# Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility

Sang-Kyu Lee, Hyunbi Kim, Jung-Il Cho, Cong Danh Nguyen, Sunok Moon, Jeong Eun Park, Hye Rang Park, Jin Hoe Huh, Ki-Hong Jung, Emmanuel Guiderdoni, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Sang-Kyu Lee, Hyunbi Kim, Jung-Il Cho, Cong Danh Nguyen, Sunok Moon, et al.. Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility. Journal of Experimental Botany, 2019, 71 (1), pp.116-125. 10.1093/jxb/erz436. hal-03128415

HAL Id: hal-03128415

https://hal.inrae.fr/hal-03128415

Submitted on 20 Jun 2022

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH PAPER

# Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility

Sang-Kyu Lee¹,†, Hyunbi Kim¹,†, Jung-II Cho¹,‡, Cong Danh Nguyen¹, Sunok Moon¹, Jeong Eun Park², Hye Rang Park², Jin Hoe Huh², Ki-Hong Jung¹, Emmanuel Guiderdoni³,⁴ and Jong-Seong Jeon¹,*

¹ Graduate School of Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 17104, Korea

² Department of Plant Science, Seoul National University, Seoul 08826, Korea

³ CIRAD, UMR AGAP, 34398 Montpellier, France

⁴ Université de Montpellier, CIRAD INRA Montpellier SupAgro, 34000 Montpellier, France

† Current address: Crop Production and Physiology Division, National Institute of Crop Science, Rural Development Administration, Wanju 55365, Korea

† These authors contributed equally to this work.

* Correspondence: jjeon@khu.ac.kr

Received 20 August 2019; Editorial decision 18 September 2019; Accepted 10 October 2019

Editor: Dabing Zhang, Shanghai Jiao Tong University, China

## Abstract

There is little known about the function of rice hexokinases (*HXKs*) in planta. We characterized *hxl5-1*, a *Tos17* mutant of *OsHXK5* that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of *hxl5-1* and reciprocal crosses between the wild-type and heterozygotes revealed that loss of *HXK5* causes male sterility. Homozygous *hxl5-1*, produced via anther culture, and additional homozygous *hxl5-2*, *hxl5-3* and *hxl5-4* lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. *In vitro* pollen germination ability and *in vivo* pollen tube growth rate were significantly reduced in the *hxl5* mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the *hxl5* male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

**Keywords:** Hexokinase, male sterility, *Oryza sativa*, pollen germination, pollen tube growth, starch.

## Introduction

In flowering plants, pollen formation and subsequent pollination and fertilization are crucial steps for sexual reproduction. Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to germinate quickly, to produce a pollen

tube derived from the vegetative cell that has polar growth, and to deliver two sperm cells into the embryo sac for double-fertilization (Johnson and Preuss, 2002; Lord and Russell, 2002; Dresselhaus et al., 2016). In the major cereal crop rice (*Oryza sativa*), sexual reproduction is the stage with the greatest sensitivity
to environmental stress. For example, low or high temperatures can lead to defects in pollen development, germination, and tube growth, which affect spikelet fertility and grain yield (Andaya and Mackill, 2003; Jagadish et al., 2010; Coast et al., 2016).

Rice pollen loses germination activity in a relatively short period of time. For example, pollen viability drops rapidly by nearly 50% between 6 min and 20 min after anther dehiscence (Khatun and Flowers, 1995; Fu et al., 2001; Song et al., 2001; Zak and Irish, 2003). Highly efficient and rapid pollen germination and tube growth are critical for normal fertilization and spikelet fertility (Endo et al., 2009; Jagadish et al., 2010; Rang et al., 2011). Therefore, it is important to produce and maintain robust pollen grains for full fertility and complete seed set in rice. Despite its importance, little is currently known about the detailed carbohydrate metabolic pathway that is active during the late stages of pollen grain development when starch biosynthesis and degradation mainly occur.

In common with the other major cereal crops maize, wheat, and barley, rice utilizes starch as the main storage reserve in mature pollen grains. Starch is degraded as soluble sugars and used for supplying energy and carbon skeletons. In addition, it is also used for osmotic homeostasis in order to ensure pollen germination and tube growth, thus supporting proper pollination and fertilization (Dickinson, 1968; Wen and Chase, 1999; Datta et al., 2002; Hirose et al., 2010; Rounds et al., 2011; Hepler et al., 2013; Lee et al., 2016; Wu et al., 2016). Insufficient starch biosynthesis during the process of pollen development is responsible for male sterility in these crop species. For example, mutations in rice *Plastidic Phosphoglucomutase* and *ADP-Glucose Pyrophosphorylase4* reduce starch content and result in male sterility (Lee et al., 2016). In addition, degradation of starch in pollen grains through the expression of α-amylase induces male sterility in maize, which provides an experimental strategy for creating male sterility (Wu et al., 2016).

Relatively few genes involved in carbohydrate metabolism whose mutations affect pollen germination and tube growth have been functionally characterized. For example, mutations in rice *Sucrose Transporter1* (*SUT1*) lead to male sterility. The pollen grains of the *sut1* mutant normally accumulate starch during development, but do not germinate and participate in fertilization with the ovule (Hirose et al., 2010; Eom et al., 2012, 2016). Disruption of rice *Sucrose Phosphate Synthase1* (*SPS1*) results in sterile pollen grains (Hirose et al., 2014). The *sps1* mutant accumulates sufficient starch in the pollen, but its germination efficiency is reduced to half that of the wild-type, which suggests that OsSPS1 is essential in pollen germination.

Hexokinase (HXK) catalyses the first irreversible step of hexose metabolism by phosphorylating hexoses into hexose-6-phosphate. In sink organs, including pollen, this reaction directs the cleavage products of imported sucrose into the starch biosynthesis pathway. The HXK catalysis reaction also initiates all the physiologically relevant hexose utilization pathways, including glycolysis (Cho et al., 2006a, 2006b, 2009; Claeyssen and Rivoal, 2007). In rice, there are 10 HXK isoforms (Cho et al., 2006a), which raises questions about the specific roles of each one. It has been shown that different HXKs are localized to different subcellular compartments, such as the cytosol, mitochondria, and chloroplasts (Cheng et al., 2011). Few OsHXK

genes have been examined in loss-of-function mutants. For example, the cytosolic hexokinase HXK7 appears to play an important role in O₂-deficient germination since the *hxl7* mutant shows slow germination in anoxic conditions (Kim et al., 2016). In addition, rice *HXK10*-RNAi lines are male-sterile, probably due to a defect in anther dehiscence (Xu et al., 2008).

The OsHXK5 protein has been detected in proteomic studies on germinating pollen grains (Dai et al., 2007), implying that it functions during pollen germination and tube growth. We have previously found that OsHXK5 is primarily localized in mitochondria, and possibly in the nucleus, with a portion functioning as a sugar sensor (Cho et al., 2009). Here, we isolated and characterized mutant alleles of OsHXK5, and genetic, phenotypic, and biochemical analysis of these mutants revealed a novel function of hexokinase that is essential for pollen development, germination, and tube growth in rice.

## Materials and methods

### Plant material

The *Oryza sativa* subsp. *japonica* wild-type rice cultivars Dongjin (DJ) and Nipponbare (NP) together with the mutants were grown in a greenhouse under a day/night cycle of 14/10 h at 30/20 °C with ~80% humidity. Anthers from wild-type (DJ) plants were harvested at different developmental stages for quantitative RT-PCR (qRT-PCR) analysis.

### Isolation and production of the OsHXK5 mutants

The OsHXK5 (LOC_Os05g44760) mutant allele *hxl5-1* was isolated from the Génoplante Insertion Line Library through a screen of the Signal RiceGE (Rice Functional Genomic Express Database; http://signal.salk.edu/cgi-bin/RiceGE) (Sallaud et al., 2004). Genotypes of the *hxl5-1* mutant were determined by genomic DNA PCR using the *Tos17*-specific T1 primer (5'-CCAGTCCATTGGATC TTGTATCTTGTATATAC-3') and the OsHXK5 gene-specific F1 (5'-TATATGGGGTCCTAAAGAAGCTA-3') and R1 (5'-AAAAAGATGGTAAGATTTCAAGG-3') primers.

The other three mutant alleles, *hxl5-2*, *hxl5-3*, and *hxl5-4*, were generated using the CRISPR/Cas9 system. To find an effective protospacer adjacent motif (PAM) and avoid any off-target effects, we screened possible target sequences using the CRISPRdirect program (http://crispr.dbcls.jp/; Naito et al., 2015). Guide RNA (5'-GCGGGGCATCTCGGACGCCA-3') was cloned into an entry vector, pOS-sgRNA, and then cloned into a destination vector, pH-Ubi-cas9-7, using the Gateway system (Miao et al., 2013; Lee et al., 2016). The resulting vector was transformed into the wild-type (DJ) by *Agrobacterium* mediation (Jeon et al., 2000). Genotypes of the *hxl5-2*, *hxl5-3*, and *hxl5-4* were determined by Sanger sequencing of the target PAM site. PCR amplicons were generated using gene-specific F2 (5'-GGTTAAAACGCCCAAGTCAGTG-3') and R2 (5'-AAGGTCAGTCGGTCCTAATCAGAG-3') primers, purified, and subsequently subjected to DNA sequencing.

### RNA isolation and RT-PCR analysis

Total RNA was prepared from leaves, pollen grains, and anthers using Trizol reagent, and was reverse-transcribed using the ReverTra Ace qPCR RT Master Mix cDNA Synthesis kit (Toyobo, Tokyo, Japan). To evaluate the expression patterns of OsHXKs, we prepared samples from anthers at four different developmental stages. First-strand cDNA was used in PCR reactions with gene-specific and control primers. All primers for qRT-PCR are listed in Supplementary Table S1 at JXB online. The *Ubiquitin5* gene (*OsUbi5*; LOC_Os01g22490) was used as an internal PCR control.
## Anther culture

Anthers were collected from spikelets at a stage when the distance between the flag leaf and the penultimate leaf was 3–5 cm. The anthers were then cultured on modified N<sub>6</sub> media to induce calli and subsequently plants, as previously described (Eom *et al.*, 2016).

## Flow cytometry analysis

Samples of ~20 mg of leaves were finely chopped with a clean razor blade in 1 ml of ice-cold Tris-MgCl<sub>2</sub> buffer (0.2 M Tris, 4 mM MgCl<sub>2</sub>, 0.5% Triton X-100, pH 7.5) in a glass Petri dish on ice (Pfosser *et al.*, 1995). Nuclei were stained in 50 µg l<sup>-1</sup> propidium iodide solution with 50 µg l<sup>-1</sup> RNase, filtered through a 40-µm cell strainer, and kept on ice. Flow cytometry was performed with a medium flow rate in a FACS Canto II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the data acquired were analysed using the BD FACSDiva software. An FL2 detector was used to measure fluorescence, and phycoerythrin, peridin chlorophyll protein, forward scatter, and side scatter parameters were used for analysis according to the manufacturer's instructions.

## Pollen staining and in vitro germination assays

Mature pollen grains were placed directly into 10% Lugol's solution for starch staining. Pollen germination was performed on artificial media containing 20% sucrose, 0.32 mM H<sub>3</sub>BO<sub>3</sub>, 1 mM CaCl<sub>2</sub>, and 1% agarose under high humidity conditions (Kariya, 1989; Hirose *et al.*, 2014). After 30 min, germinated pollen grains were observed using a BX61 microscope (Olympus), and were stained with 10% Lugol's solution.

## In vivo pollen-tube growth assays

Spikelets were collected at various times after the panicles were fully pollinated, and fixed overnight with a 3:1 (v/v) mixture of absolute ethanol and glacial acetic acid. The spikelets were then washed in a vial of distilled water. Dissected ovaries were transferred to 2 N NaOH, treated overnight to clear the tissue, and then stained for 5 h with 0.2% aniline blue in 0.1 M K<sub>2</sub>HPO<sub>4</sub>. The stained ovaries were mounted on glass slides and fluorescence was observed using a 365-nm excitation filter and a 397-nm barrier filter on a BX61 microscope (Olympus).

## Measurement of hexokinase activity

Samples of ~3 mg of anthers containing mature pollen grains were resuspended in 100 µl of lysis buffer containing 50 mM Tris-HCl (pH 7.6), 5 % (v/v) glycerol, 5 mM EDTA, 5 mM DTT, 1× proteinase inhibitors, and 1 mM PMSF. The samples were collected at the bottom of a tube by centrifugation at 1000 g for 5 min at 4 °C, and then ground on ice using a pestle in a 1.5-ml tube. Soluble proteins were obtained by centrifugation at 15 000 g for 10 min at 4 °C and used for the enzyme assay. Hexokinase activity was measured using a NAD(P)H-coupled enzymatic method (Cho *et al.*, 2009).

## Determination of starch and soluble sugars

Samples of ~3 mg of anthers containing mature pollen grains were harvested at the end of the light period from mature panicles. The soluble sugars glucose, fructose, sucrose, and insoluble starch were measured using NAD(P)H-coupled enzymatic tests in the ethanol soluble and insoluble fractions as described by Lee *et al.* (2008). The measured metabolite contents were normalized to the anther fresh weight.

## Production of transgenic rice expressing catalytically inactive OsHXK5

To introduce a catalytically inactive mutant form of OsHXK5 into the *hxlk5* null-mutant, we cloned the OsHXK5-G113D mutant form (Cho *et al.*, 2009) between the maize *Ubiquitin1* promoter and four Myc tag sequences, followed by the Nos terminator of the Gateway binary vector,

pJJ4385, which contains the BASTA resistance gene as a selection marker. The resulting vector was transformed into *hxlk5-1* by *Agrobacterium*-mediated (Jeon *et al.*, 2000). To detect the OsHXK5-G113D-Myc protein in leaves and in anthers with mature pollen, protein gel-blot analysis was performed using the Myc antibody (Rabbit anti-c-myc antibody; A190-105A, Bethyl Laboratories, Montgomery, TX, USA) (Lee *et al.*, 2008). Six lines were selected and were designated as OsHXK5<sup>G113D</sup>.

# Results

## Preferential expression of HXK5 during late stages of pollen development

We have previously identified 10 rice hexokinases and shown that they are expressed in various tissues, including leaves, roots, seed, and flowers (Cho *et al.*, 2006a). To identify HXKs that are expressed during late stages of pollen development and germination, we examined six Affymetrix rice microarray data series prepared from developing anthers and pollen grains in the NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/) (Nguyen *et al.*, 2016) (Supplementary Fig. S1). The analysis showed that OsHXK5 (LOC_Os05g44760) is expressed throughout all stages but is specifically expressed at high levels during the mature pollen and pollen germination stages. In contrast, expression of OsHXK2 (LOC_Os05g45590) was detectable only until the tricellular pollen stage, and expression of the other HXKs was barely detected during anther and pollen development. We were not able to examine expression of OsHXK6 (LOC_Os01g53930), a close homolog gene of OsHXK5, because the OsHXK6 probe was unavailable in the public array data (Supplementary Fig. S1).

To confirm expression of *HXK5*, we performed qRT-PCR analysis on anthers collected at different developmental stages from meiosis to mature pollen (Fig. 1A). As the anthers matured, expression of *HXK5* gradually increased and was highest at the mature pollen stage, which was consistent with the microarray data. In our analysis, *HXK6* appeared to be expressed at relatively low levels during all stages. In contrast, *HXK2* and *HXK10* were expressed weakly at the mature stage, and the other *HXKs* were not expressed at any of the stages. Further analysis in germinating pollen grains indicated relatively high expression of *HXK5* and relatively low expression of *HXK6* (Fig. 1B). This suggested that *HXK5* plays an important role during late pollen development and germination stages in rice.

## Isolation and progeny analysis of the OsHXK5 mutant

To understand the *in vivo* function of OsHXK5, we isolated *hxlk5-1* from the Génoplante Insertion Line Library (Sallaud *et al.*, 2004). The mutant allele harbored a Tos17 retrotransposon in the ninth exon of OsHXK5 (Fig. 2A). We performed genomic DNA PCR analyses using gene- and Tos17-specific primers to isolate homozygotes from the segregating progeny of self-pollinated heterozygous *hxlk5-1* mutant plants. This showed no homozygous Tos17 insertion mutant line (*hxlk5-1/hxlk5-1*); all appeared to be either wild-type (NP) or heterozygote (*HXK5/hxlk5-1*; He) (Fig. 2B). The progeny segregation ratios from self-pollinated heterozygous plants were nearly 1:1:0 for the wild-type, heterozygote, and homozygote plants,
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Expression of rice hexokinase *HXK* genes in anthers at different stages of pollen development. (A) Quantitative RT-PCR analysis of *HXKs* in anthers from meiosis to the mature pollen stage. (B) Quantitative RT-PCR analysis of *HXK5* and *HXK6* in mature pollen and germinating pollen. Data are means (±SE) of three replicates.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Molecular characterization of the rice *HXK5* mutants *hxl5-1*, *hxl5-2*, *hxl5-3*, and *hxl5-4*. (A) Schematic diagram of *Tos17* in *hxl5-1* and the CRISPR/Cas9-generated indel mutant alleles. The nine exons of *OsHXK5* are indicated by the numbered boxes. Primers for genotyping are indicated with arrows. *Tos17* is inserted into the ninth exon of *OsHXK5*. The mutants carrying one and two nucleotide insertions, and one nucleotide deletion are designated as *hxl5-2*, *hxl5-3*, and *hxl5-4*, respectively, and are highlighted in red. (B) PCR analysis of genomic DNA from the progeny of self-fertilized *HXK5/hxl5-1* plants. An F1/R1 primer set was used for the wild-type *HXK5* allele (top) and an F1/T1 primer set was used for the *hxl5-1* allele (bottom). NP, mutant background cv. Nipponbare genotype; He, heterozygote. (C) Sequence alignment of the CRISPR/Cas9 target sites in the *OsHXK5* mutant alleles. The target site protospacer adjacent motif is highlighted in blue.</figcaption></figure>

respectively (47:45:0; Table 1). This clearly suggested a defect in either the male or female gametophyte of the *hxl5-1* mutant allele during the process of genetic inheritance.

## Genetic transmission analysis of the *hxl5-1* mutant allele

To determine whether the gametophytic defect was caused by the male or female component, we performed reciprocal

crosses between wild-type and *hxl5-1* heterozygotes (Table 2). When the *hxl5-1* heterozygote was used as a pollen donor, none of the 45 F₁ plants yielded the heterozygous genotype; all were wild-type. However, when the heterozygous line was used as a female donor, the genotypes of 44 F₁ plants were nearly 1:1 for the wild-type (23) and the *hxl5-1* heterozygote (21). This clearly indicated that the *hxl5-1* mutant pollen was sterile and suggested a crucial role of *OsHXK5* in male fertility.
## Production and characterization of the *hxl5-1* homozygous mutant

To further examine the functions of *HXK5* during rice pollen development, pollination, and fertilization, we produced homozygous plants via anther culture of the *hxl5-1* heterozygote. Unsurprisingly, approximately half of the regenerated plants were wild-type and the other half were *hxl5-1* homozygous mutants. The regenerated *hxl5-1* homozygous plants were confirmed by genomic DNA PCR (Fig. 3A). In the RT-PCR analysis of the homozygous mutants, the *HXK5* transcript was not detected in

leaves or in anthers harboring pollen, whereas the transcript was abundant in both these tissues in the wild-type plants (Fig. 3B). Homozygous plants produced via anther culture grew normally and were similar to the wild-type during vegetative growth (Fig. 3C). In flow cytometry analysis, the *hxl5-1* homozygous plants showed the same diploid levels as the wild-type plants, representing the 2C DNA content (Supplementary Fig. S2A). However, the homozygous *hxl5-1* plants bore a limited number of seeds, resulting in fertility of ~5–10% with a mean of 7.4% in our greenhouse growth conditions (Fig. 4A, B). Notably, the self-pollinated seeds produced from the *hxl5-1* homozygous mutant were confirmed to be homozygotes (Supplementary Fig. S2B), which excluded cross-pollination with the wild-type.

<table><caption>**Table 1.** Segregation in self-pollinated rice *hxl5-1* heterozygous mutant plants</caption><thead><tr><th rowspan="2">Parent plant</th><th colspan="3">Percentage of observed/expected genotypes of progeny (actual numbers recorded)</th></tr><tr><th></th><th></th><th></th></tr></thead><tbody><tr><td rowspan="2">HXK5/hxl5-1</td><td>Wild-type</td><td>HXK5/hxl5-1</td><td>hxl5-1/hxl5-1</td></tr><tr><td>51.1/25 (47/92)</td><td>48.9/50 (45/92)</td><td>0/25 (0/92)</td></tr></tbody></table>

Genotypes of progeny plants were determined by PCR using gene- and *Tos17*-specific primers.

<table><caption>**Table 2.** Results of reciprocal crosses between the rice wild-type and *hxl5-1* heterozygous plants</caption><thead><tr><th colspan="2">Genetic cross</th><th colspan="2">Percentage of observed/<br>expected genotypes of progeny<br>(actual numbers recorded)</th></tr><tr><th>Paternal</th><th>Maternal</th><th>Wild-type</th><th>HXK5/hxl5-1</th></tr></thead><tbody><tr><td>HXK5/hxl5-1</td><td>Wild-type</td><td>100/50 (45/45)</td><td>0/50 (0/45)</td></tr><tr><td>Wild type</td><td>HXK5/hxl5-1</td><td>52/50 (23/44)</td><td>48/50 (21/44)</td></tr></tbody></table>

Genotypes of *F*₁ plants were determined by PCR using gene- and *Tos17*-specific primers.

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Molecular and phenotypic analysis of rice *hxl5-1* mutants produced via anther culture. (A) Genotype analysis of the *hxl5-1* homozygous mutant. An F1/R1 primer set was used for the wild-type (WT) *HXK5* allele (top), and an F1/T1 primer set was used for the *hxl5-1* allele (bottom). (B) Expression analysis of *HXK5* in leaf tissues and anthers harboring mature WT and *hxl5-1* pollen grains. *OsUBQ5* was used as the internal PCR control. (C) Mature WT and *hxl5-1* plants grown in soil for 8 weeks.</figcaption></figure>

## Production and characterization of additional *hxl5* homozygous mutant alleles

To further confirm the male-sterile phenotype of *hxl5-1*, we utilized the CRISPR/Cas9 system as an alternative to anther culture for producing homozygous mutant lines. A region in the first exon was selected as the guide RNA target. Of 16 independent transgenic lines obtained, three were homozygous mutants at the target site lines, namely *hxl5-2*, *hxl5-3*, and *hxl5-4*. The *hxl5-2*, *hxl5-3*, and *hxl5-4* alleles (background cv. DJ) respectively had one nucleotide (T) insertion, two nucleotide insertions (AC), and one nucleotide deletion (G) (Fig. 2A, C). The fertility of these plants ranged from ~5–10%, with mean values of 6.4%, 6.2%, and 7.2% for *hxl5-2*, *hxl5-3*, and *hxl5-4*, respectively (Fig. 4A, B). The fertility levels were comparable to that of *hxl5-1*.

## Pollen analysis of the *hxl5* homozygous mutant

To further understand male sterility in the *hxl5* mutant pollen, we examined iodine staining in *hxl5-1* and *hxl5-2* homozygous plants. We found that all of the mutant pollen grains
were stained relatively weakly compared to the wild-type (Fig. 5A). Based on Hoechst 33342 nuclear staining, all pollen grains from the wild-type (cv. NP as a representative) and the *hxl5-1* and *hxl5-2* mutant plants contained one vegetative nucleus and two generative nuclei (Fig. 5A, insets). This indicated that the *hxl5-1* and *hxl5-2* mutant pollen contained less starch, but that it underwent nuclear division normally. We also found

similar results in pollen grains from *hxl5-3* and *hxl5-4* (data not shown).

We then compared pollen germination and tube growth between the wild-type and *hxl5-1* and *hxl5-2* using *in vitro* and *in vivo* germination assays. For the *in vitro* analysis, we tested mature pollen grains collected immediately prior to pollination. This revealed that wild-type grains germinated with an

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Reduced fertility in rice *hxl5* mutants. (A) Representative images of mature *hxl5* mutant panicles. NP and DJ, Nipponbare and Dongjin background wild-type (WT) genotypes. (B) Mean fertility of the *hxl5* mutants. *Ubi::OsHXK5G113D* represents the mean of six independent transgenic lines with a catalytically inactive mutant form of OsHXK5 in the *hxl5* null mutant. Other data are means (±SE) from at least three different plants.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Pollen grain staining and germination assays in rice *hxl5-1* and *hxl5-2* homozygotes. (A) Pollen grains of the wild-type Nipponbare (NP), *hxl5-1*, and *hxl5-2* stained with an I₂/KI solution. Pollen grains stained with Hoechst 33342 are shown in the insets, where red arrowheads indicate generative nuclei and white arrowheads indicate vegetative nuclei. (B) *In vitro* germination ratio of mature pollen grains prepared from the wild-types NP and Dongjin (DJ), *hxl5-1*, and *hxl5-2*. (C) Mean length of elongated pollen tubes measured 30 min after germination from the wild-types and the mutant lines. Data in (B, C) are means (±SE) of three replicates and significant differences compared to the wild-types were determined using Student's *t*-test: \*P<0.01. (D) Representative images of *in vitro* germinated pollen grains at 10 min after germination from the wild-types and the mutant lines. Scale bars are 40 µm. (E) *In vivo* pollen germination assays on pistil stigmas after pollination. Spikelets were collected 10 min after full pollination, and the pistils from the NP wild-type and the mutant lines were stained with aniline blue solution.</figcaption></figure>
efficiency of 91.7% and 90.6% for cvs NP and DJ, respectively, whereas *hxm5-1* homozygote grains germinated with an efficiency of only 36.6% (Fig. 5B). The mean length of the pollen tube was 191.4 µm at 30 min after germination in the wild-type (cv. NP), but was only 108.6 µm in the *hxm5-1* homozygotes (Fig. 5C, D). Similarly, *hxm5-2* homozygote pollen grains germinated with an efficiency of 32.6% (Fig. 5B). The mean length of pollen-tube growth was 201.4 µm at 30 min after germination in the wild-type (cv. DJ), but only 107.5 µm in the *hxm5-2* homozygotes (Fig. 5C). In iodine staining analysis, germinated mutant pollen grains were stained much more intensely than the wild-types (Supplementary Fig. S3), indicating a defect in starch utilization in the mutant pollen.

*In vivo* pollen germination assays conducted 10 min after pollination on the stigma and followed by aniline blue staining showed efficient pollen germination and tube growth for the wild-type grains (cv. NP as a representative). In contrast, only limited numbers of *hxm5-1* and *hxm5-2* grains germinated on the stigma (Fig. 5E).

Taken together, these results demonstrated that pollen germination and tube growth were significantly reduced in the *hxm5* mutants compared to the wild-types. Thus, the pollen tubes of the mutant could not reach the ovary when competing with wild-type pollen in the heterozygous mutant. However, a few *hxm5* mutant pollen grains were able to fertilize when not competing with wild-type pollen in the homozygous mutant, and thus were able to produce some seeds.

## Biochemical analysis in anthers of *hxm5* mutants

To determine the contribution of HXK5 to the total hexokinase activity in rice anthers carrying mature pollen grains, we examined hexokinase activity in the anthers of the wild-types (cvs. NP and DJ) and the *hxm5-1*, *hxm5-2*, *hxm5-3*, and *hxm5-4* mutants at the mature stage. We found that the total hexokinase activity in the *hxm5* mutants was reduced to ~70% of that in the wild-types (Fig. 6A). We also measured starch and soluble sugars in the mature anthers and found that those of the *hxm5* mutants contained more glucose, fructose, and sucrose compared to the wild-types, but less starch (Fig. 6B): the anthers of the homozygous mutants had 75% of the starch

found in the wild-type, which was consistent with the relative weak starch staining of their pollen grains (Fig. 5A). Given that starch biosynthesis during the final phase of pollen maturation is critical for normal pollen development and starch is the main reserve energy source for pollen germination (Min et al., 2013; Zhang et al., 2013; Khan et al., 2015; Lee et al., 2016; Wu et al., 2016), our results suggested that the reduced starch content contributed to male sterility in the *hxm5* mutants.

## Production of transgenic rice expressing a catalytically inactive form of HXK5 in *hxm5-1*

It has been reported that a catalytically inactive form of OsHXK5, OsHXK5-G113D, complements the sugar-insensitive phenotype of the Arabidopsis *glucose insensitive2-1* mutant (Cho et al., 2009). To examine whether the male-sterile phenotype of rice *hxm5* mutants was due to a lack of glucose sensing, OsHXK5-G113D fused with a Myc tag was introduced into *hxm5-1* under the control of the maize *Ubiquitin1* promoter, which has been shown to drive strong gene expression in mature pollen (Cornejo et al., 1993). Among the 21 transgenic plants, six lines (designated OsHXK5G113D) were selected by protein gel-blot analysis of leaves and of anthers carrying mature pollen using an anti-Myc antibody (Supplementary Fig. S4). The mean fertility rate of the T₀ plants was 7.4% (Fig. 4A, B), which suggested that catalytically inactive OsHXK5 could not rescue the *hxm5* male-sterile phenotype.

## Discussion

In common with many major food crops, rice stores starch in pollen to provide the building blocks and energy necessary for its germination and tube growth, and hence it is commonly used as an indicator for viable pollen. However, only a few enzymes likely to be functional in the starch biosynthesis pathway during pollen development have been defined in rice (Mu et al., 2009; Lee et al., 2016). Sucrose is transported from the photosynthetic source organs to the non-photosynthetic sink anthers that carry the pollen, where it is hydrolysed by invertase into glucose and fructose (Koch, 2004). HXK then

<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Biochemical analysis of rice *hxm5* mutant anthers harboring mature pollen grains. (A) Hexokinase activity of mature anthers from the wild-types Nipponbare (NP) and Dongjin (DJ) and homozygous *hxm5* lines. Units are nmol min⁻¹ at pH 7.5 and at room temperature. (B) Starch and soluble sugar contents of mature anthers from the wild-types and homozygous *hxm5* lines. All data are means (±SE) from at least three different plants. Significant differences compared to the wild-types were determined using Student's t-test: \*P<0.01.</figcaption></figure>
catalyses the first irreversible step of sugar metabolism through
the phosphorylation of hexoses into hexose-6-phosphate.
This serial reaction therefore potentially constitutes an im-
portant regulatory step in starch biosynthesis in mature pollen
grains (Dai *et al.*, 1999; Winter and Huber, 2000). HXK ac-
tivity in carbohydrate catabolism is also critical for supplying
the building blocks and energy during pollen germination and
tube growth (Dai *et al.*, 2007).

Little is known about how HXKs regulate growth and devel-
opment in rice. In this study, among the 10 identified rice HXK
genes (Cho et al., 2006a), we found that *HXK5* is highly ex-
pressed in the late stages of pollen development when starch be-
gins to accumulate (Raghavan, 1988; Yamagata et al., 2010) (Fig. 1,
Supplementary Fig. S1). The isolated *Tos17* insertion mutant line
*hxl5-1* did not bear any homozygotes from self-pollinated het-
erozygotes (Fig. 2, Table 1). Reciprocal-cross experiments between
*hxl5-1* and wild-type plants revealed a male-sterile phenotype in
the mutant pollen (Table 2). Pollen analysis of the *hxl5* mutant
alleles showed reduced hexokinase activity, partly impaired starch
biosynthesis, and a severe sterile phenotype (Figs 3–6). A pollen
germination assay further supported the presence of a defect in
*hxl5-1* pollen germination and tube growth (Fig. 5).

It was notable that, while the heterozygous plants did not
produce homozygous progeny, anther-derived homozygous
*hxl5-1* plants and *hxl5-2*, *hxl5-3*, and *hxl5-4* plants that were
generated using the CRISPR/Cas9 system produced some
homozygous seeds (Fig. 4, Supplementary Fig. S2B). This sug-
gests that the *hxl5* mutant pollen grains could not compete
with wild-type grains. However, since the grains contained a
considerable amount of starch (nearly 75% of the wild-type
level), the mutant retained some fertilization capability des-
pite its relatively slow germination and tube growth (Figs 5, 6).
Other HXKs may have contributed to the residual hexokinase
activity that could direct starch biosynthesis. Although pollen
separated from anthers was not directly examined due to the
technical challenges involved, we believe that pollen represents
the most metabolically active component in mature anthers
and hence it contributes to a large portion of the hexokinase
activity and starch content in the anthers. Therefore, it was not
surprising that *hxl5* mutant pollen could fertilize in self-pollin-
ated homozygous lines.

It is not currently known whether the male sterility of *HXK5*-deficient pollen is primarily due to insufficient starch biosynthesis or to a defect in pollen germination and tube growth. In general, HXK functions in both the starch biosynthesis and utilization pathways (Supplementary Fig. S5). Thus, an insufficient starch content may affect subsequent processes after pollen maturation. The results from our present study suggest that OsHXK5 is a major HXK isoform that functions during pollen germination and tube growth. A deficiency in HXK5 may affect the building blocks and energy production required for that process (Supplementary Fig. S5). We hypothesize that HXK5 functioning is critical for pollen maturation, germination, and tube growth in rice.

In a previous study, Xu *et al.* (2008) found that suppression of *OsHXK10* expression using RNAi caused a reduction in transcripts in the anther walls of the transgenic plants. This reduction disrupted dehiscence due to inhibited cell-wall

thickening in the anthers, and reduced pollen germination and
fertility. In contrast, the pollen appeared to be morphologic-
ally normal with levels of cell wall polysaccharides and starch
similar to those found in the wild-type. Therefore, it is likely
that the function of HXK5 that we examined is distinct from
that of HXK10 in rice anthers. Mutation of *OsSUT1* affects
pollen germination rather than maturation, and reinforces the
necessity of sucrose in pollen germination (Hirose *et al.*, 2010).
Similarly, a mutation in *OsSPS1* results in starch being accu-
mulated normally during pollen maturation, but pollen ger-
mination is reduced by 50% (Hirose *et al.*, 2014). Therefore, it
is likely that the function OsHXK5, which affects both pollen
maturation, germination, and tube growth, is distinct from that
of OsSUT1 and OsSPS1, mutations of which only disrupt ger-
mination and tube growth.

The functions of the other OsHXKs need to be charac-
terized. In particular, OsHXK6, which is constitutively ex-
pressed at low levels throughout anther development, is a close
homolog to OsHXK5 (Fig. 1). Huang et al. (2015) found that
OsHXK6 physically associates with Restorer-of-Fertility 6
(RF6). RF6 encodes a pentatricopeptide-repeat family pro-
tein, which controls pollen development by promoting the
processing of aberrant cytoplasmic male sterility (CMS)-
associated transcripts and rescues the Honglian CMS pheno-
type. Our preliminary analysis of a rice *hxl6-1* T-DNA mutant
allele showed normal Mendelian progeny segregation ratios in
the self-pollinated heterozygous plants (data not shown), sug-
gesting that *OsHXK6* may not be critical for normal pollen
development in our wild-type cv. DJ background. It would be
of value to examine a double-mutant of *hxl5/hxl6* to under-
stand any functional redundancy.

OsHXK5, in addition to OsHXK6, is primarily present in
mitochondria, but has the potential to locate to the nucleus
using a nuclear-localization signal sequence that functions in
sugar signaling and sensing (Cho et al., 2009). We tried to com-
plement the low fertility of the *hxl5* mutant by introducing
a catalytically inactive OsHXK5-G113D, but none of the re-
sulting *OsHXK5*<sup>G113D</sup> transgenic rice plants showed normal
fertility (Fig. 4). It is likely that the kinase activity of HXK5 is
critical for pollen development, germination, and tube growth
in rice. It has recently been reported that glucose sensing via
AtHXK1 is involved in the glucose-mediated inhibition of
pollen tube growth in Arabidopsis, and that this is independent
of the catalytic function of AtHXK1 (Rottmann et al., 2018).
We have previously reported that OsHXK5 and OsHXK6
have glucose sensing functions similar to AtHXK1 (Cho et al.,
2009). It would therefore be interesting to determine whether
these HXKs have a similar role in sugar signaling and sensing in
the glucose-mediated inhibition of pollen tube growth in rice.

**Supplementary data**

Supplementary data are available at JXB online.

Fig. S1. Digital expression profiles of *OsHXK* genes using Affymetrix microarray data prepared from developing pollen of *japonica* rice and downloaded from NCBI GEO.

Fig. S2. Characterization of *hxl5-1* homozygous rice plants
derived from anther culture.
Fig. S3. Iodine staining of pollen grains of wild-type and *hxl5-1* and *hxl5-2* mutants at 30 min after germination.

Fig. S4. Protein gel-blot analysis of transgenic rice plants expressing *Ubi::OsHXK5G113D-Myc*.

Fig. S5. A model of carbohydrate metabolism in rice pollen grains.

## Acknowledgements

This work was supported by grants from the Next Generation BioGreen 21 Program of the Rural Development Administration of Korea (PJ013172012019) and from the Mid-Career Researcher Program of the National Research Foundation of Korea (NRF-2017R1A2B4009687). The authors declare no conflicts of interest.

## References

**Andaya VC, Mackill DJ.** 2003. QTLs conferring cold tolerance at the booting stage of rice using recombinant inbred lines from *a japonica × indica* cross. Theoretical and Applied Genetics **106**, 1084–1090.

**Cheng W, Zhang H, Zhou X, Liu H, Liu Y, Li J, Han S, Wang Y.** 2011. Subcellular localization of rice hexokinase (OsHXK) family members in the mesophyll protoplasts of tobacco. *Biologia Plantarum* **55**, 173–177.

**Cho JI, Ryoo N, Eom JS, et al.** 2009. Role of the rice hexokinases OsHXK5 and OsHXK6 as glucose sensors. *Plant Physiology* **149**, 745–759.

**Cho JI, Ryoo N, Ko S, et al.** 2006a. Structure, expression, and functional analysis of the hexokinase gene family in rice (*Oryza sativa* L.). *Planta* **224**, 598–611.

**Cho YH, Yoo SD, Sheen J.** 2006b. Regulatory functions of nuclear hexokinase1 complex in glucose signaling. *Cell* **127**, 579–589.

**Claeyssen E, Rivoal J.** 2007. Isozymes of plant hexokinase: occurrence, properties and functions. *Phytochemistry* **68**, 709–731.

**Coast O, Murdoch AJ, Ellis RH, Hay FR, Jagadish KS.** 2016. Resilience of rice (*Oryza* spp.) pollen germination and tube growth to temperature stress. *Plant, Cell & Environment* **39**, 26–37.

**Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE.** 1993. Activity of a maize ubiquitin promoter in transgenic rice. *Plant Molecular Biology* **23**, 567–581.

**Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, Levine A, Granot D.** 1999. Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. *The Plant Cell* **11**, 1253–1266.

**Dai S, Chen T, Chong K, Xue Y, Liu S, Wang T.** 2007. Proteomics identification of differentially expressed proteins associated with pollen germination and tube growth reveals characteristics of germinated *Oryza sativa* pollen. *Molecular & Cellular Proteomics* **6**, 207–230.

**Datta R, Chamusco KC, Chourey PS.** 2002. Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in maize. *Plant Physiology* **130**, 1645–1656.

**Dickinson DB.** 1968. Rapid starch synthesis associated with increased respiration in germinating lily pollen. *Plant Physiology* **43**, 1–8.

**Dresselhaus T, Sprunck S, Wessel GM.** 2016. Fertilization mechanisms in flowering plants. *Current Biology* **26**, R125–R139.

**Endo M, Tsuchiya T, Hamada K, Kawamura S, Yano K, Ohshima M, Higashitani A, Watanabe M, Kawagishi-Kobayashi M.** 2009. High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development. *Plant & Cell Physiology* **50**, 1911–1922.

**Eom J-S, Choi SB, Ward JM, Jeon JS.** 2012. The mechanism of phloem loading in rice (*Oryza sativa*). *Molecules and Cells* **33**, 431–438.

**Eom J-S, Nguyen CD, Lee D-W, Lee S-K, Jeon J-S.** 2016. Genetic complementation analysis of rice sucrose transporter genes in Arabidopsis SUC2 mutant *atsuc2*. *Journal of Plant Biology* **59**, 231–237.

**Fu J-H, Lei L-G, Chen L-B, Qiu G-Z.** 2001. Wall ultrastructure and cytochemistry and the longevity of pollen of three grass species. *Australian Journal of Botany* **49**, 771–776.

**Hepler PK, Rounds CM, Winship LJ.** 2013. Control of cell wall extensibility during pollen tube growth. *Molecular Plant* **6**, 998–1017.

**Hirose T, Hashida Y, Aoki N, Okamura M, Yonekura M, Ohto C, Terao T, Ohsugi R.** 2014. Analysis of gene-disruption mutants of a sucrose phosphate synthase gene in rice, *OsSPS1*, shows the importance of sucrose synthesis in pollen germination. *Plant Science* **225**, 102–106.

**Hirose T, Zhang Z, Miyao A, Hirochika H, Ohsugi R, Terao T.** 2010. Disruption of a gene for rice sucrose transporter, *OsSUT1*, impairs pollen function but pollen maturation is unaffected. *Journal of Experimental Botany* **61**, 3639–3646.

**Huang W, Yu C, Hu J, et al.** 2015. Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility. *Proceedings of the National Academy of Sciences, USA* **112**, 14984–14989.

**Jagadish SVK, Cairns J, Lafitte R, Wheeler TR, Price AH, Craufurd PQ.** 2010. Genetic analysis of heat tolerance at anthesis in rice. *Crop Science* **50**, 1633–1641.

**Jeon JS, Lee S, Jung KH, et al.** 2000. T-DNA insertional mutagenesis for functional genomics in rice. *The Plant Journal* **22**, 561–570.

**Johnson MA, Preuss D.** 2002. Plotting a course: multiple signals guide pollen tubes to their targets. *Developmental Cell* **2**, 273–281.

**Kariya K.** 1989. Sterility caused by cooling treatment at the flowering stage in rice plants. *III. Establishment of a method of in vitro* pollen germination. *Japanese Journal of Crop Science* **58**, 96–102.

**Khan MMR, Hasnunnahar M, Iwayoshi M, Ogura-Tsujita Y, Isshiki S.** 2015. Pollen degeneration in three functional male-sterile lines of eggplant with the wild *Solanum* cytoplasms. *Horticulture, Environment, and Biotechnology* **56**, 350–357.

**Khatun S, Flowers TJ.** 1995. The estimation of pollen viability in rice. *Journal of Experimental Botany* **46**, 151–154.

**Kim HB, Cho JI, Ryoo N, Shin DH, Park YI, Hwang YS, Lee SK, An G, Jeon JS.** 2016. Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism. *Journal of Integrative Plant Biology* **58**, 127–135.

**Koch K.** 2004. Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. *Current Opinion in Plant Biology* **7**, 235–246.

**Lee SK, Eom JS, Hwang SK, Shin D, An G, Okita TW, Jeon JS.** 2016. Plastidic phosphoglucomutase and ADP-glucose pyrophosphorylase mutants impair starch synthesis in rice pollen grains and cause male sterility. *Journal of Experimental Botany* **67**, 5557–5569.

**Lee SK, Jeon JS, Börnke F, et al.** 2008. Loss of cytosolic fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth retardations in rice (*Oryza sativa*). *Plant, Cell & Environment* **31**, 1851–1863.

**Lord EM, Russell SD.** 2002. The mechanisms of pollination and fertilization in plants. *Annual Review of Cell and Developmental Biology* **18**, 81–105.

**Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ.** 2013. Targeted mutagenesis in rice using CRISPR-Cas system. *Cell Research* **23**, 1233–1236.

**Min L, Zhu L, Tu L, Deng F, Yuan D, Zhang X.** 2013. Cotton GhCK1 disrupts normal male reproduction by delaying tapetum programmed cell death via inactivating starch synthase. *The Plant Journal* **75**, 823–835.

**Mu H, Ke JH, Liu W, Zhuang CX, Yip WK.** 2009. UDP-glucose pyrophosphorylase2 (*OsUgp2*), a pollen-preferential gene in rice, plays a critical role in starch accumulation during pollen maturation. *Chinese Science Bulletin* **54**, 234–243.

**Naito Y, Hino K, Bono H, Ui-Tei K.** 2015. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. *Bioinformatics* **31**, 1120–1123.

**Nguyen TD, Moon S, Nguyen VN, et al.** 2016. Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage. *Plant Molecular Biology* **92**, 71–88.

**Pfosser M, Amon A, Lelley T, Heberle-Bors E.** 1995. Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. *Cytometry* **21**, 387–393.

**Raghavan V.** 1988. Anther and pollen development in rice (*Oryza sativa*). *American Journal of Botany* **75**, 183–196.

**Rang ZW, Jagadish SVK, Zhou QM, Craufurd PQ, Heuer S.** 2011. Effect of high temperature and water stress on pollen germination and spikelet fertility in rice. *Environmental and Experimental Botany* **70**, 58–65.
Rottmann T, Fritz C, Sauer N, Stadler R. 2018. Glucose uptake via STP transporters inhibits *in vitro* pollen tube growth in a HEXOKINASE1-dependent manner in *Arabidopsis thaliana*. The Plant Cell **30**, 2057–2081.

Rounds CM, Winship LJ, Hepler PK. 2011. Pollen tube energetics: respiration, fermentation and the race to the ovule. AoB Plants **2011**, plr019.

Sallaud C, Gay C, Larmande P, et al. 2004. High throughput T-DNA insertion mutagenesis in rice: a first step towards *in silico* reverse genetics. The Plant Journal **39**, 450–464.

Song ZP, Lu BR, Chen JK. 2001. A study of pollen viability and longevity in *Oryza rufipogon*, *O. sativa* and their hybrids. IRRN **26**, 31–32.

Wen LY, Chase CD. 1999. Mitochondrial gene expression in developing male gametophytes of male-fertile and S male-sterile maize. Sexual Plant Reproduction **11**, 323–330.

Winter H, Huber SC. 2000. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Critical Reviews in Plant Sciences **19**, 31–67.

Wu Y, Fox TW, Trimnell MR, Wang L, Xu RJ, Cigan AM, Huffman GA, Garnaat CW, Hershey H, Albertsen MC. 2016. Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnology Journal **14**, 1046–1054.

Xu FQ, Li XR, Ruan YL. 2008. RNAi-mediated suppression of hexokinase gene *OsHXM10* in rice leads to non-dehiscent anther and reduction of pollen germination. Plant Science **175**, 674–684.

Yamagata Y, Yamamoto E, Aya K, et al. 2010. Mitochondrial gene in the nuclear genome induces reproductive barrier in rice. Proceedings of the National Academy of Sciences, USA **107**, 1494–1499.

Zhang H, Xu C, He Y, Zong J, Yang X, Si H, Sun Z, Hu J, Liang W, Zhang D. 2013. Mutation in CSA creates a new photoperiod-sensitive genic male sterile line applicable for hybrid rice seed production. Proceedings of the National Academy of Sciences, USA **110**, 76–81.

Zik M, Irish VF. 2003. Flower development: initiation, differentiation, and diversification. Annual Review of Cell and Developmental Biology **19**, 119–140.