relationship between cytokinins and seed development, which may help improve cereal yield. Rice PUP11 seed setting rate seed development cytokinin sugar Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key message Purine permease PUP11 is essential for rice seed development, regulates the seed setting rate, and may influence cytokinin content, sugar transport, and starch biosynthesis during grain development. INTRODUCTION Cytokinins (CKs), which can be divided into active forms (free base) and inactive forms (combined forms), are a series of plant hormones that contribute to root and shoot structure, nutrient absorption, senescence, and stress responses (Duran-Medina et al. 2017; Zurcher and Muller 2016). However, CK biosynthesis occurs in a limited number of organs, especially in the roots. Because the supply and demand of CKs in plant tissues are unbalanced, the process of CK transport from the biosynthesis site to other organs plays an important role in regulating plant growth and development (Wu et al. 2017; Zhang et al. 2023). Some combined forms of CKs, such as trans -zeatin riboside (tZR) and N 6-(Δ2-isopentenyl) adenine riboside (iPR), which are also known as storage forms because they adapt to long-distance transport, are precisely transported to other tissues, and transferred by certain enzymes to the free base form, where they completed their functions (Qi and Xiong 2013). Moreover, each type of CK has a different direction of transport in vascular tissue. In the xylem sap, the trans -zeatin (tZ) forms, especially tZR, are major CK forms, while in the phloem sap, N 6-(Δ2-isopentenyl) adenine (iP)- and cis -zeatin (cZ)-forms are predominant (Sakakibara 2021). Recently, several CK transporters with diverse functions have been identified, with some participating in the long-distance transport of special CKs, such as the A TP- b inding c assette G family proteins (ABCG) AtABCG14 and OsABCG18, which have the classic function of transporting tZ and tZR from root to shoot (Zhang et al. 2014; Zhao et al. 2019), and E quilibrative N ucleoside T ransporters (ENT), which participate in the long-distance transport of CKs with nucleosides (Hirose et al. 2005). Pu rine p ermease (PUP) transports CKs between vascular tissues/apoplasts and cells, thereby facilitating CK cell-to-cell movement (Zurcher et al. 2016). A recent study also found that the S ugars W ill E ventually be E xported T ransporter (SWEET) HvSWEET11b can also transport tZ and tZR in grains across the maternal–filial boundary, which appears to represent cell-to-cell movement (Radchuk et al. 2023). AZA-GUANINE RESISTANT (AZG) also exhibits CK transport activity between cell organs (Tessi et al. 2021). With the functional divergence of CK transporters, loss-of-function mutants show different phenotypes. Mutants that show disruptions in long-distance tZ transport from root to shoot, such as atabcg14 or osabcg18 mutants, showed smaller roots and shoots compared to the wild type because of the accumulation of CKs in roots and lack of CKs in shoots (Zhang et al. 2014; Zhao et al. 2019). In Arabidopsis, AtPUP14 downregulation reduced CK transport from the apoplast to cytosol, and the higher apoplast CK content led to ectopic CK responses; moreover, the mutants appeared to have more branches and flower primordia compared to the wild type (Zurcher et al. 2016). In contrast, the atpup7/8/21 triple mutant showed a narrower CK signaling pattern in the shoot apical meristem compared to the wild type (WT), and the rosette size was decreased (Hu et al. 2023). In rice, the overexpression of OsPUP1 reduces the tiller number, plant height, and panicle size (Xiao et al. 2020). Overexpression of OsPUP4 causes reduced shoot growth but larger grains, and ospup7 had phenotypes similar to those of an OsPUP4 overexpression line (Qi and Xiong 2013; Xiao et al. 2019). However, the functions of CK transporters remain poorly understood. Several factors can influence CK transport, such as exogenous plant hormones, nutrition, and stress. In rice, the expression of OsABCG18 is induced by exogenous CKs but inhibited by exogenous auxins (Zhao et al. 2019), and OsPUP7 is induced by exogenous CKs, abscisic acid, and drought stress (Qi and Xiong 2013). In addition, the CK content increases in the xylem and phloem sap when nitrogen is applied to Arabidopsis and rice, and this increased content implies enhanced CK transporter activity (Sakakibara 2021). The process of grain filling is important for the seed setting rate and 1000-grain weight of cereals. The sources of grain filling are photosynthesis from functional leaves after flowering and nonstructural carbohydrates in sink organs, such as the stem and sheath (Hu et al. 2022). Photosynthesis converts nonstructural carbohydrates into sucrose, and then SWEETs and sucrose transporters (SUTs) transport sucrose from the source to the grain through the phloem via transmembrane transport (Deng et al. 2021; Hu et al. 2022). CELLWALL INVERTASE 2 (CIN2) and monosaccharide transporters also participate in this process (Liu et al. 2022; Wang et al. 2008). Sucrose is transported to grains and synthesized into starch via enzymes such as sucrose synthase (SuSase), ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), and starch branching enzyme (SBE) (You et al. 2016). These processes lead to the completion of grain filling. Plant hormones have different functions during rice grain filling, and each shows fluctuating content during this process. For example, the CK content reaches a peak at an early stage and then decreases, whereas the auxin content increases at an early stage and reaches a peak approximately 10 days after pollination (Basunia and Nonhebel 2019; Liu et al. 2022). Endogenous CKs play important roles in regulating grain filling by promoting cell division in the early stages of grain filling and thus are involved in regulating the seed setting rate and grain size (Tsago et al. 2020). In this study, we investigated the function of PUP11 in rice and confirmed that the two PUP11 transcripts showed redundant positive roles in seed development, ultimately upregulating the seed setting rate. Furthermore, certain genes related to CK signaling, sucrose transport, and starch biosynthesis were differentially expressed in the developing grains of pup11 mutants compared with the Zhonghua 11 (ZH11) cultivar. Our study confirmed that PUP11 is necessary for seed development and provides insights for further research on rice grain development and cereal yield. MATERIALS AND METHODS Plant materials and growth conditions Oryza sativa L. ssp. japonica (cultivars Nipponbare (NIP) and ZH11) was the WT rice material chosen for this study. The mutants included a complete mutant of PUP11 from ZH11 and NIP and a special transcript mutant of PUP11 from NIP (Supplemental Fig. S1 A and S4A). Rice plants were grown in a field in Danyang, Jiangsu Province, China (31.907° N 119.466° E). We used 150 kg/hm 2 P 2 O 5 and 240 kg/hm 2 K 2 O as base fertilizers for the field experiment, and 200 kg/hm 2 nitrogen with a base fertilizer: tiller fertilizer: panicle fertilizer ratio of 1:1:2. Plasmid construction The mutants were constructed using CRISPR/Cas9 technology. Single guide RNA oligo targets were assigned to complete mutants or special transcript mutants in each cultivar using CRISPR/Cas9 technology, as previously described (Mao et al. 2013). The primers used for vector construction and genotyping are listed in Supplementary Table S5. To construct pPUP11-1::GUS and pPUP11-2::GUS plants, an upstream fragment of the encoding region of each transcript (> 3 kb) was amplified using PCR, and the resulting amplicon was excised with the corresponding restriction endonuclease and ligated into the pCAMBIA1300::GUS vector (Wang et al. 2020). Primers used for vector construction are listed in Supplementary Table S5. Additionally, the coding regions of the two transcripts of PUP11 were cloned into pCambia1300::35S::GFP (green fluorescent protein) or pCambia1300::35S::mCherry ( mCherry fluorescent protein ) plasmids to generate the plasmids p35S::PUP11-1-GFP , p35S::PUP11-2-GFP , p35S::PUP11-1-mCherry , and p35S::PUP11-2-mCherry for subcellular localization (Chang et al. 2021). Primers used for vector construction are listed in Supplementary Table S5. Analysis of GUS activity and subcellular localization analysis GUS reporter activity was assayed by histochemical staining using a GUS Staining Kit (Warbio, Nanjing, China, http://www.warbio.cn/ ). Various tissues were collected from the pOsPUP11-1::GUS or pOsPUP11-2::GUS transgenic plants at each developmental stage, after which they were immersed in the GUS staining solution and incubated for 6–48 h at 37°C in the dark. The samples were destained three times with 70% ethanol in a water bath for 5 min. Images were captured using a SZX16 microscope (Olympus, Tokyo, Japan; https://www.olympus-lifescience.com/en/ ). Each pOsPUP11-1::GUS or pOsPUP11-2::GUS transgenic plant had 20 independent lines, from which two or three independent lines were chosen and stained at the same position as the other lines for further studies. For the analysis of subcellular localization, p35S::PUP11-1-GFP and p35S::PUP11-2-GFP were transformed using Agrobacterium tumefaciens strain EHA105 and inoculated into 4-week-old tobacco ( Nicotiana benthamiana ) leaves, as previously described (Liu et al. 2022). GFP fluorescence was detected using a LSM800 confocal laser microscope (Zeiss, Oberkochen, Germany; https://www.zeiss.com.cn ). Histological analysis For paraffin sectioning, samples were fixed overnight at 4°C in FAA (formalin:glacial acetic acid:70% ethanol; 1:1:18) and dehydrated using a graded ethanol series. Following substitution with xylene, the samples were embedded in paraffin, sectioned into 20 µm sections using a rotary microtome, dewaxed, stained with I 2 -KI (0.33%I 2 and 0.67%KI), incubated for 5 min, and then observed using a microscope (Olympus, SZX16) (Chang et al. 2021). Sampling, RNA extraction, and gene expression analysis Developing seeds were collected 6 and 10 d after flowering from the ZH11 and pup11-1 mutants. Plant tissues were collected at different developmental stages. Total RNA was extracted using an E.Z.N.A. Plant RNA Kit (Omega Biotek Inc., Norcross, GA, USA; https://www.omegabiotek.com/ ) and subjected to reverse transcription using a PrimeScript RT Reagent Kit (Takara Biotechnology, Tokyo, Japan; https://www.takarabio.com/ ). Quantitative reverse-transcription PCR was performed on an ABI PRISM 7300 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA; https://www.thermofisher.com/ ) with SYBR Premix Ex Taq (Takara), following the manufacturer’s instructions. Relative expression analysis was performed using the UBQ gene as an internal control. The primers used are listed in Supplementary Table S5. Floral organ observation and Pollen viability assay The floral organs and grains were subsequently observed under an SZX16 microscope (Olympus, Tokyo, Japan), and more than 20 spikelets were observed for each material. We used I 2 -KI staining to determine the pollen viability of the mutant and WT (Xun et al. 2022). Pollen grains of the dehiscent anthers were placed on glass slides with I 2 -KI and incubated for 5 min. The experiments were performed in quintics for each sample. ACCESSION NUMBERS Sequence data from this article can be found in Rice Annotation Project database ( http://rice.plantbiology.msu.edu ) under the following accession numbers: Ubq (Os03g0234200), OsPUP11 (Os02g0689200), OsRR1 (Os04g0442300), OsRR2 (Os02g0557800), OsRR3 (Os02g0830200), OsRR4 (Os01g0952500), OsRR6 (Os04g0673300), OsRR9 (Os11g0143300), OsSUT1 (Os03g0170900), OsSUT2 (Os12g0641400), OsSUT4 (Os02g0827200), OsSWEET11 (Os08g0535200), OsSWEET15 (Os02g0513100), OsSSSI (Os06g0160700), OsSSSII-3 (Os06g0229800), and OsSSSIII-2 (Os08g0191433) Results PUP11 had essential roles in regulating seed setting rate To determine the specific functions of OsPUP11, we first obtained two independent pup11 mutant lines using CRISPR/Cas9 technology on a ZH11 background. Genome sequencing confirmed pup11-1 had a 1499 bp deletion and two inversion insertions while pup11-2 had a 252 bp deletion in the coding region (Supplementary Fig. S1 A, B). Previous studies have confirmed that OsPUP11 has three transcripts, while the two major transcripts encode a protein with ten transmembrane regions (Qi and Xiong 2013). Protein sequencing showed that the pup11-2 mutant exhibited a deletion of 84 amino acids in both transcript protein sequences, the 3rd to 5th transmembrane regions of each transcript were damaged (Supplementary Fig. S1 C). First, we observed the agronomic phenotypes of pup11 mutants and found that the plant height of both pup11 lines was significantly reduced compared to that of ZH11(Fig. 1A, B; Supplemental Table S1 ). Further analysis of the length of each internode of the ZH11 and pup11 mutants showed that the two mutant lines had shorter 1st and 2nd top internodes compared to ZH11, and these differences influenced plant height (Fig. 1C, D). Moreover, the flag leaves of the two pup11 lines were significantly longer and wider than those of ZH11, and both lines had wider second and third top leaves (Supplemental Table S1 ). The tiller number of pup11 mutants was similar to that of ZH11 plants (Supplemental Table S1 ). Subsequently, we focused on the yield component factors. During the three-year-long field experiment, the most notable phenotype of pup11 mutants was the extremely low seed setting rate, which was below 25% and significantly lower than that of ZH11 (Fig. 2A-D). Obvious differences were not observed between the ZH11 and pup11 mutants in terms of panicle number, primary branches, and primary spikelets, and both pup11 mutant lines showed significantly increased secondary branches and secondary spikelets; therefore, they showed more spikelets per panicle (Supplemental Table S2). In addition, the 100-grain weights of pup11-1 and pup11-2 were lower than those of ZH11, because of these mutants showed reduced grain thickness (Supplemental Fig.S2). Distribution of filled grains was changed in the pup11 mutant Observations of the phenotypes of the panicles also noticed that compared to ZH11, both pup11 mutant lines showed a special distribution of filled grains. First, we found that the seed setting rate of the primary spikelets of both pup11 mutant lines was very low (below 6%), which was significantly lower than that of ZH11 (Fig. 2E). Compared with the primary spikelets, the seed setting rate of the secondary spikelets of both pup11 mutant lines was higher than that of primary spikelets, and these differences were lower than those observed with ZH11 (Fig. 2F). Further analysis of yield contribution in ZH11 showed that the primary and secondary spikelets showed almost equal yield contributions; however, in both pup11 mutant lines, the yield was mainly from secondary spikelets (Supplemental Fig.S3A). We also noticed that in the pup11 mutants, the seed setting rate showed a decreasing trend from bottom to top. Therefore, we statistically analyzed the seed setting rates in the branches of the bottom 1/3, middle 1/3 and top 1/3 of the panicles. Although the seed setting rate in pup11 mutant was higher at the bottom, the value was lower than that of ZH11. In the top branches, the seed setting rate in pup11 was close to zero (Fig. 2, G-I). Because of this special distribution, spikelets in the bottom part showed a much higher yield contribution ratio in the pup11 mutants than in ZH11, whereas spikelets in the middle and top parts provided a much lower yield contribution in the pup11 mutants than in ZH11 (Supplemental Fig.S3B). OsPUP11 is necessary for grain development To reveal why pup11 mutants had extremely low seed setting rates, we first observed the structure of the flower organs. Both pup11 mutant lines had two glumes, two lodicules, six stamens, and one pistil with two stigmas, which were the same as in ZH11. Thus, PUP11 did not appear to influence the number of flower organs (Fig. 3A, B). We then detected pollen activity using the I 2 -KI method and found that the pollen viability of pup11 mutant was not significantly different from that of ZH11 (Fig. 3C, D). We then peeled the glumes of unfilled grains from pup11 mutants and found two mutant lines that developed fertilized grains, but their development stopped before storage product accumulation (Fig. 3E). To precisely characterize the development process, we also observed the structure of the endosperm by paraffin sectioning using I 2 -KI staining. We found that starch accumulated in aleurone six days after flowering in both the ZH11 and pup11 mutants. Nine days after flowering, we found that starch accumulated in the endosperm of ZH11 but disappeared in the aleurone and endosperm of the pup11 mutant (Fig. 3F). Moreover, seed shape was irregular in the pup11 mutant. Expression pattern of two major OsPUP11 transcripts As described above, OsPUP11 has two major transcripts encoding two proteins with 24 amino acids at the N-terminus. Although these two proteins share large sequence similarities, their expression patterns differ. First, we detected the spatial expression of both transcripts using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). In general, the expression of PUP11-2 was lower than PUP11-1 (Fig. 4A, B). While PUP11-1 showed special expression in the 0–1 mm young panicles and stems, PUP11-2 showed special expression in the 1–5 cm young panicles. To better understand the tissue distribution of these two transcripts, we constructed p PUP11-1::GUS and p PUP11-2::GUS plants to express the β-glucuronidase (GUS) gene under the control of their promoters and terminators, respectively (Fig. 4C). In vegetative organs, we found that PUP11-1 was expressed in young and flag leaves while PUP11-2 was not detected in the leaves. Both transcripts were expressed in axillary buds, shoot bases, and stems, with extremely strong expression in the region near the nodes. However, neither transcript was detected in the roots. In young panicles, we found PUP11-2 expressed higher in the early stages of panicle differentiation, at which stage PUP11-1 expresses very low. During the booting stage, PUP11-1 was highly expressed at the bottom of the spikelet, stamen primordium, and young branches, whereas PUP11-2 expression was detected throughout the spikelet. Both transcripts were weakly expressed in mature glumes. At grain maturity, both PUP11-1 and PUP11-2 were specifically expressed in the region near the embryo, with little expression at the top of the grains. In the germinated seeds, we found PUP11-1 expressed in the region near the embryo, and young leaves were wrapped in the coleoptile; however, PUP11-2 was not expressed at this stage. To learn the subcellular location of the two transcripts, we constructed 35S::PUP11-1-GFP , 35S::PUP11-1-mCherry , 35S::PUP11-2-GFP , and 35S::PUP11-2-mCherry , to investigate the subcellular localization of both transcripts. All four plasmids were transferred into Agrobacterium. We used two combinations: one with a mixture of 35S::PUP11-1-mCherry and 35S::PUP11-2-GFP injected into a leaf, and the other injected a mixture of 35S::PUP11-2-mCherry and 3 5S::PUP11-1-GFP . Both combinations confirmed that PUP11-1 was mainly localized in the plasma membrane (PM), and PUP11-2 was mainly localized in the plasma membrane and endoplasmic reticulum (ER) (Fig. 4D). Two major PUP11 transcripts have redundant function in regulating seed setting rate Because the two major PUP11 transcripts have different expression patterns and subcellular locations, we speculated that the two transcripts may have different functions. Using the NIP background, we designed two single guide RNA, each of which can edit one transcript without influencing the other. Moreover, to confirm the phenotypes of the pup11 mutant, we constructed another pup11 mutant line with loss-of-function for all transcripts in the NIP background (Supplemental Fig.S4A). We obtained three mutant lines of the PUP11-1 transcript and two mutant lines of the PUP11-2 transcript. In addition, a heterozygous mutant of all PUP11 transcripts was constructed with a C insertion and a 12 bp deletion in both chromosomes (no chromatograms of the homozygous mutant; the mutant with a C insertion can be cut by NcoI, but that with the 12 bp deletion cannot, with further studies performed to recognize the genotype by enzyme cutting) (Supplemental Fig.S4A, B). The 12 bp deletion mutant changed 4 amino acids at the third outer membrane region in both protein sequences. We named the C insertion mutant pup11-3-3-C , 12 bp deletion mutant pup11-3-3-12 , and heterozygous mutant pup11-3-3-C/12 . First, we observed the vegetative organ phenotypes of all mutants. For pup11-3 homozygous, as the segregation ratio of the C insertion from heterozygotes was extremely low, some data were not statistically significant. The plant heights of the pup11-1 and pup11-2 mutants were reduced (Fig. 5, A-B; Supplemental Table S3). For the tiller numbers, just pup11-2-1 showed more tillers compared to WT (Supplemental Table S3). Furthermore, we found reduced flag leaf length in pup11-1-2 and pup11-1-3 and reduced flag leaf width in all mutant lines except pup11-1-1 (Supplemental Table S3). After harvesting the panicles, we focused on the seed setting rate. As confirmed from two years of data, the loss-of-function of one PUP11 transcript had no effect on the seed setting rate, while pup11-3-3-C showed a very low seed setting rate because of seed development arrest, which is the same as the mutants in the ZH11 background (Fig. 5A-G). A lower seed setting rate was not observed in pup11-3-3-12 , which seemed the 4aa amino acids at the third outside membrane region had a slight effect on seed development (Fig. 5F). Moreover, we found a lower seed setting rate in pup11-3-C/12 heterozygotes, which may have been caused by the lack of development of homozygous seeds with the C insertion (Fig. 5F). We also observed other yield factors. In both years, pup11-3-3-C showed fewer spikelets per panicle, with fewer secondary branches and secondary spikelets compared to NIP, in contrast to the pup11 mutants in the ZH11 background (Supplemental Table S4). Damage to either the PUP11-1 or PUP11-2 transcripts showed no significant difference in panicle number, primary branches, and primary spikelets compared to the NIP, but reduced secondary branches, secondary spikelets, and total spikelets per panicle compared to the NIP (Supplemental Table S4). PUP11 influences the expression of genes related to cytokinin signaling and sugar metabolism Previous studies have shown that the PUP family genes function in transporting CKs. We suspected that in the grains of pup11 mutants, normal CK transport in grains was damaged, the CK content in grains was altered, and finally caused unusual grain development. Type A RR genes quickly respond to exogenous CKs and are used as marker genes to indicate CKs levels. Therefore, we measured the expression of type A RR genes in the developing grains of ZH11 and pup11-1 firstly. We found that at six days after flowering, when the phenotype of the grain did not show obvious changes, the expression of type-A RR genes did not change significantly in the pup11 mutants compared to ZH11, except RR4 (Fig. 6A). However, 10 days after flowering, we found that the expression of RR2 , RR4 , RR6 , and RR9 was significantly upregulated in the pup11 mutants compared to ZH11 (Fig. 6B). This may be due to changes in CK homeostasis in the pup11 mutants. Starch accumulation in the grains of pup11 was unusual. We suspected that the expression of some genes related to sucrose transport or starch synthesis in grains was altered in the pup11 mutants. Sucrose transport into grains is an important step in grain filling, as SUT1 , SWEET11 and SWEET15 have been reported to play essential roles in the process and influence grain filling (Deng et al. 2021). At both 6 and 10 days after flowering, the expression of SUT1 was significantly reduced in the pup11 mutant while the expression of SUT2 and SUT4 did not change or was slightly upregulated in the pup11 mutant (Fig. 7A). Compared with SUT s, the expression of SWEET11 and SWEET15 , which have sucrose efflux functions at the nucellar projection and play a role in sucrose transfer across the nucellar epidermis/aleurone interface, was upregulated in the pup11 mutant (Fig. 7B). Numerous studies have shown that many genes are involved in controlling starch synthesis from sucrose, including SSSI , SSSII-3 , and SSSIII-2 (You et al. 2016). Interestingly, we found that three SSS s genes are all downregulated in the pup11 mutant at both stages, which explains the low starch accumulation and grain formation arrest in the pup11 mutant (Fig. 7C). Discussion 1. Cytokinin transporters play different roles in regulating rice development CK transporters help adjust the CK content from the cellular organ level to the whole plant. An abnormal CK distribution in plant tissues results in different phenotypes. Recently, function of four CK transporters (ABCG18, PUP1, PUP4, and PUP7) were identified in rice. Although CK transporters in rice are poorly understood, studies have provided insights for plant breeding. Each known CK transporter in rice plays a special role throughout the rice life cycle. Although ABCG18 had a positive effect on tiller bud outgrowth, plant height, and panicle development (Zhao et al. 2019), PUP1 seems to have the opposite effect (Xiao et al. 2020). Smaller panicles with larger seeds were observed for the PUP4-overexpression line and pup7 mutant compared with the WT. However, we found that the two transcripts of PUP11 showed no obvious functions in regulating tillers, but each PUP11 transcript showed a positive role in regulating plant height (Supplemental Table S1 ; Supplemental Table S3). Previous studies have also found that several CK transporters, such as ABCG18 and PUP4, can influence rice seed setting rate (Xiao et al. 2019; Zhao et al. 2019). By observing pup11 mutant lines with the two transcripts, which showed damage under the two backgrounds, we found a severely reduced seed setting rate compared to the WT, which was caused by arrested seed development. We also found that the filled seed distribution of pup11 differed from that of ZH11 (Fig. 2H-M). This result suggests that PUP11 has special functions in seed development that are different from those of other known CK transporters. Overall, several CK transporters showed some advantages for yield as well as shortcomings. Only the ABCG18-overexpression lines showed a higher yield (Zhao et al. 2019). These results indicated the functional variations of CK transporters, which inspired us to explore the factors underlying the differences. 2. Potential causes of the functional differences in cytokinin transporters Each CK transporter exhibits a unique expression pattern. ABCG18 and ENT2 , which participate in long-distance CK transport, are mainly expressed in the vascular tissues around the whole plant (Hirose et al. 2005; Zhao et al. 2019). PUP family genes are also particularly expressed in vascular tissues; however, each PUP has a unique spatiotemporal expression (Qi and Xiong 2013; Xiao et al. 2019). In our study, the two PUP11 transcripts had different expression patterns. Moreover, the PUP11-1 transcript could be detected in most vegetative organs in the shoot while PUP11-2 could not be detected. However, the expression of both transcripts overlapped in the nodes and young flowers (Fig. 4A-C). In summary, CK transporters have unique expression patterns, influence the transport of CKs in different plant tissues and at different stages, and precisely regulate plant growth and development. CK transporters are primarily localized in the PM and ER. In our study, the two PUP11 transcripts encoding two proteins with different N-termini had different subcellular localizations and may function in different cell organs to precisely regulate cellular CK signaling (Fig. 4D). Therefore, we believe that the difference in cell organ localization is another factor affecting transporter function. Previous studies have also found that each CK transporter can only transport specific types of CKs. Although previous studies have found that the CK distribution is altered in the atpup7/8/21 triple mutant and OsPUP -overexpression lines (Hu et al. 2023; Qi and Xiong 2013; Xiao et al. 2019; Xiao et al. 2020), the CK type that is transported by PUP remains unknown. PUP transporters have different transport directions that may be determined by the amino acid sequence of PUP. In general, the expression pattern, subcellular location, substrate, and transport direction of the CK transporters provide them with unique functions. However, with the limited knowledge of cytokinin transporters recently, how these transporters regulate the CK content and distribution in rice remains unknown. 3. PUP11 influences cytokinin homeostasis and starch biosynthesis in grains Each type of plant hormone regulates grain development (Basunia and Nonhebel 2019), and the relationship between CK and grain filling is worthy of exploration. As summarized in previous studies, CKs have two directions for seed setting rates. Exogenous CK application can facilitate grain filling in large panicles (Chen et al. 2022; Panda et al. 2018). In contrast, several ckx mutants, such as ckx1 ckx2 , ckx3 , ckx4 ckx9 and ckx11 , have higher endogenous CK content, although their seed setting rate is lower than that of the WT (Huang et al. 2023; Rong et al. 2022; Zhang et al. 2021). Recent research has reported that HvSWEET11b can transport both sucrose and CKs and knocking down HvSWEET11b prevents the production of grains with normal vegetative, panicle, and pollen development (Radchuk et al. 2023), which is similar to the results for the ospup11 mutant. In HvSWEET11B-RNAi lines, tZR accumulates in several parts of the grains, such as the vascular region and nucellar projections (Radchuk et al. 2023). In our study, the grains of pup11 mutants also showed higher expression of type-A RR genes, which may imply a higher CK content (Fig. 6). But because of technological limitation, we can’t see cytokinin content in each part of developing seed. However, these results prompted us to determine the relationship between endogenous CKs and grain filling in cereals. Exogenous CK usage in the panicles helps improve soluble sugar content and starch biosynthesis in the inferior spikelets (Chen et al. 2022). However, few studies have focused on the molecular mechanisms by which endogenous CKs influence sugar transport or grain filling. In our study, we detected the expression of several sucrose transporters and starch biosynthesis genes in the grains of ZH11 and pup11 mutants. Each sucrose transporter has a special function in rice grain filling. SWEET11, SWEET14, and SWEET15 are involved in sucrose efflux at the nucleolar projections and transfer across the aleurone interface (Fei et al. 2021; Yang et al. 2018). SUT1/3/4 function in sucrose transport from the dorsal phloem to the filial aleurone. SUT1 plays a role in seed sucrose uptake, and the loss-of-function of SUT1 results in a low seed setting rate and grain weight (Deng et al. 2021; Scofield et al. 2002; Wang et al. 2021). In our study, we found extreme downregulation of SUT1 in developing grains of pup11 compared to ZH11, whereas SUT2 and SUT4 showed a slight upregulate at six days after flowering. This finding similar to that of a previous study and may be associated with the sugar transport strategy switch (Wang et al. 2021). The extremely high expression of SWEET11 and SWEET15 in the pup11 mutant may be influenced by unknown hunger signals from grains; however, sucrose transported by SWEET s cannot be transported into the endosperm. Interestingly, we found that PUP11 can positively influence the biosynthesis of starch, which is consistent with the disappearance of starch assimilation in grains of the pup11 mutant at 10 days after flowering (Fig. 3F). Except for the lower expression of SSS genes in the pup11 mutants, we also found that with grain development, SSS gene expression showed an increasing trend in the WT; however, the increasing trend was weaker in pup11 (Fig. 7). Whether the inhibition of starch biosynthesis genes was directly influenced by the loss-of-function of PUP11 or by low sugar levels in pup11 grains remains unknown. Thus, the mechanisms underlying the influence of PUP11 on sucrose transport and starch biosynthesis gene expression require further research. In conclusion, our study found that PUP11 influences several phenotypes during the rice life cycle, particularly by regulating seed development. Two major PUP11 transcripts with different expression patterns and subcellular locations showed redundant roles in seed-filling. Further studies showed that PUP11 influences CK content, sucrose transport, and starch accumulation in developing grains. Our findings establish a community resource for fully elucidating the function of PUP11 and revealed the relationship between exogenous CKs and the grain-filling process, thus providing new insights that may be used for future studies on the mechanisms underlying seed development and yield improvement. Declarations Funding: This work was supported by the Natural Science Foundation of Jiangsu Province (Grants No BK20231470). Data availability All data supporting the findings of this study are available within the paper and within its supplementary data published online. Acknowledgments We thank Jiankang Zhu and Caixia Gao for providing the vectors of the CRISPR-Cas9 system. We also thank Biogle and Biorun genome editing center for producing transgenic rice. Author contributions C.D. and Y.D. conceived the original screening and research plans; C.R., R.Z., Y.L., Z.C., Z.L., and C.D. performed the experiments and analyzed the data; C.R. and C.D. wrote the article with contributions of all the authors; C.D. agrees to serve as the author responsible for contact and ensures communication. Conflict of interest The authors declare no competing interests in relation to this work. References Basunia MA, Nonhebel HM (2019) Hormonal regulation of cereal endosperm development with a focus on rice (Oryza sativa). Funct Plant Biol 46:493-506 Chang Z, Xu R, Xun Q, Liu J, Zhong T, Ding Y, Ding C (2021) OsmiR164-targeted OsNAM, a boundary gene, plays important roles in rice leaf and panicle development. 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Journal of experimental botany 70:6277-6291 Zurcher E, Liu J, di Donato M, Geisler M, Muller B (2016) Plant development regulated by cytokinin sinks. Science 353:1027-1030 Zurcher E, Muller B (2016) Cytokinin Synthesis, Signaling, and Function--Advances and New Insights. International review of cell and molecular biology 324:1-38 Supplementary Files Supplementaldata.pdf Cite Share Download PDF Status: Published Journal Publication published 03 Apr, 2024 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Minor revisions 05 Feb, 2024 Reviewers agreed at journal 16 Jan, 2024 Reviewers invited by journal 01 Jan, 2024 Editor assigned by journal 25 Dec, 2023 First submitted to journal 24 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3801577","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264657009,"identity":"1d2ddd82-06b8-447d-8190-ce8fdc456bd2","order_by":0,"name":"Chenyu Rong","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chenyu","middleName":"","lastName":"Rong","suffix":""},{"id":264657010,"identity":"89f82220-1b26-490d-b48d-ff994675fc03","order_by":1,"name":"Renren Zhang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Renren","middleName":"","lastName":"Zhang","suffix":""},{"id":264657011,"identity":"8dbced2f-7aab-4886-889a-62d853fc7d3b","order_by":2,"name":"Yuexin Liu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuexin","middleName":"","lastName":"Liu","suffix":""},{"id":264657012,"identity":"d86697a2-864f-4e0c-ac18-b55c5f19515c","order_by":3,"name":"Zhongyuan Chang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhongyuan","middleName":"","lastName":"Chang","suffix":""},{"id":264657013,"identity":"46e43fea-d59d-4346-8ea8-4c95dd820433","order_by":4,"name":"Ziyu Liu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ziyu","middleName":"","lastName":"Liu","suffix":""},{"id":264657014,"identity":"4fa28293-675a-43e9-af43-906f1469a0ea","order_by":5,"name":"Yanfeng Ding","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanfeng","middleName":"","lastName":"Ding","suffix":""},{"id":264657015,"identity":"ff4c2ec6-d02f-41a6-a10d-cdfc75e5d0f2","order_by":6,"name":"Chengqiang Ding","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDCCw2DShoGxAUSzEa8ljRQtB5A0EqeF7zjv4Rc/ys7nMbefMWD4UHaYgX92A34tkof50ix7zt0uZuzJMWCcce4wg8SdA/i1GBzmMTNmbLud2NiQY8DM23aYwUAigSgt5xIb+98YMP8lUovxY8a2A4mNM4C2MBKjRRJoC2PPuWSglmcFB3vOpfNI3CCghe/8GeMPP8rsEjf2J2988KPMWo5/BgEtQMAmAYoOwwZIHPEQVA8EzB9AWuSJUToKRsEoGAUjEwAARXhFko3jar8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6061-1979","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Chengqiang","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2023-12-24 18:35:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3801577/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3801577/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-024-03193-z","type":"published","date":"2024-04-03T15:01:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49107437,"identity":"3bcccd63-8429-42e4-ba7b-11d3b8d37f48","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2451720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant height of Zhonghua11 (ZH11) and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e pup11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant plants in 2022.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C)\u003c/strong\u003e Habits (A), main stems (B), internode and panicle length phenotypes (C) of ZH11 and\u003cem\u003e pup11\u003c/em\u003e mutants. Images were digitally extracted and scaled for comparison (scale bar = 10 cm). \u003cstrong\u003e(D)\u003c/strong\u003eMeasurement of the length of each internode and panicle in ZH11 and \u003cem\u003epup11\u003c/em\u003emutants (n \u0026gt; 20). \u003cem\u003eP\u003c/em\u003e values indicate the level of statistical significance between ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants determined using Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/cf3973a4b265b4def85c4807.png"},{"id":49107438,"identity":"81e5a549-6117-4c75-868d-f4a67009a9ce","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1381027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSeed setting rate of Zhonghua11 (ZH11) and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e pup11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant plants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Images of the panicle phenotypes of ZH11 and \u003cem\u003epup11 \u003c/em\u003emutants. Images were digitally extracted and scaled for comparison (scale bar = 10 cm). \u003cstrong\u003e(B-D)\u003c/strong\u003e Measurement of the seed setting rate in 2020 (B), 2021 (C), and 2022 (D) in ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants (n \u0026gt; 20). \u003cstrong\u003e(E-I) \u003c/strong\u003eSeed setting rate of primary spikelets (E), seed setting rate of secondary spikelets (F), seed setting rate of bottom 1/3 (G), seed setting rate of mid 1/3 (H), and seed setting rate of top 1/3 (I) in the ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants (n \u0026gt; 20). \u003cem\u003eP\u003c/em\u003evalues indicate the level of statistical significance between ZH11 and \u003cem\u003epup11\u003c/em\u003emutants determined using Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/92752a25e29f3d5ab35c1070.png"},{"id":49107435,"identity":"eb1f4acb-54e4-473f-a4db-e66a9b41ea20","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1707242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFloral organ\u003c/strong\u003e \u003cstrong\u003ephenotypic characterization of Zhonghua11 (ZH11) and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e pup11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant plants.\u003c/strong\u003e \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C)\u003c/strong\u003e Phenotypic features of spikelets (A), floral organs (B) and pollen viability (C) of ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants. Images were digitally extracted and scaled for comparison (scale bar = 1 mm). \u003cstrong\u003e(D)\u003c/strong\u003e Measurement of pollen viability of ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants (n \u0026gt; 15). \u003cstrong\u003e(E, F)\u003c/strong\u003e Phenotypic features of unfilled grains in the \u003cem\u003epup11\u003c/em\u003e mutants (E) and transverse sections and I\u003csub\u003e2\u003c/sub\u003e-KI staining of ZH11 and \u003cem\u003epup11 \u003c/em\u003egrains at 6 days and 10 days after flowering (F). Images were digitally extracted and scaled for comparison (scale bar = 1 mm).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/cc99fbef4443c502e65c28af.png"},{"id":49107848,"identity":"fe7c93da-8936-48f7-b71c-3a27462af775","added_by":"auto","created_at":"2024-01-03 08:34:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2645891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns and subcellular of two transcripts of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePUP11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eExpression patterns of \u003cem\u003ePUP11-1\u003c/em\u003e transcript (A) and\u003cem\u003e PUP11-2\u003c/em\u003e transcript (B) investigated in the root at the vegetative stage (R), shoot base at the vegetative stage (BP), leaf blade at the vegetative stage (LB), 0–1 mm inflorescence meristem (I1mm), 1-5cm inflorescence meristem (I5cm), stem (S), flower (F), grain 3 days after flowering (G3) and grain 6 days after flowering (G6). Results are qRT-PCR data, represent means ± SD (n = 3). Total RNA was isolated from NIP. UBQ was used as an internal gene. \u003cstrong\u003e(C)\u003c/strong\u003e Histochemical GUS staining of \u003cem\u003epOsPUP11-1::GUS\u003c/em\u003e and \u003cem\u003epOsPUP11-2::GUS\u003c/em\u003e transgenic plants at various developmental stages. The scale bars were 1 mm. \u003cstrong\u003e(D) \u003c/strong\u003eSubcelluar location of PUP11-1 and PUP11-2. The combination of PUP11-1-GFP and PUP11-2-mCherry or PUP11-2-GFP and PUP11-1-mCherry were co-transformed into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves (scale bar = 20 μm).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/c3e152e6cd5bd10b4b63cd89.png"},{"id":49107441,"identity":"2303287e-afda-4394-bac4-f85bd2f7e4a8","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1780003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic characterization of Nipponbare (NIP) and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e PUP11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcript mutant plants in 2022.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C)\u003c/strong\u003e Habits for comparison between NIP and\u003cem\u003e pup11-1\u003c/em\u003e mutants (A), \u003cem\u003epup11-2\u003c/em\u003e mutants (B), and \u003cem\u003epup11-3\u003c/em\u003emutants (C). Images were digitally extracted and scaled for comparison (scale bar = 10 cm). \u003cstrong\u003e(D)\u003c/strong\u003e Image of panicles of NIP and \u003cem\u003ePUP11\u003c/em\u003e transcript mutants. Images were digitally extracted and scaled for comparison (scale bar = 10 cm).\u003cstrong\u003e (E) \u003c/strong\u003ePhenotypic features of unfilled grains in \u003cem\u003epup11-3-3-C \u003c/em\u003emutants. Images were digitally extracted and scaled for comparison (scale bar = 1 mm). \u003cstrong\u003e(F-G)\u003c/strong\u003eMeasurement of the seed setting rate from 2021 (F) and 2022 (G) in NIP and \u003cem\u003epup11\u003c/em\u003emutants (n \u0026gt; 20). \u003cem\u003eP\u003c/em\u003e values indicate the level of statistical significance between NIP and \u003cem\u003ePUP11\u003c/em\u003e transcript mutants determined using Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/bf0113ca7679d3f9f7cad18b.png"},{"id":49107436,"identity":"d3f98d3c-da70-4f17-84cf-ebff45ba163e","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":252180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of type-A \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in grains of ZH11 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epup11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eExpression of type-A\u003cem\u003e RR\u003c/em\u003e genes in ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants at 6 days (A) and 10 days (B) after flowering. Results are presented relative to ZH11. The qRT-PCR data represent means ± SD (n = 4). UBQ was used as an internal gene.\u003cem\u003e P\u003c/em\u003evalues indicate the level of statistical significance between ZH11 and \u003cem\u003epup11\u003c/em\u003emutants determined using Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/2498b19cbd8fff0b0bbde2f5.png"},{"id":49107439,"identity":"165e6fb9-cfbc-4e96-81ac-ebc7faa71de7","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":309674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of sucrose transport and starch biosynthesis genes in grains of ZH11 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epup11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C) \u003c/strong\u003eExpression of \u003cem\u003eSUT\u003c/em\u003es (A), \u003cem\u003eSWEET\u003c/em\u003es (B) and \u003cem\u003eSSS\u003c/em\u003es (C) genes in ZH11 and \u003cem\u003epup11\u003c/em\u003emutants at 6 days and 10 days after flowering. Results are presented relative to ZH11. The qRT-PCR data represent means ± SD (n = 4). UBQ was used as an internal gene.\u003cem\u003e P\u003c/em\u003e values indicate the level of statistical significance between ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants determined using Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/b6cf8e2867a0f6e286707028.png"},{"id":54304164,"identity":"6a4573d8-0855-4201-a048-8e1cd226b767","added_by":"auto","created_at":"2024-04-08 15:14:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4210909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/bcf25539-8c7d-4f90-b907-6c992e365914.pdf"},{"id":49107443,"identity":"0dc02b0e-7dd6-45af-9485-8a37e4d44a81","added_by":"auto","created_at":"2024-01-03 08:26:12","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":6727488,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3801577/v1/5cb047cf9f352ca6fdc25026.pdf"}],"financialInterests":"","formattedTitle":"Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development","fulltext":[{"header":"Key message","content":"\u003cp\u003ePurine permease\u0026nbsp;\u003cem\u003ePUP11\u003c/em\u003e is essential for rice seed development, regulates the seed setting rate, and may influence cytokinin content, sugar transport, and starch biosynthesis during grain development.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eCytokinins (CKs), which can be divided into active forms (free base) and inactive forms (combined forms), are a series of plant hormones that contribute to root and shoot structure, nutrient absorption, senescence, and stress responses (Duran-Medina et al. 2017; Zurcher and Muller 2016). However, CK biosynthesis occurs in a limited number of organs, especially in the roots. Because the supply and demand of CKs in plant tissues are unbalanced, the process of CK transport from the biosynthesis site to other organs plays an important role in regulating plant growth and development (Wu et al. 2017; Zhang et al. 2023). Some combined forms of CKs, such as \u003cem\u003etrans\u003c/em\u003e-zeatin riboside (tZR) and \u003cem\u003eN\u003c/em\u003e6-(Δ2-isopentenyl) adenine riboside (iPR), which are also known as storage forms because they adapt to long-distance transport, are precisely transported to other tissues, and transferred by certain enzymes to the free base form, where they completed their functions (Qi and Xiong 2013). Moreover, each type of CK has a different direction of transport in vascular tissue. In the xylem sap, the \u003cem\u003etrans\u003c/em\u003e-zeatin (tZ) forms, especially tZR, are major CK forms, while in the phloem sap, \u003cem\u003eN\u003c/em\u003e6-(Δ2-isopentenyl) adenine (iP)- and \u003cem\u003ecis\u003c/em\u003e-zeatin (cZ)-forms are predominant (Sakakibara 2021).\u003c/p\u003e \u003cp\u003eRecently, several CK transporters with diverse functions have been identified, with some participating in the long-distance transport of special CKs, such as the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003eTP-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eb\u003c/span\u003einding \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec\u003c/span\u003eassette \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eG\u003c/span\u003e family proteins (ABCG) AtABCG14 and OsABCG18, which have the classic function of transporting tZ and tZR from root to shoot (Zhang et al. 2014; Zhao et al. 2019), and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eE\u003c/span\u003equilibrative \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eN\u003c/span\u003eucleoside \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003eransporters (ENT), which participate in the long-distance transport of CKs with nucleosides (Hirose et al. 2005). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePu\u003c/span\u003erine \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ep\u003c/span\u003eermease (PUP) transports CKs between vascular tissues/apoplasts and cells, thereby facilitating CK cell-to-cell movement (Zurcher et al. 2016). A recent study also found that the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eugars \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eW\u003c/span\u003eill \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eE\u003c/span\u003eventually be \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eE\u003c/span\u003exported \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003eransporter (SWEET) HvSWEET11b can also transport tZ and tZR in grains across the maternal\u0026ndash;filial boundary, which appears to represent cell-to-cell movement (Radchuk et al. 2023). AZA-GUANINE RESISTANT (AZG) also exhibits CK transport activity between cell organs (Tessi et al. 2021).\u003c/p\u003e \u003cp\u003eWith the functional divergence of CK transporters, loss-of-function mutants show different phenotypes. Mutants that show disruptions in long-distance tZ transport from root to shoot, such as \u003cem\u003eatabcg14\u003c/em\u003e or \u003cem\u003eosabcg18\u003c/em\u003e mutants, showed smaller roots and shoots compared to the wild type because of the accumulation of CKs in roots and lack of CKs in shoots (Zhang et al. 2014; Zhao et al. 2019). In Arabidopsis, \u003cem\u003eAtPUP14\u003c/em\u003e downregulation reduced CK transport from the apoplast to cytosol, and the higher apoplast CK content led to ectopic CK responses; moreover, the mutants appeared to have more branches and flower primordia compared to the wild type (Zurcher et al. 2016). In contrast, the \u003cem\u003eatpup7/8/21\u003c/em\u003e triple mutant showed a narrower CK signaling pattern in the shoot apical meristem compared to the wild type (WT), and the rosette size was decreased (Hu et al. 2023). In rice, the overexpression of \u003cem\u003eOsPUP1\u003c/em\u003e reduces the tiller number, plant height, and panicle size (Xiao et al. 2020). Overexpression of \u003cem\u003eOsPUP4\u003c/em\u003e causes reduced shoot growth but larger grains, and \u003cem\u003eospup7\u003c/em\u003e had phenotypes similar to those of an \u003cem\u003eOsPUP4\u003c/em\u003e overexpression line (Qi and Xiong 2013; Xiao et al. 2019). However, the functions of CK transporters remain poorly understood.\u003c/p\u003e \u003cp\u003eSeveral factors can influence CK transport, such as exogenous plant hormones, nutrition, and stress. In rice, the expression of \u003cem\u003eOsABCG18\u003c/em\u003e is induced by exogenous CKs but inhibited by exogenous auxins (Zhao et al. 2019), and \u003cem\u003eOsPUP7\u003c/em\u003e is induced by exogenous CKs, abscisic acid, and drought stress (Qi and Xiong 2013). In addition, the CK content increases in the xylem and phloem sap when nitrogen is applied to \u003cem\u003eArabidopsis\u003c/em\u003e and rice, and this increased content implies enhanced CK transporter activity (Sakakibara 2021).\u003c/p\u003e \u003cp\u003eThe process of grain filling is important for the seed setting rate and 1000-grain weight of cereals. The sources of grain filling are photosynthesis from functional leaves after flowering and nonstructural carbohydrates in sink organs, such as the stem and sheath (Hu et al. 2022). Photosynthesis converts nonstructural carbohydrates into sucrose, and then SWEETs and sucrose transporters (SUTs) transport sucrose from the source to the grain through the phloem via transmembrane transport (Deng et al. 2021; Hu et al. 2022). CELLWALL INVERTASE 2 (CIN2) and monosaccharide transporters also participate in this process (Liu et al. 2022; Wang et al. 2008). Sucrose is transported to grains and synthesized into starch via enzymes such as sucrose synthase (SuSase), ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), and starch branching enzyme (SBE) (You et al. 2016). These processes lead to the completion of grain filling. Plant hormones have different functions during rice grain filling, and each shows fluctuating content during this process. For example, the CK content reaches a peak at an early stage and then decreases, whereas the auxin content increases at an early stage and reaches a peak approximately 10 days after pollination (Basunia and Nonhebel 2019; Liu et al. 2022). Endogenous CKs play important roles in regulating grain filling by promoting cell division in the early stages of grain filling and thus are involved in regulating the seed setting rate and grain size (Tsago et al. 2020).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the function of \u003cem\u003ePUP11\u003c/em\u003e in rice and confirmed that the two \u003cem\u003ePUP11\u003c/em\u003e transcripts showed redundant positive roles in seed development, ultimately upregulating the seed setting rate. Furthermore, certain genes related to CK signaling, sucrose transport, and starch biosynthesis were differentially expressed in the developing grains of \u003cem\u003epup11\u003c/em\u003e mutants compared with the Zhonghua 11 (ZH11) cultivar. Our study confirmed that \u003cem\u003ePUP11\u003c/em\u003e is necessary for seed development and provides insights for further research on rice grain development and cereal yield.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eOryza sativa\u003c/em\u003e L. ssp. \u003cem\u003ejaponica\u003c/em\u003e (cultivars Nipponbare (NIP) and ZH11) was the WT rice material chosen for this study. The mutants included a complete mutant of \u003cem\u003ePUP11\u003c/em\u003e from ZH11 and NIP and a special transcript mutant of \u003cem\u003ePUP11\u003c/em\u003e from NIP (Supplemental Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S4A).\u003c/p\u003e \u003cp\u003eRice plants were grown in a field in Danyang, Jiangsu Province, China (31.907\u0026deg; N 119.466\u0026deg; E). We used 150 kg/hm\u003csup\u003e2\u003c/sup\u003e P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e and 240 kg/hm\u003csup\u003e2\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eO as base fertilizers for the field experiment, and 200 kg/hm\u003csup\u003e2\u003c/sup\u003e nitrogen with a base fertilizer: tiller fertilizer: panicle fertilizer ratio of 1:1:2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003eThe mutants were constructed using CRISPR/Cas9 technology. Single guide RNA oligo targets were assigned to complete mutants or special transcript mutants in each cultivar using CRISPR/Cas9 technology, as previously described (Mao et al. 2013). The primers used for vector construction and genotyping are listed in Supplementary Table S5.\u003c/p\u003e \u003cp\u003eTo construct \u003cem\u003epPUP11-1::GUS\u003c/em\u003e and \u003cem\u003epPUP11-2::GUS\u003c/em\u003e plants, an upstream fragment of the encoding region of each transcript (\u0026gt;\u0026thinsp;3 kb) was amplified using PCR, and the resulting amplicon was excised with the corresponding restriction endonuclease and ligated into the \u003cem\u003epCAMBIA1300::GUS\u003c/em\u003e vector (Wang et al. 2020). Primers used for vector construction are listed in Supplementary Table S5.\u003c/p\u003e \u003cp\u003eAdditionally, the coding regions of the two transcripts of \u003cem\u003ePUP11\u003c/em\u003e were cloned into \u003cem\u003epCambia1300::35S::GFP\u003c/em\u003e (green fluorescent protein) or \u003cem\u003epCambia1300::35S::mCherry\u003c/em\u003e (\u003cem\u003emCherry fluorescent protein\u003c/em\u003e) plasmids to generate the plasmids \u003cem\u003ep35S::PUP11-1-GFP\u003c/em\u003e, \u003cem\u003ep35S::PUP11-2-GFP\u003c/em\u003e, \u003cem\u003ep35S::PUP11-1-mCherry\u003c/em\u003e, and \u003cem\u003ep35S::PUP11-2-mCherry\u003c/em\u003e for subcellular localization (Chang et al. 2021). Primers used for vector construction are listed in Supplementary Table S5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of GUS activity and subcellular localization analysis\u003c/h2\u003e \u003cp\u003eGUS reporter activity was assayed by histochemical staining using a GUS Staining Kit (Warbio, Nanjing, China, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.warbio.cn/\u003c/span\u003e\u003cspan address=\"http://www.warbio.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Various tissues were collected from the \u003cem\u003epOsPUP11-1::GUS\u003c/em\u003e or \u003cem\u003epOsPUP11-2::GUS\u003c/em\u003e transgenic plants at each developmental stage, after which they were immersed in the GUS staining solution and incubated for 6\u0026ndash;48 h at 37\u0026deg;C in the dark. The samples were destained three times with 70% ethanol in a water bath for 5 min. Images were captured using a SZX16 microscope (Olympus, Tokyo, Japan; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.olympus-lifescience.com/en/\u003c/span\u003e\u003cspan address=\"https://www.olympus-lifescience.com/en/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Each \u003cem\u003epOsPUP11-1::GUS\u003c/em\u003e or \u003cem\u003epOsPUP11-2::GUS\u003c/em\u003e transgenic plant had 20 independent lines, from which two or three independent lines were chosen and stained at the same position as the other lines for further studies.\u003c/p\u003e \u003cp\u003eFor the analysis of subcellular localization, \u003cem\u003ep35S::PUP11-1-GFP\u003c/em\u003e and \u003cem\u003ep35S::PUP11-2-GFP\u003c/em\u003e were transformed using \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 and inoculated into 4-week-old tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) leaves, as previously described (Liu et al. 2022). GFP fluorescence was detected using a LSM800 confocal laser microscope (Zeiss, Oberkochen, Germany; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.zeiss.com.cn\u003c/span\u003e\u003cspan address=\"https://www.zeiss.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eFor paraffin sectioning, samples were fixed overnight at 4\u0026deg;C in FAA (formalin:glacial acetic acid:70% ethanol; 1:1:18) and dehydrated using a graded ethanol series. Following substitution with xylene, the samples were embedded in paraffin, sectioned into 20 \u0026micro;m sections using a rotary microtome, dewaxed, stained with I\u003csub\u003e2\u003c/sub\u003e-KI (0.33%I\u003csub\u003e2\u003c/sub\u003e and 0.67%KI), incubated for 5 min, and then observed using a microscope (Olympus, SZX16) (Chang et al. 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSampling, RNA extraction, and gene expression analysis\u003c/h2\u003e \u003cp\u003eDeveloping seeds were collected 6 and 10 d after flowering from the ZH11 and \u003cem\u003epup11-1\u003c/em\u003e mutants. Plant tissues were collected at different developmental stages.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using an E.Z.N.A. Plant RNA Kit (Omega Biotek Inc., Norcross, GA, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omegabiotek.com/\u003c/span\u003e\u003cspan address=\"https://www.omegabiotek.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and subjected to reverse transcription using a PrimeScript RT Reagent Kit (Takara Biotechnology, Tokyo, Japan; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.takarabio.com/\u003c/span\u003e\u003cspan address=\"https://www.takarabio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Quantitative reverse-transcription PCR was performed on an ABI PRISM 7300 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.thermofisher.com/\u003c/span\u003e\u003cspan address=\"https://www.thermofisher.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with SYBR Premix Ex Taq (Takara), following the manufacturer\u0026rsquo;s instructions. Relative expression analysis was performed using the UBQ gene as an internal control. The primers used are listed in Supplementary Table S5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFloral organ observation and Pollen viability assay\u003c/h2\u003e \u003cp\u003eThe floral organs and grains were subsequently observed under an SZX16 microscope (Olympus, Tokyo, Japan), and more than 20 spikelets were observed for each material. We used I\u003csub\u003e2\u003c/sub\u003e-KI staining to determine the pollen viability of the mutant and WT (Xun et al. 2022). Pollen grains of the dehiscent anthers were placed on glass slides with I\u003csub\u003e2\u003c/sub\u003e-KI and incubated for 5 min. The experiments were performed in quintics for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eACCESSION NUMBERS\u003c/h2\u003e \u003cp\u003eSequence data from this article can be found in Rice Annotation Project database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under the following accession numbers: Ubq (Os03g0234200), \u003cem\u003eOsPUP11\u003c/em\u003e (Os02g0689200), \u003cem\u003eOsRR1\u003c/em\u003e (Os04g0442300), \u003cem\u003eOsRR2\u003c/em\u003e (Os02g0557800), \u003cem\u003eOsRR3\u003c/em\u003e (Os02g0830200), \u003cem\u003eOsRR4\u003c/em\u003e (Os01g0952500), \u003cem\u003eOsRR6\u003c/em\u003e (Os04g0673300), \u003cem\u003eOsRR9\u003c/em\u003e (Os11g0143300), \u003cem\u003eOsSUT1\u003c/em\u003e (Os03g0170900), \u003cem\u003eOsSUT2\u003c/em\u003e (Os12g0641400), \u003cem\u003eOsSUT4\u003c/em\u003e (Os02g0827200), \u003cem\u003eOsSWEET11\u003c/em\u003e (Os08g0535200), \u003cem\u003eOsSWEET15\u003c/em\u003e (Os02g0513100), \u003cem\u003eOsSSSI\u003c/em\u003e (Os06g0160700), \u003cem\u003eOsSSSII-3\u003c/em\u003e (Os06g0229800), and \u003cem\u003eOsSSSIII-2\u003c/em\u003e (Os08g0191433)\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePUP11 had essential roles in regulating seed setting rate\u003c/h2\u003e \u003cp\u003eTo determine the specific functions of OsPUP11, we first obtained two independent \u003cem\u003epup11\u003c/em\u003e mutant lines using CRISPR/Cas9 technology on a ZH11 background. Genome sequencing confirmed \u003cem\u003epup11-1\u003c/em\u003e had a 1499 bp deletion and two inversion insertions while \u003cem\u003epup11-2\u003c/em\u003e had a 252 bp deletion in the coding region (Supplementary Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). Previous studies have confirmed that \u003cem\u003eOsPUP11\u003c/em\u003e has three transcripts, while the two major transcripts encode a protein with ten transmembrane regions (Qi and Xiong 2013). Protein sequencing showed that the \u003cem\u003epup11-2\u003c/em\u003e mutant exhibited a deletion of 84 amino acids in both transcript protein sequences, the 3rd to 5th transmembrane regions of each transcript were damaged (Supplementary Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFirst, we observed the agronomic phenotypes of \u003cem\u003epup11\u003c/em\u003e mutants and found that the plant height of both \u003cem\u003epup11\u003c/em\u003e lines was significantly reduced compared to that of ZH11(Fig.\u0026nbsp;1A, B; Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Further analysis of the length of each internode of the ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants showed that the two mutant lines had shorter 1st and 2nd top internodes compared to ZH11, and these differences influenced plant height (Fig.\u0026nbsp;1C, D). Moreover, the flag leaves of the two \u003cem\u003epup11\u003c/em\u003e lines were significantly longer and wider than those of ZH11, and both lines had wider second and third top leaves (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The tiller number of \u003cem\u003epup11\u003c/em\u003e mutants was similar to that of ZH11 plants (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSubsequently, we focused on the yield component factors. During the three-year-long field experiment, the most notable phenotype of \u003cem\u003epup11\u003c/em\u003e mutants was the extremely low seed setting rate, which was below 25% and significantly lower than that of ZH11 (Fig.\u0026nbsp;2A-D). Obvious differences were not observed between the ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants in terms of panicle number, primary branches, and primary spikelets, and both \u003cem\u003epup11\u003c/em\u003e mutant lines showed significantly increased secondary branches and secondary spikelets; therefore, they showed more spikelets per panicle (Supplemental Table S2). In addition, the 100-grain weights of \u003cem\u003epup11-1\u003c/em\u003e and \u003cem\u003epup11-2\u003c/em\u003e were lower than those of ZH11, because of these mutants showed reduced grain thickness (Supplemental Fig.S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDistribution of filled grains was changed in the\u003c/b\u003e \u003cb\u003epup11\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eObservations of the phenotypes of the panicles also noticed that compared to ZH11, both \u003cem\u003epup11\u003c/em\u003e mutant lines showed a special distribution of filled grains. First, we found that the seed setting rate of the primary spikelets of both \u003cem\u003epup11\u003c/em\u003e mutant lines was very low (below 6%), which was significantly lower than that of ZH11 (Fig.\u0026nbsp;2E). Compared with the primary spikelets, the seed setting rate of the secondary spikelets of both \u003cem\u003epup11\u003c/em\u003e mutant lines was higher than that of primary spikelets, and these differences were lower than those observed with ZH11 (Fig.\u0026nbsp;2F). Further analysis of yield contribution in ZH11 showed that the primary and secondary spikelets showed almost equal yield contributions; however, in both \u003cem\u003epup11\u003c/em\u003e mutant lines, the yield was mainly from secondary spikelets (Supplemental Fig.S3A).\u003c/p\u003e \u003cp\u003eWe also noticed that in the \u003cem\u003epup11\u003c/em\u003e mutants, the seed setting rate showed a decreasing trend from bottom to top. Therefore, we statistically analyzed the seed setting rates in the branches of the bottom 1/3, middle 1/3 and top 1/3 of the panicles. Although the seed setting rate in \u003cem\u003epup11\u003c/em\u003e mutant was higher at the bottom, the value was lower than that of ZH11. In the top branches, the seed setting rate in \u003cem\u003epup11\u003c/em\u003e was close to zero (Fig.\u0026nbsp;2, G-I). Because of this special distribution, spikelets in the bottom part showed a much higher yield contribution ratio in the \u003cem\u003epup11\u003c/em\u003e mutants than in ZH11, whereas spikelets in the middle and top parts provided a much lower yield contribution in the \u003cem\u003epup11\u003c/em\u003e mutants than in ZH11 (Supplemental Fig.S3B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOsPUP11 is necessary for grain development\u003c/h2\u003e \u003cp\u003eTo reveal why \u003cem\u003epup11\u003c/em\u003e mutants had extremely low seed setting rates, we first observed the structure of the flower organs. Both \u003cem\u003epup11\u003c/em\u003e mutant lines had two glumes, two lodicules, six stamens, and one pistil with two stigmas, which were the same as in ZH11. Thus, PUP11 did not appear to influence the number of flower organs (Fig.\u0026nbsp;3A, B). We then detected pollen activity using the I\u003csub\u003e2\u003c/sub\u003e-KI method and found that the pollen viability of \u003cem\u003epup11\u003c/em\u003e mutant was not significantly different from that of ZH11 (Fig.\u0026nbsp;3C, D).\u003c/p\u003e \u003cp\u003eWe then peeled the glumes of unfilled grains from \u003cem\u003epup11\u003c/em\u003e mutants and found two mutant lines that developed fertilized grains, but their development stopped before storage product accumulation (Fig.\u0026nbsp;3E). To precisely characterize the development process, we also observed the structure of the endosperm by paraffin sectioning using I\u003csub\u003e2\u003c/sub\u003e-KI staining. We found that starch accumulated in aleurone six days after flowering in both the ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants. Nine days after flowering, we found that starch accumulated in the endosperm of ZH11 but disappeared in the aleurone and endosperm of the \u003cem\u003epup11\u003c/em\u003e mutant (Fig.\u0026nbsp;3F). Moreover, seed shape was irregular in the \u003cem\u003epup11\u003c/em\u003e mutant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExpression pattern of two major OsPUP11 transcripts\u003c/h2\u003e \u003cp\u003eAs described above, OsPUP11 has two major transcripts encoding two proteins with 24 amino acids at the N-terminus. Although these two proteins share large sequence similarities, their expression patterns differ.\u003c/p\u003e \u003cp\u003eFirst, we detected the spatial expression of both transcripts using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). In general, the expression of \u003cem\u003ePUP11-2\u003c/em\u003e was lower than \u003cem\u003ePUP11-1\u003c/em\u003e (Fig.\u0026nbsp;4A, B). While \u003cem\u003ePUP11-1\u003c/em\u003e showed special expression in the 0\u0026ndash;1 mm young panicles and stems, \u003cem\u003ePUP11-2\u003c/em\u003e showed special expression in the 1\u0026ndash;5 cm young panicles.\u003c/p\u003e \u003cp\u003eTo better understand the tissue distribution of these two transcripts, we constructed p\u003cem\u003ePUP11-1::GUS\u003c/em\u003e and p\u003cem\u003ePUP11-2::GUS\u003c/em\u003e plants to express the β-glucuronidase (GUS) gene under the control of their promoters and terminators, respectively (Fig.\u0026nbsp;4C). In vegetative organs, we found that \u003cem\u003ePUP11-1\u003c/em\u003e was expressed in young and flag leaves while \u003cem\u003ePUP11-2\u003c/em\u003e was not detected in the leaves. Both transcripts were expressed in axillary buds, shoot bases, and stems, with extremely strong expression in the region near the nodes. However, neither transcript was detected in the roots. In young panicles, we found \u003cem\u003ePUP11-2\u003c/em\u003e expressed higher in the early stages of panicle differentiation, at which stage \u003cem\u003ePUP11-1\u003c/em\u003e expresses very low. During the booting stage, \u003cem\u003ePUP11-1\u003c/em\u003e was highly expressed at the bottom of the spikelet, stamen primordium, and young branches, whereas \u003cem\u003ePUP11-2\u003c/em\u003e expression was detected throughout the spikelet. Both transcripts were weakly expressed in mature glumes. At grain maturity, both \u003cem\u003ePUP11-1\u003c/em\u003e and \u003cem\u003ePUP11-2\u003c/em\u003e were specifically expressed in the region near the embryo, with little expression at the top of the grains. In the germinated seeds, we found \u003cem\u003ePUP11-1\u003c/em\u003e expressed in the region near the embryo, and young leaves were wrapped in the coleoptile; however, \u003cem\u003ePUP11-2\u003c/em\u003e was not expressed at this stage.\u003c/p\u003e \u003cp\u003eTo learn the subcellular location of the two transcripts, we constructed \u003cem\u003e35S::PUP11-1-GFP\u003c/em\u003e, \u003cem\u003e35S::PUP11-1-mCherry\u003c/em\u003e, \u003cem\u003e35S::PUP11-2-GFP\u003c/em\u003e, and \u003cem\u003e35S::PUP11-2-mCherry\u003c/em\u003e, to investigate the subcellular localization of both transcripts. All four plasmids were transferred into Agrobacterium. We used two combinations: one with a mixture of \u003cem\u003e35S::PUP11-1-mCherry\u003c/em\u003e and \u003cem\u003e35S::PUP11-2-GFP\u003c/em\u003e injected into a leaf, and the other injected a mixture of \u003cem\u003e35S::PUP11-2-mCherry\u003c/em\u003e and 3\u003cem\u003e5S::PUP11-1-GFP\u003c/em\u003e. Both combinations confirmed that PUP11-1 was mainly localized in the plasma membrane (PM), and PUP11-2 was mainly localized in the plasma membrane and endoplasmic reticulum (ER) (Fig.\u0026nbsp;4D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTwo major\u003c/b\u003e \u003cb\u003ePUP11\u003c/b\u003e \u003cb\u003etranscripts have redundant function in regulating seed setting rate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBecause the two major PUP11 transcripts have different expression patterns and subcellular locations, we speculated that the two transcripts may have different functions. Using the NIP background, we designed two single guide RNA, each of which can edit one transcript without influencing the other. Moreover, to confirm the phenotypes of the \u003cem\u003epup11\u003c/em\u003e mutant, we constructed another \u003cem\u003epup11\u003c/em\u003e mutant line with loss-of-function for all transcripts in the NIP background (Supplemental Fig.S4A). We obtained three mutant lines of the \u003cem\u003ePUP11-1\u003c/em\u003e transcript and two mutant lines of the \u003cem\u003ePUP11-2\u003c/em\u003e transcript. In addition, a heterozygous mutant of all \u003cem\u003ePUP11\u003c/em\u003e transcripts was constructed with a C insertion and a 12 bp deletion in both chromosomes (no chromatograms of the homozygous mutant; the mutant with a C insertion can be cut by NcoI, but that with the 12 bp deletion cannot, with further studies performed to recognize the genotype by enzyme cutting) (Supplemental Fig.S4A, B). The 12 bp deletion mutant changed 4 amino acids at the third outer membrane region in both protein sequences. We named the C insertion mutant \u003cem\u003epup11-3-3-C\u003c/em\u003e, 12 bp deletion mutant \u003cem\u003epup11-3-3-12\u003c/em\u003e, and heterozygous mutant \u003cem\u003epup11-3-3-C/12\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFirst, we observed the vegetative organ phenotypes of all mutants. For \u003cem\u003epup11-3\u003c/em\u003e homozygous, as the segregation ratio of the C insertion from heterozygotes was extremely low, some data were not statistically significant. The plant heights of the \u003cem\u003epup11-1\u003c/em\u003e and \u003cem\u003epup11-2\u003c/em\u003e mutants were reduced (Fig.\u0026nbsp;5, A-B; Supplemental Table S3). For the tiller numbers, just \u003cem\u003epup11-2-1\u003c/em\u003e showed more tillers compared to WT (Supplemental Table S3). Furthermore, we found reduced flag leaf length in \u003cem\u003epup11-1-2\u003c/em\u003e and \u003cem\u003epup11-1-3\u003c/em\u003e and reduced flag leaf width in all mutant lines except \u003cem\u003epup11-1-1\u003c/em\u003e (Supplemental Table S3).\u003c/p\u003e \u003cp\u003eAfter harvesting the panicles, we focused on the seed setting rate. As confirmed from two years of data, the loss-of-function of one \u003cem\u003ePUP11\u003c/em\u003e transcript had no effect on the seed setting rate, while \u003cem\u003epup11-3-3-C\u003c/em\u003e showed a very low seed setting rate because of seed development arrest, which is the same as the mutants in the ZH11 background (Fig.\u0026nbsp;5A-G). A lower seed setting rate was not observed in \u003cem\u003epup11-3-3-12\u003c/em\u003e, which seemed the 4aa amino acids at the third outside membrane region had a slight effect on seed development (Fig.\u0026nbsp;5F). Moreover, we found a lower seed setting rate in \u003cem\u003epup11-3-C/12\u003c/em\u003e heterozygotes, which may have been caused by the lack of development of homozygous seeds with the C insertion (Fig.\u0026nbsp;5F).\u003c/p\u003e \u003cp\u003eWe also observed other yield factors. In both years, \u003cem\u003epup11-3-3-C\u003c/em\u003e showed fewer spikelets per panicle, with fewer secondary branches and secondary spikelets compared to NIP, in contrast to the \u003cem\u003epup11\u003c/em\u003e mutants in the ZH11 background (Supplemental Table S4). Damage to either the \u003cem\u003ePUP11-1\u003c/em\u003e or \u003cem\u003ePUP11-2\u003c/em\u003e transcripts showed no significant difference in panicle number, primary branches, and primary spikelets compared to the NIP, but reduced secondary branches, secondary spikelets, and total spikelets per panicle compared to the NIP (Supplemental Table S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePUP11 influences the expression of genes related to cytokinin signaling and sugar metabolism\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that the PUP family genes function in transporting CKs. We suspected that in the grains of \u003cem\u003epup11\u003c/em\u003e mutants, normal CK transport in grains was damaged, the CK content in grains was altered, and finally caused unusual grain development. Type A \u003cem\u003eRR\u003c/em\u003e genes quickly respond to exogenous CKs and are used as marker genes to indicate CKs levels. Therefore, we measured the expression of type A \u003cem\u003eRR\u003c/em\u003e genes in the developing grains of ZH11 and \u003cem\u003epup11-1\u003c/em\u003e firstly. We found that at six days after flowering, when the phenotype of the grain did not show obvious changes, the expression of type-A \u003cem\u003eRR\u003c/em\u003e genes did not change significantly in the \u003cem\u003epup11\u003c/em\u003e mutants compared to ZH11, except \u003cem\u003eRR4\u003c/em\u003e (Fig.\u0026nbsp;6A). However, 10 days after flowering, we found that the expression of \u003cem\u003eRR2\u003c/em\u003e, \u003cem\u003eRR4\u003c/em\u003e, \u003cem\u003eRR6\u003c/em\u003e, and \u003cem\u003eRR9\u003c/em\u003e was significantly upregulated in the \u003cem\u003epup11\u003c/em\u003e mutants compared to ZH11 (Fig.\u0026nbsp;6B). This may be due to changes in CK homeostasis in the \u003cem\u003epup11\u003c/em\u003e mutants.\u003c/p\u003e \u003cp\u003eStarch accumulation in the grains of \u003cem\u003epup11\u003c/em\u003e was unusual. We suspected that the expression of some genes related to sucrose transport or starch synthesis in grains was altered in the \u003cem\u003epup11\u003c/em\u003e mutants. Sucrose transport into grains is an important step in grain filling, as \u003cem\u003eSUT1\u003c/em\u003e, \u003cem\u003eSWEET11\u003c/em\u003e and \u003cem\u003eSWEET15\u003c/em\u003e have been reported to play essential roles in the process and influence grain filling (Deng et al. 2021). At both 6 and 10 days after flowering, the expression of \u003cem\u003eSUT1\u003c/em\u003e was significantly reduced in the \u003cem\u003epup11\u003c/em\u003e mutant while the expression of \u003cem\u003eSUT2\u003c/em\u003e and \u003cem\u003eSUT4\u003c/em\u003e did not change or was slightly upregulated in the \u003cem\u003epup11\u003c/em\u003e mutant (Fig.\u0026nbsp;7A). Compared with \u003cem\u003eSUT\u003c/em\u003es, the expression of \u003cem\u003eSWEET11\u003c/em\u003e and \u003cem\u003eSWEET15\u003c/em\u003e, which have sucrose efflux functions at the nucellar projection and play a role in sucrose transfer across the nucellar epidermis/aleurone interface, was upregulated in the \u003cem\u003epup11\u003c/em\u003e mutant (Fig.\u0026nbsp;7B).\u003c/p\u003e \u003cp\u003eNumerous studies have shown that many genes are involved in controlling starch synthesis from sucrose, including \u003cem\u003eSSSI\u003c/em\u003e, \u003cem\u003eSSSII-3\u003c/em\u003e, and \u003cem\u003eSSSIII-2\u003c/em\u003e (You et al. 2016). Interestingly, we found that three \u003cem\u003eSSS\u003c/em\u003es genes are all downregulated in the \u003cem\u003epup11\u003c/em\u003e mutant at both stages, which explains the low starch accumulation and grain formation arrest in the \u003cem\u003epup11\u003c/em\u003e mutant (Fig.\u0026nbsp;7C).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e1. Cytokinin transporters play different roles in regulating rice development\u003c/h2\u003e \u003cp\u003eCK transporters help adjust the CK content from the cellular organ level to the whole plant. An abnormal CK distribution in plant tissues results in different phenotypes. Recently, function of four CK transporters (ABCG18, PUP1, PUP4, and PUP7) were identified in rice. Although CK transporters in rice are poorly understood, studies have provided insights for plant breeding.\u003c/p\u003e \u003cp\u003eEach known CK transporter in rice plays a special role throughout the rice life cycle. Although \u003cem\u003eABCG18\u003c/em\u003e had a positive effect on tiller bud outgrowth, plant height, and panicle development (Zhao et al. 2019), PUP1 seems to have the opposite effect (Xiao et al. 2020). Smaller panicles with larger seeds were observed for the \u003cem\u003ePUP4-overexpression\u003c/em\u003e line and \u003cem\u003epup7\u003c/em\u003e mutant compared with the WT. However, we found that the two transcripts of \u003cem\u003ePUP11\u003c/em\u003e showed no obvious functions in regulating tillers, but each \u003cem\u003ePUP11\u003c/em\u003e transcript showed a positive role in regulating plant height (Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; Supplemental Table S3).\u003c/p\u003e \u003cp\u003ePrevious studies have also found that several CK transporters, such as ABCG18 and PUP4, can influence rice seed setting rate (Xiao et al. 2019; Zhao et al. 2019). By observing \u003cem\u003epup11\u003c/em\u003e mutant lines with the two transcripts, which showed damage under the two backgrounds, we found a severely reduced seed setting rate compared to the WT, which was caused by arrested seed development. We also found that the filled seed distribution of \u003cem\u003epup11\u003c/em\u003e differed from that of ZH11 (Fig.\u0026nbsp;2H-M). This result suggests that PUP11 has special functions in seed development that are different from those of other known CK transporters.\u003c/p\u003e \u003cp\u003eOverall, several CK transporters showed some advantages for yield as well as shortcomings. Only the \u003cem\u003eABCG18-overexpression\u003c/em\u003e lines showed a higher yield (Zhao et al. 2019). These results indicated the functional variations of CK transporters, which inspired us to explore the factors underlying the differences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2. Potential causes of the functional differences in cytokinin transporters\u003c/h2\u003e \u003cp\u003eEach CK transporter exhibits a unique expression pattern. \u003cem\u003eABCG18\u003c/em\u003e and \u003cem\u003eENT2\u003c/em\u003e, which participate in long-distance CK transport, are mainly expressed in the vascular tissues around the whole plant (Hirose et al. 2005; Zhao et al. 2019). \u003cem\u003ePUP\u003c/em\u003e family genes are also particularly expressed in vascular tissues; however, each PUP has a unique spatiotemporal expression (Qi and Xiong 2013; Xiao et al. 2019). In our study, the two \u003cem\u003ePUP11\u003c/em\u003e transcripts had different expression patterns. Moreover, the \u003cem\u003ePUP11-1\u003c/em\u003e transcript could be detected in most vegetative organs in the shoot while \u003cem\u003ePUP11-2\u003c/em\u003e could not be detected. However, the expression of both transcripts overlapped in the nodes and young flowers (Fig.\u0026nbsp;4A-C). In summary, CK transporters have unique expression patterns, influence the transport of CKs in different plant tissues and at different stages, and precisely regulate plant growth and development.\u003c/p\u003e \u003cp\u003eCK transporters are primarily localized in the PM and ER. In our study, the two \u003cem\u003ePUP11\u003c/em\u003e transcripts encoding two proteins with different N-termini had different subcellular localizations and may function in different cell organs to precisely regulate cellular CK signaling (Fig.\u0026nbsp;4D). Therefore, we believe that the difference in cell organ localization is another factor affecting transporter function.\u003c/p\u003e \u003cp\u003ePrevious studies have also found that each CK transporter can only transport specific types of CKs. Although previous studies have found that the CK distribution is altered in the \u003cem\u003eatpup7/8/21\u003c/em\u003e triple mutant and \u003cem\u003eOsPUP\u003c/em\u003e-overexpression lines (Hu et al. 2023; Qi and Xiong 2013; Xiao et al. 2019; Xiao et al. 2020), the CK type that is transported by PUP remains unknown. PUP transporters have different transport directions that may be determined by the amino acid sequence of PUP. In general, the expression pattern, subcellular location, substrate, and transport direction of the CK transporters provide them with unique functions. However, with the limited knowledge of cytokinin transporters recently, how these transporters regulate the CK content and distribution in rice remains unknown. \u003cb\u003e3. PUP11 influences cytokinin homeostasis and starch biosynthesis in grains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach type of plant hormone regulates grain development (Basunia and Nonhebel 2019), and the relationship between CK and grain filling is worthy of exploration.\u003c/p\u003e \u003cp\u003eAs summarized in previous studies, CKs have two directions for seed setting rates. Exogenous CK application can facilitate grain filling in large panicles (Chen et al. 2022; Panda et al. 2018). In contrast, several \u003cem\u003eckx\u003c/em\u003e mutants, such as \u003cem\u003eckx1 ckx2\u003c/em\u003e, \u003cem\u003eckx3\u003c/em\u003e, \u003cem\u003eckx4 ckx9\u003c/em\u003e and \u003cem\u003eckx11\u003c/em\u003e, have higher endogenous CK content, although their seed setting rate is lower than that of the WT (Huang et al. 2023; Rong et al. 2022; Zhang et al. 2021).\u003c/p\u003e \u003cp\u003eRecent research has reported that HvSWEET11b can transport both sucrose and CKs and knocking down \u003cem\u003eHvSWEET11b\u003c/em\u003e prevents the production of grains with normal vegetative, panicle, and pollen development (Radchuk et al. 2023), which is similar to the results for the \u003cem\u003eospup11\u003c/em\u003e mutant. In \u003cem\u003eHvSWEET11B-RNAi\u003c/em\u003e lines, tZR accumulates in several parts of the grains, such as the vascular region and nucellar projections (Radchuk et al. 2023). In our study, the grains of \u003cem\u003epup11\u003c/em\u003e mutants also showed higher expression of type-A \u003cem\u003eRR\u003c/em\u003e genes, which may imply a higher CK content (Fig.\u0026nbsp;6). But because of technological limitation, we can\u0026rsquo;t see cytokinin content in each part of developing seed. However, these results prompted us to determine the relationship between endogenous CKs and grain filling in cereals.\u003c/p\u003e \u003cp\u003eExogenous CK usage in the panicles helps improve soluble sugar content and starch biosynthesis in the inferior spikelets (Chen et al. 2022). However, few studies have focused on the molecular mechanisms by which endogenous CKs influence sugar transport or grain filling. In our study, we detected the expression of several sucrose transporters and starch biosynthesis genes in the grains of ZH11 and \u003cem\u003epup11\u003c/em\u003e mutants. Each sucrose transporter has a special function in rice grain filling. SWEET11, SWEET14, and SWEET15 are involved in sucrose efflux at the nucleolar projections and transfer across the aleurone interface (Fei et al. 2021; Yang et al. 2018). SUT1/3/4 function in sucrose transport from the dorsal phloem to the filial aleurone. SUT1 plays a role in seed sucrose uptake, and the loss-of-function of \u003cem\u003eSUT1\u003c/em\u003e results in a low seed setting rate and grain weight (Deng et al. 2021; Scofield et al. 2002; Wang et al. 2021). In our study, we found extreme downregulation of \u003cem\u003eSUT1\u003c/em\u003e in developing grains of \u003cem\u003epup11\u003c/em\u003e compared to ZH11, whereas \u003cem\u003eSUT2\u003c/em\u003e and \u003cem\u003eSUT4\u003c/em\u003e showed a slight upregulate at six days after flowering. This finding similar to that of a previous study and may be associated with the sugar transport strategy switch (Wang et al. 2021). The extremely high expression of \u003cem\u003eSWEET11\u003c/em\u003e and \u003cem\u003eSWEET15\u003c/em\u003e in the \u003cem\u003epup11\u003c/em\u003e mutant may be influenced by unknown hunger signals from grains; however, sucrose transported by \u003cem\u003eSWEET\u003c/em\u003es cannot be transported into the endosperm. Interestingly, we found that PUP11 can positively influence the biosynthesis of starch, which is consistent with the disappearance of starch assimilation in grains of the \u003cem\u003epup11\u003c/em\u003e mutant at 10 days after flowering (Fig.\u0026nbsp;3F). Except for the lower expression of \u003cem\u003eSSS\u003c/em\u003e genes in the \u003cem\u003epup11\u003c/em\u003e mutants, we also found that with grain development, \u003cem\u003eSSS\u003c/em\u003e gene expression showed an increasing trend in the WT; however, the increasing trend was weaker in \u003cem\u003epup11\u003c/em\u003e (Fig.\u0026nbsp;7). Whether the inhibition of starch biosynthesis genes was directly influenced by the loss-of-function of \u003cem\u003ePUP11\u003c/em\u003e or by low sugar levels in \u003cem\u003epup11\u003c/em\u003e grains remains unknown. Thus, the mechanisms underlying the influence of PUP11 on sucrose transport and starch biosynthesis gene expression require further research.\u003c/p\u003e \u003cp\u003eIn conclusion, our study found that \u003cem\u003ePUP11\u003c/em\u003e influences several phenotypes during the rice life cycle, particularly by regulating seed development. Two major PUP11 transcripts with different expression patterns and subcellular locations showed redundant roles in seed-filling. Further studies showed that PUP11 influences CK content, sucrose transport, and starch accumulation in developing grains. Our findings establish a community resource for fully elucidating the function of \u003cem\u003ePUP11\u003c/em\u003e and revealed the relationship between exogenous CKs and the grain-filling process, thus providing new insights that may be used for future studies on the mechanisms underlying seed development and yield improvement.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Jiangsu Province (Grants No BK20231470).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and within its supplementary data published online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jiankang Zhu and Caixia Gao for providing the vectors of the CRISPR-Cas9 system. We also thank Biogle and Biorun genome editing center for producing transgenic rice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.D. and Y.D. conceived the original screening and research plans; C.R., R.Z., Y.L., Z.C., Z.L., and C.D. performed the experiments and analyzed the data; C.R. and C.D. wrote the article with contributions of all the authors; C.D. agrees to serve as the author responsible for contact and ensures communication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests in relation to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBasunia MA, Nonhebel HM (2019) Hormonal regulation of cereal endosperm development with a focus on rice (Oryza sativa). 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International review of cell and molecular biology 324:1-38\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rice, PUP11, seed setting rate, seed development, cytokinin, sugar","lastPublishedDoi":"10.21203/rs.3.rs-3801577/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3801577/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe distribution of cytokinins in plant tissues determines plant growth and development and is regulated by several cytokinin transporters. Purine permease (PUP) is a cytokinin transporter found in plants. Although 13 \u003cem\u003ePUP\u003c/em\u003e genes have been identified in the rice genome, however, most of their functions remain unknown. We found that \u003cem\u003epup11\u003c/em\u003emutants showed extremely low seed setting rates and a unique filled seed distribution. Our research revealed that \u003cem\u003epup11\u003c/em\u003e mutants showed seed formation arrest because the accumulated starch disappeared 10 days after flowering. \u003cem\u003ePUP11\u003c/em\u003e has two major transcripts with different expression patterns and subcellular locations, and further studies revealed that they have redundant positive roles in regulating the seed setting rate. We also found that type-A \u003cem\u003eRR\u003c/em\u003e genes were upregulated in the developing grains of the \u003cem\u003epup11\u003c/em\u003e mutant compared with the wild type. The results also showed that PUP11 altered the expression of several sucrose transporters and significantly upregulated certain starch biosynthesis genes. In summary, our results indicate that PUP11 influences the rice seed setting rate by regulating sucrose transport and starch accumulation during grain filling. This research provides new insights into the relationship between cytokinins and seed development, which may help improve cereal yield.\u003c/p\u003e","manuscriptTitle":"Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 08:26:07","doi":"10.21203/rs.3.rs-3801577/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-02-05T05:40:31+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-01-16T05:39:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-02T00:50:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-26T04:31:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2023-12-24T05:30:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7fc13f15-260a-4e65-aa1c-a5723bcd9fbf","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-08T15:09:43+00:00","versionOfRecord":{"articleIdentity":"rs-3801577","link":"https://doi.org/10.1007/s00299-024-03193-z","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2024-04-03 15:01:32","publishedOnDateReadable":"April 3rd, 2024"},"versionCreatedAt":"2024-01-03 08:26:07","video":"","vorDoi":"10.1007/s00299-024-03193-z","vorDoiUrl":"https://doi.org/10.1007/s00299-024-03193-z","workflowStages":[]},"version":"v1","identity":"rs-3801577","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3801577","identity":"rs-3801577","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}