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First published online May 18, 2007; 10.1104/pp.107.098079 Plant Physiology 144:1531-1545 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Functional Characterization of the Arabidopsis Eukaryotic Translation Initiation Factor 5A-2 That Plays a Crucial Role in Plant Growth and Development by Regulating Cell Division, Cell Growth, and Cell Death1,[OA]State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
The eukaryotic translation initiation factor 5A (eIF-5A) is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies in mammalian and yeast (Saccharomyces cerevisiae) cells suggest that eIF-5A is mainly involved in RNA metabolism and trafficking, thereby regulating cell proliferation, cell growth, and programmed cell death. In higher plants, the physiological function of eIF-5A remains largely unknown. Here, we report the identification and characterization of an Arabidopsis (Arabidopsis thaliana) mutant fumonisin B1-resistant12 (fbr12). The fbr12 mutant shows an antiapoptotic phenotype and has reduced dark-induced leaf senescence. Moreover, fbr12 displays severe defects in plant growth and development. The fbr12 mutant plant is extreme dwarf with substantially reduced size and number of all adult organs. During reproductive development, fbr12 causes abnormal development of floral organs and defective sporogenesis, leading to the abortion of both female and male germline cells. Microscopic studies revealed that these developmental defects are associated with abnormal cell division and cell growth. Genetic and molecular analyses indicated that FBR12 encodes a putative eIF-5A-2 protein. When expressed in a yeast mutant strain carrying a mutation in the eIF-5A gene, FBR12 cDNA is able to rescue the lethal phenotype of the yeast mutant, indicating that FBR12 is a functional eIF-5A. We propose that FBR12/eIF-5A-2 is fundamental for plant growth and development by regulating cell division, cell growth, and cell death.
The eukaryotic translation initiation factor 5A (eIF-5A) has been found in all eukaryotic organisms and is structurally and functionally conserved across different kingdoms. Biochemical and molecular studies revealed that eIF-5A is synthesized as an inactive precursor that is activated by a posttranslational modification mechanism. This modification is involved in a two-step reaction to convert an absolutely conserved Lys into a Hpu. The first reaction, catalyzed by deoxyhypusine synthase (DHS; EC 1.1.1.249), is to add a butylamine group derived from spermidine to the conserved Lys to form a deoxyhypusine. Subsequently, the deoxyhypusine residue is converted into a Hpu, catalyzed by deoxyhypusine hydroxylase (EC1.14.99.29; Park and Wolff, 1988  ; Wolff et al., 1990; Park et al., 1993, 1997). The hypusination of eIF-5A is essential for viability of yeast (Saccharomyces cerevisiae) cells (Kang et al., 1993) and highly specific, as demonstrated by the observation that a Lys-to-Arg mutation abolishes hypusination of eIF-5A (Jin et al., 2003). In addition, this unusual modification thus far has only been found in eIF-5A proteins (Park et al., 1997; Thompson et al., 2004). At the cellular level, Hpu-containing eIF-5A has been shown to be involved in the control of cell proliferation and apoptosis (Park et al., 1997; Tome and Gerner, 1997; Caraglia et al., 2001; Thompson et al., 2004). More recently, eIF-5A was found to function as a regulator of p53 and p53-dependent apoptosis (Li et al., 2004).
The eIF-5A protein was initially identified as a translation initiation factor from rabbit reticulocytes in an in vitro assay (Kemper et al., 1976
In plants, genes encoding eIF-5A and DHS have been cloned from several species. Similar to its mammalian and yeast counterparts, a tomato (Lycopersicon esculentum) eIF-5A recombinant protein can be deoxyhypusine modified by a DHS recombinant protein (Wang et al., 2001
Identification and Genetic Analysis of the fumonisin B1-resistant12 Mutant
The fumonisin B1-resistant12 (fbr12) mutant was identified from a genetic screen of a T-DNA-mutagenized population of approximately 5,000 independent lines in the pga22 mutant background (Sun et al., 2003
Because fbr12 was infertile (see below), we crossed putative fbr12/+ plants (in the pga22 background) with the wild type (Ws). In F2 progeny derived from self-pollinated F1 plants, the FB1-resistant phenotype segregated in a 1:3 ratio (FB1 resistant:sensitive = 72:225;  2 = 0.055; P < 0.01). When grown under normal growth conditions in the absence of FB1, fbr12 displayed severe defects in plant growth and development (Fig. 1A; see below for details), and this phenotype also segregated in a 1:3 ratio (fbr12:wild type = 75:288; 2 = 3.58; P < 0.05). These results indicate that the fbr12 mutation is recessive in a single nuclear locus. Because of lethality of the mutation, fbr12 was maintained as heterozygous.
To investigate the cellular and molecular alterations induced by FB1 in fbr12, we first compared toxin-induced cell death by Trypan blue staining. In wild-type leaves, FB1 induced massive cell death. However, substantially reduced cell death was observed in fbr12 leaves (Fig. 1B). In some cases, no cell death was found in the mutant leaves treated by FB1 (data not shown). We next examined nuclear DNA fragmentation, a molecular hallmark of apoptotic cells, in protoplasts treated by FB1 using the TUNEL method. In untreated protoplasts derived from both the wild type and fbr12, TUNEL-positive signal was rarely detected. However, upon FB1 treatment, whereas more than 90% of wild-type protoplasts showed TUNEL-positive signals, <25% of fbr12 protoplasts displayed distinctive positive signals under the identical assay conditions (Fig. 1C). At the molecular level, FB1 is known to induce the expression of PATHOGEN-RELATED (PR) genes, and the induction is compromised in fbr1 and fbr2 mutants (Stone et al., 2000
The fbr12 mutant phenotype became apparent shortly after germination. Compared to the wild type, fbr12 was substantially smaller and more slender, with significantly shorter roots. However, all embryonic organs, including roots, hypocotyls, and cotyledons, appeared to be well defined (Fig. 2, A and B ). The initiation of true leaves was substantially delayed in fbr12 (Fig. 2B). During later growth stages, fbr12 displayed a stunted phenotype (Fig. 2, C, E, and F), producing less rosette and cauline leaves that were smaller than those of the wild type (Fig. 2, D and E). In addition, fbr12 plants also had few flowers that were abnormally developed (Fig. 2, F and G). Overall, fbr12 affected the growth rate and organogenesis characteristics of dwarfism, a reduced leaf initiation rate and reduced numbers of adult organs (Fig. 2H).
In fbr12, development of the floral organs was severely affected by the mutation. Compared to the wild type, fbr12 plants produced fewer flowers (Fig. 2, G and H) in which all floral organs displayed various developmental defects. In general, all fbr12 floral organs were smaller than those of the wild type (Fig. 3A ). Compared to that of the wild type, sepals in fbr12 flowers were often misshapen and occasionally fused together. Petals in fbr12 flowers were smaller than that of the wild type (Fig. 3A). Stamens and stigmas appeared to be morphologically normal. However, two lateral stamens were usually absent in the mutant and the gynoecium stigmatic papillae was shorter than that of the wild type (see also below for more details on germline cell development). In wild-type Arabidopsis, a flower consists of four sepals, four petals, six stamens, and a carpel. Whereas a carpel was often found in fbr12 flowers, other floral organ components had significantly reduced numbers in the mutant compared to the wild type (Fig. 3C). In particular, the number of petals and stamens was altered more dramatically by the fbr12 mutation, with <30% and 10% of flowers producing correct numbers of these organs, respectively (Fig. 3C). Approximately 50% of flowers formed the correct number (four) of sepals. As a result of these defects, fbr12 siliques were markedly shorter and did not contain any seeds (Fig. 3B).
The fbr12 Mutation Affects Cell Proliferation and Cell Growth To investigate the cellular basis of the fbr12 mutant phenotype, we analyzed the mutant by microscopy. Light microscopy revealed that the structure of the shoot apical meristem (SAM) was unaffected in fbr12, but SAM development was delayed compared to that of the wild type (Fig. 4, A–E ). Notably, 9-d-old fbr12 SAM appeared to be equivalent to that of 6-d-old wild type. A similar observation was made by scanning electron microscopy (Fig. 4, F–J). These results suggest that the fbr12 mutation may cause a slow division or growth rate of the meristem cells.
Transverse sections of stems revealed that fbr12, similar to that of the wild type, contained three distinctive layers of cell files that include, from outside to inside, the epidermis, cortex, and central cylinder. Development of epidermal cells appeared to be unaffected in the fbr12 mutant. This result suggests that radial patterning remains relatively normal in fbr12. In the cortex, however, cells were substantially enlarged in fbr12 compared to the wild type (Fig. 5, A–D ). By contrast, the mutant had smaller cells in the central cylinder than those of the wild type (Fig. 5, A–D). The central cylinder contained the vascular bundles consisting of phloem and xylem. In xylem, fbr12 had an increased number of cells that were smaller, presumably representing a group of incompletely differentiated cells. Compared to that of the wild type, no distinctive cell layers were observed in the fbr12 phloem. Moreover, both the number and the size of the phloem cells were greatly reduced (Fig. 5, C and D). Quantitative analysis also indicated that fbr12 had increased cell numbers in xylem and parenchyma, but reduced cell numbers in phloem and cortex (Fig. 5E).
Scanning electron microscopy indicated that, in fbr12 petals, whereas the cell number remains unaltered, the cell size is smaller than that of the wild type (Fig. 6, A and B ). Quantitative analysis of cell numbers in petals revealed that the total cell numbers remained nearly unaltered, but the cell size was reduced in fbr12 (Fig. 6C), thus causing smaller petals. Collectively, these observations indicate that the fbr12 mutation affects cell proliferation, cell growth, and cell differentiation in both vegetative and reproductive organs/tissues.
FBR12 Is Essential for Male and Female Sporogenesis The fbr12 mutant could grow and develop into mature plants with significantly smaller size, which could not set any seeds. Reciprocal crosses between the wild type and fbr12 did not yield any F1 seeds, suggesting that the mutant was both male and female sterile. To reveal the cellular basis of the sterile phenotype, we followed the entire reproductive developmental stages by light microscopy and scanning electron microscopy.
During male germline cell development, no apparent defects were found in fbr12 before stage 7 (anther development stages were defined according to Sanders et al. [1999]
During female gametophyte development, the fbr12 mutation also appears to express after meiosis, approximately at stage 2-III (ovule development stages are defined according to Schneitz et al. [1995] ). In the wild type at this stage, meiosis is completed to give rise to four megaspores of which only one survives and undergoes three rounds of nuclear divisions to eventually form a mature embryo sac. Meanwhile, the developing ovule grows toward the septum and the base of the carpel, whereas both the inner and outer integuments are initiated (Fig. 8, A and G
). In fbr12, no apparent defects were observed at this stage and a megaspore appeared to be normally formed in the ovule (Fig. 8, D and H). In the wild type, whereas the inner integument continues to grow symmetrically, the outer integument starts asymmetric growth throughout development (Fig. 8B), eventually leading to the formation of a hook-shaped, mature embryo sac (Fig. 8, C and G). By contrast, both inner and outer integuments stopped further growth in fbr12 ovules (Fig. 8, E and H). Therefore, ovule development was completely arrested at this stage.
We have also followed the entire reproductive development in fbr12/+ heterozygous plants. An fbr12/+ plant should give rise to haploid germline cells carrying a mutant or a wild-type allele, with approximately 50% of each genotype, respectively. On the other hand, the diploid sporophytic tissues (e.g. tapetum and ovules) should normally be developed because of the recessive nature of the mutation. No abnormally developed germline cells were observed in fbr12/+ plants, which showed no difference in fertility compared to wild type (data not shown). Moreover, in progeny derived from self-pollinated fbr12/+ plants, the mutation was segregated in a 1:3 ratio (mutant:wild type; see above) characteristic of sporophytic mutations (Yang et al., 1999 ; McCormick, 2004). These results suggest that the fbr12 mutation only affects sporogenesis, but not gametogenesis. Similarly, no abnormality was observed in developing embryos of fbr12/+ plants. Taken together, these results suggest that FBR12 is essential for sporophytic development, but dispensable for gametogenesis and embryogenesis, and that defective gametogenesis is likely caused by abnormal development of sporophytic tissues in the fbr12 mutant.
The fbr12 mutant was identified from a T-DNA-mutagenized population and the mutant genome appears to contain a single T-DNA insertion. We identified the genomic sequences flanking the left border by thermal asymmetric interlaced (TAIL)-PCR (Liu et al., 1995
To verify the identity of FBR12, we carried out a molecular complementation experiment. A 2.5-kb wild-type genomic DNA fragment, which encompassed the promoter region, 5'-untranslated region (UTR), the coding sequence, and part of the 3'-UTR of At1g26630, was cloned into binary vector pER8 (Zuo et al., 2000 ). The resulting construct was then transformed into fbr12/+ heterozygous plants via Agrobacterium tumefaciens-mediated transformation (Bechtold et al., 1993). Multiple independent transgenic lines were obtained by double selection with BASTA (carried by the fbr12 mutant) and hygromycin (carried by pER8). In the 20 tested T2 lines, all hygromycin-resistant plants displayed normal phenotype. In T3 lines derived from BASTA- and hygromycin-resistant T2 plants, we analyzed four families that did not show segregation of either the selective markers (BASTA and hygromycin) or the fbr12 phenotype. PCR analysis revealed that all tested plants (24) were homozygous for the T-DNA insertion at the At1g26630 locus. In these transgenic plants, the transgene fully rescued the developmental defects (Fig. 9, D and F) and restored the sensitivity of the mutant to FB1 (Fig. 9E). These data suggest that the At1g26630 transgene was able to fully complement the fbr12 mutant phenotype, thus representing FBR12.
Database search identified a full-length FBR12 cDNA clone (accession no. NM_102425). Comparison of the cDNA and genomic sequences revealed an open reading frame (ORF) interrupted by four introns (Fig. 9A). The ORF encodes a polypeptide of 159 amino acid residues, with a predicted molecular mass of 17.1 kD and a pI of 5.8. Sequence comparison revealed that FBR12 encodes a putative eIF-5A-1 (accession no. NP_173985; also annotated by The Arabidopsis Information Resource [TAIR]) or eIF-5A-2 (accession nos. Q93VP3 and BE039424; Thompson et al., 2004
Consistent with the pleiotropic phenotype of fbr12, FBR12 appears to be ubiquitously expressed in all examined tissues and organs (Fig. 9C). Members of the eIF-5A family are highly conserved across different kingdoms of eukaryotic organism cells (Jenkins et al., 2001
To functionally characterize FBR12, we carried out a genetic complementation experiment in a yeast mutant strain PSY1249. This strain carries a temperature-sensitive mutation in an eIF-5A gene TIF51A that allows the mutant to grow only under the permissive temperature (25°C; Valentini et al., 2002
The fbr12 Mutation Delays Dark-Induced Leaf Senescence
Previous studies showed that overexpression of antisense DHS in Arabidopsis and tomato resulted in delayed leaf senescence. Because DHS is essential for hypusination of eIF5A proteins, this transgenic phenotype was attributed to the inactivation of eIF5A proteins by knocking down DHS expression (Wang et al., 2003 Fully expanded leaves detached from fbr12 and wild-type plants were placed in the dark and their senescence rates were analyzed. Under assay conditions, leaves derived from wild-type plants showed an apparent dark-induced senescence syndrome at day 7 and became completely bleached at day 10. Under the identical assay conditions, leaves derived from fbr12 mutant plants displayed a slower senescence rate (Fig. 11A ). To quantitatively analyze the senescence rate, we measured the chlorophyll levels in fully expanded leaves of wild type and fbr12. Under normal growth conditions, the chlorophyll level was slightly lower in fbr12 leaves compared to that of the wild type. However, dark treatment caused a greater loss of chlorophyll in wild-type leaves (37% at day 4, 47% at day 7, and 80% at day 10) compared to that of fbr12 mutant leaves at the same stages (17% at day 4, 33% at day 7, and 48% at day 10; Fig. 11B). Taken together, these results suggest that FBR12/eIF5A-2 is involved in the regulation of senescence-type PCD in Arabidopsis.
In this study, we present evidence showing that the Arabidopsis FBR12 gene encodes a functional eIF5A that is involved in the regulation of cell proliferation, cell growth, and cell death. FBR12 shares significant homology with eIF-5A proteins characterized across different kingdoms. Moreover, an FBR12 cDNA clone was able to complement the temperature-sensitive lethal phenotype of yeast PSY1249 cells carrying a mutation in the TIF51A/eIF-5A gene. These results demonstrate that FBR12 is a functional eIF-5A.
The eIF-5A protein was originally characterized as a component of the translation initiation complex. However, recent studies suggest that this class of highly conserved proteins is involved in RNA metabolism and RNA trafficking (Bevec and Hauber, 1997
The extreme dwarf phenotype of the fbr12 mutant may be caused by reduced cell division and/or cell growth. Microscopic studies suggest that both cell division and cell growth are affected by the fbr12 mutation. However, the regulatory roles of FBR12 in cellular activity appear to be cell type- or tissue-specific with distinctive mechanisms. In stem development, for example, no apparent abnormality was observed in the epidermal cell layer, but various defects were found in the cortex and the central cylinder. More strikingly, the FBR12 gene appears to function differently, characteristically by inhibiting cell growth in the cortex, but promoting cell growth in the central cylinder. In a similar mode, the fbr12 mutation causes increased cell numbers in xylem and parenchyma, but reduced cell numbers in phloem and cortex. These observations suggest that FBR12 regulates cell division and cell growth in a tissue- and development-specific manner. We notice that fbr12 shows some phenotypic similarity with fbr6, whose wild-type allele encodes a transcription activator AtSPL14 (Stone et al., 2005
In addition to its role in cell division and cell growth, FBR12 also appears to play a role in regulating cell death. Similar to other fbr mutants, fbr12 shows resistance to FB1 with substantially reduced cell death induced by the toxin. Compared to the wild type, fbr12 protoplasts showed substantially less DNA fragmentation induced by FB1, suggesting that FBR12/eIF-5A-2, similar to its mammalian homologs, is likely involved in the regulation of apoptotic cell death. Because FB1 is known to inhibit ceramide synthase, thereby perturbing sphingolipid metabolism (Wang et al., 1991 In conclusion, FBR12/eIF-5A-2 plays a critical role in plant growth and development by regulating cell division, cell growth, and cell death. Identification of the fbr12 mutant provides unique materials to functionally characterize this class of highly conserved proteins in eukaryotic organisms. Clearly, identification and characterization of direct targets of FBR12/eIF-5A-2 will be critical to better understand its function.
Plant Materials, Growth Conditions, and Genetic Screen for fbr Mutants
The Ws ecotype of Arabidopsis (Arabidopsis thaliana) was used in this study unless otherwise indicated. Plants were grown under a 16-h-light/8-h-dark cycle (white light; 120 µmol m–2 s–1) at 22°C in soil or on Murashige and Skoog medium (1x Murashige and Skoog salts, 3% Suc, 0.8% agar) as described previously (Sun et al., 2003
A T-DNA-mutagenized population of approximately 5,000 independent lines was generated in the pga22 (Ws) background (Sun et al., 2003
Leaves were collected from 4- to 5-week-old seedlings and protoplasts were prepared according to Danon and Gallois (1998)
Cell death was analyzed by Trypan blue staining as described (Mou et al., 2000
Semithin sections were prepared and analyzed as described with minor modifications (Yang et al., 1999 For scanning electron microscopic analysis, samples were fixed, postfixed, and dehydrated as described above. After being critical point dried in liquid CO2 and mounted, samples were sputter-coated with gold in an E-100 ion sputter and then observed under a scanning electron microscope (model S-570; Hitachi).
For comparison of cell size and numbers, the distal portion of the petal epidermis or stem sections was analyzed as described (Mizukami and Ma, 1992
Fully expanded leaves collected from 4-week-old wild-type or fbr12 plants were used for the analysis of dark-induced leaf senescence essentially as described (Guo and Crawford, 2005
The T-DNA-tagged genomic sequence in the fbr12 genome was identified by TAIL-PCR as previously described (Liu et al., 1995
All other molecular manipulations were carried out according to standard methods (Sambrook and Russell, 2001
An FBR12 cDNA fragment was PCR amplified using primer pairs (AT1G26630F2, 5'-CCCCGGGATGTCTGACGACGAGCACCA-3'; and AT1G26630B2, 5'-CACTAGTTGACGTCCGTTGTCAAACTGGT-3'), and then cloned into a pGEM-T Easy vector (Promega). This cDNA fragment, containing the entire ORF of FBR12 and 32 bp of the 3'-UTR, was verified by DNA sequencing. The insert was released by SmaI and PstI digestion and cloned into the same sites of a pQE-82 L vector (Qiagen). The cDNA fragment released from pQE-FBR12 by SacI and SpeI was cloned into the SacI and XbaI of a pYES2 vector (Invitrogen) under the control of a GAL1 promoter. Therefore, FBR12 expression in yeast (Saccharomyces cerevisiae) cells is inducible by Gal and repressible by Glc (West et al., 1984
We would like to thank the Arabidopsis Biological Resource Center (ABRC) for providing seeds, and Dr. Allan Jacobson (University of Massachusetts Medical School) and Dr. Pamela A. Silver (Harvard Medical School) for providing yeast strains. We would also like to thank Dr. Yongbiao Xue, Dr. Shuhua Yang, and Dr. De Ye for critically reading the manuscript. We are grateful to Dr. Weicai Yang for valuable advice on microscopic studies. Received February 14, 2007; accepted May 16, 2007; published May 18, 2007.
1 This work was supported by the National Natural Science Foundation of China (NSFC; grant nos. 30330360 and 30221002), the Ministry of Science and Technology of China (grant no. 2006AA10A112), and the Chinese Academy of Sciences (grant no. KSCX2–YW–N–015). J.Z. is a recipient of the Outstanding Young Investigator Award of the NSFC (grant no. 30125025).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jianru Zuo (jrzuo{at}genetics.ac.cn).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.098079 * Corresponding author; e-mail jrzuo{at}genetics.ac.cn; fax 8610–6487–3428.
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