Taku Takahashi

Polyamines: ubiquitous polycations with unique roles in growth and stress responses

BACKGROUND Polyamines are small polycationic molecules found ubiquitously in all organisms and function in a wide variety of biological processes. In the past decade, molecular and genetic studies using mutants and transgenic plants with an altered activity of enzymes involved in polyamine biosynthesis have contributed much to a better understanding of the biological functions of polyamines in plants. POSSIBLE ROLES Spermidine is essential for survival of Arabidopsis embryos. One of the reasons may lie in the fact that spermidine serves as a substrate for the lysine hypusine post-translational modification of the eukaryotic translation initiation factor 5A, which is essential in all eukaryotic cells. Spermine is not essential but plays a role in stress responses, probably through the modulation of cation channel activities, and as a source of hydrogen peroxide during pathogen infection. Thermospermine, an isomer of spermine, is involved in stem elongation, possibly by acting on the regulation of upstream open reading frame-mediated translation. CONCLUSIONS The mechanisms of action of polyamines differ greatly from those of plant hormones. There remain numerous unanswered questions regarding polyamines in plants, such as transport systems and polyamine-responsive genes. Further studies on the action of polyamines will undoubtedly provide a new understanding of plant growth regulation and stress responses.

ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon‐induced allele suggests a non‐cell‐autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock‐dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis. We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.

Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis.

In higher plants, the outermost cell layer (L1) of the shoot apex gives rise to the epidermis of shoot organs. Our previous study demonstrated that an 8-bp motif named the L1 box functions as a cis-regulatory element for L1-specific gene expression in the shoot system of Arabidopsis. We show here that PROTODERMAL FACTOR2 (PDF2), a member of the HD-GL2 class of homeobox genes, is expressed exclusively in the L1 of shoot meristems and that recombinant PDF2 protein specifically binds to the L1 box in vitro. Although knockout mutants of PDF2 and ATML1, another L1-specific HD-GL2 class gene sharing the highest homology with PDF2, display normal shoot development, the double mutant results in severe defects in shoot epidermal cell differentiation. This suggests that PDF2 and ATML1 are functionally interchangeable and play a critical role in maintaining the identity of L1 cells, possibly by interacting with their L1 box and those of downstream target-gene promoters.

Spermidine Synthase Genes Are Essential for Survival of Arabidopsis

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.

Characterization of the Class IV Homeodomain-Leucine Zipper Gene Family in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial β-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5′-GCATTAAATGC-3′ as the preferential binding site.

The polyamine spermine protects against high salt stress in Arabidopsis thaliana

It is well known that changes in abiotic conditions such as the concentration of ions, temperature and humidity lead to modulation of polyamine contents in plants. However, little is known about the relevant parts these polyamines play in abiotic stress responses. Here we addressed a specific role of spermine during high salt stress using an Arabidopsis double knockout‐mutant plant (acl5/spms) which cannot produce spermine. The mutant showed higher sensitivity to high salt than wild type plants. This phenotype was cured by exogenous spermine but not by the other polyamines putrescine and spermidine, suggesting a strong link between spermine‐deficiency and NaCl‐hypersensitivity. The mutant was also hypersensitive to high levels of KCl but not to MgCl2 or to high osmoticum. NaCl‐hypersensitivity of the mutant was compromised by treatment with Ca2+ channel blockers. Moreover, the mutant showed poor growth on Ca2+‐depleted Murashige–Skoog agar media. The data suggest that the absence of spermine causes an imbalance in Ca2+ homeostasis in the mutant plant. Based on the data obtained, we propose a model for a role of spermine in high salt stress responses.

Spermine is not essential for survival of Arabidopsis

Spermine is the final product of the polyamine biosynthetic pathway and is ubiquitously present in most organisms. The genome of Arabidopsis thaliana has two genes encoding spermine synthase: ACAULIS5 (ACL5), whose loss‐of‐function mutants show a severe defect in stem elongation, and SPMS. In order to elucidate the function of spermine in plants, we isolated a T‐DNA insertion mutant of the SPMS gene. Free and conjugated spermine levels in the mutant, designated spms‐1, were significantly decreased compared with those in the wild‐type, but no obvious morphological phenotype was observed in spms‐1 plants. We further confirmed that acl5‐1 spms‐1 double mutants contained no spermine. Surprisingly, acl5‐1 spms‐1 was fully as viable as the wild‐type and showed no phenotype except for the reduced stem growth due to acl5‐1. These results indicate that spermine is not essential for survival of Arabidopsis, at least under normal growth conditions.

Thermospermine is Required for Stem Elongation in Arabidopsis thaliana

Loss-of-function mutants of the ACAULIS5 (ACL5) gene in Arabidopsis thaliana have severe defects in stem elongation. ACL5 was previously reported as encoding a spermine synthase. A more recent study, however, showed that the bacterial expressed recombinant ACL5 protein catalyzes the conversion of spermidine to thermospermine, a structural isomer of spermine, rather than to spermine. In the present study, we found that thermospermine was detected in wild-type seedlings but was not detectable in the acl5-1 mutant. We further examined the effect of exogenous application of these isomers on the growth of acl5-1. Daily application of 0.1 mM thermospermine onto the shoot apex partially rescued the dwarf phenotype of acl5-1, while that of spermine had no effects on the morphology of the mutant. The acl5-1 transcript level in acl5-1 seedlings, which is much higher than the ACL5 transcript level in wild-type seedlings, was reduced by exogenous thermospermine. Thus we conclude that thermospermine is indeed produced through the action of ACL5 and required for stem elongation in Arabidopsis.

The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene

Loss-of-function mutants of the Arabidopsis thaliana ACAULIS 5 (ACL5) gene, which encodes spermine synthase, exhibit a severe dwarf phenotype. To elucidate the ACL5-mediated regulatory pathways of stem internode elongation, we isolated four suppressor of acaulis (sac) mutants that reverse the acl5 dwarf phenotype. Because these mutants do not rescue the dwarfism of known phytohormone-related mutants, the SAC genes appear to act specifically on the ACL5 pathways. We identify the gene responsible for the dominant sac51-d mutant, which almost completely suppresses the acl5 phenotype. sac51-d disrupts a short upstream open reading frame (uORF) of SAC51, which encodes a bHLH-type transcription factor. Our results indicate that premature termination of the uORF in sac51-d results in an increase in its own transcript level, probably as a result of an increased translation of the main ORF. We suggest a model in which ACL5 plays a role in the translational activation of SAC51, which may lead to the expression of a subset of genes required for stem elongation.

Characterization of the spermidine synthase-related gene family in Arabidopsis thaliana

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase‐deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.