PANICLE APICAL ABORTION 3 Controls Panicle Development and Seed Size in Rice

doi:10.21203/rs.3.rs-210428/v1

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PANICLE APICAL ABORTION 3 Controls Panicle Development and Seed Size in Rice

https://doi.org/10.21203/rs.3.rs-210428/v1

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published 15 Jul, 2021

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Background

In rice, panicle apical abortion is a common phenomenon that usually results in a decreased number of branches and grains per panicle, and consequently a reduced grain yield. A better understanding of the molecular mechanism of panicle abortion is thus critical for maintaining and increasing rice production.

Results

We reported a new rice mutant panicle apical abortion 3 (paa3), which exhibited severe abortion of spikelet development on the upper part of the branches as well as decreased grain size over the whole panicle. Using mapping-based clone, the PAA3 was characterized as the LOC_ Os04g56160 gene, encoding an H⁺-ATPase. The PAA3 was expressed highly in the stem and panicle, and its protein was localized in the cytoplasm. Our data further showed that PAA3 played an important role in maintaining normal panicle development by participating in the removal of reactive oxygen species (ROS) in rice.

Conclusions

Our studies suggested that PAA3 might function to remove ROS, the accumulation of which leads to programmed cell death, and ultimately panicle apical abortion and decreased seed size in the paa3 panicle.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

rice

panicle apical abortion

programmed cell death

H+-ATPase

Rice (Oryza sativa) is one of the most important food crops in the world and the major staple food for more than half the world’s population (Takeda and Matsuoka, 2008). Rice yield is determined by three main component traits: number of panicles, number of grains per panicle, and grain weight ( Xing and Zhang, 2010). Therefore, the panicle plays a key role in contributing directly to yield, and the achievement of optimal panicle structure, size, and shape is one of the goals in high-yield breeding (Sakamoto and Matsuoka, 2004).

In the development of the inflorescence, the transition from shoot meristems to axillary meristems (AMs) determines the complexity of the inflorescence architecture (Huijser and Schmid, 2011). The rice panicle has a four-order inflorescence structure, comprising the main axis, primary branches (PBs), secondary branches (SBs), lateral spikelet (LS), and terminal spikelet (TS), where PBs, SBs, and LS all originate from the AMs. The past few years have seen the identification of a number of regulatory genes involved in the determination of panicle architecture, including lymphocyte transmembrane adaptor 1 (LAX1), LAX2, and monoculm 1. These genes participate in the initiation, formation, and maintenance of AMs in the rice panicle and are regulators of panicle architecture. They all encode transcription factor and the loss-of-function mutants display greatly reduced numbers of branches and spikelets (Komatsu et al., 2003; Li et al., 2003; Tabuchi et al., 2011). The transition from branch meristem to spikelet meristem is also considered a key process related to panicle architecture. Reticulocalbin 1 (RCN1) and RCN2 maintain the branch meristem and control its fate in rice, and their overexpression leads to the delayed transition from branch meristem to spikelet meristem, and then to more branches and spikelets (Nakagawa et al., 2002). Abberant panicle organization 2 (APO2)/RFL can be activated by the DNA-binding one zinc finger DOF-domain transcription activator short panicle 3 (SP3), and interacts with APO1 to delay the transition from branch meristem to spikelet meristem. In the mutants apo1, apo2 and sp3, the numbers of branches and spikelets are all significantly reduced (Rao et al., 2008; Ikeda-Kawakatsu et al., 2012). In addition, TAW1 encodes a nuclear protein that participates in determining the fate of the branch meristem. In the gain-of-function mutant taw1-d, the branch meristem activity is increased and the differentiation of the spikelet meristem is delayed, which promotes the formation of more SBs and spikelets (Yoshida et al., 2013). Plant hormones such as cytokinin also play crucial roles in panicle architecture. Grain number 1a (Gn1a), a major quantitative trait locus of grain number-per-panicle, encodes cytokinin oxidase 2, which is responsible for the degradation of cytokinin. In the natural allelic variant with low expression of Gn1a, the accumulation of cytokinin leads to greatly increased numbers of branches and spikelets, and thus grain yield (Ashikari et al., 2005). Multiple genes such as DROUGHT AND SALT TOLERANCE (DST), VIN3-LIKE 2 (VIL2) and LARGER PANICLE (LP)(Li et al., 2011; Li et al., 2013) (Yang et al., 2019) participate in panicle development by regulating the expression of Gn1a. The microRNA156-ideal plant architecture 1-dense and erect panicle 1 (MicroRNA156-IPA1-DEP1) pathway probably regulates panicle development through cytokinin, in which IPA1 can directly activate DEP1 expression by interacting with its promoter, and then the high expression of DEP1 represses Gn1a (Huang et al., 2009; Lu et al., 2013).

After the panicle architecture is established, normal spikelet growth is very important for the final yield. Panicle abortion occurs at either the top or basal parts of the panicle. Spikelet growth often stops at the apex of the panicle and/or branches under unfavourable climatic conditions (malnutrition, extreme temperatures, shading, and water stress) and genetic alteration, which is usually termed “panicle apical abortion” (Kobayasi et al., 2001; Kato et al., 2008; Bai et al., 2015). To date, only a few studies have set out to characterize the molecular mechanism of panicle abortion. The TOTOU1 gene was the first cloned pleiotropy gene associated with panicle degeneration, through its encoding of cyclic adenosine monophosphate receptor protein inhibitors. TOTOU1 mutation leads to panicle apical abortion and other defects including tiller reduction, leaf tip degeneration, and dwarfing (Bai et al., 2015). Squamosa promoter-binding protein-like 6 (SPL6) functions as a transcriptional repressor of inositol-requiring enzyme (IRE1), and acts as an essential survival factor for the suppression of persistent or intense stress in the endoplasmic reticulum, leading ultimately to cell death in rice. The spl6-1 mutant displays hyperactivation of IRE1, leading to cell death in spikelets in the panicle apex (Wang et al., 2018). Aluminum-activated malate transporter 7 (OsALMT7) is a malate transporter that functions in the development of panicle apical portions, and its mutation also results in reduced malate and cell death, particularly at the apical portion of the panicle (Heng et al., 2018). The paa1019 mutant is specifically defective in panicle development. PAA1019 encodes OsCIPK31, a calcineurin B-like-interacting protein kinase that affects the development of panicle apical spikelets (Peng et al., 2018). The degenerated panicle and partial sterility 1 (dps1) mutant also shows panicle apical degeneration and reduced fertility in middle spikelets. In addition, the amounts of cuticular wax and cutin are reduced significantly in dps1 anthers, and the accumulation of reactive oxygen species (ROS), lower antioxidant activity, and increased programmed cell death (PCD) have all been observed (Zafar et al., 2019).

In this study, we report on a novel rice mutant panicle apical abortion 3 (paa3), which exhibits the degeneration of spikelets at the tops of panicles during the late stage of panicle development. The results of gene cloning and complementation tests indicate that PAA3 is LOC_Os04g56160, encoding an H⁺-ATPase. Our data further suggest that PAA3 might function to remove peroxides, and the accumulation of ROS leads to PCD and ultimately panicle apical abortion in the paa3 mutant.

The paa3 Mutant Displayed a Semi-Dwarf Phenotype and Severe Panicle Apical Abortion

The paa3 mutant exhibited decreased plant height and grain defects (Fig. 1). The dwarf phenotype in the paa3 mutant was first observed at the tillering stage (Fig. 1a), and a semi-dwarf phenotype was observed at the maturation stage (Fig. 1b). Detailed analyses show that the lengths of both the internodes (from 1 to 4) and the panicle in the paa3 mutant were all significantly reduced compared to the wild type (WT), and internode 5 was not elongated in the paa3 mutant but was elongated in the WT (Fig. 1c, i). Both the grain number and size in the paa3 mutant were also affected (Fig. 1e–h). The numbers of PBs and SBs were all sharply reduced compared with the WT (Fig. 1l, m), which resulted in a significant reduction in the spikelet number per panicle (Fig. 1n). The number of grian in the paa3 panicles was also significantly decreased compared to the WT (Fig. 1o). In addition, both grain length and grain width, as well as 1000-grain weight in the paa3 mutant were all significantly reduced compared to those in the WT (Fig. 1e–h, p). In particular, unlike the WT plants in which all the spikelets developed normally, obvious panicle apical abortion was found in the paa3 panicle, in which the spikelets in the apical part of the panicle showed termination of development and were unable to seed (Fig. 1d). To determine when the abortion occurred, we investigated a series of mutant panicles at nine stages according to panicle length (~1, ~3, ~5, ~7, ~9, ~11, ~13, ~15, and ~17 cm). The paa3 spikelets at the top of the panicles began to show developmental delay at 11 cm, and the abortion phenotype became increasingly obvious from ~13 to ~17 cm (Fig. 2a1–a9). We further observed in detail the spikelets at the top of the paa3 panicles from ~9 to ~17 cm (Fig. 2b1–b10). At the ~9 cm panicles, there were no obvious differences between the WT and the paa3 spikelets (Fig. 2b1, b2). However, some of the spikelets at the top of the ~11 and ~13 cm panicles displayed white hulls and smaller sized paa3 mutants, whereas the WT had green hulls and were larger in size (Fig. 2b3–b6). In about ~15 cm paa3 panicles, the spikelet organs at the top of the panicles began to browning (Fig. 2b7, b8), and in the ~17 cm paa3 panicles, the spikelets were completely dry (Fig. 2b9, b10). We further used scanning electron microscopy to observe the hull development in the ~11 cm panicles and found the size of the epidemic cells in both the lemma and palea were significantly smaller than those in the WT (Fig. 2d1–d6). Trypan blue and DAB staining were also used to detect the level of cell death and H₂O₂ accumulation at ~13 cm in the WT and paa3 spikelets at the top of the panicles, and significantly deeper staining was observed in the paa3 spikelets than in the WT (Fig. 2c1-c4). Therefore, these results indicated that the panicle apical abortion related to PCD in spileklet development began to occurred in about ~11 cm paa3 panicles.According to these results, we speculated that the spikelets located at the top of the paa3 panicles stopped growing at ~9 to 11 cm, after which cell death occurred gradually. In addition, we observed the degree of spikelet abortion among the PBs for paa3 by statistical analysis of 15 paa3 panicles from 15 individual plants at the heading stage. The degree of spikelet abortion increased gradually from the lower to the upper PBs, and the upper spikelets were degraded at a higher rate than the lower ones (Fig. 2e1, e2).

PCD Occurs in the Spikelets of paa3 Panicles

To examine further if the panicle abortion phenotype was related to the PCD, we performed a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay, which showed nuclear DNA fragmentation at the single-cell level. In the ~7 cm panicles, no obvious TUNEL signal was detected in either the WT or the paa3 spikelet (Fig. 3a1–f1, a2–f2). In the ~11 cm panicles, however, strong TUNEL signals were observed in the anthers of the paa3 spikelets, and parts of the hull cells in the paa3 spikelets also showed clear TUNEL signals (Fig. 3j1–l1, j2–l2), whereas no TUNEL signal was seen in the WT spikelets (Fig. 3g1-i1,i2-i2). In the ~15 cm panicles, there was still no TUNEL signal in the WT spikelets (Fig. 3m1–o1, m2–o2), but the whole of the spikelet (including hulls, stamens, and sterile lemma) in the paa3 mutant showed very strong TUNEL signals (Fig. 3p1–r1, p2–r2). These results suggest that DNA fragmentation and cell death started to occur in the paa3 spikelets between the stages of ~7- ~11 cm, and reached a limition at the stage of ~15 cm, consistent with the results of phenotypic analysis.

Map-based Cloning of the PAA3 Gene

The paa3 mutant was crossed with the sterile line 56S. All F₁ hybrids displayed a WT phenotype, and the F₂ progeny showed segregation between WT and mutant phenotypes in a 3:1 ratio, indicating that the mutant trait was controlled by a single recessive gene. In the F₂ progeny, 58 individuals exhibiting a mutant phenotype were used as the mapping population. Using bulked segregant analysis, the PAA3 gene was mapped to the long arm of chromosome 4 within an approximately 88 Kb region between the simple-sequence repeat (SSR) markers CHR4-P118-6 and CHR4-P118-9. In this region, a single-nucleotide substitution from G to A (Gly to Asp) was identiﬁed in the 13th exon of LOC_Os04g56160 (Fig. 4a).The complementary expression vector containing the LOC_Os04g56160 coding sequence (6406 bp), the 3251 bp upstream sequence from the start codon, and the 1068 bp downstream sequence from the stop codon was then transformed into the paa3 mutants. In total, 28 transgenic plants were obtained, of which 15 showed rescue of the mutated phenotypes (Fig. 4b, c). We used two pairs of primers for amplification to detect the 15 transformants (comF1-GUSR1 for exogenous vector; comF2-comR2 for endogenic sites) (Fig. 4d). The sequencing results showed that the comF1-GUSR1 fragment was homozygous WT genotype and the comF2-comR2 fragment was homozygous mutation genotype, indicating that the exogenous complementary plasmid had been transformed successfully with the paa3 mutant (Fig. 4e). Therefore, these results toghther indicated that LOC_Os04g56160 was the PAA3 gene.

Expression Pattern Analysis and Subcellular Location of the PAA3

To explore the spatiotemporal expression of PAA3 in rice, we firstly applied quantitative PCR (qPCR) to examine PAA3 expression in the WT plants. When its expression was detected in all rice organs analysed, PAA3 was relatively highly expression in the stems, roots, and panicles (Fig. 5a). Next, we generated stable transgenic rice plants expressing the β-glucuronidase (GUS) reporter gene driven by a 3138 bp promoter sequence of PAA3 (Fig. 5b). Strong GUS staining was detected in the roots, stems, young panicles, and spikelets (Fig. 5c–e, h), while faint staining was observed in the leaves and sheathes (Fig. 5f, g), similar to the qPCR results. Notably, a strong GUS signal was detected in the hulls of the spikelets and stems (Fig. 5i–j).

PAA3 encoded a H⁺-ATPase, which was involved in blue light (BL)-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species (Toda et al., 2016). However, its subcellular localization was still unclear. In this study, to clarify its subcellular location, we fused PAA3 with the green fluorescent protein (GFP) reporter gene and expressed the PAA3::GFP fusion protein in rice protoplasts. In cells expressing PAA3::GFP protein, the GFP signal was observed mainly in the cytoplasm but not in the vacuole (Fig. 5k). These results suggest that PAA3 protein might be localized in the cytoplasm.

Overaccumulation of ROS Induces PCD in Panicle Apical Spikelets

ROS act as an important trigger of PCD, and excessive accumulation of hydrogen peroxide (H₂O₂) can trigger cell death (Mittler, 2017). In the paa3 panicles, DAB staining had revealed higher levels of ROS accumulation than that in the WT (Fig. 2c1–c2). Here, We further measured H₂O₂ content in panicles of WT and paa3, and found an H₂O₂ blast at the ~11 cm stage in the paa3 panicle (Fig. 6a). Malondialdehyde (MDA) accumulation is considered to be an indicator of lipid peroxidation and cell death (Chen and Murata, 2002). We therefore measured MDA content and found that the MDA levels were raised significantly in the 11 and 15 cm paa3 panicles compared to the WT (Fig. 6b).

Next, some of the genes related to ROS or PCD were investigated. Vacuolar processing enzymes (VPEs) are involved in PCD in Arabidopsis ( Kuroyanagi et al., 2005). There are only four VPE homologs (OsVPE1,OsVPE2, OsVPE3, OsVPE4) in rice, of which OsVPE2 and OsVPE3 play crucial roles in H₂O₂-induced PCD (Deng et al., 2011). We therefore measured the expression of both OsVPE2 and OsVPE3 in the WT and the paa3 panicle at the ~7, ~11, and ~15 cm stages. The expression of OsVPE2 in paa3 was similar to that of the WT at the ~7 cm stage, but was increased significantly at the ~11 and ~15 cm stages comprared to those of the WT (Fig. 6c). The expression of OsVPE3 in the paa3 panicles was lower than that in the WT panicles at the ~7 cm stage but was higher than that at the ~11 and ~15 cm stages (Fig. 6d). Catalase (CAT) is the key peroxidase in the biological defence system, playing a role in converting excessive H₂O₂ into oxygen. OsCATA, OsCATB, and OsCATC encode CAT isozymes in rice (Zhang et al., 2016). Our results show that all three genes were expressed at higher levels in paa3 than those in the WT panicles (Fig. 6e–g).

To provide further clarification of the mechanisms underlying ROS accumulation in the paa3 panicle, we conducted transcriptome analysis. A total of 1075 differentially expressed genes (DEGs) were characterized, including 991 upregulated and 84 downregulated genes. The number of upregulated DEGs was 11.79 times the number of downregulated DEGs (Fig. S1). Next, Gene Ontology (GO) analysis showed that DEGs related to the oxidation response (GO: 0006979) and oxidoreductase activity (GO: 0016705) were enriched significantly, most of which were upregulated in the paa3 mutant (Fig. 7a, b). Further qPCR was used to verify the expression of some of these DEGs including Os04g10160, Os01g43750, Os11g29290, and Os02g36030 in “oxidoreductase activity”; and Os04g59190, Os04g59150, Os01g73200, and Os04g59260 in response to “oxidative stress”. Compared to the WT, expression of Os04g10160 and Os04g59190 was upregulated nearly 20-fold in the paa3 mutant, and expression of the other genes was upregulated more than 4-fold (Fig. 7c). These results suggested that PAA3 exactly played a key role in the removal of ROS in rice.

In this study, we characterized a novel panicle development mutant paa3, which exhibited serious spikelet degeneration at the apical portion of panicle. Along with a reduced number of branches and 1000-grain weight, panicle apical portion resulted in a severe reduction of grain yield in the paa3 mutant. During rice growth and development, panicle development is crucial for grain yield in rice. Panicle apical portion is a common reason for the low seed-setting rate in rice (Heng et al., 2018). A number of environmental, physiological, and genetic factors can cause panicle apical portion and reduction in grain yield including climatic conditions (Yao et al., 2000), hormonal imbalance (Wang et al., 2018), nutrient deficiency (Durbak et al., 2014), and mutations of genes including tut1, paab1-1, spl6, paa1019, and dps1 (Bai et al., 2015; Heng et al., 2018; Peng et al., 2018; Wang et al., 2018; Zafar et al., 2019). When panicle apical portion in tut1, spl6, and paa1019 occurred before the heading stage, it occurred in late panicle development in the paab1-1 and dps1 mutants, similar to paa3 in this study. With the exception of the abortion phenotype, similar to tut1, the seed size of the paa3 also became smaller, while it did not change in the paab1-1, spl6, paa1019, and dps1 mutants. In addition, a series of agronomy traits, such as panicle length, number of spikelets per panicle, setting percentage, and 1,000-grain weight were all decreased in these mutants. Therefore, these studies suggested that the genes related to panicle apical portion might have a wide effect on panicle development.

PCD induced by ROS accumulation in the panicle might play a key role in most panicle abortion mutants in rice. OsALMT7 encoded an aluminum-activated malate transporter in rice. Malate was a central metabolite in the plant cell and was involved in the mitochondrial tricarboxylic acid and glyoxylate cycles in plant species, and can participate in redox reactions to produce NAD (H) or NADP (H) to maintain the balance of intracellular redox. In the paab1-1 mutant, the loss-of-function mutant of OsALMT7, reduced malate might disrupted the redox balance in the panicle cells, leading to the accumulation of ROS and the death of panicle cells (Heng et al., 2018). DPS1 encoded a CBSDUF protein, and could interact with Trx proteins (Trx1 and Trx20) to regulate ROS homeostasis in rice panicle development. Loss-of-function DPS1 accumulated more ROS in defective panicles, and also induced cell death and panicle apical degeneration (Zafar et al., 2019). In this study, our results also strongly supported the involvement of PAA3 in ROS removal, and overaccumulation of ROS triggers PCD in the apical portion of the paa3 panicle.

PAA3 encoded OSA7, a plasma membrane H⁺-ATPase that was a member of the ATPase superfamily. The structure of the plasma membrane H⁺-ATPase was highly conserved from fungi to higher plants, with the exception of the C-terminal region (Wang et al., 2014). Depending on the structure of the C-terminal region, the plasma membrane H⁺-ATPase could be divided into two types: the penultimate threonine (Thr)-containing H⁺-ATPase (pT H⁺-ATPase) and the non-penultimate Thr-containing H⁺-ATPase (non-pT H⁺-ATPase) (Okumura et al., 2012); OSA7 belonged to the former group. The plasma membrane H⁺-ATPases were involved in many aspects of biology including BL-induced stomatal opening of dumbbell-shaped guard cells in the monocotyledon species, the uptake of phosphorus by the roots, and sustained pollen tube growth and fertilization (Chang et al., 2008; Toda et al., 2016; Hoffmann et al., 2020). A Tos17 insertion mutant of OSA7 resulted in impairment of the BL-induced stomatal opening, and ultimately a reduced transpiration rate, suggesting that OSA7 was involved in the BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species (Toda et al., 2016). However, to date only a few H⁺-ATPase isoforms had been identified and their physiological roles have proven difficult to analyse, given that no phenotypes of H⁺-ATPase mutant have been reported. In this study, the PAA3 mutation resulted in excessive accumulation of ROS followed by PCD in the panicle, providing a novel perspective to explore the function of H⁺-ATPases in the future.

A novel paa3 mutant was identified in rice and showed severe panicle apical apportion and semi-dwarf. PAA3 encoded OSA7, a plasma membrane H⁺-ATPase and was highly expressed in stems and panicle. The TUNEL assay showed that the DNA fragmentation and cell death in paa3 spikelets started to occur between the stages of ~ 7 and ~ 11 cm panicles. DAB staining and measurement of H₂O₂ and MDA content showed overaccumulation of ROS in the paa3 spikelets at the ~ 11 cm stage. Staining with trypan blue showed cell death in paa3 spikelets at the stage of ~ 13 cm. The expression of genes involving in H₂O₂-induced PCD also indicated over-accumulation of ROS in the paa3 spikelets. Taken together, the results of this study indicate that PAA3 play a key role in mainting the panicle development through ROS removal.

Plant Materials and Growth Conditions

In this study, the paa3 was derived from the ethyl methane sulfonate mutant library of the maintainer XIDA1B (1B). The paa3 mutant was crossed with the sterile line 56S to obtain the F₁ generation, and the F₁ generation was self-crossed to obtain the F2 population. All plant materials were grown in the experimental fields of the Rice Research Institute of Southwest University (Chongqing, China).

Scanning Electron Microscopy

The panicles in both the WT and the paa3 mutant were examined using a scanning electron microscope (SU3500; Hitachi, Tokyo, Japan) with a -20°C cooling stage under a low-vacuum environment, when the paa3 panicle began to show a panicle abortion phenotype. At the flowering stage, spikelets from paa3 and WT plants were observed using a stereomicroscope (SMZ1500; Nikon, Tokyo, Japan).

TUNEL Assay

Apical spikelets of the WT and paa3 panicles at the panicle lengths 7, 11, and 15 cm were collected and fixed in formalin-acetone-alcohol solution for 48 h, soaked in paraffin, embedded, sliced, and baked for 3 days. The spikelets were then dewaxed with xylene, dried, and incubated with protease K for 10 min, soaked in phosphate-buffered saline (PBS) for 5 min, and soaked in 4% methanol-free formaldehyde solution for 5 min. After adding 50 mL rTdT, the specimen was incubated at 37°C for 3 h, followed by incubation with 2x SSC solution in the dark for 15 min and with PBS for 5 min; this procedure was repeated three times. Specimens were incubated with propidium iodide (PI) solution for 15 min to prevent infiltration, and soaked in water for 5 min; this procedure was repeated three times. Finally, the tablets were sealed with sealant (PBS and glycerin 1:1) for observation. The green ﬂuorescence of ﬂuorescein (TUNEL signal) and red ﬂuorescence of propidium iodide were analysed at 488 nm (excitation) and 520 nm (detection), and 488 nm (excitation) and 610 nm (detection), respectively, under a confocal laser scanning microscope (LSM710; Zeiss, Jena, Germany).

Staining and Quantitative Measurement of ROS and Measurement of MDA Content

We monitored cellular ROS levels in apical degenerated spikelets using DAB staining to detect H₂O₂. According to a method described previously (Wu et al., 2017), we used DAB to stain the top spikelets of WT and paa3, and quantified ROS by measuring H₂O₂. Fresh panicles (1g) were collected and then measured using the H₂O₂ assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). To measure MDA content, we collected 1g spikelets from the apical part of the WT and paa3 panicles. Then, according to the instructions of the MDA Assay Kit (Nanjing Jiancheng Bioengineering Institute), we measured the MDA content. All experiments were conducted on panicles at the tillering stage.

Trypan Blue Staining

Trypan blue staining was used to examine PCD. The panicles of mutant and WT at the 13 cm stage were immersed in a boiling solution of trypan blue for 10 min, and then removed and left at room temperature for 12 h. This was then immersed in a solution of chloral hydrate at a concentration of 2.5 mg/mL to allow decolourization. Finally, the decolourized samples were stored at 50% glycerin. Staining was observed using a stereomicroscope.

Map-Based Cloning

The paa3 mutant was crossed with the sterile line “56S” to generate F₁. The 58 F₂ plants that exhibited a mutant phenotype were selected as the mapping population. SSR repeat markers from publicly available rice databases, including Gramene (http://www. gramene.org), the Rice Genomic Research Program (http://rgp.dna.affrc. go.jp/public data/caps/index.html), and In/Del markers designed by our group according to re-sequence of XD1B and 56S genome, were used for fine-mapping of PAA3. The primer sequences used for mapping and identification of transgenic plants are listed in Table S1.

Vector Construction and Transformation

To construct the complementation plasmid, a 10725 bp genomic fragment that contained the PAA3 coding sequence, coupled with the 3251 bp upstream and 1068 bp downstream sequences, was ampliﬁed using the primers PAA3-com-F (EcoR1) and PAA3-com-R (HindIII). The fragment was inserted into the binary vector pCAMBIA1301 using the pEASY^®-Uni Seamless Cloning and Assembly Kit (TransGen, Beijing, China). The recombinant plasmids were transformed into the paa3 mutant using the Agrobacterium tumefaciens-mediated transformation method as described previously (Hui et al., 2020). For the PAA3P::GUS assays, the promoter of PAA3 gene (3138 bp) was ampliﬁed using the primers PAA3P-GUS-F (EcoR1) and PAA3P-GUS-R (Nco1). The fragment was inserted into the binary vector pCAMBIA1301 using the pEASY^®-Uni Seamless Cloning and Assembly Kit (TransGen, Beijing, China). The recombinant plasmids were transformed into the japonica cultivar ZHONGHUA 11 (ZH11) using the A. tumefaciens-mediated transformation method as described previously (Hui et al., 2020). The primers used for vector construction are listed in Table S1.

PAA3P::GUS Staining

For promoter activity analysis, a 3138 bp genomic fragment, which is the promoter of the PAA3 gene, was PCR-ampliﬁed from WT genomic DNA with the primer pair PAA3P-GUS-F and PAA3P-GUS-R (Table S1), and fused to the GUS reporter gene in the vector pCAMBIA1301. GUS staining was performed on PAA3P::GUS T₀ generation transgenic plants in accordance with a previous method (Jefferson, 1989). After bleaching with ethanol, photographs were taken using a stereomicroscope.

Subcellular Localization of the PAA3 Protein

The full-length coding region (without the termination codon) of PAA3 was amplified using the SL-PAA3-F (spe1) and SL-PAA3-R (sma1) primers. The fragment was cloned into the expression cassette pAN580-35S:: GFP to generate the pAN580-35S::PAA3-GFP fusion vector. The pAN580-35S::GFP and pAN580-35S:: PAA3-GFP plasmids were then transformed into rice protoplasts. After incubation for 12–16 h at 28°C, GFP ﬂuorescence was detected using a confocal laser scanning microscope (LSM710; Zeiss, Jena, Germany). Primers used for subcellular localization are listed in Table S1.

DNA Extraction, RNA Isolation, and qPCR

Total DNA from WT and paa3 mutant was extracted using the cetyltrimethylammonium bromide method. Total RNA from root, stem, leaf, sheath, panicle, bud, and shoot was isolated using the RNA prep Pure Plant Kit (Tiangen, Beijing, China). The ﬁrst-strand complementary cDNA was synthesized from 2 µg total RNA using oligo(dT)₁₈ primers in a 20 µL reaction volume using the PrimeScript^® Reagent Kit with gDNA Eraser (Takara, Dalian, China). The qPCR analysis was performed using the SYBR^® Premix Ex Taq™ II Kit (Takara) in the ABI 7500 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). ACTIN (OsRac1, LOC_Os01g12900) was used as the endogenous control. At least three replicates were performed. Primers used for qPCR are listed in Table S1.

RNA-Sequencing Analysis

Analysis of RNA sequencing (RNA-seq) data was performed using a standard protocol (Trapnell et al., 2012). For RNA-seq analysis, RNA was extracted from WT and paa3 panicles of 11 cm in length that corresponded to the developmental stage just after the start of panicle abortion. All sequencing samples were treated. RNA sequencing was performed by Novogene Biotechnology (Beijng, China), and sequencing data were retrieved through the standard Illumina pipeline with custom and default parameters. HTSeq software was used to analyse the original sequences of known genes for all the samples (Novogene Biotechnology, Beijing, China), and the expression of known genes was calculated using the fragments per kilobase of transcript per million fragments mapped (FPKM). HTSeq was used to estimate gene expression levels. DEGs were identified considered P ≤ 0.05 and a log2 fold-change ≥ 1. Clusters were analysed by principal component analysis, and DEGs were analysed by DESeq, with a cut-oﬀ P ≤ 0.05 and fold change ≥ 2. GO analysis and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the signiﬁcantly enriched biological processes in paa3.

PAA3:PANICLE APICAL ABORTION 3; WT: wild-type; paa3: panicle apical abortion 3; AMs: Axillary meristems; PB: Primary branches; SB: Secondary branches; LS: Lateral spikelet; TS:Terminal spikelet; QTL:Quantitative trait locus; ROS: Reactive oxygen species; PCD: Programmed cell death; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; BSA: Bulked segregant analysis; MDA: Malondialdehyde; SSR: Simple sequence repeat; DAB: 3,3’-Diaminobenzidine; EMS: Ethyl methane sulfonate; CTAB: cetyltrimethylammonium bromide; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GFP: Green fluorescent protein; GUS: β- glucuronidase; qPCR: Quantitative PCR; TEM: Transmission electron microscopy.

Acknowledgements

We thank Benoit Lefebvre for critical reading of the manuscript.

Authors’ Contributions

YFL and FYY designed the research; FYY and MJH performed the mapping-based clone and phenotype analyses; FYY and ZCL performed the TUNEL assay; FYY, HZ, and RC performed the experiments to determine the subcellular localization of PAA3 protein; FYY and MX performed the qPCR; JT ,YW, QLC, ZYW, HHZ and LL contributed to the data analysis; FYY ,YFL and MX wrote the manuscript; All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31971919), the Natural Science Foundation Project of Chongqing Science and Technology Commission (No. cstc2020jcyj-jqX0020), and Chongqing Graduate Research and Innovation Project funding (No. CYS20123)

Availability of Data and Materials

The datasets supporting the conclusions of this article are included within

the article and its additional files.

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.

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STable1Allprimersforthisstudy.xlsx
SFigure1.pptx
Supplemental Figure 1, transcriptome analysis of paa3 a, Showed that there were 1075 differential genes in WT and paa3 samples, among which 991 genes were up-regulated and 84 genes were down-regulated. b The volcano figure showed an overall overview, including 28,317 genes with no change in expression, 991 up-regulated genes and 84 down-regulated genes.

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PANICLE APICAL ABORTION 3 Controls Panicle Development and Seed Size in Rice

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