Current iodine nutrition status and progress toward elimination of iodine deficiency disorders in Jazan, Saudi Arabia

Rashad Mohammed
Ali Alsanosy
Abdelrahim Mutwakel Gaffar
Husam Eldin
Elsawi Khalafalla
Mohamed Salih Mahfouz
Abdel Naser
Shaaban Zaid
Ibrahim Ahmed Bani
Current iodine nutrition status and progress toward elimination of iodine deficiency disorders in Jazan, Saudi Arabia
R E S E A R C H A R T I C L E Open Access
Background: The term iodine deficiency disorders (IDD) refers to all the effects of iodine deficiency on growth and development in human and animal populations that can be prevented by correction of the iodine deficiency. The objective of this paper was to determine the iodine nutrition status among schoolchildren in the Jazan Region of the Kingdom of Saudi Arabia (KSA), by measuring urinary iodine concentrations and by clinical assessments of goiter rate. Methods: A school-based cross-sectional survey was conducted in the Jazan region of southwestern KSA from May to November 2010. A total of 311 children, aged 6-13 years, drawn from 12 schools, were selected by a three-stage cluster random sampling method. Data on sociodemographic characteristics were collected using a structured questionnaire. Urine samples were collected and physical examinations were conducted to determine the presence or absence of goiter. Data were analyzed using SPSS version 17.0. Chi square and independent t-tests were used for proportions and mean comparisons between groups. Results: Out of 360 selected children, 311 were examined. There were 131 males (42%) and 180 females (58%). The median urinary iodine concentration (UIC) of the study group was 421 μg/L. The study population proportion with UIC > 300 μg/L was 74% with a higher proportion among males and urban populations. The proportion of children with UIC of 100-300 μg/L was only 21% and was significantly higher among females compared with males (p < 0.001). Only about 3% of the children had a median UIC less than 50 μg/L. The prevalence of total goiter rate (TGR) among the sample of schoolchildren in Jazan was 11%, with significant variations between rural and urban populations and by gender.Conclusions:The present study demonstrates a remarkable achievement in Universal Salt Iodization (USI) and IDD elimination goals in the Jazan area. However, UIC levels reflect excessive iodine intake and may put the population at risk of adverse health consequences like iodine-induced hyperthyroidism and autoimmune thyroid diseases.

Background

The term iodine deficiency disorders (IDD) refers to all the effects of iodine deficiency on growth and development in human and animal populations that can be prevented by correction of the iodine deficiency [1]. IDD in the most severe form includes cretinism, stillbirth and miscarriage, and increased infant mortality [1]. In developing countries about 38 million newborns every year remain unprotected from the lifelong consequences of brain damage associated with IDD [2]. A recent study estimated that 266 million school-age children and two billion of the general population worldwide have insufficient iodine intake [3].

In the Middle East and North Africa region the situation of IDD control varies considerably between countries. Only the Islamic Republic of Iran and Tunisia have achieved IDD control goals [4]. Iraq, Afghanistan and Pakistan were classified as suffering from severe IDD while Morocco, Sudan and the Kingdom of Saudi Arabia (KSA) were considered as suffering from moderate IDD problems [4,5].

Universal Salt Iodization (USI) is a strategy to ensure sufficient intake of iodine by all individuals was recommended by the WHO and UNICEF Joint Committee on Health Policy in 1994 [6]. Some experts believe that universal salt iodization may be the most successful public health effort of the past two decades [2] and a remarkably cost-effective public health goal [7]. UNICEF estimates that less than 20% of households in the developing world were using iodized salt in the early 1990s, and by 2000, the average had jumped to some 70%. By 2006, around 120 countries were implementing salt iodization programs [2]. In 2007, worldwide 12 countries have optimal iodine status and iodine intake is more than adequate, or even excessive, in 34 countries [3], an increase from 27 in 2003 [8]. In the Middle East and North Africa region, 64% of households consume adequately iodized salt with wide variation between countries [5].

The targets for sustainable IDD elimination include process and impact indicators. The process indicators are; (1) 100% of all salt produced and imported is iodized; (2) 95% of all iodized salt imported and produced is adequately iodized; (3) 90% of households are using adequately iodized salt; and (4) 100% of all districts have reached the goal of USI, i.e. > 90% of households are using adequately iodized salt. The impact indicators are: (1) population median urinary iodine concentration (UIC) is 100-300 μg/L; (2) proportion of samples with UIC levels below 100 μg/L is < 50%; and (3) proportion of samples with UIC levels below 50 μg/L [6].

In the KSA a national cross-sectional epidemiological survey for studying iodine status was conducted among Saudi schoolchildren aged 8-10 years in 1997 [9]. The median national UIC was 180 μg/L and the total goiter rate (TGR) ranged from 8% to 30%. Nationally the proportion of the population with UIC less than 100 was 23%. The southern province of Jazan had the lowest median UIC (110 μg/L) and the highest percentage (45%) of subjects with a UIC < 100 μg/L [9]. The IDD control program using the USI strategy started in 1997, and the Saudi Standards, Metrology and Quality Organization (SASO) recommend that iodine content of salt must be 70-100 ppm in all food salt [10]. Since the national survey in 1997, no follow up survey or monitoring system has taken place [4] to assess the iodine nutrition status in the population neither nationally or in the Jazan region.

The objective of this paper is to determine the iodine nutrition status among schoolchildren in the Jazan Region of the KSA, by measuring the UIC and by clinical assessment of the goiter rate.

Methods

Setting

This was a cross-sectional, observational study conducted in the Jazan region during the period from May to November 2010. The Jazan region is one of the 13 regions of the KSA, located on the tropical Red Sea coast in the southwestern KSA. Jazan covers an area of 11,671 km 2 , including some 5,000 villages and towns. Adjacent to the region are 100 islands, including the largest island of Farasan. The Jazan region runs along the Red Sea coast for almost 300 km and is a highly relatively populated region with a total population of 1.5 million.

Sampling method

A three-stage cluster random sampling procedure was adopted to select target children from 12 schools representing different governorates of the Jazan region. The 12 schools were randomly selected from the list of schools of the target age group in the region and then one class was randomly selected in each of the selected schools. Proportionate to Population Size (PPS) sampling technique was used to determine the required number of children from classes as recommended by the WHO for iodine nutrition surveys. We aimed to obtain a sample of 300 children for this study [6].

Data collection tools and techniques

Data regarding participants' sociodemographic characteristics were collected from the children using a structured questionnaire. A casual urine sample of~10 mL also was collected in a plain tube from each child participating in the survey and stored in a refrigerator (4°C). An aliquot of urine (5-6 mL) was transferred to a tube with a tight cap and was shipped to the International Council for the Control of Iodine Deficiency Disorders (ICCIDD) iodine reference laboratory in the Nutritional Intervention Research Unit (NIRU) of the Medical Research Council (MRC), Cape Town, South Africa, for determination of iodine concentrations. Another aliquot of urine (5-6 mL) was kept as a reference material at the Jazan University Medical Research Centre.

Urine samples were analyzed using a modified microplate method based on manual digestion with ammonium persulfate followed by the colorimetric determination of the Sandel-Kolthoff reaction by using 96-multiwell plates and an absorbance microplate reader at 405 nm. The absolute iodine value is expressed in μg/L [6,11].

Quality control

Internal quality control (QC) samples were prepared by collecting random urine samples over a 24-hour period, which were either diluted with deionized water or spiked with a known potassium iodate solution to obtain samples at low, median and high iodine concentrations. These QC samples are normally used to determine the inter-and intra-assay precision of the method. During the analysis of study samples the analytical intra-assay precision of the method was determined by calculating the coefficient of variation (% CV) of four analyses of six urine pools ranging from 49.6 to 239.8 μg/L. Inter-assay precision was established by assaying three urine pools (low, medium and high) in 8 to 12 microplate runs. The intra-assay CVs were 5.0%, 2.2%, 0.5%, 1.4%, 0.6% and 6.6% at 49.6, 100.0, 120.7, 129.2, 175.2 and 239.8 μg/L iodine, respectively. The inter-assay CVs were 7.8%, 4.8% and 7.1% at 47.9, 124.6 and 230.4 μg/L iodine, respectively.

Clinical assessment

Neck examination for goiter was done by trained medical doctors for all children. The ICCIDD uses a simplified goiter classification: grade 0, no palpable or visible goiter; grade 1, palpable but not visible when neck is in normal position; and grade 2, palpable and visible when the neck is in normal position [6].

Statistical analysis

Results are presented as means ± standard deviation (SD), medians and frequencies as well as percentages. Comparisons between two means were conducted using Student's t-test for continuous variables. The chi-square test was used to compare some selected categorical variables. Before the t-test was used, normality assumptions were assessed using the Kolmogorov-Smirnov test and homogeneity of variances were tested using the Levene's test. All statistical analyses were performed using SPSS, version 17 (SPSS Inc., Chicago, IL, USA). P-value less than 0.05 was considered statistically significant.

Ethical considerations

Ethical approval was obtained from the Research Ethics Committee in the Medical Research Center-Jazan University, and the study was approved by the regional education authorities. Written informed consent was obtained from the parents of participating children.

Results

Out of 360 selected children 311 were examined and provided salt and urine samples (response rate = 86%). The mean age of the children was 10 years (SD 1.6), and ranged between 6 to 13 years. There were 131 males (42.1%) and 180 females (57.9%). Rural dwellers formed the biggest proportion of children (72.2%); the urban population constituted (24.8%) of the children. Table 1 presents the number of participants, median, mean and SD of UIC and percentage of participants with UIC > 300 μg/L. The median UIC was 517 μg/L and was significantly higher among male participants (P-value < 0.001). However, no significant difference (P = 0.230) was observed between the mean UIC for rural and urban children. The proportion of the population with UIC > 300 μg/L was 74% with a higher proportion among males and urban populations. Table 2 shows the UIC (μg/L) of school children according to residence and gender in the Jazan region. The proportion of the population with UIC 100-300 μg/L was only 21% and was significantly higher among females than males (P < 0.001). Only 3% of the population had a UIC less than 50 μg/L. The proportion of the population with a UIC more than 200 μg/L was 87%. Table 3 shows the distribution of schoolchildren according to goiter prevalence. The prevalence of TGR among schoolchildren in Jazan was 11% with significant variations between rural and urban populations and gender. The TGR was higher among rural than urban Table 1 The sample size, median, and mean (SD) urinary iodine concentration (UIC) and percentage of subjects with urinary iodine concentration > 300 μg/L population (P = 0.004) and higher in female than male participants (P = 0.012).

Discussion

The three most important indicators used in IDD surveillance, as well as in initial problem assessment, are goiter prevalence, urinary iodine excretion in a population, and thyroid function tests [6]. The WHO advises researchers to combine at least two indicators, one morphological and one laboratory test. A 1997 survey found that 23% of the national population and 45% of the Jazan region population had UIC below 100 μg/L, although their median urinary iodine concentration was within the normal range. Additionally, goiter prevalence ranged from 25% to 30% in costal and mountain areas in the southern region. The authors recommend that the country needs to implement an IDD control program [9]. In this study the researchers used both urinary iodine excretion and goiter prevalence to assess the iodine nutrition status.

Urinary iodine

In our study the median UIC among the studied population in the Jazan area was 421 μg/L compared with 110 μg/L in the 1997 survey in Jazan, and 180 μg/L at the national level [9]. This median UIC reflects excessive iodine intake in the population based on WHO cut-off points [6]. The median UIC is significantly higher among male participants (484 μg/L) than female participants (369 μg/L) (P-value < 0.001). The median UIC in Jazan, KSA, is the highest in the Gulf and Arab region and similar to that reported in Ecuador (420 μg/L), Uganda (464 μg/L) and the Democratic Republic of the Congo (495 μg/L) [5]. Based on the UIC cut-off points recommended by the WHO regarding iodine nutrition status, only 8% of the population has adequate iodine intake (UIC 100-199 μg/L), 13% above requirements (UIC 200-299 μg/L), and 74% with excessive iodine intake [6]. Only 5% of children have UIC less than 100 μg/L compared with 45% in 1997 [9]. The population of Jazan was subjected to excessive iodine intake with a risk of adverse health consequences like iodine-induced hyperthyroidism and autoimmune thyroid diseases [6,[12][13][14]. A similar phenomenon was observed in 34 countries after the implementation of USI, which emphasized the importance of establishing and strengthening the monitoring system to guide USI to provide optimal iodine nutrition and protect against excessive intake [6,15].

Goiter rate

Goiter prevalence in the studied region ranged from 0% in urban areas to 14% in rural areas. The prevalence of goiter was significantly higher (P-value = 0.012) among female participants (14%) than male participants (11%). Al-Nuaim et al. in 1997 reported a goiter prevalence rate of 4% in Gizan or Jazan and 22% in the Aseer area [9]. Similar prevalence in the Aseer region (24%) was reported by Abu-Eshy et al. in 2001 [16]. Goiter prevalence in Jazan is much less than what is reported by many countries in the Eastern Mediterranean region. In Yemen the TGR was 16.8% [5,17]. In the Gulf region the TGR in 2010 was 40.4% in the United Arab Emirates, 10% in Oman and 1.7% in Bahrain [5]. On the other hand in other Arab countries the TGR is 65% in Algeria, 36.3% in Tunisia, 33.5% in Jordan, 30.9% in Mauritania, 25.7% in Lebanon, and 21.4% in Egypt [5]. In Iran, the TGR was reduced from 68.0% in 1989 to only 5.7% in 2007 through implementation of a comprehensive IDD control program [18].

The salt iodization standard in the KSA as issued by SASO recommends that the amount of iodine to be between 70-100 ppm in the form of potassium iodate. This recommendation is the highest in the Gulf region [10] and higher than that recommended by the WHO which is 15-40 ppm [6]. Since the start of the salt iodization program in 1997, now more than 99% of the study population in Jazan uses iodized salt. Three hundred and eleven random salt samples were tested to determine the iodine content; 44.1% had iodine concentrations higher than 40 ppm.

Al-Attas quantified the iodine content of foods in the KSA and concluded that food consumed by Saudis  appears to have an adequate iodine concentration [19].

The high level of UIC may be explained, in light of the previous study finding by Al-Attas, by the high level of iodine concentrations in salt, increased consumption of dairy products, and ready-prepared salty meals that may have substantially changed the amount of salt and iodine consumed by the school population.

There is a need to reconsider the level of salt iodization in the KSA following the latest WHO recommendation in light of local dietary patterns. It will be important to provide optimal iodine nutrition and to protect the population from the adverse health consequences of excessive iodine intake.

Conclusion

The present study demonstrates a remarkable progress in achieving USI and IDD elimination goals in the Jazan area of the KSA. However, the UIC reflects excessive iodine intake and may put the population at risk of adverse health consequences like iodine-induced hyperthyroidism and autoimmune thyroid diseases. The levels of iodine in the salt were substantially higher than the WHO recommendation and revision of salt specifications is highly recommended. Establishing surveillance and monitoring systems will protect the population and help in guiding the implementation of USI in the country.

Study limitations

Logistical issues of the study including send samples to South Africa for analysis. For this reason the sample size is small to detect the differences between clusters, although it is quite reasonable for the study. Now that the capacity of the Medical Research Center is developed, further studies could be done and samples analysis will be conducted locally. The schools in the country do not provide meals for their students. Also, no school nutrition program is implemented in the country and the food industry is required to use iodized salt for all processed food. Thus, the results reflect the iodine nutrition in the community.

Table 2
2Urinary iodine concentration (μg/L), according to residence and gender in JazanUIC
range

Residence no. (%)
Gender no. (%)
Total

Rural
Urban
Male
Female

n=234
n=77
n=131
n=180

0-49
8 (3.4)
-
-
8 (4.4)
8 (2.6)

50-99
7 (3.0)
-
1 (0.8)
6 (3.3)
7 (2.3)

100-199
22 (9.4)
3 (3.9)
5 (3.8)
20 (11.1)
25 (8.0)

200-299
30 (12.8)
10 (13.0)
10 (7.6)
30 (16.7)
40 (12.9)

≥300
167 (71.4)
64 (83.1)
115 (87.8)
116 (64.4)
231(74.3)

P-value
0.085
< 0.001

Table 3
3Distribution of schoolchildren according to goiter prevalenceGoiter
rate

Residence no. (%)
Gender no. (%)
Total

Rural
Urban
Male
Female

Grade 0
202 (86.3)
73 (100)
121 (95.3)
145 (85.6)
275(89.6)

Grade 1
18 (7.7)
-
5 (3.9)
13 (7.2)
10 (5.9)

Grade 2
14 (6.0)
-
1 (0.8)
13 (7.2)
14 (4.6)

Total
234 (100)
73 (100)
127(100)
180 (100)
307(100)

P-value
0.004
0.012

http://www.biomedcentral.com/1471-2458/12/1006
AcknowledgementsWe would like to thank Prof. David Haxton (ICCIDD Executive Director), Dr Izzeldin Alsharief (ICCIDD, Gulf Regional Coordinator), and Prof. Pieter Jooste (Nutritional Intervention Research Unit of the Medical Research Council, Cape Town, South Africa) for their collaboration in the analysis of the urine samples. We appreciate the efforts of the Education Directorate of the Jazan Region, administrative staff, teachers and school health professionals for coordination of data collection.Author detailsCompeting interestThe authors have no conflicts of interest to declare.Authors' contribution RMA, HEE, ASZ and IAB prepared the project proposal; RMA, AMG and MSM designed this research paper; MSM and AMG performed data analysis; AMG and RMA wrote the manuscript. HEE, IAB, and ASZ provided significant input on the manuscript. All authors read and approved the final manuscript.Sources of funding
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Current iodine nutrition status and progress toward elimination of iodine deficiency disorders in Jazan, Saudi Arabia. Alsanosy , BMC Public Health. 121006Cite this article asCite this article as: Alsanosy et al.: Current iodine nutrition status and progress toward elimination of iodine deficiency disorders in Jazan, Saudi Arabia. BMC Public Health 2012 12:1006.


Ostkpr1 functions in anther cuticle development and pollen wall formation in rice

Dawei Xu
Shuying Qu
Matthew R Tucker
Dabing Zhang
Wanqi Liang
Jianxin Shi
Ostkpr1 functions in anther cuticle development and pollen wall formation in rice
10.1186/s12870-019-1711-4R E S E A R C H A R T I C L E Open Access
Background: During pollen wall formation in flowering plants, a conserved metabolon consisting of acyl-CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide α-pyrone reductase (TKPR), is required for sporopollenin synthesis. Despite this, the precise function of each of these components in different species remains unclear. Results: In this study, we characterized the function of OsTKPR1, a rice orthologue of Arabidopsis TKPR1. Loss of function of OsTKPR1 delayed tapetum degradation, reduced the levels of anther cuticular lipids, and impaired Ubisch body and pollen exine formation, resulting in complete male sterility. In addition, the phenylpropanoid pathway in mutant anthers was remarkably altered. Localization studies suggest that OsTKPR1 accumulates in the endoplasmic reticulum, while specific accumulation of OsTKPR1 mRNA in the anther tapetum and microspores is consistent with its function in anther and pollen wall development.Conclusions:Our results show that OsTKPR1 is indispensable for anther cuticle development and pollen wall formation in rice, providing new insights into the biochemical mechanisms of the conserved sporopollenin metabolon in flowering plants.

Background

Pollen grains of land plants are surrounded by a complex multilayered cell wall, the so called pollen wall, which offers protection from biotic and abiotic environmental stresses and plays an important role during the interaction between male and female organs [1,2]. The typical pollen wall in flowering plants is composed of two distinct layers: the outer exine and inner intine, with the pollen coat deposited on the surface of the exine [2,3]. The exine can be further divided into the sexine (outer) and nexine (inner), and the sexine itself also contains two layers: an outer tectum layer and an inner layer of vertical regular arrested bacula between the tectum and the nexine [4]. Mutants with defective pollen wall structures often show reduced levels or complete loss of fertility [5,6].

The highly sculptured exine is mainly composed of sporopollenin, whose chemical composition is still an enigma [1]. In general, sporopollenin is a complex biopolymer consisting of fatty acids and phenolic compounds, covalently connected by ester or ether linkages [5,7]. Precursors of sporopollenin are known to be produced and secreted from the tapetal cells to the surface of pollen grains [7]; however, some evidence also demonstrates that pollen grains themselves contribute to sporopollenin biosynthesis and exine formation [8]. In tapetal cells, the de novo biosynthesis of sporopollenin precursors occurs in plastids, and the deposition of sporopollenin precursors begins soon after the completion of meiosis when the temporary callose wall is degraded and the primexine is formed (stage 9). The deposition of sporopollenin precursors continues until microspore vacuolation, and is largely completed by the time of pollen mitosis (stage 12) [9]. Sporopollenin biosynthesis and deposition coincides with tapetum programmed cell death (PCD), which occurs from stage 8a until stage 10 [4], suggesting a possible relationship between these two events. Immature or delayed tapetum PCD usually disrupts pollen wall formation and causes defects in male fertility [10][11][12].

Recent genetic and biochemical studies in plants, mainly from two model plants Arabidopsis and Oryza sativa (rice), have significantly increased our understanding of pollen wall formation [6,13]. Because the biopolymer sporopollenin is found to be conserved in flowering plants, moss, ferns and even fungi [14,15], the biosynthesis and deposition of sporopollenin monomers therefore represents one of the most ancient biochemical pathways in flowering plants [16][17][18]. Consistent with this, related orthologous genes involved in pollen wall formation are found in dicots, monocots, gymnosperms, and even the moss, such as MS2 (Male Sterile 2)/DPW (Defective Pollen Wall) [19,20], CYP703A2/CYP703A3 [18,21], CYP704B1/CYP704B2 [22,23], ABCG26/ OsABCG15 (PDA1) [24,25], ACOS5 (Acyl-CoA Synthetase 5)/OsACOS12/NtACOS1 [12,26,27], LAP6 (Less Adhesive Pollen 6)/PKSA (Polyketide Synthase A)/ OsPKS1/NtPKS1, LAP5/PKSB/OsPKS2 [16,17,[27][28][29][30], TKPR1 (Tetraketide α Pyrone Reductase 1)/NtKPTR1/ OsTKPR1 [27,31], and AMS (Aborted Microspores)/TDR (Tapetum Degeneration Retardation) [10,11]. The fact that most of these reported orthologous genes are mainly expressed in tapetum and/or microspores suggests that there is a conserved male organ tissue-specific regulatory network controlling pollen wall formation in flowering plants.

In Arabidopsis, the plastid de novo synthesized fatty acids are first activated by ACOS5 to generate fatty acyl CoAs [26], and the resulting fatty acyl CoAs are then condensed with malonyl-CoA by PKSA/LAP6 and/or PKSB/LAP5 to produce α-pyrone products [16,17], which are further reduced by TKPR1 and TKPR2 to yield hydroxylated tetraketide compounds [31]. All intermediate and final metabolites in this pathway, including fatty acyl CoAs, α-pyrones and hydroxylated tetraketides, serve as important resources for sporopollenin precursors. Genetic and biochemical studies reveal that ACOS5, PKSA/B (LAP6/LAP5), and TKPR1 form a sporopollenin metabolon in the Arabidopsis tapetum for pollen wall formation [32]. Although this metabolon is also found to be present in tobacco [27], rice [27], moss [33], Hypericum perforatum [34] and canola [35], detailed genetic and biochemical evidence is needed to elucidate the specific or common roles of the components in pollen wall formation in various plant species.

The rice genome contains single copies of genes encoding ACOS5 (OsACOS12), PKSA/LAP6 (OsPKS1), PKSB/LAP5 (OsPKS2), and TKPR1 (OsTKPR1). Mutation of any one of these genes significantly affects male fertility in rice; for example, osacos12 [12,36,37], ospks2 [28,30], and ostkpr1 [27] are completely male sterile, while ospks1 is partially fertile [27]. This suggests that the ACOS5-PKSA/B-TKPR1 sporopollenin metabolon is likely to be conserved between rice and Arabidopsis. Nevertheless, some reports also indicate that each component of this metabolon may have divergent functions in different species. For example, unlike ACOS5 in Arabidopsis, OsACOS12 affects tapetum PCD and anther cuticle formation in rice [12,36]. Moreover, PKSB/LAP5 and OsPKS2 have different effects on anther phenylpropanoid metabolism [16,28]; pksb/lap5 in Arabidopsis are partially male sterile, while ospks2 in rice is completely male sterile [28]. A previous study reported that the osptkpr1 mutant (carrying a T-DNA insertion in the second exon of LOC_Os09g32020) lacks mature pollen grains and does not produce seeds [27], however, the mechanism by which OsTKPR1 controls fertility remains unresolved.

In this study, we characterized a novel mutant of OsTKPR1 in rice, namely ostkpr1-2, which was completely sterile, displaying delayed tapetum degradation, impaired Ubisch body patterning, reduced anther cuticular profiles, and aborted pollen grains with defective exine. The phenylpropanoid profile of ostkpr1-2 anthers was remarkably altered, which was distinct from that in oslap5/ospks2 anthers. Our studies suggest that OsTKPR1 functions in anther cuticle development and pollen wall formation, and reveal some interesting putative differences compared to TKPR1 from Arabidopsis.

Results

Isolation and characterization of the ostkpr1-2 mutant

Screening of a 60 Co γ-ray radiated mutant library in the background of 9522, a cultivar of O. sativa ssp japonica, led to the isolation of a male sterile mutant, ostkpr1-2, which was named based on the results presented below. The mutant exhibited normal vegetative development (Fig. 1a) with normal inflorescences and spikelets (Fig. 1b, c), but was completely male sterile with smaller and paler anthers containing neither mature nor viable pollen grains as evidenced by iodine potassium iodide (I 2 -KI) staining ( Fig. 1d-g). All F1 plants of crosses between WT and the mutant using WT pollen were fertile, and resulting F2 plants exhibited a segregation ratio of approximately 3:1 for fertility: sterility (fertile: sterile = 93:29, χ 2 = 0.132, P > 0.05, χ 2 test used), indicating that ostkpr1-2 is a recessive, fully penetrant male sterile mutant.

Phenotypic analysis of ostkpr1-2 Semi-thin cross sections from anthers of various developmental stages were compared to identify possible reproductive developmental defects in ostkpr1-2. No obvious defects in anther development could be observed in ostkpr1-2 compared to WT at tetrad stage (stage 8b), where typical four anther wall layers and tetrads are formed in both anther locules (Fig. 2a, f ). At stage 9, WT microspores were regular in shape (oval), evenly distributed in the anther locule, and tapetal cells became condensed (Fig. 2b), while ostkpr1-2 microspores seemed to be less compact with irregular shape and closely aggregated in the anther locule, and the tapetum cells had not expanded (Fig. 2g). At stage 10,  WT microspores expanded and became highly vacuolated, and the tapetum appeared much thinner due to degradation (Fig. 2c), while ostkpr1-2 microspores were shrunken without vacuolation and the tapetum appeared to have persisted without degradation (Fig. 2h). From stage 11 to stage 12, WT mature pollen grains formed in the anther locule after two rounds of mitotic division, and the tapetum had degraded completely into cellular debris (Fig. 2d, e). In contrast, the microspores of ostkpr1-2 had degraded, leaving only debris inside the shriveled anther locules (Fig. 2i, j).

At stage 13, scanning electron microscopy (SEM) revealed additional defects in anther and pollen development. The outer surface of WT anthers was typically covered with a thick layer of sculptured nanoridges (Fig. 3a), while the inner surface of WT anthers was covered with plenty of evenly distributed Ubisch bodies (Fig. 3c). In contrast, the outer surface of ostkpr1-2 anthers was covered with a much thinner layer of smooth nanoridges (Fig. 3b), while the inner surface of ostkpr1-2 anthers was covered with fewer and irregularly distributed Ubisch bodies (Fig. 3d). While an elaborate exine pattern appeared on the surface of WT pollen grains (Fig. 3e, g), aggregated irregularly-distributed particles were present on the shrunken ostkpr1-2 pollen surface ( Fig. 3f, h). SEM observation indicated that OsTKPR1 is indispensable for anther cuticle and pollen exine formation in rice.

To further examine the early morphological changes occurred in ostkpr1-2 anthers and microspores, transmission electron microscopy (TEM) was used. At stage 9, a dark-stained cuticle was present at the surface of the WT anther (Fig. 4a), and typical Ubisch bodies had formed on the WT anther inner surface (Fig. 4b). At this stage, WT microspores with regular pollen walls had been released from tetrads (Fig. 4c), and the sexine together with bacula appeared on the microspore plasma membrane (Fig. 4d). At the corresponding stage in ostkpr1-2 mutants, the cuticle showed weak staining (Fig. 4e), while fewer and smaller Ubisch bodies (Fig. 4f) were present on the outer and inner surfaces of ostkpr1-2 microspores, respectively. The released ostkpr1-2 microspores had a weakly-stained pollen wall ( Fig. 4g) with fewer bacula and sexine structures (Fig. 4h). At stage 10, the WT tapetum had degenerated, microspores were highly vacuolated ( Fig. 4i), and Ubisch bodies exhibited a characteristic shape with high electron density (Fig. 4j). By contrast, in ostkpr1-2 anthers the tapetum remained present, the microspores did not become vacuolated (Fig. 4m), and Ubisch bodies seemed to be normally shaped but with less electron density ( Fig. 4n). At this stage, the exine of the WT pollen was thick ( Fig. 4k) with two typical, well-organized electron-dense layers (Fig. 4l), while the exine of ostkpr1-2 pollen was very thin ( Fig. 4o) with two thin electron-dense layers (Fig. 4p). The TEM results support the results obtained from semi-thin section and SEM analyses, highlighting the important roles of OsTKPR1 in formation of the rice anther cuticle, Ubisch bodies, and pollen exine, and for the timely tapetum degradation. The OsTKPR1 gene was first mapped between two indel molecular markers chr9-3485 and chr9-6308 on chromosome 9 and eventually mapped between 69.5 and 92.1 centimorgans (CMs) with molecular markers chr9-4979 and chr9-6308 (Fig. 5a). Using a high throughput re-sequencing technique and PCR, a 15 nucleotide deletion was identified in the fourth exon of LOC_Os09g32020, which is predicted to result in the removal of 5 amino acids (serine, leucine, serine, histidine and glutamic acid) and the change of one amino acid from proline into glutamine (Fig. 5b). LOC_Os09g32020 was previously annotated as OsDFR2 (Dihydroflavonol-4-reductase-like protein) and identified as an anther-specific gene down-regulated in a thermo-sensitive male sterile mutant in rice [38]. An allelism test with a homozygous ostkpr1 T-DNA insertion mutant PFG_2B-00257 confirmed that the mutation in LOC_Os09g32020 almost certainly causes the developmental defects in ostkpr1-2 (Additional file 1: Table S1). The ostkpr1 mutant displayed similar reproductive defective phenotypes to that of ostkpr1-2, namely, complete male sterility with a defective anther cuticle and abnormal pollen exine (Additional file 2: Figure S1).

Expression pattern of OsTKPR1

In silico analysis using a publically available microarray database (http://ricexpro.dna.affrc.go.jp/data-set.html) revealed that OsTKPR1 is specifically expressed in anthers at early developmental stages (Additional file 3: Figure S2). This digital expression pattern of OsTKPR1 was confirmed by quantitative RT-PCR with anthers at different developmental stages and other tissues of WT plants, which revealed that OsTKPR1 expression could only be detected in anthers at stage 9 and 10 (Fig. 6a). The precise spatial and temporal expression pattern of OsTKPR1 was further confirmed by in situ hybridization using WT anther sections, in which OsTKPR1 signal was observed mainly in the microspores and, to a less extent, in the tapetum (Fig. 6b, c). These expression results confirm that OsTKPR1 is closely associated with the development of anther and pollen in rice at early stages.

Subcellular location of OsTKPR1 protein

Arabidopsis TKPR1, the orthologous protein of OsTKPR1, was previously reported to be an important part of the sporopollenin metabolon, acting in the ER of tapetal cells to influence sporopollenin biosynthesis and pollen formation [31]. Given the similarity of these two proteins (63% identity revealed using Clustal X; Additional file 4: Figure S3) and the comparable deficiencies in pollen exine formation in both ostkpr1-2 and tkpr1 mutants, we considered whether OsTKPR1 protein might also localize to the ER. To test this hypothesis, confocal microscopy was used to observe Nicotiana benthamiana (tobacco) leaves that were transiently transformed with an OsTKPR1-GFP construct under the control of the CaMV35S promoter. The results confirmed that the GFP signal of OsTKPR1-GFP was mainly observed in ER-like structures (Fig. 7d). The ER localization of OsTKPR1 protein was further verified by Agrobacterium mediated co-transformation, which showed OsTKPR1-GFP signal overlapping with that of the ER-marker CD3-959 [39] tagged with mCherry (Fig. 7f).

Reduced cuticular lipid components in ostkpr1-2 anthers

The abnormal anther cuticle and pollen exine in ostkpr1-2 anthers suggested the mutant may be defective in the biosynthesis of lipidic precursors for sporopollenin and cutin. To assess this, waxes and cutin monomers in both WT and ostkpr1-2 anthers at stage 12 were measured by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID) as previously described [28]. The results indicated that the levels of both wax and cutin monomers were significantly reduced in ostkpr1-2 anthers as compared with those of the WT (Fig. 8a). The total amount of wax measured in ostkpr1-2 was 0.061 μg/mm 2 , a reduction of approximately 30% compared to WT (0.093 μg/mm 2 ) (Fig. 8a). This reduction in total wax content in ostkpr1-2 anthers was attributed mainly to a significant reduction of the major rice anther wax components, such as fatty acids and saturated alkanes, although amounts of alkenes (C29:1, C31:1, C35:1) and sterols (campesterol and stigmasterol) were increased (Fig. 8c; Additional file 5: Table S2). The total amount of cutin monomers detected in WT was 0.372 μg/mm 2 , which also reflected a 30% reduction of the levels detected in ostkpr1-2 (0.265 μg/mm 2 ) (Fig. 8a). The reduction of cutin monomers was due to a significant reduction in the levels of fatty acids and ω-hydroxylated fatty acids, although levels of other 2-hydroxylated fatty acids (2HFAs, including length of C21:0, C22:0, C24:0, C25:0) and ferulic acid were increased ( Fig. 8b; Additional file 6: Table S3). Given that the rice tapetum utilizes a common biosynthetic pathway to provide precursors of the anther cuticle and sporopollenin [6,10,40], our chemical analysis suggests that OsTKPR1-2 plays an essential role in synthesizing

Altered phenylpropanoid metabolism in ostkpr1-2 anthers

Previous studies reported that components of sporopollenin metabolon, such as PKSB/LAP5 and OsPKS2, are also involved in phenylpropanoid metabolism, contributing phenolic precursors for pollen wall formation [16,28]. To investigate whether the loss of OsTKPR1 function also has effects on phenylpropanoid metabolism, phenolic profiles were generated for extracts of stage 11 anthers from wild type and ostkpr1-2 using an untargeted UHPLC-MS/MS approach. This analysis identified 238 metabolites in total; among them, 67 were flavonoids and 22 were hydroxycinnamyl derivatives.

When focusing on the phenylpropanoid pathway, levels of phenylalanine (the precursor for phenylpropanoids), naringenin chalcone and naringenin (important intermediate metabolites in the phenylpropanoid pathway), kaempferol-3-Oglucoside and tricin-O-hexosidedeoxyhexoside (sugar-conjugated derivatives of downstream metabolites) were decreased in mutant anthers. In contrast, levels of luteolin and dihydroquercetin (sugar-conjugated derivatives of downstream metabolites of apigenin and quercetin, respectively) were significantly increased in ostkpr1-2 mutant anthers (Fig. 9). The inhibitory effect of OsTKPR1 on the anther phenylpropanoid pathway is distinct from that observed in OsPKS2 [28], but similar to that of PKSB/LAP5 or PKSA/LAP6 [16], suggesting that disruption of individual sporopollenin metabolon components might have different effects on phenylpropanoid metabolism in flowering plants.

Discussion

In Arabidopsis, the "sporopollenin metabolon" consists of ACOS5-PKSA/B-TKPR1 and plays essential roles in pollen wall formation [32]. Although this ancient metabolon is found to be present in many plants, including tobacco and rice [27], increasing evidence highlights both conserved and diversified functions of each component between dicots and monocots [12,28,30,37]. In this study, we characterized OsTKPR1, the orthologue of Arabidopsis TKPR1, and revealed its vital roles in male reproduction in rice. Our results confirm the conserved function of OsTKPR1 in pollen wall formation [27] and highlight potentially additional functions in tapetum PCD, anther cuticle patterning and Ubisch body formation.

OsTKPR1-2 is indispensable for anther cuticle development and pollen exine formation

The anther-specific TKPR1 gene in Arabidopsis was first thought to be involved in flavonoid pathway, but was later found to act downstream of PKS, catalyzing the reduction of the carbonyl group of tetraketides generated by PKS to secondary alcohols, thus functioning in sporopollenin formation [27,31]. In Arabidopsis, TKPR1 is important for male fertility, and the tkpr1 mutant displays a completely disorganized thin exine without bacula and tectum but a normal anther surface [31,41]. In rice, OsTKPR1 exhibits the same enzyme activity as tobacco TKPR1 proteins, participating in the reduction of tetraketides, and ostkpr1 mutants produce no mature pollen or seeds (Additional file 2: Figure S1) [27]. In this study we found that in addition to defective exine (Fig.  4g, h), ostkpr1-2 mutants also display defective anther cuticle and Ubisch bodies (Fig. 3b, d; Fig. 4f ), indicating that OsTKPR1 may contribute to the formation of the anther surface and pollen wall. Although stage 9 and 10 anthers were not directly analyzed due to difficulties in tissue collection, chemical analyses of stage 11 anthers were consistent with cytological observations, which revealed that levels of anther cuticular lipids (Fig. 8) and anther phenolic profiles were significantly altered in mutants (Fig. 9). These results are consistent with previous findings in rice that both the anther cuticle and pollen exine share common lipid and phenolic metabolic pathways [6,20,40]. The spatio-temporal expression pattern of OsTKPR1 in the tapetum and microspores (Fig. 6 b,  c), and its similar ER localization (Fig. 7) to that of TKPR1 or NtTKPR1 [27,31] provide further support for a role of OsTKPR1 in both anther and pollen development.

OsTKPR1-2 is possibly involved in tapetum degradation

The successful formation of anther cuticle and pollen wall depends largely on the timely degradation of the tapetum, and disruption of tapetum degradation usually leads to male sterility with defective anther cuticle and pollen wall [10-12, 21, 25, 42]. Although the function of TKPR1 on tapetum degradation is not reported in Arabidopsis or tobacco [27,31], our data suggests that OsTKPR1 contributes to tapetum degradation in rice (Fig. 2), which is consistent with the spatial and temporal expression pattern of OsTKPR1 mRNA (Fig. 6) in the initial stage of tapetum degradation [4]. Additional evidence suggests that other components of the sporopollenin metabolon may impact tapetum PCD in rice. For example, a recent study revealed the role of OsA-COS12 in tapetum PCD by both cytological and TUNEL analyses [12], which is clearly different from its ortholog ACOS5 in Arabidopsis [26] or NtACOS1 in tobacco [27]. Conversely, there is no report of the effect of OsPKS2 or OsPKS1 on tapetum PCD in rice [28][29][30].

The mechanism underlying OsTKPR1's function on tapetum degradation was not investigated in this study. It is possible that OsTKPR1 fulfils a similar role in Arabidopsis and tobacco, but it has been overlooked in previous studies; alternatively, the role of sporopollenin metabolon components in tapetum PCD might be more prominent in monocots. Nevertheless, without additional evidence, it is also possible that the delayed tapetum degradation in ostkpr1-2 is an indirect effect.

Diverse interactions between sporopollenin metabolon components influence rice male fertility

In the sporopollenin metabolon, fatty acids synthesized de novo in plastids are modified by CYP703A2/ OsCYP703A3 [18,21] to produce hydroxylated fatty acids, which are then activated by ACOS (ACOS5/OsA-COS12/NtACOS1) to generate fatty acyl CoAs [12,26]. Fatty acyl CoAs are condensed first by PKS (PKSA/ LAP6/OsPKS1 and PKSB/LAP5/NtPKS1/OsPKS2) [16,17,28], and the resultant triketides and tetraketides (also called α-pyrones) are then reduced by TKPR (TKPR1/ OsTKPR1/NtTKPR1) to produce sporopollenin precursors, such as hydroxylated tetraketides and secondary alcohols [27,31]. In Arabidopsis, ACOS5 directly interacts with PKSA, PKSB and TKPR1 in the ER of the tapetum to form a multi-enzyme association [32]. ACOS5 also interacts with CYP703A2 [32], a conserved enzyme involved in the biosynthesis of precursors for anther cuticle and pollen exine formation [18]. In Brassica napus, BnPKSB directly interacts with BnPKA and BnA-COS5, but does not interacts with BnTKPR [35]. Notably, the interaction between BnPKSA and BnPKSB has not been reported for their orthologues in Arabidopsis. Similarly, interactions among these individual metabolon components have not been reported in rice and tobacco [29]. Although further studies are required, these differences may indicate there is a degree of flexibility in the interactions between different metabolon components in different plant systems.

Genetic evidence also suggests a degree of specialization in the role of metabolon components. The acos5, osacos12 and ntacos1 mutants all produce pollen that apparently lack exine [12,26,27,36], but unlike acos5 and ntacos1, osacos12 displays additional defects including smooth anthers with a reduced anther cuticle [12,36], abnormal Ubisch bodies and delayed tapetum degradation [12]. Expression of OsACOS12 under the control of the ACOS5 promoter partially restores male fertility of acos5 mutants [36], suggesting at least some degree of functional conservation. In addition, while pksa/lap6 and pksb/lap5 single mutants produce fertile pollen with irregular exine patterning, the double mutant is completely male sterile with pollen lacking exine deposition [16,17]. By contrast, tobacco plants expressing NtPKS1-RNAi [27] and the single mutant of ospks2 [28,30] are complete sterile, producing aborted pollen with disorganized exine and abnormal anther surfaces. OsPKS2 driven by the PKSB/LAP5 promoter can partially rescue the abnormal exine patterning of pksb/lap5 [28].

Additional evidence supporting functional diversity is derived from the in vitro activity of metabolon components. ACOS belongs to the 4-coumarate CoA ligase (4CL) family, however, in vitro enzyme assays indicate that ACOS5, NtACOS1 and OsACOS12 are not 4CL, but rather fatty acyl-CoA synthetases. The preferential substrates of these enzymes are hydroxylated fatty acids (C8 to C18), fatty acids (C10 to C16) and fatty acid (C18:1), respectively [12,26,27,43]. Plant specific type III PKS is thought to have chalcone synthase (CHS) activity, but an in vitro CHS activity test confirms that PKSA/B (LAP6/5), NtPKS1 and OsPKS1 are not CHS enzymes, but rather are true polyketide synthases. These enzymes have preferential substrates of fatty acyl-CoAs with different carbon length, C6 to C12, C10 to C16, and C16 to C18, respectively, generating corresponding  (2); cis-9,10epoxy C18:0 ω-HFA, cis-9,10-epoxy 18-hydroxy-stearic acid; cis-9,10epoxy C18:1 ω-HFA, cis-9,10-epoxy 18-hydroxyoleic acid; C26 FA, cerotic acid; ALK, alkane; C20:2 ω-HFA, 20-hydroxy-eicosadienoic acid; 9,10,16 Tri-OH C16 FA, 9,10,16-hydroxy-hexadecanoic acid; C20:0 2HFA, 2-hydroxyeicosanoic acid; C21:0 2HFA, 2hydroxyheneicosanoic acid; C22:0 2HFA, 2-hydroxydocosanoic acid; C24:0 2HFA, 2-hydroxytetracosanoic acid; C25:0 2HFA, 2hydroxypentacosenoic acid; ALK, alkane. UI, unknown chemical. *P < 0.05; **P < 0.01 (Student's t test)

α-pyrone lipids [16,27,28]. Although ACOS and PKS in Arabidopsis, tobacco and rice have similar functions, they differ clearly in substrate preference. Unfortunately, in vivo activity of ACOS5/NtACOS1/OsAOC12 has yet to be reported and metabolic analysis failed to identify pyrenes in developing anthers [16], meaning that in planta substrates of ACOS and PKS remain to be determined. Previous studies revealed that neither NtTKPR1 nor OsTKPR1 has DFR activity, because typical substrates including dihydrokaempferol and dihydroquercetin can't be metabolized by NtTKPR1 or OsTKPR1 [27]. However, the significant accumulation of dihydroquercetin in ostkpr1-2 anthers (Fig. 9) could be a clue that OsTKPR1 has in vivo DFR activity in rice. Alternatively, given the significant accumulation of several 2-hydroxylated fatty acids and ferulic acid in anther cutin profile (Fig. 8), it is plausible that both 2-hydroxylated fatty acids and ferulic acid could be the in vivo substrates of OsTKPR1. Our previous study implies that ferulic acid is important for the polymerization of anther cutin, and pollen sporopollenin [3]. Upon the loss of OsTKPR1 activity, the conjugates between aliphatic 2-hydroxylated fatty acids and aromatic ferulic acid in mutant anthers can't be reduced to corresponding secondary alcohols, thus, after de-polymerization, corresponding individual monomers accumulate significantly. At this point in time we can't provide additional evidence to distinguish above hypotheses, which merits further investigations in future studies.

The final evidence for distinct functions of metabolon components comes from the different effects of these proteins on phenylpropanoid metabolism. Results from this study and others [16,28] suggest that phenylpropanoid metabolism is equally important as lipid metabolism in plant male fertility. The inhibition of flavonoid metabolism in ostkpr1-2 anthers was also reported in pksa/lap6 and pksb/lap5 mutants [16]. In contrast, flavonoid metabolism in ospks2 is significantly activated [28]. Therefore, although ostkpr1-2 is male sterile, similar to that of ospks2 [28], ostkpr1-2 showed a different pollen surface and anther inner surface to those of ospks2 [28] (Fig. 3), which may be the consequence of the different effects of these two proteins on the flavonoid pathway ( Fig. 9) [28]. In addition, different proteins differentially impact ferulic acid metabolism. It is reported that the level of free aromatic lipid ferulic acid is increased in ospks2 [28], while the level of polymerized ferulic acid was found to be increased in ostkpr1-2 (Fig. 8 b). This difference could contribute to the different phenotypes observed in ospks2 and ostkpr1-2 (Fig. 4). Our previous study implies that ferulic acid is important for the polymerization of anther cutin, and pollen sporopollenin [3].

Taken together, our studies suggest that OsTKPR1 is a conserved sporopollenin metabolon component but with possibly unique biochemical functions important for male fertility in rice. Some of these unique functions could form the basis of future comparative studies of pollen development between monocot and dicot species.

Conclusions

Our functional characterization results suggested that OsTKPR1, an orthologue of Arabidopsis TKPR1, is indispensable for male fertility in rice. OsTKPR1 affected not only the formation of pollen but also the formation of anther cuticle, which provided new insights into the conserved sporopollenin metabolon in flowering plants. WT and ostkpr1-2 (9522 background, japonica) heterozygous rice seeds were sourced from our lab seeds stock while T-DNA insertion line PFG_2B-00257 (ostkpr1) heterozygous seeds were a gift from Professor Clive Lo (The University of Hong Kong). All rice plants, including wild-type (WT), ostkpr1, and ostkpr1-2 plants, were grown in the paddy field of Shanghai Jiao Tong University (31.03°N, 121.45°E), Shanghai, China. Map-based cloning was performed as previously described [44] with F2 populations generated from crosses between ostkpr1-2 and Guang Lu Ai 4 (indica). All primers used for mapping are listed in Additional file 7: Table S4.

Phenotypic observation

Images of the whole plant and reproductive organs were captured with a Nikon E995 digital camera and a Leica M205A microscope, respectively. Routine analysis of I2-KI staining, semi-thin cross sections, SEM (scanning electron microscopy) and TEM (transmission electron microscopy) were performed as described previously [3,10]. The classification of various stages of developing rice anthers were carried out as described [4].

Quantitative RT-PCR analysis

Total RNA was isolated from various rice tissues using Trizol reagent (Tiangen, Beijing, China), and analyzed with a Nanodrop 1000 spectrophotometer (Thermo Scientific, UT) for assessment of quality and quantity. For RT-PCR, first-strand cDNA was reverse transcribed from total RNA with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa). Real-time RT-PCR was performed with iQ SYBR Green Supermix (Bio-rad), using the real-time PCR system (Bio-Rad C1000 CFX96). The rice actin gene was used as an internal control. Primers used to quantify the expression of OsTKPR1 are listed in Additional file 7: Table S4.

In situ hybridization RNA hybridization and immunological detection of the hybridized probes were performed according to the protocol as previously [45]. The primers used for generation of in situ hybridization probes are listed in Additional file 7: Table S4.

Subcellular localization of OsTKPR1

For transient expression in tobacco (Nicotiana benthamiana) leaves, full-length OsTKPR1 CDS was PCR amplified using OsTKPR1CDS-F and OsTKPR1CDS-R (Additional file 7: Table S4), and introduced into the 1301-GFP vector digested with Bgl II and Spe I to generate 1301-35S pro :OsTKPR1CDS-GFP. The GFP construct was transformed into Agrobacterium strain GV3101 and infiltrated into leaves of 4-week-old tobacco plants. After two day's dark culture, the tobacco leaf epidermis with 1301-35S pro :OsTKPR1CDS-GFP was mounted in water and observed under a confocal microscope (Leica TCS SP5).

Fig. 1
1Phenotypic comparisons between WT and the ostkpr1-2 mutant. a Plants after bolting. b Rice panicles at the heading stage. c Spikelets at the heading stage. d Spikelets after removal of the lemma and palea. e Anther at stage 13 of WT. f Stage 13 WT pollen grains stained with 2% I 2 -KI solution. Mature pollen grains are stained. g Anther at stage 13 of ostkpr1-2 mutant. h Contents of stage 13 anthers of ostkpr1-2 stained with 2% I 2 -KI solution showing no pollen grains. gl, glume; le, lemma; pa, palea; st, stamen. Bars = 2 mm in c, 1 mm in d and e, and 100 μm in f and g

Fig. 2
2Transverse thin-section comparison of anther development between WT and ostkpr1-2 showing locules from the anther section of WT (ae) and ostkpr1-2 (f-j). a and f stage 8b. b and g stage 9. c and h Stage 10. d and i early stage 11. e and j stage 12. E, epidermis; En, endothecium; M, middle layer; T, tapetal layer; Msp, microspores; DMsp, degraded microspore; Mp, mature pollen; Deb, debris. Bars = 15 μm

Fig. 3
3Scanning electron microscopic examination of anthers and pollen in WT and the ostkpr1-2 mutant. a and b, Anther outer surface of WT (a) and ostkpr1-2 (b) at stage 13. c and d, Anther inner surface of WT (c) and ostkpr1-2 (d) at stage 13. e and f, The morphology of a mature WT (e) and ostkpr1-2 (f) pollen. g and h, Enlarged image of the mature pollen surface of WT (g) and ostkpr1-2 (h). Bars = 20 μm in e and f; 10 μm in a to d; 5 μm in g and h Map-based cloning of OsTKPR1

Fig. 4
4Transmission electron microscopy (TEM) analysis of anthers, microspores and pollen exine in WT and ostkpr1-2. a and e Anther at stage 9 of WT and ostkpr1-2. Bars: 5 μm. b and f Tapetal cells at stage 9 of WT and ostkpr1-2. Bars: 1 μm. c and g Microspores at stage 9 of WT and ostkpr1-2. Bars: 2 μm. d and h, Pollen exine at stage 9 of WT and ostkpr1-2. Bars: 0.5 μm. i and m Anther at stage 10 of WT and ostkpr1-2. Bars: 5 μm. j and n Tapetal cells at stage 10 of WT and ostkpr1-2. Bars: 0.5 μm. k and o Microspores at stage 10 of WT and ostkpr1-2. Bars: 2 μm. l and p Pollen exine at stage 9 of WT and ostkpr1-2. Bars: 0.5 μm. Ba, Bacula; C, cuticle; E, epidermis; En, endothecium; Ex, exine; Ml, middle layer; Msp, microspores; Ne, Nexine; T, tapetal layer; Te, Tectum; Ub, Ubisch body

Fig. 5
5Molecular identification of OsTKPR1. a Fine mapping of the OsTKPR1 gene on chromosome 9. Names and positions of molecular markers and the recombinants are indicated. The OsTKPR1 locus was mapped to a 22.6 centimorgan region between two molecular markers (chr9-4979 and chr9-6308). b Schematic representation of the exon and intron organization of OsTKPR1. The mutant sequence has a 15-nt deletion in the fourth exon (indicated by red colored characters in the wild-type sequence and a red arrow in the schematic figure below), causing the deletion of five amino acids (Ser, Leu, Ser, His, Glu) and alteration of one amino acid (from Pro to Gln). Blue boxes indicate exons, intervening lines indicate introns lipidic precursors for the formation of both the anther cuticle and pollen exine in rice.

Fig. 6
6Expression analysis of OsTKPR1. a qPCR analysis of the OsTKPR1 transcript. St 7 to St 13 correspond to anthers at stages 7 to stage 13, respectively. The result is shown as the mean ± standard derivation of three biological replicates. b-e In situ localization of OsTKPR1 expression with digoxigenin-labelled antisense (b and c) and sense (d and e) RNA probe in the WT anther sections at stage 9 (b and d) and stage 10 (c and e). Bars = 20 μm

Fig. 7
7Confocal images showing the subcellular localization of the OsTKPR1-GFP protein in tobacco leaf epidermal cells. Images taken from the GFP fluorescence (Green; a and d), bright field (b), mcherry fluorescence (Red; e) and the merged image (c and f). a-c Localization of the control GFP protein in the nucleus and cytoplasm. d and f Localization of the OsTKPR1-GFP protein in the ER. e Co-localization of OsTKPR1-GFP with the ER-marker of mcherry. Bars = 50 μm

Fig. 8
8Analysis of anther wax and cutin in WT and ostkpr1-2. a Total anther cutin and wax content. b Profile of anther cutin monomers c Profile of anther wax constituents. The results are expressed as mean ± standard derivation of four biological replicates. Compound names are abbreviated as follows: C16:0 FA, palmitic acid; C18:0 FA, stearic acid; C18:1 FA, oleic acid; C18:2 FA, linoleic acid; C18:3 FA, linolenic acid; C20:0 FA, eicosanoic acid; C20:1 FA, gadoleic acid; C22:0 FA, docosanoic acid; C16:0 ω-HFA, 16-hydroxy-hexadecanoic acid; C18:1 ω-HFA, 18-hydroxy-oleic acid; C18:2 ω-HFA, 18-hydroxylinoleic acid; C18:2 ω-HFA(2), 18-hydroxy-linoleic acid

Fig. 9
9Analysis of the phenylpropanoid pathway in WT and ostkpr1-2 anthers. Red and Green colors denote increased and decreased abundance of compounds. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate ligase; CHS, chalconesynthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase. Numbers in the bracket show the altered fold changes of corresponding compounds. The # symbol in some compounds means isomerides Methods Plant materials and molecular cloning of OsTKPR1-2

Funding

AcknowledgementsThe authors would like to thank Dr. Guorun Qu and Qian Luo for their assistance in anther cuticle analysis; we thank Zhijin Luo and Mingjiao Chen for mutant screening and generation of F2 populations for the mapping. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. The gene sequence of OsTKPR1 is deposited in the Phytozome 12 (https://phytozome.jgi.doe.gov/ pz/portal.html) under accession number LOC_Os09g32020.Analysis of anther wax and cutinAnther wax and cutin were analyzed as described previously[3]. The measurement of the anther surface was carried out by plotting the calculated anther surface area against anther dry weight as described previously (Additional file 8:Figure S4)[20].Metabolite profiling and data analysisFour biological replications of rice anthers at stage 11 were collected, immediately frozen in liquid nitrogen, and lyophilized for metabolomics analysis. Methanol extracts from 10 mg of anther powder per sample were analyzed by UHPLC-MS/MS as described previously[46]. Metabolite identification and data analysis were performed as described previously[46].Additional filesAdditional file 1:Table S1. Allelism test of ostkpr1-2 with ostkpr1. Ethics approval and consent to participate Not applicable.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Author details
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