Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Gene therapy of the human retina wikipedia , lookup
Designer baby wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Microevolution wikipedia , lookup
Genetically modified crops wikipedia , lookup
History of genetic engineering wikipedia , lookup
Genetically modified organism containment and escape wikipedia , lookup
JIPB Journal of Integrative Plant Biology Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana FA State Key Laboratory for Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China † These authors contributed equally to this work. *Correspondence: Xiongbo Peng ([email protected]) doi: 10.1111/jipb.12536 Abstract The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase (NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed in most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically complementation NAGK in pollen or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Specific expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss-of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, the results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development. INTRODUCTION nitrogen storage, arginine is a precursor of compounds that act as second messengers in developmental processes; such compounds include polyamines (Takahashi and Kakehi 2010) and nitric oxide (Crawford 2006; Grun et al. 2006; Neill et al. 2008). Arginine plays a major metabolic role in seed maturation and germination, and in phloem and xylem transport, and accumulates under stress conditions (Kalamaki et al. 2009a, 2009b). Therefore, arginine and its metabolism are of central importance in plant biology, but the genes regulating its biosynthesis are only partially known in plants. Arginine synthesis and its regulation have been characterized in prokaryotes, fungi and animals (Cunin et al. 1986; Davis 1986; Caldovic and Tuchman 2003). The biosynthesis of arginine in microorganisms is Edited by: Li-Jia Qu, Peking University, China Received Feb. 10, 2017; Accepted Mar. 14, 2017; Online on Mar. 15, 2017 FA: Free Access, paid by JIPB © 2017 Institute of Botany, Chinese Academy of Sciences www.jipb.net XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX Free Access Nitrogen is commonly the limiting essential element in plant growth (Cleland and Harpole 2010). It becomes increasingly important to investigate the mechanisms of nitrogen uptake, storage and recycling and to understand the interplay of these processes with the regulation of plant development. Due to its high nitrogen to carbon ratio among the amino acids necessary for proteins biosynthesis, arginine is an ideal element for nitrogen storage (Llacer et al. 2008). Arginine provides a significant portion of the stored nitrogen in proteins or as free amino acid in seeds, bulbs, and other plant parts (Micallef and Shelp 1989). In addition to being an important amino acid required in protein synthesis and an intermediate for Research Article Jie Huang†, Dan Chen†, Hailong Yan, Fei Xie, Ying Yu, Liyao Zhang, Mengxiang Sun and Xiongbo Peng* 2 Huang et al. accomplished in eight enzymatic steps and can be divided in two processes (Cunin et al. 1986; Caldovic and Tuchman 2003). First, ornithine is synthesized from glutamate either in a cyclic or a linear pathway, followed by the synthesis of arginine from ornithine. The first reaction in arginine biosynthesis is N-acetylation of glutamate by N-acetylglutamate synthase (NAGS). The second reaction is the phosphorylation of N-acetylglutamate by N-acetylglutamate kinase (NAGK) to produce N-acetylglutamate-5-phosphate which is subsequently converted to N-acetylglutamate-5-semialdehyde, a reaction catalyzed by N-acetylglutamate-5-phosphate reductase (NAGPR). In the fourth reaction, an amino group is transferred to N-acetylglutamate-5-semialdehyde by N2-acetylornithine aminotransferase (NAOAT) to produce N2-acetylornithine which is subsequently converted to ornithine by N2-acetylornithine: glutamate acetyltransferase (NAOGAcT) or N2-acetylornithine deacetylase (NAOD). Then ornithine is converted to citrulline by ornithine transcarbamylase (OTC). Argininosuccinate is then formed from citrulline and aspartate by argininosuccinate synthase (ASSY). In the last reaction, fumarate is cleaved from argininosuccinate by argininosuccinate lyase (ASL) to produce arginine (Slocum 2005). The enzymes involved in plant arginine biosynthesis have partly been identified in silico and biochemically (Slocum 2005; Winter et al. 2015), but less is known about the specific functions of the genes encoding these enzymes (Winter et al. 2015). Only some of the Arabidopsis genes involved in arginine biosynthesis have been studied with the use of mutants. The TUMORPRONE5 (TUP5, At1g80600) gene of Arabidopsis encodes a NAOAT (Fremont et al. 2013). tup5 loss-offunction mutant lines showed a strongly reduced free arginine content and a short root growth phenotype. Molesini et al. analyzed NAOD’s activity in Arabidopsis after downregulation of a putative NAOD gene (At4g17830) using T-DNA insertion mutants and RNA silencing (Molesini et al. 2015). All NAOD-suppressed plants showed consistently reduced ornithine content, early flowering and impaired seed sets. The Venosa3 (VEN3, At1g29900) and Venosa6 (VEN6, At3g27740) genes encode for the large and small subunits of the carbamoyl phosphate synthetase, respectively (MollaMorales et al. 2011). Carbamoyl phosphate and ornithine are substrates of OTC to produce citrulline (Slocum 2005). The ven3 and ven6 mutants displayed a reticulate XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX Figure 1. Putative arginine biosynthesis pathway in plants The enzymes catalyzing the reaction steps of arginine biosynthesis are indicated beside arrows. ASL, argininosuccinate lyase; ASSY, argininosuccinate synthase; NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-phosphate reductase; NAOAT, N2-acetylornithine aminotransferase; NAOGAcT, N2-acetylornithine, glutamate acetyltransferase; NAOD, N2-acetylornithine deacetylase; OTC, ornithine transcarbamylase. leaf phenotype that was correlated with a defect in mesophyll development (Molla-Morales et al. 2011). A T-DNA insertion in the OTC gene caused increased sensitivity to ornithine (Quesada et al. 1999). In addition, the rice ASL mutant osred1 showed a short root phenotype (Xia et al. 2014). These findings suggest that the genes that involved in specific step of arginine biosynthesis play specific roles in particular plant developmental processes. NAGK catalyzes the second reaction of arginine biosynthesis (Figure 1) and is the target of arginine in the negative feedback loop of the arginine biosynthetic pathway (McKay and Shargool 1981). Besides its important role in arginine biosynthesis, NAGK has an important function in the balance of nitrogen and carbon by interacting with PII signaling proteins (Winter et al. 2015). PII signaling proteins interact with www.jipb.net NAGK is critical for gametophytes and embryo development enzymes, transcription factors, and ammonia channels, and regulate their activities and carbon/nitrogen homeostasis (Forchhammer 2008; Uhrig et al. 2009; Huergo et al. 2013). A high nitrogen level will be sensed by the PII protein, which favors PII-NAGK complex formation leading to arginine synthesis and nitrogen storage, while limited nitrogen levels prevent the formation of the PII-NAGK complex, resulting in decreased enzyme activity of NAGK and an increase in the feedback inhibition of the complex by arginine (Burillo et al. 2004; Chen et al. 2006; Feria Bourrellier et al. 2009; Winter et al. 2015). Although plant NAGK have been identified in silico, biochemical and structural analyses (Burillo et al. 2004; Slocum 2005; Chen et al. 2006; Winter et al. 2015), little is known about plant NAGK including its gene expression and function in plant development. In the present investigation, we studied the role of the AT3G57560 gene, a putative Arabidopsis NAGK. Heterologous expression of the Arabidopsis NAGK restored NAGK-deficient Escherichia coli growth on arginine-deficient medium. Loss-of- function of NAGK resulted in slow growth of male gametophytes, decreasing the ability of female gametophytes for pollen tube guidance and abortion of early embryos. The results indicated the essential role of NAGK in these reproductive processes. RESULTS A T-DNA insertion in NAGK affected seed development To screen genes required for gametes and embryo development in Arabidopsis thaliana, we established a T-DNA insertion mutant library that contains one insertion site with hygromycin resistance in the genomic DNA of qrt1 plant background (Preuss et al. 1994). A heterozygous mutant with aborted seeds was isolated (Figure 2B). We identified the flanking sequence of the T-DNA insertion site of the mutated gene by thermal asymmetrical interlaced PCR (Liu et al. 1995) and found that the T-DNA was inserted in AT3G57560, a gene predicted to encode the NAGK, which catalyzes the second reaction of arginine biosynthesis. The T-DNA insertion site is 47bp upstream of the ATG start codon, and thus we designated the mutant nagk (Figure 2A). The authenticity of the T-DNA insertion site was confirmed by PCR www.jipb.net 3 Figure 2. Embryo developmental processes are impaired in nagk/NAGK plants (A) Diagram of the NAGK genomic DNA and the insertion sites for the T-DNA. The T-DNA in nagk/NAGK plants inserted into the promoter at 47 bp upstream of the ATG start codon of NAGK gene (AT3g57560). (B–D) Micrographs of siliques of wild type plants (B), nagk/ NAGK plants (C) and genomic complemented lines (D). Siliques of wild type and genomic complemented line show only normally fertilized seeds (B and D), while nagk/NAGK plant siliques showing normally fertilized ovules, aborted fertilized ovules (indicated by stars) and aborted unfertilized ovules (indicated by arrows) (C). (E) and (F) Micrographs of normally fertilized ovules and aborted fertilized ovules 5 d after fertilization in nagk/NAGK plant. When embryo develops to torpedoshaped embryo in normally fertilized ovules (E), the mutant embryo in aborted fertilized ovules remained in four-celled stage (F). Scale bar ¼ 1 mm for (B) to (D), 50 mm for (E) and 20 mm for (F). using DNA from nagk. nagk/nagk mutant plants could not be recovered in the offspring of nagk/NAGK plants, suggesting the nagk/nagk embryos or/and nagk gametes are lethal. Only normally fertilized ovules were found in wild type siliques (Figure 2B), while in the siliques of nagk/ NAGK plants, three types of ovules were observed: normally fertilized ovules, aborted fertilized ovules and aborted unfertilized ovules (Figure 2C). The ratio of these three types ovules are shown in Table 1. The aborted fertilized ovules were expected to contain the homozygous nagk/nagk embryos. To verify this, we crossed nagk/ NAGK plants with wild type plants. The crosses resulted in no aborted fertilized ovules in the hybrid siliques XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX 4 Huang et al. Table 1. Ovule phenotype of self-pollination and reciprocal cross between nagk/NAGK plants and wild type plants Female Male Normally fertilized ovules (ratio) Aborted fertilized ovules (ratio) Unfertilized ovules (ratio) Wild type nagk/NAGK nagk/NAGK Wild type Wild type nagk/NAGK Wild type nagk/NAGK 438 (97.33%) 656 (70.69%) 799 (79.66%) 357 (100%) 0 (0%) 91 (9.81%) 0 (0%) 0 (0%) 2 (0.67%) 181 (19.50%) 204 (20.34%) 0 (0%) (Table1), proving that the small aborted fertilized ovules enclosed the homozygous nagk/nagk embryos. Normally fertilized ovules and aborted fertilized ovules were separated from nagk/NAGK plants 5 d after fertilization and then observed by whole-mount clearing technique. In normally fertilized ovules, embryos had developed into torpedo-shaped embryos (Figure 2E), while in small aborted fertilized ovules the embryos remained at the four-celled stage (Figure 2F), indicating nagk is embryo lethal. To further confirm that defective seed development phenotype was caused by the loss of function of AT3G57560, a genomic fragment of AT3G57560 was cloned in a kanamycin resistant vector and introduced into nagk/NAGK plants. The seedlings showing both kanamycin resistance and hygromycin resistance were designated complementation lines, and were transplanted to soil. In the complementation lines, normal seed development was restored (Figure 2D). These results indicated that the AT3G57560 genomic fragments could successfully complement the nagk/NAGK plants phenotype. NAGK is a plastid-localized protein expressed in the majority of developmental processes in Arabidopsis The iPSORT prediction program predicted that NAGK is targeted to plastid (Bannai et al. 2002). To confirm NAGK localization, plants co-expressing 35S::NAGK-GFP and the plastid marker 35S::Plastid-RFP were produced by crossing and examined with confocal laser scanning microscopy. In root cells, NAGK-GFP was found to colocalize with the plastid-localized RFP (Figure 3A–D), indicating that NAGK is a plastid-localized protein. In petal cells, NAGK-GFP was found to locate to sub-plastid (Figure 3E–H), indicating that arginine synthesis catalyzed by NAGK is compartmented within the plastid of petal cells. The results indicated that NAGK is a nuclearencoded and plastid-localized protein. XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX Real time RT–PCR analysis showed that transcripts for NAGK are expressed in the root, shoot, leaf and flower (Figure 4A). To further analyze the expression pattern of NAGK, we fused a 2010 bp fragment of its native promoter to the GUS reporters and produced pNAGK::GUS. The transgenic T2 progeny of pNAGK::GUS homozygous lines showed high GUS activity in the root of seedlings and relative weaker signal in the leaf (Figure 4B). Regarding inflorescence developmental stages, the GUS signal was observed in the whole inflorescence (Figure 4C) and mature pollens (Figure 4D). To further verify the expression pattern of NAGK in plants, we analyzed transgenic Arabidopsis plants containing a protein fusion construct in which the NAGK promoter and the entire NAGK coding sequence were fused to GFP (pNAGK::NAGK-GFP). This construct was sufficient to rescue the mutant phenotype of the nagk/NAGK plants, suggesting that expression of this construct mimics that of the endogenous gene. As shown in Figure 4E, pNAGK::NAGK-GFP expression was detected in the root. To determine whether pNAGK:: NAGK-GFP is expressed in reproductive organs, we analyzed its expression in flowers at stage 12c or after fertilization. pNAGK::NAGK-GFP expressed in pollens (Figure 4F), female gametophytes (Figure 4G) and embryos (Figure 4H), suggesting NAGK plays a role in these tissues. AtNAGK rescued a NAGK-Deficient E. coli mutant To test whether AtNAGK has the predicted function of a NAGK, we examined its ability to restore arginine autotrophy in the NAGK-deficient E. coli mutant strain JW5553-1 (Baba et al. 2006). For heterologous expression of the Arabidopsis NAGK, the coding region of AtNAGK cDNAs with or without the transit peptide sequences (NAGKDTP) were amplified and cloned into the expression vector pET-28a to produce a NAGK plasmid and a NAGKDTP plasmid. As expected, the www.jipb.net NAGK is critical for gametophytes and embryo development 5 Figure 3. Localization of NAGK-GFP protein is in the plastid Plant coexpressing NAGK-GFP and the chloroplast marker chloroplast-RFP were examined with confocal laser scanning microscopy. (A–D) Colocalization in root cells. (A) Bright field of root cells. (B) Red fluorescent signal from the plastid marker plastid-RFP. (C) Green fluorescent signal from NAGK-GFP. (D) Merged picture with green and red signals showing colocalization. (E–H) Colocalization in petal cells. (E) Bright filed of petal cells. (F) Red fluorescent signal from the plastid marker chloroplast-RFP. (G) Green fluorescent signal from NAGK-GFP. (H) Merged picture with green and red signals showed that NAGK-GFP located into sub-chloroplast. Scale bar ¼ 40 mm. JW5553-1 and JW5553-1 transformed with the empty vector pET-28a or NAGK plasmid or NAGKDTP plasmid were able to grow on medium containing arginine (Figure 5A). The JW5553-1 and JW5553-1 transformed with the empty vector pET-28a were unable to grow on medium lacking arginine (Figure 5B). In contrast, the JW5553-1 transformed with the NAGK plasmid or NAGKDTP plasmid became arginine autotrophic (Figure 5B), indicating that Arabidopsis NAGK is an evolutionarily conserved enzyme that functions as a NAGK to catalyzes the synthesis of N-acetylglutamate-5-phosphate from N-acetylglutamate. nagk mutation affected both male and female gametophytes Our study showed that 44.14% (n ¼ 1,330) of nagk/NAGK plants progeny seeds showed hygromycin resistance (Hygr) (Table 2). The ratio was much lower than the expected 75% ratio. In addition, nagk/NAGK plants siliques showed aborted unfertilized ovules (Figure 1C). These www.jipb.net results suggested that nagk mutation affected the development of gametophytes in addition to embryos. To determine whether nagk mutation affected the female gametophyte or the male gametophyte, we assessed the nagk mutant transmission efficiency through male/female gametophyte by reciprocal crosses of nagk/NAGK plants and wild type plants. As shown in Table 2, the Hygr ratio of the crossed line’s seeds was 30.57% (n ¼ 700) when nagk/NAGK plants were used as female parent, indicating a defect exists in female gametophytes. However, the Hygr ratio dropped to 4.05% (n ¼ 1,286) when nagk/NAGK plants were used as male parent, demonstrating a stronger defect occurred in the male gametophyte (Table 2). nagk mutation lowered pollen tube competitiveness We carried out a semi-in vitro pollen growth assay to determine the defects occurring in the male gametophytes of nagk/NAGK plants. We pollinated pollen grains of nagk/NAGK plants on wild type stigma. Pollinated XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX 6 Huang et al. 6.57% (n ¼ 198) of the pollen tubes were nagk pollen tubes (marked by GFP) (Figure 5C). These results indicated that nagk lowered pollen tube competitiveness due to nagk pollen tubes growing much slower than wild type pollen tubes. As a result, few nagk mutant pollen tubes could enter the ovules, leading to a decrease of nagk mutant transmission efficiency through male gametophytes. Figure 4. The expression pattern of NAGK (A) Real time RT-PCR analysis showed that transcripts for NAGK were expressed in roots, shoot and flower, with lower expression in leave. (B–D) GUS expression pattern in different plant parts of transgenic pro NAGK:: GUS lines. (B) 14-d-old seedling. Strong GUS signal was observed in root and shoot meristems. Leave showed a relative weaker GUS signal. (C) The inflorescence showed a strong GUS signal. (D) Pollens showed a strong GUS signal. (E–H) GFP expression pattern in different plant parts of transgenic pNAGK::NAGK-GFP. (E) pNAGK::NAGK-GFP line showed GFP signal in plastids of root. (F) pNAGK::NAGK-GFP line showed GFP signal in plastids of pollens. (G) pNAGK::NAGK-GFP line showed GFP signal in plastids of the ovule. Note female gametophytes (surrounded by white line) showed GFP signal. (H) pNAGK::NAGK-GFP expressed in plastids of the embryo. Scale bar ¼ 4 mm for B; 2 mm for C; 50 mm for D; 100 mm for E and 20 mm for F–H. pistils were cut 1 h later, and cultured on solid medium. Pollen tubes could grow out of the cut end of the pistils for several millimeters 6 h after pollination, but only XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX Specific expression of NAGK in pollens and exogenous application of arginine rescued the nagk pollen tube growth phenotype To test whether the decreased competitiveness of mutant pollen tubes could be recovered by specific expression of NAGK in nagk pollens, we transformed nagk/NAGK plants with the pLAT52::NAGK construct. T2 pLAT52 complementation lines (nagk/NAGK, pLAT52:: NAGK/pLAT52::NAGK) were screened for the semi-in vitro pollen growth assay. pLAT52 complementation line (Figure 5D, 44.69%, n ¼ 179) showed significant difference (P < 0.01, Student’s t-test) in the ratio of GFP pollen tubes penetrated through style when compared to nagk mutant (Figure 5C, 6.57%, n ¼ 198). We further crossed wild type with the pollens of the two pLAT52 complementation lines, respectively, to determine the transmission efficiency of Hygr through the male gametophytes. The results showed that the male transmission efficiency of Hygr increased from 4.05% (Table 2, data of the first arrow) to about 48% in both two pLAT52 complementation lines (Table 2, data of the fourth and fifth arrows). Together, these results confirmed that nagk mutant lowered pollen tube competitiveness in the style, and the decreased competitiveness of nagk pollen tubes could be recovered by specifically compensating NAGK in pollens. Since NAGK is a key enzyme for arginine biosynthesis, it is reasonable to conclude that the insufficient arginine in nagk pollen tubes decreases their competitiveness. We investigated whether exogenous applications of arginine (1 mmol/L) could rescue the defect of the nagk pollens. The ratio of GFP pollen tubes (nagk background) that penetrated through style increased to 39.13% (n ¼ 207) when arginine was added into the medium (Figure 5E), which is much higher than that of nagk pollen tubes without exogenous arginine (6.57%, n ¼ 198, Figure 5C). The results indicated that arginine deficiency is the major cause for the defect of nagk pollen tubes. www.jipb.net NAGK is critical for gametophytes and embryo development 7 Figure 5. Arabidopsis NAGK is an evolutionarily conserved enzyme that functions as a NAGK involved in arginine synthesis (A, B) AtNAGK restores arginine autotrophy in the NAGK-deficient Escherichia coli mutant strain JW5553-1. (A) Escherichia coli on medium containing arginine. (B) Escherichia coli on medium lacking arginine. 1, JW5553-1; 2, JW5553-1 transformed with the empty expression vector pET-28a; 3, JW5553-1 transformed with vector containing the coding region of AtNAGK cDNAs. 4, JW5553-1 transformed with vector containing the coding region of AtNAGK cDNAs without the transit peptide sequences. (C–E) nagk mutant weakens pollen tube competitiveness. Pollens from different lines were pollinated to the stigma of wild type. CLSM images of pollen tubes growth were obtained after 6 h on semi-in vitro conditions. (C) Pollinated with pollens from nagk/NAGK plants showed pollen tubes of nagk mutant (marked by GFP) are less and shorter than wild type pollen tubes (without GFP). (D) Pollinated with pollens from LAT52 complement line (nagk/NAGK plants transformed with pLAT52::NAGK) showed that pollen tubes of nagk mutant (marked by GFP) grew similar to that of wild type (without GFP). (E) Pollinated with pollens from nagk/NAGK plants and supplemented with arginine showed that pollen tubes of nagk mutant (marked by GFP) grew similar to that of wild type (without GFP). Scale bar ¼ 100 mm for (C) to (E). nagk/NAGK plants were defective in micropylar pollen tube guidance Aborted unfertilized ovules were found in the siliques of nagk. To determine the mechanism through which nagk mutant affects female gametophytes, we investigated whether pollen tube guidance was impaired in nagk/ NAGK plants. By aniline blue staining, we found that 100% of the ovules (n ¼ 199) had pollen tube entry in Table 2. Segregation of the nagk1 mutation in selfed, reciprocally crossed with wild type or pLAT52 complementation lines offspring populations Parental genotype Progeny genotype Female Male Hygr Total seeds Hygr ratio nagk/NAGK nagk/NAGK wild type wild type wild type nagk/NAGK wild type nagk/NAGK LAT52::NAGK-1 LAT52:: NAGK 2 587 214 50 174 221 1,330 700 1,286 358 467 44.14% 30.57% 4.05% 48.6% 47.30% www.jipb.net XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX 8 Huang et al. Figure 6. nagk/NAGK plants formed morphologically normal but dysfunctional female gametophytes (A, B) Aniline blue staining showed pollen tube guidance in ovules. Ovules attracted pollen tubes (indicated by arrows) in wild type (A), but some ovules of nagk/NAGK plants lost their ability to guide pollen tubes entering the micropyle and were unfertilized. (B) The stars in (A) and (B) indicated micropylar end of the ovules. (C, D) Cleared whole mounts of female gametophytes. (C) A wild type ovule with mature embryo sac at stage FG7. Note the polar nuclei fused to form a diploid central nucleus and antipodal nuclei were degenerated. (D) A mutant ovule with normal mature embryo sac at stage FG7. (E–H) Expression of FGR 7.0 in wild-type (E and G) and nagk (F and H) female gametophytes. Scale bar ¼ 20 mm. wild-type pistils (Figure 6A), while 17.71% of ovules (n ¼ 288) had no pollen tube entry in mutant pistils (Figure 6B). These observations revealed that some mutant ovules lost their ability to guide pollen tube growth to the micropyle, resulting in aborted unfertilized ovules in nagk/NAGK plants. nagk/NAGK plants formed morphologically normal but dysfunctional female gametophytes To determine the mechanism leading to the loss of ability to guide pollen tubes in mutant ovules, we firstly analyzed female gametophytes at the terminal developmental stage (stage FG7) (Christensen et al. 1997). We emasculated flowers at stage 12c (Smyth et al. 1990; Christensen et al. 1997), waited 24 h, and observed ovules by whole-mount clearing. Wild-type female gametophytes at this stage have one egg cell, two synergid cells and one central cell (Figure 6C); the three antipodal cells undergoing cell death are hardly detected (Christensen et al. 1997). In nagk female gametophytes, the egg cell, two synergid cells and one XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX central cell appeared similar to those of wild type plants (Figure 6D). The results indicated that nagk female gametophytes could reach the terminal developmental stage morphologically. It is possible that these morphologically normal female gametophytes have alterations in cell fate specification, thus losing their ability of guiding pollen tube growth. To test the possibility, we crossed a €lz triple marker of female gametophytes FGR7.0 (Vo et al. 2013) with nagk/NAGK plants and analyzed their segregation patterns in the F2 generations. Typical marker patterns in FGR7.0 ovules are shown in Figure 6E and 6G. In transgenic plant lines heterozygous for the nagk mutation and homozygous for the FGR7.0, 75.68% of ovules (n ¼ 370) displayed the same fluorescent pattern as FGR7.0, while 12.97% of ovules showed a weak fluorescent signal (Figure 6F, H), and 11.35% of ovules showed no fluorescent signal. The above phenotypic analysis indicated that nagk specifically affects cell fate specification in female gametophytes. www.jipb.net NAGK is critical for gametophytes and embryo development 9 Figure 7. Aborted ovules in nagk/NAGK plants could be rescued by specific expression of NAGK in the synergid cells (A) An ovule expressing pDD31::NAGK-GFP in the synergids. NAGK-GFP is localized to plastid. (B) Ovules phenotype in wild type plants, nagk/NAGK plants, two independent transformant lines of pDD31::NAGK-GFP in the nagk/NAGK plants background (DD31 line 1 and DD31 line 2), and nagk/NAGK plants was watered with ornithine or arginine supplementation. Scale bar ¼ 10 mm. Specific expression of NAGK in the synergid cells rescued the mutant phenotype Synergid cells play an important role in pollen tube guidance (Higashiyama et al. 2001; Kasahara et al. 2005; Okuda et al. 2009). Since nagk specifically affected female gametophytes cell fate specification and pollen tube guidance, it was reasonable to investigate whether synergid cells were dysfunctional in nagk/NAGK plants. We restricted the expression of NAGK to the synergid cell using a synergid cell–specific DD31 promoter (Steffen et al. 2007). We introduced the transgene pDD31::NAGK-GFP into the nagk/NAGK plants (Figure 7A). Two independent transgenic plants (DD31 lines 1 and 2) were obtained and they showed significant decrease of the unfertilized ovules (5.2% and 7.1%) compared with nagk (19.50%) (Figure 7B). Our results suggested that NAGK was required for the synergid cell functional specification, which was necessary for pollen tube guidance. Aborted ovules could not be rescued by watering nagk/NAGK plants with ornithine or arginine supplementation We investigated whether supplemental arginine could rescue aborted ovules in nagk/NAGK plants. Solution containing 1mM arginine was supplied to the roots of nagk/NAGK plants during daily watering. We detected www.jipb.net the development of ovules in siliques that were formed after arginine being added. The results showed the ratio of aborted ovules in the nagk/NAGK plants watered with arginine is similar to that of nagk/NAGK plants without exogenous arginine (Figure 7B), indicating that the exogenous application of arginine could not rescue the aborted ovules of nagk/NAGK plants. As ornithine is an intermediate in arginine biosynthesis, we also tested its ability to rescue nagk/NAGK plants. The results showed that the ratio of aborted ovules in the nagk/NAGK plants watered with 1mM ornithine is similar with that of nagk/NAGK plants (Figure 7B), indicating that the exogenous application of ornithine could not rescue the aborted ovules of nagk/NAGK plants. DISCUSSION AtNAGK encodes a plastid-localized N-acetylglutamate kinase NAGK catalyzes the second step in the arginine biosynthetic pathway. The reaction catalyzed by NAGK involves two substrates, ATP and NAG, in which NAGK transfers the phosphate group from ATP to NAG to form N-acetylglutamyl-phosphate. Based on its sequence similarity to NAGK from other species, XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX 10 Huang et al. AtNAGK has been proposed to function in the arginine biosynthetic pathway and be localized to plastid in Arabidopsis (Slocum 2005). We showed that AtNAGKGFP fusion protein was localized to plastid. This is consistent with the database prediction of a plastid localization of NAGK and immuno-histological staining observations (Chen et al. 2006). We showed that the phenotype of our T-DNA mutant line was indeed due to an insertion mutation in a gene encoding the Arabidopsis NAGK required for arginine biosynthesis. The validity of our mutant and the function of AtNAGK were supported by the finding that the nagk pollen tube phenotype could be complemented by supplementation with arginine, which is synthesized downstream of NAGK. Furthermore, we showed that Arabidopsis NAGK could rescue a NAGK-deficient E. coli mutant. Recombinant AtNAGK can catalyze ATP and NAG to form N-acetylglutamyl-phosphate in vitro (Chen et al. 2006). Together, these results indicated that Arabidopsis NAGK is an evolutionarily conserved enzyme that functions as a plastid-localized NAGK to catalyze the synthesis of N-acetylglutamate5-phosphate from N-acetylglutamate. AtNAGK is required for embryo development Phenotype analysis of nagk/NAGK plants revealed the requirement of NAGK for early embryo development. The homozygous nagk/nagk plants could not be recovered, and in small aborted fertilized ovules the embryos remained at the four-celled stage nagk/NAGK plants. These results indicated that arginine was essential for embryo development. The embryo lethal phenotype of nagk/NAGK plants indicated that surrounding maternal tissues were unable to provide sufficient arginine to support continued embryo development, suggesting embryos at least need a certain degree of arginine autotrophy. Embryo lethality had been also reported in other amino acid biosynthesis mutants, including mutants in lysine biosynthesis (Song et al. 2004; Hudson et al. 2006), histidine biosynthesis (Muralla et al. 2007) and proline kely et al. 2008). Mutants of biosynthesis genes (Sze four different histidine biosynthetic genes (HISN2, HISN3, HISN4, HISN6A) were shown to exhibit an embryo-defective phenotype that could be rescued by watering heterozygous plants with histidine supplementation. In contrast, nagk embryo-defective phenotype could not be rescued by watering heterozygous XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX plants with arginine or ornithine. These results suggested that Arabidopsis embryos are completely arginine autotrophic and little arginine was transported by the maternal tissue to the early embryos. Arginine is required for synergid functional specification for pollen tube guidance In the sexual reproduction of flowering plants, successful double fertilization depends on the delivery of two immotile sperm cells through the tip growth of pollen tube to the female gametophyte. Early studies based on mutant analysis showed that the pollen tube is precisely guided by female gametophyte to achieve fertilization (Hulskamp et al. 1995; Ray et al. 1997; Shimizu and Okada 2000). Cell ablation experiment indicated that two synergid cells are essential for the attraction of the pollen tube to the ovule in Torenia fournieri (Higashiyama et al. 2001). Then the pollen tube attractants, defensin-like cysteine-rich LURE peptides, secreted from the synergid cell were identified in Torenia fournieri and Arabidopsis thaliana (Okuda et al. 2009; Takeuchi and Higashiyama 2012). Recently two groups identified several molecules located on the pollen tube membrane as the receptors of AtLURE1 (Takeuchi and Higashiyama 2016; Wang et al. 2016). Aniline blue staining showed that 17.71% ovules lost their ability to guide pollen tubes in nagk/NAGK plants. We further showed that 24.32% of ovules displayed abnormal female gametophyte marker FGR7.0 in nagk/ NAGK plants. These results indicated that about 20% female gametophytes cannot fulfill their functional specification in nagk/NAGK plants. The female gametophytes in nagk/NAGK plants, however, were morphologically normal compared to those of the wild type, suggesting functional specification of female gametophytes is uncoupled from their morphology. Loss of function of NAGK resulted in a pollen tubes guidance defect, while restricting the expression of NAGK to the synergid cell in nagk/NAGK plants rescued the phenotype. Regarding NAGK’s role in arginine synthesis, we suggested that arginine was required for synergid functional specification, which appeared necessary for pollen tube guidance. This may provide a novel clue for the establishment of signals in synergid cells for male and female gametophyte interaction. It is interesting to know how the arginine plays its regulatory role in these critical developmental processes. Based on the work described above arginine does not www.jipb.net NAGK is critical for gametophytes and embryo development only act as an essential nutrient, since other amino acid biosynthesis mutants are embryo lethality but do not show synergid cell defects (Song et al. 2004; Hudson kely et al. 2008). et al. 2006; Muralla et al. 2007; Sze However, we don’t have sufficient evidence yet to confirm its function as a signaling molecule in these processes. Obviously, further much more works are required to clarify whether arginine serves as an essential nutrient or a signaling molecule in plant reproduction. MATERIALS AND METHODS Plant materials and growth conditions The nagk allele was isolated from our mutant library with hygromycin resistance (Wu et al. 2012; Xie et al. 2016; Yan et al. 2016). The transgenic line FGR7.0, a reporter that combines the marker genes for synergids, €lz et al. egg cell, and central cell on one plasmid (Vo 2013), was kindly provided by Professor Rita Groß-Hardt (University of T€ ubingen, Germany). Seeds were surface sterilized with 20% bleach for 10 min, and washed three times with sterile distilled water. Seeds were stratified for 3 d at 4 °C, and then sown on 1/2 MS plates with 1.0% (w/v) sucrose. Agar plates were placed in a growth room with a photoperiod of 16 h light/8 h dark. For kanamycin selection, 50 mg/L of kanamycin (Sigma) was supplemented to the media. Similarly, 50 mg/L of hygromycin (Roche) was added for hygromycin selection. For Basta selection, 10-d old seedlings were selected by spraying them with 0.1% BASTA herbicide in the greenhouse, and repeated two times at 4-d intervals. Plants were grown in soil in a greenhouse under long-day conditions (16 h light/8 h dark) at 22 °C. Cloning of the T-DNA flanking sequence of the nagk The T-DNA flanking sequence in the nagk mutant was cloned by TAIL-PCR (Liu et al. 1995). The authenticity of the cloned sequence was confirmed by PCR using a pair of primers located around the T-DNA border region (nagk-T1: CTGTTTTATTTCCCGCTACAAGATG; LB-S: CCAA AATCCAGTACTAAAATCCAG). nagk-T1 is a gene-specific primer and LB-S is a T-DNA specific primer. Vector construction and plant transformation Plasmid P092, P093, P094, 35S::EGFP construct and 35S:: RFP construct were produced as previously described (Xie et al. 2016; Yan et al. 2016). To generate the www.jipb.net 11 genomic complementation construct, a 3,533-bp wildtype genomic sequence containing the AT3g57560 gene, 2,010-bp upstream of the ATG codon and 479bp downstream of the TAA codon sequences, was PCRamplified with primers NAGK-F1: NNNNGGTACCTTCCAACAAGAAGAGAGCAGTAGAG and NAGK-R1: NNNNGAATT CCAGAGCTAAACAAACAAACAAATGAG from genomic DNA and then was cloned into the P092 plasmid. To investigate the expression pattern of NAGK, we amplified NAGK promoter with primers NAGK-F1 and NAGK-R2: NNNNCTCGAGTCCTGAACCTTACCGGAGAAGG and clone it upstream of GUS in P093 to generate pNAGK::GUS. To produce the pNAGK::NAGK-GFP construct, we amplified the NAGK promoter and the entire NAGK coding sequence with primers NAGK-CDS2: NNNNCTCGAGTCCAGTAATCATAGTTCCAGCTCCTTC and NAGK-F1 from genomic DNA and cloned it into P094. To examine the subcellular localization of NAGK, the NAGK ORF was amplified with primers NAGK-CDS1: NNNNGGTACCGCCACCATGGCCACCGTCACATCC and NA GK-CDS2 from genomic DNA and cloned into 35S::EGFP plasmid to generate a 35S::NAGK-EGFP construct. To produce the plastid marker line, we amplified and cloned the 168bp DNA fragment containing the plastid-targeted pre-sequence of RECA1 gene AT1G79050 (Cerutti et al. 1992) with primers RECA-1: NNNN GGTACCGCCACCATGGATTCACAGCTAGTCTTGTCTC and RECA-2: NNNNCTGCAGGAGTTTCTTCGCGGCGTAG into 35S::RFP to generate 35S:: Plastid-RFP construct. To specifically express NAGK in the pollens, the nopaline synthase (NOS) terminator was amplified with primers NOST-1: NNNNAAGCTTACCAGCTCGAATTTCCCCG and NOST-2: NNNNGAATTCCCGATCTAGTAACATAGATGACACC and cloned into P092 to produce P105. Then the promoter of pollen specific gene LAT52 (Twell et al. 1989) was amplified with primers LAT-23: NNNN CCAACGCGTTGGTGTCGACATACTCGACTCAGAAG LAT-24: NNNNGAGCTCTTTAAATTGGAATTTTTTTTTTTGG and cloned into P105 to generate P175. Then the NAGK ORF was cloned into P175 to generate and pLAT52::NAGKNosT construct To specifically express NAGK in synergid cells, we amplified a synergid cell–specific DD31 promoter (Steffen et al. 2007) using primers DD31-1: NNNNC CAACGCGTTGGACCCACACGAAGAATCGGAC and DD31-2: NNNNGAGCTCTTTTTTTATGGATGTAAGAATACTT TTAGTATTG and cloned it into P094 to produce pDD31::GFP. Then the NAGK ORF was cloned into pDD31::GFP to generate and pDD31::NAGK-GFP construct. XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX 12 Huang et al. All constructs were transformed into Agrobacterium tumefaciens strain GV3101, and then transformed into Arabidopsis plants by floral dipping (Clough and Bent 1998). Phenotype characterization of embryo and female gametophyte development To detect the embryo development, seeds of 5 d after fertilization were dissected from siliques and cleared using a chloral hydrate solution (Breuninger et al. 2008). To detect the female gametophyte development, we emasculated flowers at stage 12c (Christensen et al. 1997), waited 24 h, and observed ovules by wholemount clearing (Wu et al. 2012). The cleared seeds were placed under a microscope (Olympus) fitted with differential interference contrast optics for imaging. Semi-in vitro pollen tube growth assays Semi-in vitro pollen tube growth assay was performed as previously described (Palanivelu and Preuss 2006). Pollen germination medium containing 1 mmol/L CaCl2, 1 mmol/L Ca(NO3)2, 1 mmol/L MgSO4, 0.01% H3BO4, 18% sucrose and 1% agarose, which was modified from recent reports (Dou et al. 2016; Liao et al. 2016). After hand-pollination, pistils were cut at the shoulder region of the ovaries. Cut pistils were incubated on pollen tube growth medium with or without arginine (1 mmol/L) at 28 °C. synthesized using oligo-dT and M-MLV reverse transcriptase (Invitrogen). Quantitative PCR analysis was performed using FastStart Essential DNA Green Master (Roche) on a CFX ConnectTM Real-Time System (BioRad). Each experiment was repeated three times and samples were normalized using UBQ10 expression. Data acquisition and analyses used the Bio-Rad CFX Manager software; relative expression levels were measured using the 2(-DDCt) analysis method and the error bars represent the variance of three replicates. The primers used for detection of NAGK mRNA expression are NAGK–D1: TCGTCTTCTCACAGCACGAC and NAGK–D2: AGCGGACGTAACACAGATGG. The primers used for detection of UBQ10 mRNA expression are UBQ10-D1: GGCCTTGTATAATCCCTGATGAATAAG and UBQ10–D2: AAAGAGATAACAGGAACGGAAACATAGT. Analysis of subcellular localization of NAGK The iPSORT prediction program predicted that NAGK is targeted to the plastid. To confirm its plastid localization, transgenic plants containing p35S::NAGK-EGFP construct were crossed with a transgenic mitochondrial marker line expressing 35S::Plastid-RFP. The root cells and petal cells of the F1 progeny were visualized using a FV1000 confocal laser-scanning microscope (CLSM; Olympus). GFP fluorescence was detected with excitation at 488 nm and emission at 510–530 nm; RFP fluorescence was detected with excitation at 568 nm and emission at 590–620 nm. Histochemical analysis of GUS activity The histochemical analysis of GUS activity was performed as previously described (Vielle-Calzada et al. 2000). Plant tissues were incubated at 37 °C in GUS staining solution (2 mmol/L 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50 mmol/L sodium phosphate buffer, pH 7.0) containing 0.1% Triton X-100, 2 mmol/ L K4Fe(CN)6 and 2 mmol/L K3Fe(CN)6. The stained tissues were then transferred to 70% (v/v) ethanol solution. Samples were mounted with clearing solution and placed under a microscope (Olympus) fitted with differential interference contrast optics for imaging. Aniline blue Staining of Pollen Tubes To visualize pollen tubes, siliques were fixed immediately in Carnoy’s fixative (acetic-acid/methanol, 1:3) twice. The fixed sample was rinsed twice with distilled water and then put in 5M NaOH overnight. Siliques were then rinsed twice with distilled water and subsequently stained with 0.1% aniline blue (Sigma–Aldrich) for 4 h. The ovary walls of the stained siliques were removed with a fine needle under a stereoscope. Then the samples were observed using a microscope (Olympus) equipped with an epifluorescence UV filter set. Quantitative RT-PCR Total RNA of root, shoot, inflorescence and leaf were individually extracted using the RNAqueous Phenol-free total RNA Isolation kit (Ambion) according to the manufacturer’s protocol. After digestion with RNasefree DNase I (Promega), the first strand of cDNA was Analysis of the expression pattern of pNAGK:: NAGK-GFP For fluorescent marker line analysis, ovules of Arabidopsis at specific development periods were collected and put on a 30 mm diameter culture plate with a drop of 10% glycerin added to 80 mmol/L sorbitol. A sharp XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX www.jipb.net NAGK is critical for gametophytes and embryo development capillary glass tube was used as a dissection tool to extract the embryos from maternal tissues (Yan et al. 2016). The isolated embryos, pollens, ovules and roots of pNAGK::NAGK-GFP lines were visualized using a FV1000 confocal laser-scanning microscope (CLSM; Olympus). GFP fluorescence was detected with excitation at 488 nm. Analysis of FGR7.0 in nagk female gametophytes To analyze FGR7.0 expression in the nagk mutant background, nagk/NAGK plants acted as females and were crossed with plants homozygous for the reporter construct FGR7.0. F1 plants heterozygous for the nagk (hygromycin resistance) and hemizygous for FGR7.0 (Basta resistance) were allowed to self-cross. F2 plants heterozygous for the nagk and homozygous for FGR7.0 were selected for fluorescence analysis. One-quarter of these plants should also be homozygous for FGR7.0. Analysis of FGR7.0 expression was performed at stage FG7. Flowers were emasculated at stage 12c and pistils were collected at 24 h after emasculation. The ovules were dissected and visualized using a CLSM (Leica). GFP fluorescence was detected with excitation at 488 nm and emission at 510–550 nm; RFP fluorescence was detected with excitation at 568 nm and emission at 590–620 nm. Functional complementation of an E. coli mutant defective in NAGK The E. coli strain JW5553-1 with a deletion in argB (homologous gene of NAGK) was provided by the Coli Genetic Stock Center (CGSC) at Yale. The coding region of AtNAGK cDNAs was amplified with the primers Q5-R1: NNNNCCATGGGACGAGGTAAAACCATAGTTGTCAAAT and Q5-R3: NNNNCTCGAGTTATCCAGTAATCATAGTTCCAGCTC CTTC. In addition, the coding region of AtNAGK cDNAs lacking the transit peptide sequences (NAGKDTP) was amplified with the primers Q5-R2: NNNNCCATGGGAGCCACCGTCACATCCAATG and Q5-R3: NNNNCTCGAGTTATCCAGTAATCATAGTTCCAGCTCCTTC. The two fragments were cloned into expression vector pET-28a respectively to produce NAGK plasmid and NAGKDTP plasmid. The two plasmids and the empty vector pET-28a were transformed into JW5553-1. Transformed E. coli strains and JW5553-1 were grown on synthetic defined medium containing 20 mg arginine or without arginine to test arginine autotrophy. www.jipb.net 13 ACKNOWLEDGEMENTS We thank Professor Rita Groß-Hardt for providing the transgenic line FGR7.0. This work was supported by the Fund of Key Basic Theory Research of Ministry of Science and Technology of China (2013CB945100) and the National Natural Science Foundation of China (31570317, 31270362). AUTHOR CONTRIBUTIONS J.H., D.C., H.Y., F.X., Y.Y., L.Z. and X.P. performed the experiments. X.P. and M.S. wrote the manuscript. X.P. and M.S. designed the experiments. X.P. revised the manuscript. REFERENCES Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Mol Syst Biol 2: 1–11 Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18: 298–305 Breuninger H, Rikirsch E, Hermann M, Ueda M, Laux T (2008) Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo. Dev Cell 14: 867–876 Burillo S, Luque I, Fuentes I, Contreras A (2004) Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis. J Bacteriol 186: 3346–3354 Caldovic L, Tuchman M (2003) N-acetylglutamate and its changing role through evolution. Biochem J 372: 279–290 Cerutti H, Osman M, Grandoni P, Jagendorf AT (1992) A homolog of Escherichia coli RecA protein in plastids of higher plants. Proc Natl Acad Sci USA 89: 8068–8072 Chen YM, Ferrar TS, Lohmeier-Vogel EM, Morrice N, Mizuno Y, Berenger B, Ng KK, Muench DG, Moorhead GB (2006) The PII signal transduction protein of Arabidopsis thaliana forms an arginine-regulated complex with plastid N-acetyl glutamate kinase. J Biol Chem 281: 5726–5733 Christensen CA, King EJ, Jordan JR, Drews GN (1997) Megagametogenesis in Arabidopsis wild type and the Gf mutant. Sex Plant Reprod 10: 49–64 Cleland EE, Harpole WS (2010) Nitrogen enrichment and plant communities. Ann N Y Acad Sci 1195: 46–61 Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–7430 XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX 14 Huang et al. Crawford NM (2006) Mechanisms for nitric oxide synthesis in plants. J Exp Bot 57: 471–478 Cunin R, Glansdorff N, Pierard A, Stalon V (1986) Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 50: 314–352 Davis RH (1986) Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae. Microbiol Rev 50: 280–313 Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8: 457–463 Llacer JL, Fita I, Rubio V (2008) Arginine and nitrogen storage. Curr Opin Struct Biol 18: 673–681 McKay G, Shargool PD (1981) Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons. Biochem J 195: 71–81 Dou XY, Yang KZ, Ma ZX, Chen LQ, Zhang XQ, Bai JR, Ye D (2016) AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis. J Integr Plant Biol 58: 679–692 Micallef BJ, Shelp BJ (1989) Arginine metabolism in developing soybean cotyledons: III. Utilization. Plant Physiol 91: 170–174 Feria Bourrellier AB, Ferrario-Mery S, Vidal J, Hodges M (2009) Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase. Biochem Biophys Res Commun 387: 700–704 Molesini B, Mennella G, Martini F, Francese G, Pandolfini T (2015) Involvement of the putative N-acetylornithine deacetylase from Arabidopsis thaliana in flowering and fruit development. Plant Cell Physiol 56: 1084–1096 Forchhammer K (2008) P(II) signal transducers: Novel functional and structural insights. Trends Microbiol 16: 65–72 Molla-Morales A, Sarmiento-Manus R, Robles P, Quesada V, Perez-Perez JM, Gonzalez-Bayon R, Hannah MA, Willmitzer L, Ponce MR, Micol JL (2011) Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development. Plant J 65: 335–345 Fremont N, Riefler M, Stolz A, Schmulling T (2013) The Arabidopsis TUMOR PRONE5 gene encodes an acetylornithine aminotransferase required for arginine biosynthesis and root meristem maintenance in blue light. Plant Physiol 161: 1127–1140 Grun S, Lindermayr C, Sell S, Durner J (2006) Nitric oxide and gene regulation in plants. J Exp Bot 57: 507–516 Muralla R, Sweeney C, Stepansky A, Leustek T, Meinke D (2007) Genetic dissection of histidine biosynthesis in Arabidopsis. Plant Physiol 144: 890–903 Higashiyama T, Yabe S, Sasaki N, Nishimura Y, Miyagishima S, Kuroiwa H, Kuroiwa T (2001) Pollen tube attraction by the synergid cell. Science 293: 1480–1483 Neill S, Bright J, Desikan R, Hancock J, Harrison J, Wilson I (2008) Nitric oxide evolution and perception. J Exp Bot 59: 25–35 Hudson AO, Singh BK, Leustek T, Gilvarg C (2006) An LLdiaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants. Plant Physiol 140: 292–301 Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, Kawano N, Sakakibara T, Namiki S, Itoh K, Otsuka K, Matsuzaki M, Nozaki H, Kuroiwa T, Nakano A, Kanaoka MM, Dresselhaus T, Sasaki N, Higashiyama T (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458: 357–361 Huergo LF, Chandra G, Merrick M (2013) P(II) signal transduction proteins: Nitrogen regulation and beyond. FEMS Microbiol Rev 37: 251–283 Hulskamp M, Schneitz K, Pruitt RE (1995) Genetic evidence for a long-range activity that directs pollen tube guidance in Arabidopsis. Plant Cell 7: 57–64 Kalamaki MS, Alexandrou D, Lazari D, Merkouropoulos G, Fotopoulos V, Pateraki I, Aggelis A, Carrillo-Lopez A, RubioCabetas MJ, Kanellis AK (2009a) Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses. J Exp Bot 60: 1859–1871 Kalamaki MS, Merkouropoulos G, Kanellis AK (2009b) Can ornithine accumulation modulate abiotic stress tolerance in Arabidopsis? Plant Signal Behav 4: 1099–1101 Kasahara RD, Portereiko MF, Sandaklie-Nikolova L, Rabiger DS, Drews GN (2005) MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis. Plant Cell 17: 2981–2992 Liao HZ, Zhu MM, Cui HH, Du XY, Tang Y, Chen LQ, Ye, Zhang XQ (2016) MARIS plays important roles in Arabidopsis pollen tube and root hair growth. J Integr Plant Biol 58: 927–940 XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX Palanivelu R, Preuss D (2006) Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol 6: 7 Preuss D, Rhee SY, Davis RW (1994) Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264: 1458–1460 Quesada V, Ponce MR, Micol JL (1999) OTC and AUL1, two convergent and overlapping genes in the nuclear genome of Arabidopsis thaliana. FEBS Lett 461: 101–106 Ray SM, Park SS, Ray A (1997) Pollen tube guidance by the female gametophyte. Development 124: 2489–2498 Shimizu KK, Okada K (2000) Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance. Development 127: 4511–4518 Slocum RD (2005) Genes, enzymes and regulation of arginine biosynthesis in plants. Plant Physiol Biochem 43: 729–745 Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2: 755–767 Song JT, Lu H, Greenberg JT (2004) Divergent roles in Arabidopsis thaliana development and defense of two www.jipb.net NAGK is critical for gametophytes and embryo development homologous genes, aberrant growth and death2 and AGD2-LIKE DEFENSE RESPONSE PROTEIN1, encoding novel aminotransferases. Plant Cell 16: 353–366 Steffen JG, Kang IH, Macfarlane J, Drews GN (2007) Identification of genes expressed in the Arabidopsis female gametophyte. Plant J 51: 281–292 kely G, Abraham E, Cseplo A, Rigo G, Zsigmond L, Csiszar J, Sze Ayaydin F, Strizhov N, Jasik J, Schmelzer E, Koncz C, Szabados L (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53: 11–28 Takahashi T, Kakehi J (2010) Polyamines: Ubiquitous polycations with unique roles in growth and stress responses. Ann Bot 105: 1–6 Takeuchi H, Higashiyama T (2012) A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biol 10: e1001449 Takeuchi H, Higashiyama T (2016) Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis. Nature 531: 245–248 Twell D, Wing R, Yamaguchi J, McCormick S (1989) Isolation and expression of an anther-specific gene from tomato. Mol Gen Genet 217: 240–245 Uhrig RG, Ng KK, Moorhead GB (2009) PII in higher plants: A modern role for an ancient protein. Trends Plant Sci 14: 505–511 €lz R, Heydlauff J, Ripper D, von Lyncker L, Groß-Hardt R Vo (2013) Ethylene signaling is required for synergid Scan using WeChat with your smartphone to view JIPB online www.jipb.net 15 degeneration and the establishment of a pollen tube block. Dev cell 25: 310–316 Vielle-Calzada JP, Baskar R, Grossniklaus U (2000) Delayed activation of the paternal genome during seed development. Nature 404: 91–94 Wang T, Liang L, Xue Y, Jia PF, Chen W, Zhang MX, Wang YC, Li HJ, Yang WC (2016) Corrigendum: A receptor heteromer mediates the male perception of female attractants in plants. Nature 536: 3600 Winter G, Todd CD, Trovato M, Forlani G, Funck D (2015) Physiological implications of arginine metabolism in plants. Front Plant Sci 6: 534 Wu JJ, Peng XB, Li WW, He R, Xin HP, Sun MX (2012) Mitochondrial GCD1 dysfunction reveals reciprocal cell-tocell signaling during the maturation of Arabidopsis female gametes. Dev Cell 23: 1043–1058 Xia J, Yamaji N, Che J, Shen RF, Ma JF (2014) Normal root elongation requires arginine produced by argininosuccinate lyase in rice. Plant J 78: 215–226 Xie T, Chen D, Wu J, Huang X, Wang Y, Tang K, Li J, Sun M, Peng X (2016) Growing slowly 1 locus encodes a PLS-type PPR protein required for RNA editing and plant development in Arabidopsis. J Exp Bot 67: 5687–5698 Yan H, Chen D, Wang Y, Sun Y, Zhao J, Sun M, Peng X (2016) Ribosomal protein L18aB is required for both male gametophyte function and embryo development in Arabidopsis. Sci Rep 6: 31195 Scan with iPhone or iPad to view JIPB online XXX 2017 | Volume XXXX | Issue XXXX | XXX-XX