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Role of polyamines in plant vascular development
Plant Physiology and Biochemistry 48 (2010) 534e539
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Role of polyamines in plant vascular development Francisco Vera-Sirera a, Eugenio G. Minguet a, b, Sunil Kumar Singh c, Karin Ljung d, Hannele Tuominen c, Miguel A. Blázquez a, Juan Carbonell a, * a
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), 46022 Valencia, Spain Laboratoire de Physiologie Cellulaire Végétale, UMR CNRS 5168 e CEA e INRA e UJF, 38054 Grenoble, France c Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden d Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, SLU, SE-90183 Umeå, Sweden b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 October 2009 Accepted 14 January 2010 Available online 22 January 2010
Several pieces of evidence suggest a role for polyamines in the regulation of plant vascular development. For instance, polyamine oxidase gene expression has been shown to be associated with lignification, and downregulation of S-adenosylmethionine decarboxylase causes dwarfism and enlargement of the vasculature. Recent evidence from Arabidopsis thaliana also suggests that the active polyamine in the regulation of vascular development is the tetraamine thermospermine. Thermospermine biosynthesis is catalyzed by the aminopropyl transferase encoded by ACAULIS5, which is specifically expressed in xylem vessel elements. Both genetic and molecular evidence support a fundamental role for thermospermine in preventing premature maturation and death of the xylem vessel elements. This safeguard action of thermospermine has significant impact on xylem cell morphology, cell wall patterning and cell death as well as on plant growth in general. This manuscript reviews recent reports on polyamine function and places polyamines in the context of the known regulatory mechanisms that govern vascular development. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Thermospermine Xylem Cell death
1. Introduction Putrescine, spermidine, and spermine have been described as the most common polyamines in eukaryotes. However, in plants, recent evidence has shown that spermine is found only in angiosperms while thermospermine is likely present throughout the whole plant kingdom [33]. The importance of polyamines has become clear from the changes in polyamine levels that accompany certain developmental transitions or exposure to stress conditions. In addition, exogenous applications of polyamines have frequently been shown to affect plant growth and as well as the response against various stress factors [14,30]. Loss of function mutations in polyamine metabolism genes have shown that the diamine putrescine and the triamine spermidine are essential for life in all organisms evaluated [6,16,24,25,46]. With respect to tetraamines, although they have been proven to be dispensable for strict survival in plants, they have been shown to be involved in stress tolerance [2,48,49]. Moreover, recent results have highlighted the regulation of vascular
* Corresponding author. Tel.: þ34 963 877 872; fax: þ34 963 877 859. E-mail address: [email protected] (J. Carbonell). 0981-9428/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2010.01.011
differentiation by the tetraamine thermospermine, as reviewed in the next sections [7,20,27,34]. 2. Brief overview of vascular development All vascular tissues are derived from undifferentiated, meristematic cells (e.g. the procambium and cambium) [50]. The body plan for the vasculature in the adult plant is already established in the embryo. Certain cells along the embryo axis acquire procambial identity, and they will sustain primary growth of the vasculature. In a later developmental stage, the vascular cambium is formed giving rise to the secondary, radial growth of the vasculature in the stem. Procambial and cambial cells are polar in the sense that depending on their position, they give rise either to the phloem or the xylem. The initiation of xylem differentiation depends on the concerted action of several players [45]. Auxin plays an important role as a trigger of the developmental program, while the activity of a family of homeodomain transcription factors (HD-ZIP III) provides the necessary spatial information for the acquisition of xylem identity. Among these genes is ATHB8, which is an early marker for procambial activity [3,4]. Overexpression of ATHB8 provokes overproliferation of xylem cells [4]. Downregulation of another member of the HD-ZIP III family, ATHB15, results in plants with increased
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vascular tissue, while its overexpression leads to smaller vascular bundles [28,41]. This suggests that not all HD-ZIP III genes act as positive regulators of cambial cell specification and proliferation. Xylem is composed of the water-transporting tracheary elements, such as tracheids and the vessel elements, and the xylem fibers that provide the necessary support to the growing tissue [35]. The xylem elements typically undergo a differentiation program that involves, in a sequential manner, cell expansion, deposition of secondary cell wall material including lignin, and cell death. Cell death of tracheary elements is a well recognized example of programmed cell death during plant development, in which specific genes are upregulated to trigger destruction. This process involves the collapse of the vacuole followed by hydrolysis of the nuclear DNA and the cytoplasmic contents as a result of the activation or the release of the hydrolytic enzymes released from the vacuole [12,26]. Even though the process of xylem cell death is well described, little is known about the molecular mechanism that regulates it. 3. Broad range effects of polyamines on vascular development An early link between polyamines and plant vasculature was the identification of relatively high concentrations of putrescine, spermidine and spermine in xylem exudates of sunflower, grapevine, mung bean, and orange trees [10]. However, this finding was logically interpreted in the key of long-distance translocation of polyamines and their role in the regulation of plant development along the whole organism. This idea was pursued later by careful examination of the levels of the different polyamines along the axis of the plant, establishing correlations between the concentrations of putrescine, spermidine and spermine, and the size of the cells and the age of the tissues [38]. More relevant to vascular development, polyamine metabolism has been related to the formation of the reactive oxygen species (ROS) that play a key role in cell wall expansion and growth, vascular differentiation, and lignin polymerization [40]. During vascular differentiation, lignin is formed with the concurrence of H2O2 and cell wall bound peroxidases [9]. Enzymes that may be responsible for H2O2 generation include diamine oxidase (DAO) [42,47], and polyamine oxidase (PAO) [8]. The strong upregulation of DAO and PAO in vascular tissues and their direct correlation with peroxidase gene expression in tobacco [39] could make amine oxidases an attractive candidate for a role in the generation of H2O2 for protein cross-linking and lignification. The availability of decarboxylated S-adenosylmethionine (dcSAM) for the synthesis of longer-chain polyamines like spermidine and spermine also seems to be an important limiting step in the correct development of the vasculature. Compared with wildtype, the morphology of the vascular bundles of the bud2 mutant, defective in SAMDC4, one of the four SAM decarboxylase genes in Arabidopsis, is greatly affected, due to a significant increase in bundle size [13]. Moreover, the lignin content in bud2 mutants was at least 30% higher than in wild-type inflorescences, although cellulose content did not vary. On the other hand, the bushy phenotype of bud2 suggests a possible involvement of polyamines in signal transduction of auxin and cytokinin. 4. Thermospermine and xylem formation 4.1. ACL5 and vascular development Beyond the influence of polyamine synthesis and degradation on the formation of cell walls and lignin biosynthesis through the interference with redox metabolism, the most solid evidence available for the involvement of a polyamine in the formation of the
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vasculature was derived from molecular genetic analyses of Arabidopsis mutants impaired in stem elongation. In an attempt to identify genes controlling the architecture of the aerial part of the plant, five acaulis mutants were isolated with a defect in stem elongation which couldn't be rescued by the application of hormones known to control organ growth, such as gibberellins and brassinosteroids [19,44]. Particular attention was devoted to ACAULIS5 (ACL5), whose loss of function caused severe shortening of internodes, smaller mature leaves, and, more importantly, overproliferation of xylem vessel elements in the vascular bundles of the inflorescence stems. Given that the general cell size in elongating organs was smaller in acl5 mutants compared to the wild-type, and that the orientation of cortical microtubules in epidermal cells of the acl5 mutant was mostly longitudinal instead of transversal, as it is the case in wild-type cells, ACL5 was initially attributed a role in signaling during cell elongation in inflorescence stems. ACL5 was cloned and proposed to encode a spermine synthase based on the similarity with aminopropyl transferases and the ability of the ACL5 protein expressed in Escherichia coli to catalyze the conversion of spermidine into a tetraamine identified as spermine by HPLC analysis [20]. Interestingly, an apparent paradox was raised when another gene encoding a spermine synthase (SPM1) was identified in Arabidopsis [37], the corresponding knockout mutant did not display any defect in stem elongation [24]. But in fact, this contradiction is explained because the spermidine aminopropyl transferase encoded by ACL5 in fact catalyzes the in vitro formation of thermospermine, and not spermine [29]. These two tetraamines cannot be distinguished with the HPLC method used in previous studies, although they are easily separated by alternative procedures, such as thin-layer chromatography. Thermospermine had been detected at low concentrations mainly in archaea, bacteria and certain aquatic plants [15,36], and its presence in Arabidopsis wild-type (but not acl5) extracts has been recently confirmed in a qualitative manner [27]. Therefore, the most likely cause of the stem growth defect displayed by acl5 mutants is the lack of thermospermine, although exogenous supply of this polyamine to mutant plants allowed only very partial rescue of the wild-type phenotype [27]. Although it has been suggested that the xylem defects associated with acl5 were the result of deficient auxin transport [7], two other pieces of evidence make this a less likely possibility: a slight increase in the concentration of the natural auxin, indole-3-acetic acid (IAA), and increased expression of the IAA marker line DR5::GUS in the hypocotyls of young acl5 seedlings (Fig. 1), which must be the result of IAA transport from the apical meristem and the youngest leaves where it is synthesized. Therefore, it seems that IAA can be transported in young acl5 seedlings. It is possible that the reduced auxin transport capacity demonstrated earlier in excised pieces of the inflorescence stem is a secondary effect due to altered xylem specification and impaired growth of the acl5 mutant. Apart from the overproliferation of vascular cells observed in the stems of acl5 mutants, additional pieces of evidence suggest that thermospermine has an important role in the correct development of the vasculature. First, expression of the ACL5 gene is upregulated upon auxin treatments [20]. This regulation might be highly significant because, as indicated above, auxin serves as a signal for the initiation of cell differentiation at the vascular cambium. Second, ACL5 transcripts are detected only associated to the vascular cambium, in cells that initiate differentiation [7,34]. And third, thickvein (tkv) mutants are allelic to acl5, and they present an overall increase in leaf vascularization and in vein thickness [7]. Besides, it is noteworthy that the morphological defects of the knockout mutants in Arabidopsis BUD2/SAMDC4, with lower capacity to synthesize one of the substrates for thermospermine synthesis, closely resemble those displayed by acl5 mutants [13].
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thermospermine in guaranteeing adequate duration of vessel element differentiation. Interestingly, certain genes encoding enzymes involved in the execution of programmed cell death during xylem differentiation, such as the ribonuclease BFN1 and the cysteine protease XCP1, were among the genes with higher expression in acl5 compared to wildtype seedlings (our unpublished results). This misregulation very likely reflects premature death of xylem vessels. The final confirmation of the involvement of ACL5 in the control of the duration of xylem vessel differentiation was provided by the phenotype of plants expressing the diphteria toxin A under the control of the ACL5 promoter. In these plants, premature death of the ACL5expressing cells reproduced the stem growth defect of acl5 mutants and, more importantly, the anatomical and morphological defects in xylem formation [34]. Therefore, a likely role for thermospermine is to maintain differentiating xylem elements alive until the differentiation process has culminated. 4.3. Events downstream of ACL5
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Fig. 1. Auxin transport in acl5 mutants. (A) Concentration of indole-3-acetic acid (IAA) in the root, hypocotyl and cotyledons of 7-day-old seedlings grown in vitro. No statistically significant differences were observed in acl5 compared to the wild-type, using a KruskaleWallis test. (B,C,D) Histochemical GUS staining in hypocotyls of DR5:: GUS seedlings. (C) is a higher magnification photo from (B). (E,F,G) Histochemical GUS staining in hypocotyls of DR5::GUS acl5 seedlings. (F) is a higher magnification photo from (E). Seedlings were grown in vitro for seven days (B,C,E,F) or for one day in light followed by 2.5 days in darkness and an additional seven days in light, to ensure the optimal induction of adventitious roots in the hypocotyls (D,G). (H) Adventitious root formation in DR5::GUS (control) and DR5::GUS acl5. An increased capacity of acl5 to form adventitious roots in response to the appropriate stimulatory conditions is indicative of increased IAA levels in the acl5 hypocotyl. Scale bars: 50 mm.
4.2. Control of xylem differentiation by ACL5 A detailed analysis of the relative proportion of cell types associated with the vasculature revealed that, although phloem development was largely unaffected, xylem fibers and the elaborate type of vessels elements with pitted secondary cell wall thickenings were practically absent both in the inflorescence stems (Fig. 2) and in the hypocotyl of the acl5 mutant [34]. Their place was occupied by the simple type of vessel elements with spiral or reticulate secondary cell wall thickenings. This phenotype together with analysis of cytological and molecular markers showed that the cell death of the xylem vessel elements proceeded too fast in the acl5 mutant, and that the time required for xylem cell maturation, including the extensive deposition of the cell wall material, was inadequate [34]. The data therefore supported a safeguard role for
Even before the unequivocal identification of ACL5 as a thermospermine synthase, the search for targets that delineate the involvement of this enzyme in the regulatory pathway that controls stem growth and xylem differentiation e had been approached from different angles. The reduced size of the cells in acl5 mutants led to the hypothesis that ACL5 would control the expression of genes that influence the mechanical properties of the cell wall, given that cell enlargement is strongly dependent on extensibility of the cell wall and also turgor pressure inside the cell. In fact, the expression of several endo-xyloglucan transferases (EXGTs) is altered in acl5, as well as in other acl mutants [1,19]. These enzymes have been shown to participate as cell wall-loosening activities necessary for cell expansion [11], so the correlation between reduced size of acl5 cells and lower expression of this set of genes has physiological meaning. However, the effect on EXGT expression might also be a consequence rather than the primary cause of phenotype of acl5 mutants, considering the misregulation of cell death in these mutants [34]. The same reasoning holds true for the expression of the gene encoding tonoplast intrinsic protein g-TIP, found to be reduced at least 100-fold in the acl5 mutant [19]. This protein is one of the aquaporins responsible for water transport through the vacuolar membrane and the increase in turgor pressure during cell expansion [32], but the significance of the reduction in expression level has not been unequivocally established in relation with the acl5 phenotype. A less biased, genetic approach has also provided important additional information on the molecular mechanism that underlies ACL5 action. In an attempt to identify possible functional targets of ACL5, four extragenic suppressors of the acl5 mutation were isolated in Arabidopsis [23]. One of them, sac51-d, was found to suppress, in a dominant manner, all the defects described to date associated to acl5 loss of function, such as stem growth, vascular development, and the expression of EXGT and g-TIP genes. SAC51 encodes a bHLH transcription factor that belongs to subfamily 13 in Arabidopsis [23]. The similarity thus extends to the rest of over 140 bHLH transcription factors in the Arabidopsis genome [21,43], but it is restricted to the bHLH domain necessary for DNA binding and dimerization. In fact, the main distinctive characteristic of SAC51 is the presence of an unusual long 50 -UTR in the corresponding mRNA, preceding the bHLH ORF. More interestingly, this 50 -UTR contains five overlapping uORFs, and the dominant mutation of the sac51-D allele truncates the reading frame of the fourth uORF. Small uORFs are usually implicated in translational control of the corresponding main ORF [31], and this is also the case in SAC51. Expression of the GUS mRNA under the
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Fig. 2. Vascular defects caused by loss of thermospermine synthase activity in Arabidopsis. The photographs show the transverse (A, B) and longitudinal sections (C, D) of the inflorescence stems of 35-day-old plants. The vascular bundle is larger in acl5 than in the Ler wild-type. Xylem in acl5 consists of spiral and reticulate types of vessel elements and parenchymatic cells, while the wild-type contains in addition pitted vessel elements and high abundance of fibers. Even though xylary fibers are missing in the acl5 mutant, the presence of heavily lignified interfascicular fibers is greatly enhanced. IF, interfascicular fibers; PX, protoxylem; MX, metaxylem; Fs, xylary fibers; Vs, vessel elements. The horizontal bar indicates 200 mm.
control of the SAC51 promoter and either the wild-type or the sac51-D leader sequences was the same, but the GUS activity was much higher when the GUS ORF was preceded by the sac51-D 50 UTR, indicating that the wild-type uORF controls the translation of the SAC51 bHLH protein [23]. The dominant nature of the sac51-D allele supports a model in which the growth defect of acl5 mutants can be rescued by allowing (or enhancing) the production of the SAC51 bHLH protein. Additional evidence supporting this model came from the identification of a second dominant extragenic suppressor, sac52-D. SAC52 turned out to encode ribosomal protein L10 (RPL10A), and the mutant allele causes the change of Gly14 to Ser14 [22]. Although this residue is not conserved in L10 proteins from other organisms, the mutant allele of SAC52 indeed allows enhanced translation of the bHLH transcription factor encoded by SAC51 in an acl5 mutant background. This result strongly suggests that both suppressors coincide in their suppression mechanism: that the production of the SAC51 bHLH protein rescues the defects caused by loss of thermospermine synthase activity in acl5 mutants.
4.4. A model for thermospermine action The involvement of ACL5 in regulation of xylem differentiation and the identification of the SAC51 bHLH transcription factor as a suppressor of the acl5 mutant phenotype prompts the design of at
least one hypothetical model for the involvement of thermospermine in vascular differentiation (Fig. 3). The model contemplates thermospermine synthesis as a safeguard mechanism triggered at the cambium when the xylem differentiation program is initiated. Support for this idea comes from the regulation of ACL5 expression by auxin, and a prediction to be tested would be whether ACL5 expression is also under the control of patterning genes in the cambium, such as those encoding HD-ZIP family III transcription factors. In any case, an important role of thermospermine is to prevent cell death of xylem vessel elements before their differentiation is complete. An attractive scenario is that SAC51 acts as a negative regulator of the expression of genes, such as XCP2 and BFN1, which control xylem vessel maturation. The cell-autonomous production of thermospermine in vessel elements undergoing differentiation could then maintain a high level of translation of the SAC51 protein by interfering with the repression of translation imposed by the uORF in the corresponding 50 -UTR. According to this model, the lack of thermospermine would decrease the production of the SAC51 bHLH, which would result in derepression of cell death genes. The model is supported by previous reports on the function of polyamines as modulators of translation in plants [5]. For instance, the 50 -UTR region of plant SAMDC genes contains two uORFs, a tiny uORF and a small uORF, whose sequence is highly conserved in the plant kingdom, including Chlamydomonas [18]. The small uORF inhibits the translation of the main ORF encoding SAMDC [17],
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acquired only recently. Nevertheless, it is possible that the biochemical function of thermospermine in this process e be it the regulation of translation efficiency of a set of target genes, or an alternative one e reflects an equivalent activity in the ancestor which is possibly conserved in non-vascular plants. This issue could be experimentally addressed by examining the phenotype of Physcomitrella patens defective mutants in the orthologous ACL5 gene. 5. Conclusions
Fig. 3. An hypothetical model for the regulation of xylem differentiation by thermospermine. Genes encoding HD-ZIP III transcription factors mark the procambial/ cambial cells that will undergo xylem development, which is initiated in an auxin dependent manner. The differentiation program culminates with the formation of mature xylem vessels. Concurrently, a safeguard mechanism is activated (in red) by the same signals, to prevent premature death of developing xylem vessels. This mechanism includes the upregulation of ACL5 expression resulting in increased thermospermine concentration. This polyamine hypothetically interferes with the translational control of SAC51, therefore ensuring an appropriate level of the bHLH transcription factor that acts as a negative regulator of cell death related genes (for instance the ones encoding the ribonuclease BFN1 or cysteine protease XCP1). Currently there is no experimental evidence for the regulation of translation efficiency by thermospermine, or for the direct regulation of cell death genes by SAC51 (dotted lines).
and this inhibition is counteracted by translation of the preceding tiny uORF. When polyamine levels are high, translation of the tiny uORF decreases, and the small uORF accumulates, thus reducing translational efficiency of the main ORF. A likely mechanism in this case would be leaky ribosomal scanning, because the AUG of the tiny uORF is located in a poor context of a Kozak sequence, which makes it more sensitive to the general inhibitory effect of polyamines upon translation [18]. An alternative possibility which cannot be ruled out at this point is that thermospermine regulates xylem differentiation through the interaction with yet unidentified elements. In that case, SAC51 would not be a physiological target for thermospermine, as suggested above, but suppressor mutations in this gene and in RPL10A would merely compensate the defect in vascular development through a parallel pathway. One way to distinguish between these two possibilities and obtain information on the physiological role of SAC51 in xylem differentiation and stem growth would be to analyze the phenotype of knockout mutations in this bHLH gene. Moreover, ACL5 has also been proposed to regulate polar auxin transport, and therefore vascular development [7]. Again, it is difficult to assess if impaired polar transport in acl5 mutants is a cause or a consequence of the defect in the formation of the vasculature. 4.5. Evolutionary implications Although no spermine synthase gene has been described in plants outside the angiosperms, thermospermine synthase genes appeared very early in the plant lineage, possibly as a result of horizontal transfer from ancient prokaryotes [33]. Thus, the presence of thermospermine is not exclusively associated to tracheophytes, and its role in the control of xylem differentiation has been
Considering the dramatic effect of the loss of thermospermine synthase activity on plant development, the question raises of whether the different alterations are all linked to the same primary defect, or thermospermine has multiple roles in development. In other words, is the stem growth defect a consequence of the alteration of xylem differentiation? The lack of mature xylem vessels could hinder the transport of water necessary for plant growth, thus explaining the dwarfism of acl5 mutants. Moreover, the lack of xylary fibers could result in the lack of physical support for growth. In any case, the analysis of acl5 suppressors indicates that the ability to restore normal size is, so far, always linked to the capacity to rescue the defects in the formation of mature xylem elements. If this is the case, thermospermine would stand out as the polyamine responsible for the correct development of a vast amount of land plants. References [1] T. Akamatsu, Y. Hanzawa, Y. Ohtake, T. Takahashi, K. Nishitani, Y. Komeda, Expression of endoxyloglucan transferase genes in acaulis mutants of Arabidopsis. Plant Physiol. 121 (1999) 715e722. [2] N. Bagni, K. Ruiz-Carrasco, M. Franceschetti, S. Fornale, R.B. Fornasiero, A. Tassoni, Polyamine metabolism and biosynthetic gene expression in Arabidopsis thaliana under salt stress. Plant Physiol. Biochem. 44 (2006) 776e786. [3] S. Baima, F. Nobili, G. Sessa, S. Lucchetti, I. Ruberti, G. Morelli, The expression of the athb-8 homeobox gene is restricted to provascular cells in Arabidopsis thaliana. Development 121 (1995) 4171e4182. [4] S. Baima, M. Possenti, A. Matteucci, E. Wisman, M.M. Altamura, I. Ruberti, G. Morelli, The Arabidopsis athb-8 hd-zip protein acts as a differentiationpromoting transcription factor of the vascular meristems. Plant Physiol. 126 (2001) 643e655. [5] J. Carbonell, M.A. Blázquez, Regulatory mechanisms of polyamine biosynthesis in plants. Genes Genomics 31 (2009) 107e118. [6] M.K. Chattopadhyay, C.W. Tabor, H. Tabor, Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (schizosaccharomyces pombe). Proc. Natl. Acad. Sci. U S A. 99 (2002) 10330e10334. [7] N.K. Clay, T. Nelson, Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport. Plant Physiol. 138 (2005) 767e777. [8] A. Cona, F. Cenci, M. Cervelli, R. Federico, P. Mariottini, S. Moreno, R. Angelini, Polyamine oxidase, a hydrogen peroxide-producing enzyme, is up-regulated by light and down-regulated by auxin in the outer tissues of the maize mesocotyl. Plant Physiol. 131 (2003) 803e813. [9] A. De Marco, K.A. Roubelakis-Angelakis, The complexity of enzymic control of hydrogen peroxide concentration may affect the regeneration potential of plant protoplasts. Plant Physiol. 110 (1996) 137e145. [10] R. Friedman, N. Levin, A. Altman, Presence and identification of polyamines in xylem and phloem exudates of plants. Plant Physiol. 82 (1986) 1154e1157. [11] S.C. Fry, R.C. Smith, K.F. Renwick, D.J. Martin, S.K. Hodge, K.J. Matthews, Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem. J. 282 (Pt 3) (1992) 821e828. [12] V. Funk, B. Kositsup, C. Zhao, E.P. Beers, The Arabidopsis xylem peptidase xcp1 is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiol. 128 (2002) 84e94. [13] C. Ge, X. Cui, Y. Wang, Y. Hu, Z. Fu, D. Zhang, Z. Cheng, J. Li, Bud2, encoding an s-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development. Cell Res. 16 (2006) 446e456. [14] M.D. Groppa, M.P. Benavides, Polyamines and abiotic stress: recent advances. Amino Acids 34 (2007) 35e45. [15] K. Hamana, M. Niitsu, K. Samejima, Unusual polyamines in aquatic plants: the occurrence of homospermidine, norspermidine, thermospermine, norspermine, aminopropylhomospermidine, bis(aminopropyl)ethanediamine, and methylspermidine. Can. J. Bot. 76 (1998) 130e133. [16] N. Hamasaki-Katagiri, C.W. Tabor, H. Tabor, Spermidine biosynthesis in saccharomyces cerevisae: polyamine requirement of a null mutant of the spe3 gene (spermidine synthase). Gene 187 (1997) 35e43.
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Research article
Role of polyamines in plant vascular development Francisco Vera-Sirera a, Eugenio G. Minguet a, b, Sunil Kumar Singh c, Karin Ljung d, Hannele Tuominen c, Miguel A. Blázquez a, Juan Carbonell a, * a
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), 46022 Valencia, Spain Laboratoire de Physiologie Cellulaire Végétale, UMR CNRS 5168 e CEA e INRA e UJF, 38054 Grenoble, France c Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden d Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, SLU, SE-90183 Umeå, Sweden b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 October 2009 Accepted 14 January 2010 Available online 22 January 2010
Several pieces of evidence suggest a role for polyamines in the regulation of plant vascular development. For instance, polyamine oxidase gene expression has been shown to be associated with lignification, and downregulation of S-adenosylmethionine decarboxylase causes dwarfism and enlargement of the vasculature. Recent evidence from Arabidopsis thaliana also suggests that the active polyamine in the regulation of vascular development is the tetraamine thermospermine. Thermospermine biosynthesis is catalyzed by the aminopropyl transferase encoded by ACAULIS5, which is specifically expressed in xylem vessel elements. Both genetic and molecular evidence support a fundamental role for thermospermine in preventing premature maturation and death of the xylem vessel elements. This safeguard action of thermospermine has significant impact on xylem cell morphology, cell wall patterning and cell death as well as on plant growth in general. This manuscript reviews recent reports on polyamine function and places polyamines in the context of the known regulatory mechanisms that govern vascular development. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Thermospermine Xylem Cell death
1. Introduction Putrescine, spermidine, and spermine have been described as the most common polyamines in eukaryotes. However, in plants, recent evidence has shown that spermine is found only in angiosperms while thermospermine is likely present throughout the whole plant kingdom [33]. The importance of polyamines has become clear from the changes in polyamine levels that accompany certain developmental transitions or exposure to stress conditions. In addition, exogenous applications of polyamines have frequently been shown to affect plant growth and as well as the response against various stress factors [14,30]. Loss of function mutations in polyamine metabolism genes have shown that the diamine putrescine and the triamine spermidine are essential for life in all organisms evaluated [6,16,24,25,46]. With respect to tetraamines, although they have been proven to be dispensable for strict survival in plants, they have been shown to be involved in stress tolerance [2,48,49]. Moreover, recent results have highlighted the regulation of vascular
* Corresponding author. Tel.: þ34 963 877 872; fax: þ34 963 877 859. E-mail address: [email protected] (J. Carbonell). 0981-9428/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2010.01.011
differentiation by the tetraamine thermospermine, as reviewed in the next sections [7,20,27,34]. 2. Brief overview of vascular development All vascular tissues are derived from undifferentiated, meristematic cells (e.g. the procambium and cambium) [50]. The body plan for the vasculature in the adult plant is already established in the embryo. Certain cells along the embryo axis acquire procambial identity, and they will sustain primary growth of the vasculature. In a later developmental stage, the vascular cambium is formed giving rise to the secondary, radial growth of the vasculature in the stem. Procambial and cambial cells are polar in the sense that depending on their position, they give rise either to the phloem or the xylem. The initiation of xylem differentiation depends on the concerted action of several players [45]. Auxin plays an important role as a trigger of the developmental program, while the activity of a family of homeodomain transcription factors (HD-ZIP III) provides the necessary spatial information for the acquisition of xylem identity. Among these genes is ATHB8, which is an early marker for procambial activity [3,4]. Overexpression of ATHB8 provokes overproliferation of xylem cells [4]. Downregulation of another member of the HD-ZIP III family, ATHB15, results in plants with increased
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vascular tissue, while its overexpression leads to smaller vascular bundles [28,41]. This suggests that not all HD-ZIP III genes act as positive regulators of cambial cell specification and proliferation. Xylem is composed of the water-transporting tracheary elements, such as tracheids and the vessel elements, and the xylem fibers that provide the necessary support to the growing tissue [35]. The xylem elements typically undergo a differentiation program that involves, in a sequential manner, cell expansion, deposition of secondary cell wall material including lignin, and cell death. Cell death of tracheary elements is a well recognized example of programmed cell death during plant development, in which specific genes are upregulated to trigger destruction. This process involves the collapse of the vacuole followed by hydrolysis of the nuclear DNA and the cytoplasmic contents as a result of the activation or the release of the hydrolytic enzymes released from the vacuole [12,26]. Even though the process of xylem cell death is well described, little is known about the molecular mechanism that regulates it. 3. Broad range effects of polyamines on vascular development An early link between polyamines and plant vasculature was the identification of relatively high concentrations of putrescine, spermidine and spermine in xylem exudates of sunflower, grapevine, mung bean, and orange trees [10]. However, this finding was logically interpreted in the key of long-distance translocation of polyamines and their role in the regulation of plant development along the whole organism. This idea was pursued later by careful examination of the levels of the different polyamines along the axis of the plant, establishing correlations between the concentrations of putrescine, spermidine and spermine, and the size of the cells and the age of the tissues [38]. More relevant to vascular development, polyamine metabolism has been related to the formation of the reactive oxygen species (ROS) that play a key role in cell wall expansion and growth, vascular differentiation, and lignin polymerization [40]. During vascular differentiation, lignin is formed with the concurrence of H2O2 and cell wall bound peroxidases [9]. Enzymes that may be responsible for H2O2 generation include diamine oxidase (DAO) [42,47], and polyamine oxidase (PAO) [8]. The strong upregulation of DAO and PAO in vascular tissues and their direct correlation with peroxidase gene expression in tobacco [39] could make amine oxidases an attractive candidate for a role in the generation of H2O2 for protein cross-linking and lignification. The availability of decarboxylated S-adenosylmethionine (dcSAM) for the synthesis of longer-chain polyamines like spermidine and spermine also seems to be an important limiting step in the correct development of the vasculature. Compared with wildtype, the morphology of the vascular bundles of the bud2 mutant, defective in SAMDC4, one of the four SAM decarboxylase genes in Arabidopsis, is greatly affected, due to a significant increase in bundle size [13]. Moreover, the lignin content in bud2 mutants was at least 30% higher than in wild-type inflorescences, although cellulose content did not vary. On the other hand, the bushy phenotype of bud2 suggests a possible involvement of polyamines in signal transduction of auxin and cytokinin. 4. Thermospermine and xylem formation 4.1. ACL5 and vascular development Beyond the influence of polyamine synthesis and degradation on the formation of cell walls and lignin biosynthesis through the interference with redox metabolism, the most solid evidence available for the involvement of a polyamine in the formation of the
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vasculature was derived from molecular genetic analyses of Arabidopsis mutants impaired in stem elongation. In an attempt to identify genes controlling the architecture of the aerial part of the plant, five acaulis mutants were isolated with a defect in stem elongation which couldn't be rescued by the application of hormones known to control organ growth, such as gibberellins and brassinosteroids [19,44]. Particular attention was devoted to ACAULIS5 (ACL5), whose loss of function caused severe shortening of internodes, smaller mature leaves, and, more importantly, overproliferation of xylem vessel elements in the vascular bundles of the inflorescence stems. Given that the general cell size in elongating organs was smaller in acl5 mutants compared to the wild-type, and that the orientation of cortical microtubules in epidermal cells of the acl5 mutant was mostly longitudinal instead of transversal, as it is the case in wild-type cells, ACL5 was initially attributed a role in signaling during cell elongation in inflorescence stems. ACL5 was cloned and proposed to encode a spermine synthase based on the similarity with aminopropyl transferases and the ability of the ACL5 protein expressed in Escherichia coli to catalyze the conversion of spermidine into a tetraamine identified as spermine by HPLC analysis [20]. Interestingly, an apparent paradox was raised when another gene encoding a spermine synthase (SPM1) was identified in Arabidopsis [37], the corresponding knockout mutant did not display any defect in stem elongation [24]. But in fact, this contradiction is explained because the spermidine aminopropyl transferase encoded by ACL5 in fact catalyzes the in vitro formation of thermospermine, and not spermine [29]. These two tetraamines cannot be distinguished with the HPLC method used in previous studies, although they are easily separated by alternative procedures, such as thin-layer chromatography. Thermospermine had been detected at low concentrations mainly in archaea, bacteria and certain aquatic plants [15,36], and its presence in Arabidopsis wild-type (but not acl5) extracts has been recently confirmed in a qualitative manner [27]. Therefore, the most likely cause of the stem growth defect displayed by acl5 mutants is the lack of thermospermine, although exogenous supply of this polyamine to mutant plants allowed only very partial rescue of the wild-type phenotype [27]. Although it has been suggested that the xylem defects associated with acl5 were the result of deficient auxin transport [7], two other pieces of evidence make this a less likely possibility: a slight increase in the concentration of the natural auxin, indole-3-acetic acid (IAA), and increased expression of the IAA marker line DR5::GUS in the hypocotyls of young acl5 seedlings (Fig. 1), which must be the result of IAA transport from the apical meristem and the youngest leaves where it is synthesized. Therefore, it seems that IAA can be transported in young acl5 seedlings. It is possible that the reduced auxin transport capacity demonstrated earlier in excised pieces of the inflorescence stem is a secondary effect due to altered xylem specification and impaired growth of the acl5 mutant. Apart from the overproliferation of vascular cells observed in the stems of acl5 mutants, additional pieces of evidence suggest that thermospermine has an important role in the correct development of the vasculature. First, expression of the ACL5 gene is upregulated upon auxin treatments [20]. This regulation might be highly significant because, as indicated above, auxin serves as a signal for the initiation of cell differentiation at the vascular cambium. Second, ACL5 transcripts are detected only associated to the vascular cambium, in cells that initiate differentiation [7,34]. And third, thickvein (tkv) mutants are allelic to acl5, and they present an overall increase in leaf vascularization and in vein thickness [7]. Besides, it is noteworthy that the morphological defects of the knockout mutants in Arabidopsis BUD2/SAMDC4, with lower capacity to synthesize one of the substrates for thermospermine synthesis, closely resemble those displayed by acl5 mutants [13].
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thermospermine in guaranteeing adequate duration of vessel element differentiation. Interestingly, certain genes encoding enzymes involved in the execution of programmed cell death during xylem differentiation, such as the ribonuclease BFN1 and the cysteine protease XCP1, were among the genes with higher expression in acl5 compared to wildtype seedlings (our unpublished results). This misregulation very likely reflects premature death of xylem vessels. The final confirmation of the involvement of ACL5 in the control of the duration of xylem vessel differentiation was provided by the phenotype of plants expressing the diphteria toxin A under the control of the ACL5 promoter. In these plants, premature death of the ACL5expressing cells reproduced the stem growth defect of acl5 mutants and, more importantly, the anatomical and morphological defects in xylem formation [34]. Therefore, a likely role for thermospermine is to maintain differentiating xylem elements alive until the differentiation process has culminated. 4.3. Events downstream of ACL5
E
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Fig. 1. Auxin transport in acl5 mutants. (A) Concentration of indole-3-acetic acid (IAA) in the root, hypocotyl and cotyledons of 7-day-old seedlings grown in vitro. No statistically significant differences were observed in acl5 compared to the wild-type, using a KruskaleWallis test. (B,C,D) Histochemical GUS staining in hypocotyls of DR5:: GUS seedlings. (C) is a higher magnification photo from (B). (E,F,G) Histochemical GUS staining in hypocotyls of DR5::GUS acl5 seedlings. (F) is a higher magnification photo from (E). Seedlings were grown in vitro for seven days (B,C,E,F) or for one day in light followed by 2.5 days in darkness and an additional seven days in light, to ensure the optimal induction of adventitious roots in the hypocotyls (D,G). (H) Adventitious root formation in DR5::GUS (control) and DR5::GUS acl5. An increased capacity of acl5 to form adventitious roots in response to the appropriate stimulatory conditions is indicative of increased IAA levels in the acl5 hypocotyl. Scale bars: 50 mm.
4.2. Control of xylem differentiation by ACL5 A detailed analysis of the relative proportion of cell types associated with the vasculature revealed that, although phloem development was largely unaffected, xylem fibers and the elaborate type of vessels elements with pitted secondary cell wall thickenings were practically absent both in the inflorescence stems (Fig. 2) and in the hypocotyl of the acl5 mutant [34]. Their place was occupied by the simple type of vessel elements with spiral or reticulate secondary cell wall thickenings. This phenotype together with analysis of cytological and molecular markers showed that the cell death of the xylem vessel elements proceeded too fast in the acl5 mutant, and that the time required for xylem cell maturation, including the extensive deposition of the cell wall material, was inadequate [34]. The data therefore supported a safeguard role for
Even before the unequivocal identification of ACL5 as a thermospermine synthase, the search for targets that delineate the involvement of this enzyme in the regulatory pathway that controls stem growth and xylem differentiation e had been approached from different angles. The reduced size of the cells in acl5 mutants led to the hypothesis that ACL5 would control the expression of genes that influence the mechanical properties of the cell wall, given that cell enlargement is strongly dependent on extensibility of the cell wall and also turgor pressure inside the cell. In fact, the expression of several endo-xyloglucan transferases (EXGTs) is altered in acl5, as well as in other acl mutants [1,19]. These enzymes have been shown to participate as cell wall-loosening activities necessary for cell expansion [11], so the correlation between reduced size of acl5 cells and lower expression of this set of genes has physiological meaning. However, the effect on EXGT expression might also be a consequence rather than the primary cause of phenotype of acl5 mutants, considering the misregulation of cell death in these mutants [34]. The same reasoning holds true for the expression of the gene encoding tonoplast intrinsic protein g-TIP, found to be reduced at least 100-fold in the acl5 mutant [19]. This protein is one of the aquaporins responsible for water transport through the vacuolar membrane and the increase in turgor pressure during cell expansion [32], but the significance of the reduction in expression level has not been unequivocally established in relation with the acl5 phenotype. A less biased, genetic approach has also provided important additional information on the molecular mechanism that underlies ACL5 action. In an attempt to identify possible functional targets of ACL5, four extragenic suppressors of the acl5 mutation were isolated in Arabidopsis [23]. One of them, sac51-d, was found to suppress, in a dominant manner, all the defects described to date associated to acl5 loss of function, such as stem growth, vascular development, and the expression of EXGT and g-TIP genes. SAC51 encodes a bHLH transcription factor that belongs to subfamily 13 in Arabidopsis [23]. The similarity thus extends to the rest of over 140 bHLH transcription factors in the Arabidopsis genome [21,43], but it is restricted to the bHLH domain necessary for DNA binding and dimerization. In fact, the main distinctive characteristic of SAC51 is the presence of an unusual long 50 -UTR in the corresponding mRNA, preceding the bHLH ORF. More interestingly, this 50 -UTR contains five overlapping uORFs, and the dominant mutation of the sac51-D allele truncates the reading frame of the fourth uORF. Small uORFs are usually implicated in translational control of the corresponding main ORF [31], and this is also the case in SAC51. Expression of the GUS mRNA under the
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Fig. 2. Vascular defects caused by loss of thermospermine synthase activity in Arabidopsis. The photographs show the transverse (A, B) and longitudinal sections (C, D) of the inflorescence stems of 35-day-old plants. The vascular bundle is larger in acl5 than in the Ler wild-type. Xylem in acl5 consists of spiral and reticulate types of vessel elements and parenchymatic cells, while the wild-type contains in addition pitted vessel elements and high abundance of fibers. Even though xylary fibers are missing in the acl5 mutant, the presence of heavily lignified interfascicular fibers is greatly enhanced. IF, interfascicular fibers; PX, protoxylem; MX, metaxylem; Fs, xylary fibers; Vs, vessel elements. The horizontal bar indicates 200 mm.
control of the SAC51 promoter and either the wild-type or the sac51-D leader sequences was the same, but the GUS activity was much higher when the GUS ORF was preceded by the sac51-D 50 UTR, indicating that the wild-type uORF controls the translation of the SAC51 bHLH protein [23]. The dominant nature of the sac51-D allele supports a model in which the growth defect of acl5 mutants can be rescued by allowing (or enhancing) the production of the SAC51 bHLH protein. Additional evidence supporting this model came from the identification of a second dominant extragenic suppressor, sac52-D. SAC52 turned out to encode ribosomal protein L10 (RPL10A), and the mutant allele causes the change of Gly14 to Ser14 [22]. Although this residue is not conserved in L10 proteins from other organisms, the mutant allele of SAC52 indeed allows enhanced translation of the bHLH transcription factor encoded by SAC51 in an acl5 mutant background. This result strongly suggests that both suppressors coincide in their suppression mechanism: that the production of the SAC51 bHLH protein rescues the defects caused by loss of thermospermine synthase activity in acl5 mutants.
4.4. A model for thermospermine action The involvement of ACL5 in regulation of xylem differentiation and the identification of the SAC51 bHLH transcription factor as a suppressor of the acl5 mutant phenotype prompts the design of at
least one hypothetical model for the involvement of thermospermine in vascular differentiation (Fig. 3). The model contemplates thermospermine synthesis as a safeguard mechanism triggered at the cambium when the xylem differentiation program is initiated. Support for this idea comes from the regulation of ACL5 expression by auxin, and a prediction to be tested would be whether ACL5 expression is also under the control of patterning genes in the cambium, such as those encoding HD-ZIP family III transcription factors. In any case, an important role of thermospermine is to prevent cell death of xylem vessel elements before their differentiation is complete. An attractive scenario is that SAC51 acts as a negative regulator of the expression of genes, such as XCP2 and BFN1, which control xylem vessel maturation. The cell-autonomous production of thermospermine in vessel elements undergoing differentiation could then maintain a high level of translation of the SAC51 protein by interfering with the repression of translation imposed by the uORF in the corresponding 50 -UTR. According to this model, the lack of thermospermine would decrease the production of the SAC51 bHLH, which would result in derepression of cell death genes. The model is supported by previous reports on the function of polyamines as modulators of translation in plants [5]. For instance, the 50 -UTR region of plant SAMDC genes contains two uORFs, a tiny uORF and a small uORF, whose sequence is highly conserved in the plant kingdom, including Chlamydomonas [18]. The small uORF inhibits the translation of the main ORF encoding SAMDC [17],
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acquired only recently. Nevertheless, it is possible that the biochemical function of thermospermine in this process e be it the regulation of translation efficiency of a set of target genes, or an alternative one e reflects an equivalent activity in the ancestor which is possibly conserved in non-vascular plants. This issue could be experimentally addressed by examining the phenotype of Physcomitrella patens defective mutants in the orthologous ACL5 gene. 5. Conclusions
Fig. 3. An hypothetical model for the regulation of xylem differentiation by thermospermine. Genes encoding HD-ZIP III transcription factors mark the procambial/ cambial cells that will undergo xylem development, which is initiated in an auxin dependent manner. The differentiation program culminates with the formation of mature xylem vessels. Concurrently, a safeguard mechanism is activated (in red) by the same signals, to prevent premature death of developing xylem vessels. This mechanism includes the upregulation of ACL5 expression resulting in increased thermospermine concentration. This polyamine hypothetically interferes with the translational control of SAC51, therefore ensuring an appropriate level of the bHLH transcription factor that acts as a negative regulator of cell death related genes (for instance the ones encoding the ribonuclease BFN1 or cysteine protease XCP1). Currently there is no experimental evidence for the regulation of translation efficiency by thermospermine, or for the direct regulation of cell death genes by SAC51 (dotted lines).
and this inhibition is counteracted by translation of the preceding tiny uORF. When polyamine levels are high, translation of the tiny uORF decreases, and the small uORF accumulates, thus reducing translational efficiency of the main ORF. A likely mechanism in this case would be leaky ribosomal scanning, because the AUG of the tiny uORF is located in a poor context of a Kozak sequence, which makes it more sensitive to the general inhibitory effect of polyamines upon translation [18]. An alternative possibility which cannot be ruled out at this point is that thermospermine regulates xylem differentiation through the interaction with yet unidentified elements. In that case, SAC51 would not be a physiological target for thermospermine, as suggested above, but suppressor mutations in this gene and in RPL10A would merely compensate the defect in vascular development through a parallel pathway. One way to distinguish between these two possibilities and obtain information on the physiological role of SAC51 in xylem differentiation and stem growth would be to analyze the phenotype of knockout mutations in this bHLH gene. Moreover, ACL5 has also been proposed to regulate polar auxin transport, and therefore vascular development [7]. Again, it is difficult to assess if impaired polar transport in acl5 mutants is a cause or a consequence of the defect in the formation of the vasculature. 4.5. Evolutionary implications Although no spermine synthase gene has been described in plants outside the angiosperms, thermospermine synthase genes appeared very early in the plant lineage, possibly as a result of horizontal transfer from ancient prokaryotes [33]. Thus, the presence of thermospermine is not exclusively associated to tracheophytes, and its role in the control of xylem differentiation has been
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